Biology

I. Problem How does the absence of stomata in leaves affect oxygen production in plants

II. Hypothesis Oxygen production in plants is due to stomatal function and photosynthesis.

III. Related Literature of the Study
Photosynthesis is the process of food production in plants. All energy comes from the sun. Animals unlike plants do not have the capability of converting the energy of the sun to food. The whole planet is dependent on the plants to produce food. When grass is consumed by herbivores like cows or chicken, this is the chance how carnivores can then be nourished. Photosynthesis takes place due to the pigment chlorophyll. The equation for photosynthesis is

6 CO2  6 H2O ------sunlight and chlorophyll----------------- C6H12O6 6 O2

This means that 6 molecules of carbon dioxide and 6 molecules of water is transformed into glucose molecules of oxygen using sunlight and chlorophyll intervention. Plants use carbon dioxide for photosynthesis. In this way, unclean air with carbon dioxide is processed by plants to transform deadly carbon monoxide to oxygen.

The planet does not only get food from plants. They also produce oxygen that the planet needs which is essential for life on the Earth. Oxygen production is found in the leaves of plants. Oxygen production occurs in the granum of the choloroplast where light is absorbed by cholorophyll. Chlorophyll is a type of photosynthetic pigment that converts the light energy to chemical energy. This chemical energy reacts with water. The output of this reaction is the production of oxygen and glucose.

This oxygen output is one of the crucial benefits in tree planting and reforestation. With more trees in the environment, oxygen is secured. Urban areas that do not have trees suffer excessive pollution due to carbon dioxide accumulation in the surroundings. The oxygen in the earths atmosphere is entirely the product of green plants If all the plants were to die, or the earth be cut off from sunlight, the oxygen in the atmosphere would soon be gone. Even if all life were removed from the earth in a moment, the oxygen in the atmosphere would combine with rocks and soil and other inorganic materials in a fairly short time. Interestingly, the very oxygen produced by green plants is what allows life to live out of the water. Until there was oxygen in the atmosphere, there could be no ozone layer, since ozone (O3) is another form of oxygen (O2). And the ozone layer is what protects life on the surface of the earth from powerful, damaging ultraviolet radiation from the sun.

Transpiration occurs in the leaves of the plants. Transpiration is affected by different factors. It is affected by the amount of light available to the plant. The light helps the stomata to open. It also warms the leaf that speeds up respiration. Since transpiration is the evaporation of water from plant, if the plant is subjected to high temperatures, water evaporates more rapidly. To illustrate this, transpiration can occur three times faster if with a 10 percent increase in degrees centigrade temperatures. Humidity is another factor in transpiration. Transfer of liquids from two regions flow from a region that has high humidity to an area with low humidity. Water inside the plant will go out of the plant faster if the air outside the plant is drier. Wind is also a factor because wind speed affects humidity thereby affecting substance transfer. Humid air is replaced by drier air if there is available wind or breeze. Another factor that affects transpiration is the rate of how the plant water is replaced by available water in the soil. If roots fail to absorb water, there is loss of turgor pressure that brings about the closing of the stomata. Immediately, the rate of transpiration which occurs in the stomata closes, and the plant begins to wilt. The volume of water lost in transpiration can be very high. It has been estimated that over the growing season, one acre of corn plants may transpire 400,000 gallons of water. As liquid water, this would cover the field with a lake 15 inches deep. An acre of forest probably does even better.

IV. Importance of the Study
The study attempts to replicate proof that stomata presence helps plants in photosynthesis. Oxygen production of plants is a byproduct of photosynthesis. It also attempts to illustrate the transpiration process. Understanding the processes found in plants helps in the appreciation of their role in the protecting the environment and life.

Photosynthesis is one of the major reasons why the Earth is the planet for all living things. It is imperative that people understand the role of plants in photosynthesis and how photosynthesis is enhanced or hindered. Many activities by human beings lead to the environments oxygen depletion such as cutting of trees, water pollution and soil degradation. Using this experiment as guide, the young scientist can devise ways that can sustain oxygen production by taking importance in the role of plants in the environment.

V. Materials
A. Prepare the following materials
Two pop bottle terrariums.
2 bunch of plants where stems and leaves are still intact with about the amount of leaves
Well-lighted and safe area where there is sunlight
Colorless Nail Polish to coat the whole leaf.

VI. Methodology
Label the bottles A and B. A will be the control.
Get 2 stems of leaves from a nearby bush.
Apply colorless nail polish on the leaves in one bunch of plants. The whole leaf must be coated with nail polish.
Put the plants in two separate bottle containers.
Leave the containers under direct sunlight for three hours.
Observe for dewdrop accumulation in the glass jar.
Observe the color of leaves.
Observe the quality appearance of leaves.

Bottle A -  Control Bottle B     (nailpolish)

V. Results
Table 1. Oxygen Production Observation after four hours in the sunlight.
Pop Bottle Terrarium APop Bottle Terrarium BWater Drops AccumulationWater drops can be seen around the glass bottle.There are no water drops found around the glass bottle.Color of LeavesGreenPale yellowAppearance of LeavesHealthy and looking normalCurled up, wilted
After exposing the bottles under sunlight for four hours, it was observed that Control Bottle A had more moisture accumulation observed on the glass bottle compared to B. In terms of color of leaves, A had more green leaves compared to B. The leaves in bottle B started to wilt and most of the leaves were wilted. The leaves were all curled up and had started to wilt.

VI. Discussion
Coating the leaves with colorless nail polish maintained the green colors of the leaves but essentially blocked the stomata found in the leaves. Without the stomata, photosynthesis and transpiration could not take place in Bottle B.

The water found around the water bottle came from the release of water by the plants to the atmosphere. This is possible if photosynthesis is allowed to occur. Photosynthesis occurs when sunlight, air and carbon dioxide and water is present in the plant. Even though the plants used for the experiment were just cuttings for nearby bushes, the plants have existing water found inside their stems and leaves. Turgor pressure is still working at the whole duration of the experiment. Since the stomata in the control bottle was still working, and with enough sunlight, transpiration occurred transferring the water in the plant to be released toward the immediate surroundings of the plant. Since water vapor was trapped inside the water bottle, it accumulated on the glass as water droplets.

The presence of water droplets on the glass bottle signifies the amount of oxygen that was produced by the plant. Transpiration occurs in the stomata. The presence of the water droplets signify that the stomata of the leaves are working. If the stomata is present, there is a good chance to not that with enough sunlight, photosynthesis is at work. And if photosynthesis is present, oxygen which is a by product of photosynthesis is present.

On the other hand, the plant that had nail polish coated leaves had their stomata closed and inactivated. Due to this, transpiration cannot occur, that is why no moisture droplets were found on the bottle of B. Instead, the wilting of the leaves can be observed. The wilting is due to loss in turgor pressure brought about by the inability of the stomata to do its function.

VII. Conclusion
I therefore conclude that stomata presence helps oxygen production in plants.

The word cancer refers to any malignant tumor of any part of the body. According to Willis, a cancer, or a neoplasm is an abnormal mass of tissue the growth of which exceeds and is uncoordinated with that of the normal tissues and persists in the same excessive manner after the cessation of the stimuli which evoked the change. A cancer can therefore be said to be a disorder of cell growth and behavior. Cancers are one of the major causes of deaths occurring from medical conditions. Overall, it is estimated that about 1.4 million new cancer cases will occur in 2006, and 565,000 people will die of cancer in the United States. Recent estimates also from the World Health Organization suggests that cancer leads to about 12 of all human deaths, claiming over 6,000,000 lives each year, and that cancer is the second leading cause of death specifically in the United States, being responsible for approximately one in every four deaths (Aggarwal  Shishodia). Being then a serious condition, the ultimate cause of cancer needs to be found. Because cancer occurs at the cellular level, the cause has to be found at this level.

The Multistep Carcinogenesis Theory (Molecular Basis)
It has been postulated that carcinogenesis is a multistep process at both the phenotypic and the genetic levels, resulting from the accumulation of multiple mutations. Continuous buildup of somatic mutations in a particular single cell line over a long period of time results in a morphologic progression from a normal epithelium to hyperproliferative, dysplastic and finally the development of neoplastic epithelium.

Figure 1. Molecular model for the evaluation of colorectal cancers through the adenoma-carcinoma sequence.

Fig. 1 above clearly illustrates this diagrammatically with respect to the evolution of colorectal cancers through the adenoma-carcinoma sequence. DNA damage from accumulated exposure to carcinogens become obvious during the development of neoplasia. Several researchers have discovered the role of some specific genes in neoplastic transformation. These genes, when altered, ultimately result in cancer. There are four classes of these genes

Growth-promoting proto-oncogenes  which encode growth factors, growth factor receptors, proteins involved in signal transduction, cell cycle regulators and nuclear regulatory proteins.

Growth-inhibiting tumor suppressor genes  encoding proteins that regulate nuclear transcription and cell cycle such as the Rb gene, p53, BRCA-1 and BRCA-2, NF-1 and NF-2, APC, WT-1, TGF- receptor, and p16(INK4a).

Apoptotic genes (genes that regulate programmed cell death) such as bcl-2, bcl-xS, bcl-xL, bad, bid, and bax.

DNA-repair genes.

These genes affect all the stages of tumor progression. The initiation stage is fast and irreversible. It is at this stage that the cells DNA is bound and damaged, that is, mutation occurs at this stage. Promotion, which involves epigenetic mechanisms, leads to premalignancy and is generally irreversible while progression, which is due to genetic mechanisms, is the period between premalignancy and the cancer and is also generally irreversible.

