Polycystic Ovarian Syndrome (PCOS)

• PCOS is heterogenous disorder of uncertain cause. Some evidence shows it is a genetic disease (autosomal dominant) with high genetic penetrance but variable expressivity in females. It is a health problem that affects 1 in 10 women. It is the most common endocrine disorder which is caused by an imbalance of reproductive hormones which creates problems in ovary. In PCOS, the egg from the ovaries may not develop as it should or it may not be released during ovulation. This causes missed or irregular menstrual periods. Irregular periods can lead to : (a) Infertility (b) development of cysts (small fluid-filled sacs) in the ovaries.
• PCOS encompasses hyperandrogenism which has internal effects on ovarian function and metabolism and external manifestation on skin. Ovarian dysfunction is associated with erratic menses and anovulation.
•  Cysts are a symptom instead of the cause. Cysts are the immature follicles whose development has arrested at early antral stage. Polycystic ovaries develop when the ovaries are stimulated to produce excessive amounts of androgenic hormones, mainly testosterone.
• Women with PCOS experience an increased frequency of hypothalamic GnRH pulses, which in turn results in an increase in LH/FSH ratio. Hyperinsulinemia amplifies hyperandrogenism . Majority of women are obese and insulin-resistant. Hyperinsulinemia increases GnRH pulses frequencies. LH over FSH dominance increased ovarian androgen production, decreased follicular maturation.  Adipose tissue possesses aromatase which convert androstenedione to estrone and testosterone. Hence, obese women will have high androgen production.

CAUSES: 

(a) Excess insulin = when cells become resistant to the action of insulin, blood sugar as well as insulin levels arises. This leads to increase in androgen production.

(b) Low grade inflammation = this stimulates polycystic ovaries to produce androgen in PCOS women.

(c) Other causes are hypothyroidism, Cushing syndrome and hyperprolactinemia.

MECHANISM:

(a) Androgen control the development of male traits. Women with PCOS have more androgens than normal. High levels prevent the ovaries from releasing an egg (ovulation) and cause extra hair growth and acne (signs of PCOS).

(b) The action of insulin on the liver leads to decrease production of sex hormone binding globulin which results in increased free testosterone.

SIGNS AND SYMPTOMS:

1. Chronic anovulation 
2. Reduced menstrual bleeding
3. Absence of menstrual bleeding (Amenorrhea)
4. Hirsutism (increased facial and body hair)
5. Alopecia (baldness)
6. Obesity (high waist to hip ratio)
7. Infertility or recurrent miscarriages.
8. Anxiety\
9. Oily skin, severe and late-onset acne
10. pelvic pain
11. patches of thickened, dark, velvety skin- a condition called as Acanthosis nigricans 

DIAGNOSIS:

1. Physical examination : BP, BMI, WHR, extra hair on body, acne, skin discoloration, enlarged thyroid gland.
2. Pelvic exam: if ovaries are enlarged or swollen.
3. Ultrasound: cysts in ovaries or check the endometrium lining of uterus
4. Blood tests: androgen level, cholesterol level etc.
5. Anti-Mullerian hormone (AMH) is increased in PCOS, and become a best diagnostic criteria.

TREATMENT:

1. Medical:  Metformin is used to treat type II diabetes. It improves insulin levels and bring back the normal cycle. Clomiphene is ovulation inducer and improves fertility. Oral contraceptives decrease the action of insulin and hence increase the production of sex hormone binding globulin which leads to low levels of free testosterone.
2. Taking estrogen and progestin daily regulate ovulation, relieve symptoms and protect against cancer.
3. Lifestyle changes: exercise, eating healthy and avoid junk.

Differences between diabetic coma and insulin shock.

DIABETIC COMA:

1. It is more fatal.
2. It occurs to the patient of high blood sugar- hyperglycemia.
3. Causes are- ketoacidosis, hyperosmolar syndrome
4. Experienced by diabetic patients generally.
5. Symptoms: dry mouth, frequent urination, sweating, anxiety, shakiness.

INSULIN SHOCK:

1. Comparatively less fatal.
2. It is the body reaction to too little sugar- hypoglycemia
3. It occurs due to the result of too much insulin.
4. Even people without diabetes can also experience.
5. Symptoms: fast breathing, rapid pulse, dizziness, numbness and hunger.

How to overcome the problem of oxygen sensitivity of nitrogenase?