Cancer Prevention Strategies
A lot of scientists and researchers believe that most cancers can be prevented, if not cured. As the popular maxim goes, Prevention is better than cure, what then are the prevention modalities in existence There are three major cancer prevention strategies the primary prevention strategy, the secondary prevention strategy and the tertiary prevention strategy. The ultimate aim of these prevention strategies is to reduce the incidence rate of cancers and therefore the fatalities caused by this condition. Primary prevention strategies involve the elimination of the etiologic and risk factors associated with the development of cancers. They prevent de novo cancer formation in an otherwise healthy individual. This may involve lifestyle modifications and the avoidance of some causative agents such as carcinogens being encountered in every individuals everyday life. Secondary prevention strategies involve the use of natural or synthetic substances to actually reduce the risk or spread of malignant tissue transformation in individuals with known premalignant lesions. One major secondary prevention strategy is cancer chemoprevention. Tertiary prevention focuses on the prevention of SPTs in patients cured of their initial cancer or individuals definitively treated for their premalignant lesions.

Cancer chemoprevention is defined as the use of natural, synthetic, or biologic chemical agents to reverse, suppress, or prevent carcinogenic progression to invasive cancer. This mode of prevention employs the use of a pharmacological approach to intervene in the process of carcinogenesis and the basis of this trial is a hypothesis that interruption of any stage of the process of cancer formation will reduce the incidence rates of cancers. Although the carcinogenic process may be driven by mutation, there are clearly many epigenetic variables, particularly those relating to the action of autocrine, paracrine, and endocrine regulatory molecules, which can also be important determinants during the twenty year or more latent period before invasion and metastasis occur. It has been found out that alterations or modulations of these regulatory pathways involved in cancer development by the use of agents that block mutations of DNA presents huge potentials for the prevention of cancer.  The concept of cancer chemoprevention, based on the successful outcome of many clinical trials, presents an attractive therapy for impeding or reversing malignant transformation.

Cancer chemoprevention trials, conducted to test chemopreventive substances, are usually conducted on high risk groups in order to test the efficacy of these agents. According to Sporn and Suh (2000), the credibility of the use of cancer chemoprevention as a serious and practical approach to the control of cancer has been greatly enhanced by the publication, over the past two years, of the results of three randomized clinical trials in the field of breast cancer. These trials were conducted using three different agents which were found to be effective in the control of cancer. These agents are raloxifene, tamoxifene, and 4-hydroxyphenylretinamide (fenretinide).

One of the most important factors in conducting chemopreventive trials is the use of intermediate markers. Because cancer progression takes a long time (years) to become obvious, the evaluation of these agents might become tedious. Monitoring intermediate markers that correlate with a reduction in cancer incidence would allow a more expeditious evaluation of potentially active chemopreventive agents. A good potential source of intermediate markers is the premalignant lesions that appear before the full-blown cancer becomes obvious. The disappearance of these premalignant lesions, during the process of conducting these trials, can be said to be proportional to the efficacy of the chemopreventive agents. According to Tsao et al (2004), they may serve as intermediate endpoints for chemoprevention trials. One of these intermediate markers is intraepithelial neoplasia (IEN), which is defined as a noninvasive lesion that has genetic abnormalities, loss of cellular control functions, and some phenotypic characteristics of invasive cancer, and that predicts a substantial likelihood of developing invasive cancer. The American Association of Cancer Research Task Force defined prevention and regression of IEN as being an important clinical trial endpoint.

As mentioned earlier, before a full blown cancer can develop, there will be the alterations of a series of processes. Some of these defects will be discussed below. The essence of this is to understand the pathways of cancer formation how they can be tackled by the use of chemopreventive agents to prevent or delay the development of neoplasia.

Molecular Basis of Cancer
It has been found out that nonlethal genetic damage lies at the heart of carcinogenesis. The genetic damage (mutations) may be as a result of the actions of environmental agents such as radiation, chemicals, biologic agents (viruses), or hereditary. The genetic hypothesis of cancer implies that a tumor mass results from the clonal expansion of a single progenitor cell that has incurred the damage (i.e., tumors are monoclonal). This means that just a single cell is required to mutate before it begins mass replication to form a tumor mass.

Also, a mutation in any member of the four classes of regulatory genes  protooncogenes (growth-promoting), tumor suppressor genes (growth inhibiting), apoptotic genes, and DNA repair genes  is required for tumor development. Unlike the tumor suppressor genes that require damages in both alleles before transformation can occur, mutant alleles of the growth promoting factors are said to be dominant because only an allele is required to become mutated in order to cause transformation. Apoptotic genes are also dominant, just like the protooncogenes. They may also behave like tumor suppressor genes. On the other hand, damage of the DNA repair genes influences the survival and proliferation of cells. This damage also influences the ability of the cell to repair nonlethal damage in other genes. And like the tumor suppressor genes, two hits are needed (i.e., damage in both alleles) in order to induce genetic instability.

The process of carcinogenesis involves many steps at both the morphologic (phenotypic) and genetic levels. At the genetic level, tumor progression results from the buildup of genetic damage that is favored in most instances by alterations in DNA repair.

Basis of Cancer Chemoprevention
Just like every other pharmacological agent, the use of chemopreventive agents depends on a number of factors. One major factor that has to be considered is the mechanism of action of the agent at all levels, that is, the genetic (molecular) level up to the systemic level, and even the organism as a whole. Based on this, there are different classes of chemopreventive agents. Some of them are

Class 1  Selective inhibitors of cyclooxygenase (COX-2). Cyclooxygenases are responsible for the synthesis of prostaglandins from arachidonic acid. The fact that inflammation has a role in carcinogenesis (causes a premalignant change) has led to the development of many new pharmacologic agents. One of these agents is Celecoxib. Celecoxib has been shown to prevent colon carcinogenesis caused by azoxymethane in a standard rat model, and is now in clinical trial in cohorts of patients at high risk, such as those with familial adenomatous polyposis, or hereditary non-polyposis colorectal cancer syndromes.

Class 2  These are the Selective Estrogen Receptor Modulators (SERMs). Examples include raloxifene and tamoxifene.

Class 3  Retinoids. These substances are normal regulators of the cellular differentiation and proliferation. The retinoids bind selectively to retinoid X receptors. These receptors have the ability to heterodimerize with most other components of the nuclear receptor superfamily. Some of the members of the nuclear receptor superfamily apart from the retinoid acid receptors are thyroid receptors, vitamin D receptors, and the peroxisome proliferator-activated receptor- (PPAR-)   also called the  orphan  receptor. The major role of the retinoid X receptors is to modulate the effects of most other receptors. These receptors act as ligand-activated transcription factors when bound to retinoids and their mechanisms essentially depend on the retinoids capacity to regulate gene expression through nuclear transduction signal modulation mediated by nuclear retinoid receptors.

Class 4   Peroxisome proliferator-activated receptor- (PPAR-) ligands. Because of the ability of PPAR- to bind both fatty acids and prostaglandins, it has also become a target for investigation in colon carcinogenesis (Sporn  Suh, 2000). The PPAR- has been exploited in the treatment of Diabetes mellitus type 2 because its ligands can stimulate cells, particularly, adipocytes, to the adipogenic effect of insulin. However, recent researches have indicated the effect of troglitazone on the differentiation of human liposarcoma cells.

Most of these classes of drugs show synergism among each other, such that, they encourage the use of combination chemoprevention therapy. Combination chemoprevention is said to be a state whereby one achieves significant synergism of two drugs to obtain a desired preventive effect, while minimizing the toxic side effects of the individual components of the combined regimen. Most new classes of chemopreventive agents that are being developed today combine the effects of different drug categories to produce a single synergistic, efficacious, and less toxic agent. It is appreciated that an effective and acceptable chemopreventive agent should have certain properties (a), little or no toxic effects in normal and healthy cells (b), high efficacy against multiple sites (c), capability of oral consumption (d), known mechanism of action (e), low cost and (f), acceptance by human population (Aziz, Kumar). One of such chemopreventive agents is Resveratrol.

Cancer Chemoprevention by Resveratrol
Resveratrol is a naturally occurring substance found in grapes, red wine, berries, and some other food products. It is a phytoalexin (polyphenolic antioxidant) found to have cancer chemopreventive activities in a number of trials. Historically, the ancient Chinese and the Japanese have used root mixtures of Plygonum cuspidatum (which is now known to contain Resveratrol) to combat skin and liver disease. The cancer chemopreventive activities was first brought to light by Jang et al who demonstrated that demonstrated that Resveratrol possesses cancer chemopreventive activity against all the three major stages of carcinogenesis, i.e., initiation, promotion and progression (Cai, Udeani, Slowing et al, 1997.).

    trans-Resveratrol        cis-Resveratrol
Figure 2. The chemical structure of cis- and trans-Resveratrol
Resveratrol, also known as trans-resveratrol, has the chemical name of 3,5,4-trihydroxystilbene. The chemical structure of this compound is relevant in its mechanism of action. Resveratrol has two forms  the cis and the trans form. Because of the presence of multiple OH groups on the structure, resveratrol is classified as a polyphenol. Polyphenols are antioxidants that can react with a free radical to produce a more stable compound.

According to Jang et al (1997), Resveratrol has the following properties (i), acts as an antioxidant and antimutagen (ii), induces phase II drug-metabolizing enzymes (anti-initiation activity) (iii), mediates anti-inflammatory effects (iv), inhibits cyclooxygenase and hydroperoxidase functions (anti-promotion activity) and (iv), induces human promyelocytic leukemia cell differentiation (anti-progression activity). Apart from these properties, resveratrol also has antiproliferative properties on epithelial cells.

Resveratrol is also associated with a new concept  the French paradox. It has been discovered that the incidence rate of some co-morbid conditions such as coronary heart disease and some types of cancers is very low among members of the French population. This is in spite of the fact that the average French diet consists of high fat-containing foods such as butter, cheese, cream, etc. When the average French diet was compared to a typical American diet, it was found out that a typical French diet contains approximately 15 more saturated fat than an American diet. This coupled with the fact that Americans exercise more than the French, it should be expected that more heart conditions should be recorded. However, the reverse is the case. This paradox has been attributed to the consumption of red wine by French people.

Role of Resveratrol in Cancer
A lot of epidemiological studies have been conducted on the effect of resveratrol in preventing cancers. This effect varies among different types of cancers. Each of these cancers will be discussed below, along with the resveratrol effect.

1. Resveratrol and Breast Cancer
Breast cancer is one of the most prevalent and deadly cancers. It a major cause of deaths worldwide. It is also the most common cancer in women. High incidence rates of breast cancers have also been reported among the male population. It is estimated that 203,500 new cases of invasive breast cancer will be diagnosed in 2002, and that 39,600 women will die from the disease. The associated risk factors include older age, higher body mass index, alcohol consumption, hormone replacement, prior radiation exposure, nulliparity, family history, gene carrier status of BRCA1 and BRCA2, and prior history of breast neoplasia.

Resveratrol has been found to exert a direct antiproliferative role on the rapidly proliferating breast cancer cells in spite of the presence of estrogen receptors on the cells. Several in vitro studies have shown that resveratrol inhibited growth of 4T1 breast cancer cells in a dose- and time dependent manner, however, resveratrol was found to have no effect on the growth of 4T1-implanted tumors or its metastasis when administered intraperitoneally daily (1, 3, or 5 mgkg) for 23 days starting at the time of tumor inoculation.

The controversy exists that resveratrol is either an estrogen receptor (ER) agonist or antagonist. It is however presumed that resveratrol works with the estrogen receptor to block its activation. Also, resveratrol has been found to inhibit the proliferation of a particular ER negative human breast carcinoma cell line MDAMB-468. In addition, resveratrol significantly elevated the expression of the growth inhibitor TGF-2 mRNA without changes in TGF-1 and TGF-3 expression. These data suggested that resveratrol inhibits proliferation by altering autocrine growth modulator pathways in breast cancer cells.

2. Resveratrol and Prostate Cancer
Prostate cancer has become the most frequently diagnosed cancer and the second leading cause of cancer-related death for men in the United States. In the United States, the estimated incidence for the year 2004 was 230,110 new cases and 29,900 deaths while the lifetime risk of developing prostate cancer is 19. The risk factors for developing prostate cancer include older age, family history, race and ethnicity, and possibly dietary fat. Because of the increasing incidence rates of prostate cancer, several therapies have been sought in order to combat this cancer.

Several researches have evaluated the effect of resveratrol on prostate cancer development. The major effect of this agent is to inhibit the proliferative abilities of the malignant cells. Hsieh and Wu (1999) investigated the effects of resveratrol on growth, induction of apoptosis, and modulation of prostate-specific gene expression using DU-145, PC-3, and JCA-1 human prostate carcinoma cells. The results of this study have suggested that resveratrol inhibits the growth of CaP cells by inducing apoptosis, and also by inhibiting mitogenesis in a specific way.

Recent studies showed the unique ability of resveratrol to exert opposing effects on two important processes in cell cycle progression, induction of S phase and inhibition of DNA synthesis in prostate carcinoma cells. A mechanistic basis for the efficacious role of resveratrol in prostate cancer has been developed by Seeni et al (2008). The study was conducted using the Transgenic Rat for Adenocarcinoma of Prostate (TRAP) model.  This in vivo experiment featured the rat probasin promoterSV T 40 antigen. It was found out that resveratrol suppressed prostate cancer growth and induction of apoptosis through androgen receptor (AR) down-regulation, without any sign of toxicity (Seeni et al, 2008). Resveratrol not only downregulated androgen receptor (AR) expression but also suppressed the androgen responsive glandular kallikrein 11 (Gk11), known to be an ortholog of the human prostate specific antigen (PSA), at the mRNA level (Seeni et al, 2008). The results of the experiment are stated below

Table 2. Incidences of Prostate Adenocarcinomas in TRAP Rats Treated with Resveratrol
Source Seeni et al (2008)

TreatmentNo. of ratsIncidence of Adenocarcinoma ()VentralLateralDorsalAnteriorControl1211 (92)4 (33)00Resveratrol 50gml1210 (83)2 (17)00Resveratrol 100gml1210 (83)5 (42)00Resveratrol 200gml129 (75)2 (17)003. Resveratrol and Lung Cancer

The leading cause of cancer deaths in the world is lung cancer. For the year 2004, 173,770 new cases and 160,440 deaths are anticipated in the United States (Tsao et al, 2004). Lung cancer is also one of the most preventable forms of cancer because its most important risk factor, smoking, can easily be avoided in both youths and adults. Other risk factors for lung cancer, apart from smoking, includes exposure to carcinogenic agents such as polycyclic aromatic hydrocarbons, asbestos, radon, nickel, benzoapyrene, etc exposure to ionizing radiation and infectiousinflammatory conditions of the lungs such as chronic obstructive pulmonary diseases (COPD)  chronic bronchitis and emphysema.

A study carried out by Kimura and Okuda (2000) evaluated the effects of stilbene glucosides extracted from some grapes on tumor growth and lung metastasis in mice bearing highly metastatic Lewis lung carcinoma (LLC) tumors. It was found out that tumor growth in the right hind paw and lung metastasis were inhibited by oral administration of resveratrol 3-O-D-glucoside and 2,3,5,4-tetrahydroxystilbene-2-O-D-glucoside for 33 consecutive days, in LLC-bearing mice (Kimura  Okuda, 2000). The results of the study are as follows as stated by Aziz and Kumar (2003)

Inhibition of DNA synthesis in LLC cells by Resveratrol 3-O-D-glucoside at a concentration of 1000M, but not at lower concentrations.

Inhibition of DNA synthesis in LLC cells by 2,3,5,4-Tetra-hydroxystilbene- 2-O-D-glucoside also (IC50  81M).

In addition, both stilbene glucosides were found to inhibit the formation of capillary-like tube networks (angiogenesis) of HUVECs at concentrations of 100-1000M.

The conclusion of the authors of this study shows that the activities of these resveratrol variants may be as a result of inhibition of DNA synthesis in LLC cells and the inhibition of angiogenesis in HUVECs.

4. Resveratrol and Skin Cancer
According to the World Cancer Report, skin cancer constitutes 30 of all newly diagnosed cancers in the world and solar ultraviolet (UV) radiation, particularly its ultraviolet B (UVB) component (290320nm), is an established cause of 90 of skin cancers (Aziz, Reagan-Shaw  Wu et al, 2005). Associated risk factors for skin cancer include childhood and chronic sun exposure, individual susceptibility with red or blond hair and fair-skinned phenotype, older age, polycyclic aromatic hydrocarbon, immunocompromised status, or xeroderma pigmentosum.

A study carried out by Aziz et al (2003) evaluated the chemopreventive effects of resveratrol on the development of skin cancers in hairless mice. SKH-1 hairless mice were subjected to chronic UVB exposure (180mJcm2, twice weekly) for a period of 28 weeks resulting in the development of a variety of skin tumors. Two protocols were observed in conducting this experiment. The results, as shown in Figure 3 below, clearly demonstrate that topical application of resveratrol on mouse skin (pre- and post-treatments) resulted in a highly significant inhibition of tumor incidence ( mice with tumors) and a significant delay in the onset of tumorigenesis (Aziz et al, 2003). Also, resveratrol treatment also significantly reduced tumor multiplicity (tumorsmouse), measured as a function of time, compared with UVB alone group.

In another recent study, the involvement of the nuclear transcription factor B pathway in the development of skin cancer and the chemoprevention of UV damage by resveratrol was demonstrated. Based on this research on normal human epidermal keratinocytes, resveratrol was found to block UVB (40mJcm2)-mediated activation of NF-B in a dose (5, 10 and 25M resveratrol for 24 h)- as well as time- (5M resveratrol for 12, 24 and 48 h) dependent fashion.

Resveratrol treatment of keratinocytes was also found to inhibit UVB-mediated (i), phosphorylation and degradation of IB and (ii), activation of IKK. Based on these data, we suggested that NF-B pathway plays a critical role in the chemopreventive effects of resveratrol against the adverse effects of UV radiation including photocarcinogenesis.

Figure 3. Effect of resveratrol treatments on UVB exposure-mediated skin tumorigenesis in SKH-1 hairless mice. Source Aziz et al (2003).

5. Resveratrol and Colorectal Cancers
Cancers of the colon are rated to be the third leading cause of cancer-related death in both men and women. Epidemiological studies have shown that colon cancers are more prevalent in areas where diets low in fiber, folate and calcium are consumed. Other risk factors associated with colon cancers include obesity, sedentary lifestyle, male gender, alcohol, smoking, fatty foods, etc. Stronger, albeit less prevalent, risk factors that are more significant include inflammatory bowel disease and genetic disorders such as familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC).