To overcome the problem of O2 sensitivity of nitrogenase, following strategies are involved:

1. A number of free- living nitrogen fixing organisms have mechanical, temporal, or biochemical barriers to separate the process of N2 fixation from aerobic metabolism i.e., compartmentalization.
2. Some organism like Cyanobacterium (oxygenic photosynthetic bacteria) have specialized cells called heterocyst that lack the O2 generating steps of photosynthesis.
3. Symbiotic diazotrophs like rhizobia associate with legumes. O2 is bound to leghemoglobin in the root nodules and thus supplied at a low rate that will not harm the nitrogenase.
4. In some plants, nodules can adjust their resistance to O2 diffusion by increasing or decreasing the number of cell layers around the nodulated roots.
5. In few rhizobial species, an O2 sensitive hemoprotein kinase, FixL which phosphorylates FixJ, which then activates and controls the expression of nif and fix genes. Thus when conditions are correct, Bacteroides will express nitrogenase. 

Differences between C3, C4 and CAM plants.

C3 PLANTS:

• Typical Species : Wheat, Rice
• Carboxylating enzyme: Rubisco
• First product of CO2 fixation: 3PGA (Phosphoglycerate)
• Leaf anatomy: Photosynthesis occurs in mesophyll. Bundle sheath may present.
• Present in: in temperate region, in all photosynthetic plants
• Initial CO2 acceptor: RuBP
• Photorespiration: High

C4 PLANTS:

• Typical Species : Maize, Sugarcane
• Carboxylating enzyme: Rubisco and PEP carboxylase (both active in dark, but at different compartments)
• First product of CO2 fixation: OAA (oxaloacetic acid)
• Leaf anatomy: Photosynthesis occurs in mesophyll and bundle sheath (Kranz anatomy)
• Present in: tropical plants
• Initial CO2 acceptor: PEP (phosphoenolpyruvate)
• Photorespiration: less

CAM PLANTS:

• Typical Species : Cactus, Pineapple
• Carboxylating enzyme: Rubisco in light and PEP carboxylase in dark
• First product of CO2 fixation: 3 PGA in light and OAA in dark.
• Leaf anatomy: Photosynthesis occurs in mesophyll contain large vacuoles for malic acid storage
• Present in: Semi-arid condition or in tropical rainforest
• Initial CO2 acceptor: PEP
• Photorespiration: least

SPECIFICITY OF AN ENZYME:

The same binding energy that provides energy for catalysis also gives an enzyme its specificity. It is the ability to discriminate between a substrate and a competing molecule. Specificity is easy to distinguish from catalysis, but this distinction is much more difficult to make experimentally, because catalysis and specificity arise from the same phenomenon. If an enzyme active site has functional groups arranged optimally to form a variety of weak interaction with a particular S in the transition state, the enzyme will not be able to interact to the same degree with any other molecule. In general, specificity is derived from the formation of many weak interaction between the E and its specific S molecule.

Consider what needs to occur for a reaction to take place.

• Prominent physical and thermodynamic factors contributing to change in G++, the barrier to reaction, may include:
• the entropy (freedom of motion) of molecules in solution, which reduces the possibility that they will react together.
• the solvation shell of hydrogen- bonded water that surrounds and helps to stabilize most biomolecules in aqueous solution.
• the distortion of substrates that must occur in many reactions.
• the need for proper alignment of catalytic functional groups on the enzyme. 

Binding energy can be used to overcome all these barriers:

1. First, a large restriction in the relative motions of two substrates that are to react, or entropy reduction is one obvious benefit of binding them to an enzyme. B.E. holds the substrates in the proper orientation to react- a substantial contribution to catalysis, because productive collisions between molecules in solution can be exceedingly rare. S can be precisely aligned on the enzymes, with many weak interaction between each substrate and strategically located groups on the enzyme clamping the substrate molecule into the proper positions. Studies have shown that constraining the motion of two reactants can produce rate enhancements of many orders of magnitude. 
2. Second, formation of weak bonds between S and E results in desolvation of the S. E-S interaction replace most or all of the H-bonds between the S and water.
3. Third, binding energy involving weak interaction formed only in the reaction transition state helps to compensate thermodynamically for any distortion, primarily electron redistribution, that the S must undergo to react.
4. Finally, the enzyme itself undergoes a change in conformation when the S binds induced by multiple weak interaction with the S. This is referred to as induced fit , a mechanism postulated by Daniel Koshland in 1958. The motions can affect a small part of the enzyme near the active site, or can involve changes in the  positioning of entire domains. Typically, a network of coupled motions occurs throughout the enzyme that ultimately brings about the required changes in the active site. Induced fit serves to bring specific functional groups on the enzyme into the proper position to catalyze the reaction. The conformational change also permits formation of additional weak bonding interaction in the transition state. In either case, the new enzyme conformation has enhanced catalytic properties. Induced fit is also important in the interaction of almost every enzyme with its substrate.