Studies conducted by Schneider et al (2000) investigated the effects of resveratrol on the growth and polyamine metabolism of CaCo-2 human colon cancer cells. These CaCo-2 human colon cancer cells were treated with 25M resveratrol and were found to cause a 70 growth inhibition. The cell cycle was halted between the synthesis (S) and the gap-2 (G2) phase. Also, resveratrol caused a significant decrease of ornithine decarboxylase (ODC) activity, a key enzyme of polyamine biosynthesis (which is enhanced in cancer growth), indicating that polyamines might represent one of several targets involved in the anti-proliferative effects of resveratrol.

In another study, vaticanol C, a resveratrol tetramer isolated from the stem bark of Vatica rassak, was found to markedly suppress growth of cancer cells by an induction of apoptosis, which was characterized by nuclear changes and DNA ladder formation, in three different human colon cancer cell lines.

Again, resveratrol has been found to increase caspase-3 (apoptotic protein) activities at 24 and 48 hour post-treatments. This study, conducted by Wolter et al (2001), investigated the effect of resveratrol on the human colonic adenocarcinoma CaCo-2 cells and was found to inhibit growth and proliferation of CaCo-2 cells in a dose-dependent manner (12.5-200M) (Wolter et al, 2001). Resveratrol also, during the course of this experiment, disturbed cell cycle progression from the S to G2 phase at 50M, whereas higher concentrations led to reversal of the S phase arrest. Levels of cyclin D1 and cyclin-dependent kinase (cdk) 4 proteins were found to decrease by resveratrol-treatment. The conclusion of this study is that resveratrol exerts its chemopreventive activities by inhibiting the cell cycle.

6. Resveratrol and Other Cancers
Resveratrol exerts chemopreventive effects on almost all cancers. Apart from the effects listed above, resveratrol also inhibits the development of some other cancers.

The role of resveratrol in preventing blood cancers was tested by Surh et al (1999). In this study conducted on cultured human promyelocytic leukemic cells, the growth inhibitory and antiproliferative properties of resveratrol were suggested to be attributable to its induction of apoptotic cell death as determined by morphological and ultrastructural changes, internucleosomal DNA fragmentation, and increased proportion of the sub-diploid cell population coupled with the fact that resveratrol treatment resulted in a gradual decrease in the expression of anti-apoptotic Bcl-2.

Another study has shown that resveratrol also exerts its antiproliferative effects on thyroid cancer cell lines. The experiment studied the effects of resveratrol on two papillary thyroid carcinoma (PTC) and two follicular thyroid carcinoma (FTC) cell lines. The results showed activation and nuclear translocation of mitogen activated protein kinase (MAPK) viz. extracellular signal-regulated kinase (ERK)-1 and -2. Cellular abundance of the oncogene suppressor protein p53, serine phosphorylation of p53, and abundance of c-fos, c-jun, and p21 mRNAs were also increased by resveratrol. It was therefore concluded that resveratrol induces the apoptosis of papillary and follicular cell lines in thyroid carcinomas.

Not much has been done as regards the role of resveratrol in preventing liver cancers. However, Carbo et al (1999) demonstrated that resveratrol administration to rats inoculated with a fast growing tumor (the Yoshida AH-130 ascites hepatoma) caused a very significant decrease (25) in the tumor cell content. This was presumed to be due to the inhibition of the gap-2 (G2)  Mitosis phase of the cell cycle. Also, flow cytometric analysis of the tumor cell population revealed the existence of an aneuploid peak (representing 28 of total), which suggested that resveratrol causes apoptosis in the tumor cell population resulting in a decreased cell number. Sun et al (2002) demonstrated that resveratrol inhibited the growth of hepatoma cells line H22 in a dose- and time-dependent manner via the induction of apoptosis.

Conclusion
Several experiments have confirmed the fact that resveratrol, a phytoalexin (polyphenolic antioxidant), indeed has antiproliferative effects on cancers. They have also shown that apart from its role in preventing cardiovascular diseases, it inhibits all the three stages of malignant transformation initiation, promotion and progression. At the present, epidemiological studies on resveratrol have suggested that it merits human consumption in food sources such as red wine, berries, grapes, etc. The success of resveratrol as a chemopreventive agent depends on vigorous and extensive testing on suitable human populations. Its pharmacodynamics and pharmacokinetics needs to be extensively tested. Resveratrol has shown remarkable potentials in the fight against cancer. However, there is still a lot to be done as regards its use as a potent and efficacious chemopreventive agent against cancer.

1. Introduction
When we talk about ethics in medical science what we refer to are the practices followed in delivering a suitable solution that would not only help the patient but would also not affect or jeopardise the life of any other living being. There is a very thin line demarcating ethical or unethical practices. Bioethics is the study of the moral principles with reference to the advancement in biotechnology and medicine and often delves into politics, law and theology.

Genetics is the latest branch of science that promises solutions for countless diseases that are known to have caused deaths or trauma to the patient as well as family members in the years before the advent of genetics. This branch of science goes deep into the matter that causes these diseases and tries to cure them from the root. Study of genomics deals with the constitution or the make up of every living body.

The term bioethics was coined by Fritz Jahr in 1927 from Greek bios  life and ethos- behaviour. This incorporates human values as well as our contribution towards the preservation of our biosphere. But since the middle of the last decade there has been a surge in this branch of studies. What can be defined as ethical and what cannot, and why not has been subjected to debates along political, religious and socio-economic concerns.

One must keep in mind that genetics has been used by man since time immemorial to create hardy pest-resistant and high-yielding varieties of crops and cattle. However, since the advancement in genetics has got a sudden boost due to the other advancements in technology like electroplating and electron microscopes, geneticists have received an impetus in the form of new equipment and motivation in the discovery of the secrets of life. In more recent years, the concerns that have been coming to the forefront, have been regarding the disclosure of the results concerning populations in a particular region and more specifically of individuals and how those results may lead to a bias in judgement.

2. Contents
This paper will deal with some of the immediate issues of involving genomics in our daily lives.
Artificial fertilization in reproduction
Human genomics
Sex selection during prenatal diagnosis
Privacy of genetic data and screening
Human gene therapy
Eugenics
Biotechnology
Cloning
Genetically Modified Organisms
Xenotransplant

2.1. Artificial fertilization in reproduction
The scientific developments that have taken place in the field of in-vitro fertilization and its use in reproduction and gene therapy, since the publishing of the Ethics, Reproduction and Genetic Control in 1987, have led to an increased interest in social and ethical aspects of tampering with nature. The fact that there has been a drastic increase in the number of couples who have problems with fertility, gives the chance of modern techniques like in-vitro fertilization and artificial insemination to prove their worth and help these couples conceive and reproduce. How these methods could be used to manipulate traits in progeny and other factors is voiced by those who study ethics in genomics. Human Fertilisation and Embryology Act that was passed in the year 1990, Chadwick, 1987 has been a significant step towards legislation of the applications of human genomics. It makes allowance for the issuing of licences for storage of gametes, treatment and research in fertility and conception disorders.

2.2 Human Genomics
Human genome analysis or genomics is the identification and analysis of the sequence of genes in the genetic make up of an individual. Decoding the genetic sequence of an individual will enable us to analyse the parts of genetic material that contribute towards a particular disorder and would provide opportunity for rectification of those that have been researched. However, the success rates of these processes and how much authority is to be given to the individual to change the genetic constitution of a living being are to be given serious thought.

Sydney Brenner, the Nobel laureate from South Africa, has said that this is the most important, most interesting and the most challenging scientific project that we have 1990, cited in Chadwick, 1992 with regard to pursuing the human genome project as he feels that this will help us understand the nature, structure and functions of our species. However, Roger Lewin disagrees and thinks it would be inappropriate allocation of research funds as others too believe that sequencing human genome will encourage stereotyping of individuals which might ignore the influences of environment on an individual. But, since the completion of this project in 2003,  man has benefitted immensely. The first instance of gene therapy was tried on a young girl in 1990, being treated at National Institutes of Health for ADA or adenosine deaminase deficiency www.history.nih.gov who is now leading a normal life. The possibilities of accurate diagnosis of diseases or prediction of the kind of diseases that a patient may fall prey to are of immense interest. On the other hand, the moral implications include causing excessive anxiety among individuals who have been tested for certain disorders. Because of the results, parents whose children have been diagnosed for vulnerability towards certain ailments may end up blaming themselves for their condition, leading to guilt and other related psychological conflicts.

2.3 Sex selection during prenatal diagnosis
The advantages attributed to prenatal diagnosis are also fraught with other kinds of dangers. One must keep in mind what kind of information is to be disclosed to the individuals or their family and what can be kept away from them. For instance, in a feudal economy where preference is given to a male child Kumar, 1985 disclosure of the gender of the foetus may lead to the unethical practise of foeticide. He mentions the presence of three groups the strict anti-abortionists, modified anti-abortionists and neutralists depending on their intensity of opposition to abortion. The neutralists believe that there is nothing wrong with the act of abortion itself, though unlimited free choice of individuals in this matter may lead to dire social consequences. The role of Church in disapproving some of these practises may be mentioned here, as it has raised several issues regarding humans playing god and going against nature.  However, one cannot do away with these tests altogether as banning these tests would also mean being left completely unaware of the presence of other grave conditions in the foetus. Pre-natal diagnosis has been one of the major breakthroughs in diagnosing human ailments and has been widely-known to prevent health complications in the foetus.

2.4. Human Gene Therapy
Gene therapy is a technique that will find wide application once it gains popularity. According to the Report on Human Genome Therapy by Prof. Harold Edgar and Prof. Thomas Tursz, UNESCO Edgar  Tursz, 1994 human gene therapy is defined as the deliberate alteration of the genetic material of living cells to prevent or treat diseases. This report mentions the cases in which gene therapy has been approved and some cases in which somatic cell gene therapy was disapproved to enhance human traits. The International Bioethics Committee engages in promoting human rights and creates awareness regarding the benefits of genetic engineering and the dangers of manipulating them for selfish gains.

2.5. Eugenics
Eugenics has been defined as an effort to improve the gene pool of a population Miller, 1997. Advocates of eugenics propound that the practise of selective breeding in humans can help in improving genetic qualities like congenital defects and overall intelligence. Chan in his paper on Eugenics Chan, 1993 writes how Singapore Ex-Premier Lee Soo-sung, blatantly expressed his belief in the endowed few who have sustained the impressive economic growth of Singapore. How this belief can affect human population and society can only be open for debate.

2.6. Biotechnology
Biotechnology is a field of biology that involves using modern technology to engineer the modification of living cells to provide some definite positive results. Biotechnology, in recent years, has been used extensively in the field of medical care, agriculture, crop production, animal rearing and environmental uses. Genetic manipulation of certain microorganisms has led to the development of many vaccines or cures for other diseases. For instance, in a very recent work by S. Lacombe and others Lacombe et al., 2010, it has been shown that interfamily transfer of plant recognition receptor bestows broad spectrum resistance to bacterial diseases.

A new development in this field has been the discovery of the immortality gene. Nanog, the master gene that allows the pluripotent stem cells to multiply limitlessly, while retaining their ability to differentiate, is predicted to help in developing embryonic stem cells for medical treatments. Bhattacharya, 2003 Even with its potential for great achievements, the use of biotechnology has been impeded time and again by certain sections of society who consider themselves the upholders of morality. The reason cited for opposition is usually that biotechnology is unnatural and blasphemous as it seeks to emulate and thereby defy god.

2.7. Cloning
Cloning may be defined as the creation of an identical genetic copy of a cell, tissue or an individual. Joshua Lederberg, the Nobel Prize winning geneticist advertised the use of genetic engineering in American Naturalist in 1966. The ethics revolving around cloning have become highly controversial. Cloning has come under the scanner because of its alleged transgression of the natural laws. The Vatican especially, has expressed its displeasure at the proliferation of research in stem-cell technology. On December 12, 2008, the Vatican released a document that opposed among other things, designer babies, human cloning and embryonic cell-research Pullela, 2008. With some people paying hundreds of dollars to create copies of their lost pets, others are wondering how long the process of cloning can be restricted before its use on humans.

John Snow felt that Cholera was caused by polluted water in which water supplies were contaminated with feces.  In supporting his hypothesis, he opted to conduct his research by the application of logical reasoning.  He was not skilled in the art of scientific matters of a preliminary basis.  However, his conductive reasoning was logical in following a pattern of evidence that proposed to him the findings leading to the cause of Cholera, and its subsequent spread to such a vast percentage of the population.

In his testing of the water from the two companies who provided the water supply, his use of silver nitrate to test levels of salt found in water was ingenious.  It confirmed his suspicions there was something in the water that caused Cholera.  The other portion of his research was performed on the symptoms of those with Cholera and with the remains of those who had succumbed to the disease.  For those living he investigated the physical symptoms that were suffered.  Main symptoms included abdominal pain with gastrointestinal ailments.  Snows thoughts were that the first symptoms of Cholera began with abdominal discomfort.  He thought it to be caused by a morbid material or poison, which acted locally as an irritant on the surface of the stomach (Eyler, 2001).  He felt that this irritant continued in its aggravation affecting the intestines, resulting in vomiting, diarrhea, and dehydration (Eyler, 2001).
 
Snow was not well versed in epidemiology.  He therefore enlisted the assistance of Farr.  Farr had performed ingenious research on stratagems in accordance with the Cholera outbreak and in supporting his work he documented all confirmed avenues, even creating a law of averages.  This law of averages was a mathematical wonder at depicting percentiles of households that would be affected and the deaths that would ensue in relation to contracting Cholera (Morabia, 2001).

The scientific method that Snow used was to provide a navigational road map in relation to water supplies in and around towns.  He documented regions of locale in which persons were exposed to or dying from Cholera and he earmarked their water source (Eyler, 2001).  This may have seemed a rudimentary design of science for those times.  But it is much like the process Police use when strategically pinpointing suspects per serial killings, to originate the source (or killer in this case).

Morabia (2001) tells us that William Farr (18071883). As Superintendent of the
General Register Office, Englands center for vital statistics was monumental in finding the cause for Cholera.  His sophisticated system involving the collection, classification, analysis, and report on causes of death.  Farr compiled a 300 page report on the 1848-49 Cholera outbreak, tracing the cholera epidemic over time and space and investigated the roles of sex, age, seasons, day of the week and soil elevation (Morabia, 2001).

Farr performed what is known today as surveillance of disease.  This was due to the law of mathematics that could predict the human mortality rate according to soil elevation (Morabia, 2001).  In his surveillance, he was incorrect in his first theory that the disease was caused by fermentation.  Farrs surveillance provided the proof in supporting Snows theory of contaminated water consumption.   The elaborate array of facts which Dr Farr has set forth with so much skill, as the result of great labor and research, will render irresistible the conclusions at which he has arrived in regard to the influence of the water supply in the causation of the epidemic (Morabia, 2001).

Farr contributed the spread of Cholera to the entrance of Zymotic materials entering the body through the lungs.  He stood by this theory in addition to Snows theory of the consumption of contaminated water.  Snow would not have been able to prove his theory on ideas alone, and without Farrs surveillance system, Snows theory would not have been accepted.  Working together yet apart, the works of Snow and Farr resulted in the discovery of the causes of Cholera.  It was deemed that Cholera was transmitted in four ways, that of personal contact, by air, by sewer vapor, and by water (Eyler, 2001).

Snow provided the correct hypothesis yet was unable to prove his theory.  Snow was lacking in appropriate documentation in the overall aspects of all conditions concerned, as his focus was primarily on investigating the routes of the contaminated water supplies.  Although his hypothesis was correct, he had no way of supporting his findings.  Farr was working just as hard as Snow on his own theory of Zymotic conditions, which was not the cause of Cholera, but was a source of exposure.  It turns out that Farrs research and documentation proved invaluable to Snows work.  Working adjacent to each other, they shared their ideas and in a cumulative effort, the cause of Cholera was found.

While Farr did not have a problem in accepting Snows theory of the consumption of contaminated water supplies, Snow acclaimed that Farrs research was simply coincidental by all accounts.  The realization of the importance of Farrs work was overshadowed as Snows theory was accepted when presented with the supporting factors of Farrs work.

This oversight was corrected through time and now Farrs work is recognized as being ingenious in creating a surveillance system of monumental capacity as far as science goes.  This system is the best in existence today.  It has assisted scientists in the continued fight against disease.

NMR is a technique vastly used for studying biological molecules, crystalline structures,
molecular physics and non-crystalline structures. It is based on the fact that all nuclei that have odd number of protons have a magnetic field  and electromagnetic pulse or pulses. Nuclei absorb energy from these electromagnetic pulses and then rediate it back in the form of resonance frequency. The strength of resonance frequency is directly proportional to the strength of applied electromagnetic pulses. So, this allows scientists to exploit this property of magnetic nuclei to suty quantum mechanical magnetic properties of an atomic nucleus. NMR spectrometry is also used in advanced medical imaging techniques such as magnetic resonance imaging (MRI).

The principle of NMR usually involves two sequential steps
The alignment (polarization) of the magnetic nuclear spins in an applied, constant  magnetic
fieldH0.

The perturbation of this alignment of the nuclear spins by employing an electro-magnetic,
usually radio frequency (RF) pulse. The required perturbing frequency is dependent upon
the static magnetic field (H0) and the nuclei of observation.

Both the fields are set perpendicular to each other to give a better NMR signal. Both
use intense magnetic fields to deliver better quality spectra result of which is
detailed in zeeman effect, chemical shifts and knight shifts (in metals).

Analytical Utracentrifuge
Analytical Ulracentrifuge is widely used in studying molecular biology, proteins and long
polymers since long. A sample is taken in an Ultracentrifuge and accelerated to a very high speed of about 3600 kms2. The sample is spun with such a high acceleration and optical detectors are used to examine the sample using ultraviolet absorption or interference optical refractive index sensitive system. The sample is monitored in real time by using the above two methods. Two types of experiments are usually performed on these instruments

Sedimentaion velocity Experiments

Sedimentaion equilibrium Experiments
Sedimentation velocity experiments are concerned the concentration distribution to find the molar mass and size of the dissolved biological macromolecules. The size resolution for this method is the square of the radii of the particle being examined,or it can be adjusted by rotor speed of experiment.

Sedimentation equilibrium Experiments are performed without having regard to concentration
ditribution of the particle dissolved but are, rather concerned with the final  steady-state of
experiment, where sedimentation is balanced by diffusing opposed to concentration gradient in
sedimentation velocity experiments.