Catalytic power and specificity of enzymes:

Enzymes are highly effective catalysts, commonly enhancing reaction rates by a factor of 10^5 to 10^17.

What is the energy source for the dramatic lowering of the A.E for specific reactions?

• The rearrangement of the covalent bonds during an enzyme- catalyzed reaction. Chemical reactions of many types take place between substrates and enzymes' functional groups (specific amino acid side chains, metal ions and coenzymes). Catalytic functional groups of an enzyme may form a transient covalent bond with a substrate and activate it for reaction, or a group may be transiently transferred from the substrate to the enzyme. In many cases, these reactions occur only in the enzyme active site. Covalent interaction between enzymes and substrates lower the A.E by providing an alternative, low energy reaction path.
• The non-covalent interactions between enzyme and substrate, formation of each weak interactions in the ES complex is accompanied by release of a small amount of free energy that stabilizes the interaction. The energy derived from enzyme- substrate interaction is called binding energy. Binding energy is a major source of free energy used by enzymes to lower the A.E of reactions. The interaction between substrate and enzyme in ES complex is mediated by the factors including hydrogen bonds and hydrophobic and ionic interactions, weak and non-covalent interactions.

Two fundamental and interrelated principles provide a general explanation for how enzymes use non-covalent binding energy.

• Much of the catalytic power of enzyme is ultimately derived from the free energy released in forming many weak bonds and interactions between an enzyme and its substrate. This B.E contributes to specificity as well as to catalysis.
• Weak interactions are optimized in the reaction transition state; enzyme active sites are complementary not to the substrates per but to the transition states through which substrates pass as they are converted to products during an enzymatic reaction.

Weak interactions between enzyme and substrate are optimized in the transition state:

How does an enzyme use binding energy to lower the A.E of a reaction?                                  Emil Fischer, in 1894, carried out the studies on enzyme specificity and proposed the lock and key method. 

• In case (a), Before the stick is broken, it must first be bent (transition state). In both example, magnetic interaction takes the place of weak bonding interaction between enzyme and substrate. 
• In case (b), a stickase with a magnet-lined pocket complementary in structure to the stick (substrate). Bending is impeded by the magnetic attraction between stick and atickase.
• In case (c), An enzyme with a pocket complementary to the reaction transition state helps to destabilize the stick, contributing to catalysis of the reaction. The B.E of the magnetic interaction compensates for the increase in free energy required to bend the stick.

The bound substrate must still undergo the increase in free energy needed to reach the transition state. Interaction between the stickase and non-reaching parts of the stick provide some of the energy needed to catalyze stick breakage. This "energy payment" translates into a lower net A.E and a faster reaction rate.

Real enzyme work on an analogous principle.

Some weak interaction formed in the ES complex, but the full complement of such interaction between S and E is formed only when the S reaches the transition state. The free energy (B.E) released by the formation of these interaction partially offsets the energy required to reach the top of the energy hill. The summation of the unfavorable (+ve) A.E delta G++ and the favorable (-ve) B.E delta G^B results in  a lower net activation energy.

The important principle is that weak binding interaction between the E and S provide a substantial driving force for enzymatic catalysis. The groups on the S that are involved in these weak interaction can be at some distance from the bonds that are broken or changed. The weak interaction formed only in the transition state are those that make the primary contribution to catalysis.

Why enzymes or coenzymes are so large?

Because of the requirement for multiple weak interaction to drive catalysis. An enzyme must provide functional groups for ionic, hydrogen-bond and other interaction, and must also precisely position these groups so that B.E is optimized in the transition state. Adequate binding is accompanied most readily by positioning a substrate in a cavity (active site) where it is effectively removed from water.

 

HOW ENZYMES WORK?

• Active site : The enzyme- catalyzed reaction takes place within the confines of a pocket on the enzyme called the active site.
• Substrate:  The molecule that is bound in the active site and acted upon by the enzyme is called the substrate.
• The surface of the active site is lined with amino acid residues with substituent groups that bind the substrate and catalyzed its chemical transformation.
• The Enzyme-Substrate complex (ES), was first proposed by Charles- Adolphe Wurtz in 1880.
• A simple enzymatic reaction might be written : E + S = ES = EP = E + P

Catalysts do not affect reaction equilibrium. The function of a catalyst is to increase the rate of a reaction. The starting point for either the forward or the reverse reaction is called the ground state. 