Basic Electron Microsopy
Electron microscopy is the most powerful device for examining cellular objects, biological
molecules and other small objects that are not easily imageable using optical microscopy. In this
case, a high beam of charged electron is projected on a specimen to illuminate and find its image to a very high resolution ever possible by 1,000,000X whereas optical microsopy is only able to 1,000X. It is because of the fact that electrons ar too small as compared to photons that are used in optical microscopy.
Cyro-Electrom microscopy

Cyro-Electrom microscopy is a type of electron microscopy but in cyrogenic
temperatures,usually in liquid nitrogen temperatures. Cyro-Electrom microscopy is helpful in
studying proteins and other biochemical processes in its actual physiological environment unlike X-rat crystallography which have a retrospective impact on the sample studied because of the
articficially created environment.. Thats why cyro-Electrom microscopy is more valuable than
other technigues.

Negative Staining
Negative staining is an established method especially in diagnostic microscopy where there
is a thin specimen to be contradted with an optically opaque fluid. A negative staining technique
uses an ink to be sprinked over the sample like nigrosin and then to dry the sample,after which it is undergone an electromicroscopic treatment. The target material in the sample is seen light in contrast to the dark background.

Single Particle Analysis
Single particle analysis and electron tomography are the tools used in the structural biological analysis of proteins and other organic macromolecules. Single particle analysis in
transmission electron microscopy examines macromolecules and large proteins as opposed to
electron tomography which observes the sub-cellular portions of the organism. Electron
tomography when used as a single particle anaysis will bridge the gap between optical analysis andelectron microscopic analysis of macromelecules and other sub-cellular organelles.

Mass Spectrometry
Mass spectrometry is an analytical techniques used in the determination of biological
molecules structures and the elemental composition of organic biological moecules such as protein  etc. It employs the method of ionizing the sample and then passing it through an electric field. The charged ionized particles are deflected in the electric field based on their mass to charge ratio.

Newton second law of motion is applied in the deflection movement phenomenon.

Circular Dichroism
CD spectroscopy is relatively new technique which is based on the difference in the
absorption of left and right circularly polarized light. The phenomenon is seen in the absorption
bonds of optically chiral molecules.It has wide range of applications including structural analysis
of biological molecules etc.

SDS-PAGE
Sodium dodecyle sulphate polyacrylamide gel electrophoresis is a technique widely used in
biological forensics,protein isolation based on protein electrophoretic mobility. Electrophoretic
mobility is a function of length of polypeptide or weight of polyprotein.

Conclusion
In a nutshell, there are growing numbers of techniques in use today by reseachers and
biologists to delve deep into the protein structure and function to get fully acquainted with the life
most important constituent, the Protein. Research is currently under process to know more about the protolytic enzyme that cause Hapatitus C Virus using multiple methodologies. Although we were not able to mention the full set of techniques used in proteinology, but we did make an excellent effort to describe the overall structural based and non structural based techniques to a great deal. Yet  more and more techniques are being discovered to cover the subject of protein studies in great detail. We wish good luck to all the researchers in this field.

Phalaenopsis has established itself as the most valuable potted plants (18 million pots in sold in 2003, compared to mere 50,000 plants in 1983 in Dutch flower auctions Lopez and Runkle, 2005). In the USA Phalaenopsis alone tops with 70 - 90 of the entire Orchid group (Griesbach, 2002 Nash, 2003). Aesthetic beauty appeal, possibly olfactory attractions and the distinctly unique shelf life of these flowers even for as much as 4 long months and in a very wide spectrum of floral colors and color combinations are some of the suggested reasons. Increasing trends of Phalaenopsis domination is likely to be sustained in future if not going down.

Many market oriented breedersresearches have examined several external and recently even internal genetic factors for maximizing the flower production in Phalaenopsis in faster times because orchids in general require 4-6 years of juvenile phase from the seed stage to the stage of maturity to flower (Goh and Arditti, 2005), but most commercial orchids take only about 2-3 years probably because they are mostly tissue culture propagated. Environmental and nutrient factors then become critical for induction of flowering and therefore appropriate conditions have to be provided. This paper examines these issues.

In general there is an ongoing discussion regarding the best relative proportions of vegetative growth in relation to maximum flowering responses particularly in floriculture crops. There is a consensus that plant has to be culture in the best environment with most effective nutrient supply for the best consequences flowering. With over about 50 or so species in the genus Phalaenopsis (Pridgeon, 2000 cited by Lopez and Runkle, 2005)  and the ease of creating new hybrids coupled with ultra-rapid large scale in vitro micropropagation of  the selected hybrids provide a comfortable situation for rearing of plants. But the species diversity in floral characters such as the color combination cut flower shelf life, length of the spike itself and also more importantly the environmental and nutrient factors required for good vegetative growth just adequate for best flowering responses are quite variable. These and their combinations prevent any generalized scheme of adjusting these for maximum benefits. Further, results so far do not provide a comprehensive information on the interactions of the two broad factors and majority of studies have been confined only either to vegetative (juvenile) stage specific to different popular genera or hybrids, or to early and pre-flowering phase of this genus.

CAM in Phalaenopsis
Phalaenopsis is an epiphytic orchid characterized by Crassulacean Acid Metabolism. After the first comprehensive review on Crassulacean Acid Metabolism (CAM) was published by Wolf in 1960), many extensive researches have been carried out and reviewed periodically (Lttge, 2004). But in most of these orchids in general and Phalaenopsis in particular have been generally ignored. Lttge (2004) describes CAM as given by Osmond (1978) characterized by nocturnal uptake of CO2 via open stomata, fixation by phosphoenolpyruvate carboxylase (PEPC) and vacuolar storage of CO2 in the form of organic acids, mainly malic acid (phase I  HYPERLINK httpaob.oxfordjournals.orgcgicontentfull936629 l MCH087C205 Osmond, 1978), and (2) daytime remobilization of vacuolar organic acids, decarboxylation and refixation plus assimilation of CO2 behind closed stomata in the Calvin-cycle (phase III) .

Crassulacean Acid Metabolism (CAM) is an ecophysiological modification of photosynthetic carbon acquisition as a deviation from the well known C3 and C4 Plants. In all of these types carbon fixation pathways, six major environmental parameters CO2, H2O, light, temperature, nutrients and salinity participate in a coordinated manner at the whole plant level and also as affected by some or all these parameters. Each of these is significant in their different qualitative forms such as diurnal variations in temperature, light duration, quality and radiance and so on. 57 of all epiphytes are CAM plants.

Water use efficiency (WUE) might be the major ecophysiological property of CAM. CAM is a CO2-concentrating mechanism for adaptation to water stress because stomatal water loss through open stomata during dark is reduced by CO2 mobilization fixation (Luttge, 2004). In a CAM plant water is the most important factor as an adaptation to water-stress. Epiphytic species are especially more sensitive water stresses, in addition to particular problems of other types of plants. In a CAM plant water is the most important factor as an adaptation to water- stress because moisture loss through leaf stomata is reduced by CO2 absorption during dark via open stomata and subsequent fixation when stomata close during day time.

That Phalaenopsis is an obligate type of CAM plant is strengthened further by the experiments of Chen et al. (2008). They showed that the two groups of mature Phalaenopsis aphrodite subsp. formosana plants showed a clear diurnal oscillation of stomatal conductance, net CO2 uptake rate, malate and starch levels, and the phosphoenolpyruvate carboxylase and NAD-malic enzyme activities.

Growth and flowering have been generally excluded from each other. A discussion of environmental factors has to consider various parameters necessary for both roots zone and shoot region. Humidity and moisture, temperatures during days and night and their relations, photoperiod lengths, light intensity and quality are the major factors for both the roots and also the leaves and shoots.  

Root Zone, Potting Media, Water levels and Electrical Conductivity
Moisture in the root zone is particularly important. Humidity in the shoot zone is to be maintained around 60 - 70 and has to be critically monitored especially because Phalaenopsis is a monopodial kind of orchid. It may suffice to mention here that Wang et al. (2007) stress that inducing Phalaenopsis into flowering become easy if a good and a healthy root system is ensured. They suggest an ideal composition of a useful medium and efficient watering and fertilizer management in the early stages. Watering is crucial as Phalaenopsis is succulent unlike others which have pseudo-bulbs such as Cattleyas. Watering should be done more carefully in Phalaenopsis because of its succulent leaves. Roots should be allowed to nearly dry before rewatering can be started But can tolerate water deficiency for long periods but not more than two weeks. Also, in prolonged water deficient situation leaf photosynthetic carbon dioxide fixation goes down gradually. Long roots with light greenish tips indicate active plants.  

In the early studies such as by Sheehan (1960) examined two potting media, a specialty of orchids. This was among the first few papers which emphasized the need for well designed experiments to produce consistent results rather than just report only observations. These results also point out the importance of two commercial bark samples used due to their structure and differentially abilities to hold or release moisture and nutrients for availability to the roots.

Doritaenopsis cv. Labios and White Castle were grown with three different growing media Perlite, perlite  coir dust (31 vv) and perlite  rockwool granule (31 vv). Regardless of cultivars, plant growth on perlite  rockwool granule (31) was greatest. Perlite  rockwool granules had the best combinations of key physical characteristics such as total porosity and bulk density in addition to desirable combinations of high moisture capacity and container capacity with good air space, and electrical conductivity (Table 1). This was further proven by growing the Phalaenopsis plantlets (Table 2). Leaf pigments, chlorophylls and carotenoids were maximum on sphagnum moss followed by peat moss  perlite. Phalaenopsis plants were grown on this potting medium with three levels of media moisture content (50, 70 and 90 maximum water holding capacity) were examined. Nutrient solution with EC 1.0 dSm-1 was supplied during the culture. Plants grown at 90 showed highest growth rate but N, P, K, Ca and Mg contents in leaves and roots were fluctuating indicating that the growth rate was not related to these.

Table 1 Physical and chemical characteristics of growing media used in the experiment
         
Parameter      
MediaPerlitePerlite  coir dust Perlite  Rockwool granules (31, vv)
Total porosityz70.675.278.6
Container capacity y35.348.245.0
Air space ()35.227.032.6
Moisture capacity64.473.369.2
Bulk density (g.ml -10.180.180.16EC (dS.m-10.050.120.08Ph7.785.248.87z  
Percentage volume in container
y Predicted percentage volume at drainage

Table 2 Effect of growing medium on chlorophyll content in leaves of Doritaenopsis Tinny Tender after nine months of culture

Growing
mediumChlorophyll content (g. g -1FW)Chlorophyll abCarotenoids (g. g -1FW)   a     b A  bCoconut chips260.8 bz89.4 b350.2 b2.9 a85.7 bBark273.8 ab98.2 b372.0 ab2.8 b96.9 abPeat moss  perlite286.3 ab103.6 b389.9 ab 2.7 b93.3 abSphagnum moss297.5 a125.6 a423.1 a2.4 c101.9 az Mean separation within columns by Duncans multiple range test,   0.05

When EC of the nutrient solution maintained at 0.8 dSm-1, this potting medium resulted in the highest growth with higher number of roots than others. N, P, K, Ca and Mg contents in leaves and roots were also higher in plants grown on perlite  rockwool granule (31) than in perlite and perlite  coir dust (31).

Among four different growing media coconut husk, bark, peat moss-perlite (11) and sphagnum moss, fresh and dry weights of the White - flowered Doritaenopsis Tinny Tender plants grown in peat moss  perlite (11) mix and sphagnum moss were more than those in bark and coconut chip media. The same media resulted in better leaf growth, chlorophyll content, and N and P levels.  These media however showed lower Ca and Mg contents than in bark and coconut chips. The same two media also resulted in more flowers per plant, longer inflorescences, higher number of flowers per inflorescence, larger diameter of flowers.  Inflorescence emergence was unaffected by growing medium. Similar results were observed in another cv Labios

Doritaenopsis plants planted in culture bed filled with perlite  rock wool granule (31, vv) mix and 4 EC levels of nutrient solution were supplied. The nutrient solution for the experiment was developed by Chungbuk National University (CBNU-Phl2). At an early stage, in both cultivars, leaf growth increased with increasing EC level, of the nutrient solution, while root growth decreased with increasing EC levels. At the end of the experiment, plants grown on EC 2.0 dSm-1 showed lowest growth rate, while EC 0.8 dSm-1 resulted in the greatest plant growth. N, P, K and Mg contents in leaves increased with increasing levels of the nutrient solution but Ca contents decreased. During the first flowering season, in both cultivars, EC 1.6 dSm-1 induced larger number and size of flowers and longer stalks compared to other EC levels. Flowering was delayed by increasing EC level. During the second and third flowering season, higher flower quality was obtained at EC 0.8 dSm-1 in both cultivars. Doritaenopsis cv. Labios and White Castle were grown with three different growing media Perlite, perlite  coir dust (31 vv) and perlite  rockwool granule (31 vv). Regardless of cultivars, plant growth in perlite  rockwool granule (31) was greatest. EC of the nutrient solution maintained at 0.8 dSm-1 resulted in the highest growth with higher number of roots than others. N, P, K, Ca and Mg contents in leaves and roots were also higher in perlite  rock wool granule (31) than in perlite and perlite  coir dust (31).

Inflorescence length, number of flowers per inflorescence and flower size were greatest in perlite  rockwool granule (31) medium but inflorescence emergence was unaffected by the growing media.

Phalaenopsis plants were grown in perlite  rockwool granule (31 vv) and three levels of media moisture content (50, 70 and 90 maximum water holding capacity) were examined. Nutrient solution with EC 1.0 dSm-1 was supplied during the culture. Plants grown at 90 showed highest growth rate. On the other hand, N, P, K, Ca and Mg contents in leaves and roots were not related to the growth rate, showing fluctuations in each element. The relative water content (RWC) increased with increasing media moisture content. Chlorophyll a, chlorophyll b, and total chlorophyll content (fresh weight mgg) were highest at 90 media moisture content. Net CO2 uptake, stomatal conductance and transpiration in leaves during dark period increased progressively with increasing media moisture content, which became more significant with the duration of experimental period.

Effects of Environmental Factors and Composition of Nutrient Solution

Drought
Five-month-old in vitro cultured Doritaenopsis orchids transplanted and maintained under ex vitro condition in a greenhouse for 8 months were supplied with four nutrient solutions (at 12S, 1S, 32S and 2S with electrical conductivities of 0.9, 1.4, 1.9 and 2.4 dSm1, respectively) in ebb and flood irrigation system. Drought was imposed by withholding the supply of nutrient solution. These plants were analyzed for photosynthesis, stomatal conductance, transpiration, reducing sugar, total sugar, starch, protein, phosphoenolpyruvate carboxylase, superoxide dismutase, and peroxidase activity

Under light and dark condition photosynthesis, stomatal conductance and transpiration decreased with increasing the duration of drought. With the imposition of drought reducing sugar content increased while total sugar and starch content decreased. Protein content increased at 12S nutrient solution while decreased maximally at 32S. Drought also caused increase in phosphoenolpyruvate carboxylase and peroxidase activities but reduced superoxide dismutase activity.  Rewatering after 15 days of drought resulted in sudden revival from the stress related effects.

The relative water content (RWC) increased with increasing media moisture content. Chlorophyll a, chlorophyll b, and total chlorophyll content (fresh weight mgg) were highest at 90 media moisture content. Net CO2 uptake, stomatal conductance and transpiration in leaves during dark period increased progressively with increasing media moisture content, which became more significant with the duration of experimental period. On perlite  rockwool (31) medium, while spike emergence was unaffected, inflorescence length, number of flowers per inflorescence and flower size were greatest.

Doritaenopsis plants planted in culture bed filled with perlite  rockwool granule (31, vv) mix were fed with 4 EC levels of nutrient solution supplied for the experiment and developed by Chungbuk National University (CBNU-Phl2). At an early stage, in both cultivars, leaf growth increased with increasing EC level, of the nutrient solution, while root growth decreased with increasing EC levels.

Pre-flowering Vegetative Stem Growth
The vigor of a plant is generally reflected in its response in flowering. The vigor of a plant is generally reflected in its response in flowering. Cool night temperatures promote spike formation (Chen et al., 2008). Under constant prolonged duration of a high day and night temperature of 280C failed to induceproduce the flowering spikes but cooler temperature for at least 12 hoursday for about 6-8 weeks promoted inflorescence development in P. amabilis. This confirms Wents earlier report of observing more flower formation during cooler (20 C) at short day than at long day photo-periods. Similar observations were reported by Yoneda et al. (1991). Conversely, a diurnal fluctuation of high day and low night temperature promoted spike induction of Phalaenopsis (Lee and Lin 1984). Going by analogy provided by Sudoi and Tsutsui (1978), during night carbon dioxide fixation is more and reduces with the onset of the sunrise. In these reports, cooler temperature also stimulates flower initiation at cooler temperatures. Therefore if cooler temperatures promote greater Carbon fixation during it is very likely the spike initiation is the result of this greater carbon fixation. Sakanishi et al (1980) suggest that short day conditions may supplement to the positive lower temperature effects on spike formation and flower formation.  

Effects of Environmental factors on Physiological Characteristics of Doritaenopsis
Development of photosynthetic properties are very important for in vitro plantlets, which could be improved by altering their environmental growth conditions, such as increasing light intensity, humidity, air temperature or CO2 concentration. Humidity and temperature are crucial not only for ex vitro acclimatization of plantlets but also for a very rapid development of all normal physiological functions in a high value ornamental crop such as Phalaenopsis.

In vitro propagated Doritaenopsis New Candy plantlets acclimatized for one month. They were treated with three humidity levels (50, 70 and 90 251 C, at 12 h12 h photoperiod was used under fluorescent light (10010_molm2 s1 PPFD). In another experiment plantlets were maintained at five different temperatures 201, 251, 351 and 351 C, under 10010_molm2 s1 (PPFD). They were analyzed for different physiological characteristics including CO2 assimilation (An), stomatal conductance (Sc), transpiration rate (Tr), chlorophyll content, fresh and dry weight, leaf length, leaf area, leaf width, formation of new roots and survival rate.

Increasing humidity promoted plantlet growth as observed in fresh and dry weights, leaf dimensions, and survival rates but the number of new roots was not different. At 90 humidity fresh weight increased about 30 compared to 50 humidity.

RWC was reduced significantly after second day of transfer from in vitro to the growth chamber at 50 and 70 humidity compared to 90 humidity but as the acclimatization day progressed, RWC was completely recovered at the end of acclimatization period under all humidity stress.

Table 7 Leaf width, leaf length, leaf area of leaves, number of new roots, fresh weight, dry weight and survival rate of Doritaenopsis as affected by relative humidity (RH) and air Temperature after 30 days of acclimatizationx
RHair temperatureFresh weight (g)Dry weight (g)LeafNumber of new rootSurvival rate ()
Width (cm)Length (cm)Area (cm2Humidity506.63 b0.46 c4.0 a7.4 b9.17 b2.3 a62.2 b706.32 b0.51 b4.0 a7.7 b9.49 b2.3 a66.7 b909.46 a0.69 a4.3 a11.7 a11.17 a2.6 a92.2 aTemperature15 C    4.19 c0.30 c3.2 b6.0 b9.2 b1.0 b84.4 b
20 C7.48 ab0.54 a4.5 a6.6 a11.6 a2.3 ab91.1 a
25 C8.32 a0.59 a4.9 a6.9 a12.6 a3.0 a93.3 a
30 C6.42 b0.43 b5.1 a6.7 a12.7 a2.7 a92.2 a
35 C5.07 c0.36 c4.9 a6.6 a12.2 a2.8 a90.0 a

Mean separations within columns by different letters are significantly different according to Duncan s multiple range tests at 5 level x From Jeon et al., 2006

At 15 and 35 C, fresh weight decreased about 50 and 40, respectively compared to 25 C. Shoot dry weight decreased at both the low temperatures (at and below 20 C) and high temperature (35 C ) compared to 25 C. At low temperature (15 C), leaf area reduced more than 27 while it was less affected at high temperature (35 C) compared to 25 C grown plantlets (Table 7).

While at higher humidity (90), about 11 more new root was formed at 90 humidity compared to 50 humidity, higher temperature (25 C and above) promoted more root formation than at lower temperature (15 C).

In contrast, in temperature treated plantlets, dehydration became more serious at 15 C and RWC decreased after second day of transfer from in vitro to the growth chamber significantly and could not recover at the end of acclimatization period compared to other temperature (Fig. 2B). The dehydration was indicated by decrease of RWC.

Photosynthetic efficiency
During acclimatization on the second day itself, 50 and 70 humidity and low (15 C) and high temperature (30 and 35 C) FvFm was inhibited but recovered completely under all humidity at the end of acclimatization period (Fig. 3A). Similarly, at 15 C, FvFm decreased significantly and did not recover completely compared to 25 and 30 C grown plantlets.

Pigment content
Chlorophyll content was reduced at both low and high temperature compared to 25 C grown plantlets. About 12 chlorophyll a, 9 chlorophyll b and 12 total chlorophyll contents were decreased at 50 humidity compared to 90 humidity. At low temperature, 12 chlorophyll a, 17 chlorophyll b and 14 total chlorophyll and at high temperature 13 chlorophyll a, 11 chlorophyll b and 11 total chlorophyll were reduced compared to 25 C. The chlorophyll ab ratio was neither affected by humidity nor by temperature stress Jeon et al., 2006).

CO2 assimilation (An), stomatal conductance (Sc) and transpiration rate (Tr)
 
Under all humidity and temperatures conditions Doritaenopsis showed a typical CAM pattern of gas exchange. Both temperature and humidity stress, CO2 uptake (An) increased rapidly just after beginning of darkness and as it is a CAM orchid. An was found at night period only at both temperature and humidity stress. After 5 days of transfer, An, Sc, and Tr were decreased more at 50 humidity than 70 and 90 humidity (data not shown) and after 25 days of acclimatization, An, Sc, and Tr were increased even at 50humidity. In contrast, at 15 C, An, Sc, and Tr were highly reduced after 5 days of transfer (data not shown) and could not recover even after 25 days of acclimatization. However, under temperature, An, Sc, and Tr were higher at both 25 and 300 C compared to other temperatures. An, Sc, and Tr reached a maximum at 1900 and 0500 h under all humidity stress and all air temperatures treated plantlets. The most striking result of the present study is that increase in CO2 uptake was found in daytime at 150 C, which is also associated with the increase in stomatal conductance and transpiration except the remaining temperature.

Peroxidase activity (POD)
Peroxidase activity was determined after 30 days of acclimatization under both humidity and temperature in root. POD activity was not induced or inhibited under all humidity stresses (Fig. 2A) whereas at different temperature POD activity was highly induced and at 150 C POD activity was induced about 40 compared to 250 C (Fig.2B). However, at 20, 30 and 350 C, POD activity remained same but showed higher activity than at 250 C. Surprisingly, POD activity was not detected in leaves of Doritaenopsis (Jeon et. al., 2006).

Fig. 1. Diurnal changes in net CO2 assimilation (A), stomatal conductance (B) and transpiration (C) under humidity and net CO2 assimilation (D), stomatal conductance (E) and transpiration (F) under air temperature in Doritaenopsis grown in growth chamber.  Values are mean  S.E. (n  5).

Fig. 2 The peroxidase activity under humidity (A) and air temperature (B) stressed Doritaenopsis grown in growth chamber. Different letters are significantly different between the treatments at 5 level according to DMRT. Values are mean  S.E. (n  3).
x From Jeon et al., 2006

Effect of Nutritional and Environmental Factors on Flowering
The popularity of Phalaenopsis is just due to its attractive flowersinflorescences with many colorscolor combinations, shapes, scent, and prolonged cut flower shelf life only for its flowers. Interest in cultivating Phalaenopsis is justified and critical care has to be taken to provide appropriate environmental as well as nutritional conditions for promoting flowers in large marketable numbers in short time. Marketability is important because Blanchard et al (2007) observed that only about 50 of the flowers produced really have good market value.

Only recently there has been some activity in working out the necessary parameters accurately for producing repeatable results from a growers point. Until recently it was just accepted that temperatures higher than 25 C inhibited reproductive growth and lower temperatures than 25 C promote flowering (Ichihashi, 2003). Ichihashi studied light, carbon-nitrogen balance and cool temperatures and still concluded that more studies were required.

Jeon et al (2006) studied carbon metabolism in relation to spiking. Cool night temperatures promoted accumulation of free sugars. cool night temperatures (15  20 C ) also promote flowering  and also that temperature differences in night and day were critical (Chen et al.,  2008).

Recently Newton et al., (2009) investigated six different varieties of Phalaenopsis and found that there were cultivar specific differences with respect to exposure to 12 hours and longer at higher temperatures but still for majority of the varieties, cool temperature for less than nine hours seemed necessary for flower induction and formation. Earlier Sakanishi et al (1980) had reported that a short day condition may add to low temperature effects on flower induction.

Higher nitrogen levels promoted longer flower stalk length, inflorescence length and number of flowers per stem than lower nitrogen levels (Ruamrungsri et al., 2007).

Conclusions
Orchids are extremely in species diversity with genomic flux and still evolving. Many intergeneric hybrids are still possible. Orchids have a genome which is considered the shored of all. Angiosperms (Luttge, 2004). Yet number of varieties released by breeders is very high
In such a situation, it may not be out of context to mention that a generalized mechanism of cultural conditions for best and consensus responses from Phalaenopsis cultivars may be unexpected. It is worth mentioning that the kind of CAM plasticity observed in different eighteen individual species across Cymbidium reported recently by Motomura et al. (2008) due to weak andor strong CAM habits related to their respective habitats may exist in Phalaenopsis.

It is just the beginning of investigating into physiological and ecophysiological factors needed for this crop in general and for all its new and old popular varieties. Even with the limited amount of information, it is to be noted that there are possible and observed variations in responses to the environmental factors and nutritional aspects investigated so far. A lot more of information would be necessary to even hypothesize a theoretical scheme of factors for optimum productivity in Phalaenopsis plants and inflorescences. There is some new information on molecular aspects of some functional genes   including flowering phenomena. With the new and latest tools breeding for trait related factors may become easier.

Hepatitis is the injury to the liver with inflammation of the liver cells.1 It is commonly caused by a virus and the usual etiologic agents are named hepatitis A, B, C, D, E viruses.2 Drugs or alcohol use can also lead to hepatitis.3 However, most liver damage is caused by hepatitis A, B, and C viruses.1 Serious cases can lead to life-threatening complications like liver cirrhosis (scarring), liver failure and liver cancer.4  The typical symptoms of hepatitis are jaundice (the yellowish discoloration of eyes and skin), fatigue, abdominal pain, headache, low grade fever and even loss of appetite, to name a few. Each hepatitis disease has its own mode of transmission, prevention and treatment.

Hepatitis A, caused by the hepatitis A virus (HAV) is spread primarily through food or water contaminated by feces from an infected person. 2 Hepatitis A usually resolves itself over several weeks. Hepatitis B is caused by the hepatitis B virus (HBV) and is spread through contact with infected blood, sex, and from mother to child during childbirth.2 People who are infected with HBV can develop the disease either acutely or chronically. If you suffer from acute hepatitis B infection, you can be treated with drugs known as interferon, which slow the replication of the virus in the body. Those who have acute HBV infections are generally not treated with antiviral drugs since they resolve on their own.2 Hepatitis C is caused by the hepatitis C virus (HCV), and is spread primarily through contact with infected blood and less extensively through sexual contact and childbirth.2 Drugs known as interferon are the treatment of choice for HCV infections.

In order to protect our safety, it is recommended to undergo vaccination, particularly for Hepatitis A and B. Although no vaccines are available for hepatitis C, D, and E2 reducing ones exposure to these is the surest means of gaining protection from these debilitating viruses.