• The equilibrium between S and P reflects the difference in the free energies of their ground states. The free energy of the ground state of P is lower than that of S, so change in G^10 (biochemical standard free- energy change, pH=7, conc. of solute is low, mM, nM) for the reaction is negative and the equilibrium favors P. The position and direction of equilibrium are not affected by any catalyst.
• A favorable equilibrium does not mean that the S = P conversion will occur at a detectable rate.
• There is an energy barrier between S and P, the energy required for alignment of reacting groups, formation of transient unstable charges, bond rearrangements and other transformations required for the reaction to proceed in either direction.
• To undergo reaction, the molecules must overcome the barrier and therefore must be raised to a higher energy level. At the top of the energy hill is a point at which decay to the S or P state is equally probable. This is called the transition state.  It is not a chemical species with any significant stability and should not be confused with a reaction intermediate (EP or ES). It is simply a fleeting molecular moment.
• Activation Energy, G++ = The difference between the energy levels of the ground state and the transition state. The rate of a reaction reflects this activation energy: higher A.E corresponds to slower reaction.
• Reaction rates can be increased by raising the temperature, pressure thereby increasing the no. of molecules with sufficient energy to overcome the energy barrier.
• The activation energy can be lowered by adding a catalyst. Catalysts enhance reaction rates by lowering A.E.
• 
• Any reaction may have several steps, involving the formation and decay of transient chemical species called reaction intermediates. A reaction intermediate is any species on the reaction pathway that has a finite chemical lifetime. The ES and EP complexes can be considered intermediates.
• When several steps occur in a reaction, the overall rate is determined by the step with a highest activation energy; this is called the rate limiting step which is the slowest step.
• In practice, the rate limiting step can vary with reaction conditions, and for many enzymes several steps may have similar A.E, which means they are all partially rate- limiting.  

What causes cancer? (pt.3)

The uncontrolled proliferation of cancer cells, combined with their ability to metastasize to distant sites, makes cancer a potentially life-threatening disease.

Many chemicals can cause cancer, often after metabolic activation in the liver.

Carcinogens are the cancer causing agents. 2-naphthylamine is a potent carcinogen that causes bladder cancer in industrial workers. When it is inhaled or ingested by animals, it passes through the liver and is metabolized into other chemicals that are the actual causes of cancer. Placing 2- naphthylamine directly in an animal's bladder bypasses this metabolic activation and so cancer does not arise.

Many carcinogens share this need for metabolic activation before they can cause cancer. Substances exhibiting such behavior are more accurately called precarcinogens, a term applied to any chemical that is capable of causing cancer only after it has been metabolically activated. Members of liver cytochrome P450 enzyme family activate these procarcinogens. They catalyze the oxidation of ingested foreign chemicals, such as drugs and pollutants, to make the molecules less toxic and easier to excrete from the body.  

DNA Mutations triggered by chemical carcinogens lead to cancer.

Carcinogenic chemicals inflict DNA damage in several ways, including binding to DNA and disrupting normal base-pairing; generating crosslinks between the two strands of the double helix; creating chemical linkages between adjacent bases; hydroxylating or removing individual DNA bases; and causing breaks in one or both DNA strands.  In some cases, the mutational role of specific chemicals in causing cancer has been linked to effects on specific genes. For example, the polycyclic aromatic hydrocarbons found in tobacco smoke preferentially bind to specific regions of the p53 gene and trigger unique mutations in which the base T is substituted to base G.

Ames Test for identifying potential carcinogens. 

The Ames Test is based on the rationale that most carcinogens are mutagens. The ability of chemicals to induce mutations is measured in bacteria that cannot synthesize the amino acid histidine. When placed in a growth medium lacking histidine, the only bacteria that can grow are those that have acquired a mutation allowing them to make histidine. The number of bacterial colonies that grow is therefore related to the mutagenic potency of the substance being tested. Chemicals studied with the Ames test are first incubated with the liver homogenate because many chemicals become carcinogenic only after they have undergone biochemical modifications in the liver.

Cancer arises through a multistep process involving initiation, promotion and tumor progression.

During initiation, normal cells are converted to a precancerous state, and promotion then stimulates the altered cells to divide and form tumors. Promotion is a gradual process requiring prolonged or repeated exposure to a promoting agent. 

When a cell with an initiating mutation is exposed to a promoting agent (or natural growth regulator) that causes the initiated cell to proliferate, the number of mutant cell increases. As proliferation continues, natural selection tends to favor cells exhibiting enhanced growth rate and invasive properties, eventually leading to the formation of a malignant tumor.

Initiation and promotion are followed by a third stage, known as tumor progression. During this, tumor cells properties gradually change over time as cells acquire more aberrant traits and become increasingly aggressive. Tumor progression is made possible by a combination of DNA mutations and epigenetic changes that don't require mutation, accompanied by natural selection of those cells that acquire advantageous properties through these mechanisms.

Main stages in Cancer development : Cancer arises by a multistep process involving (1) an initiation event based on DNA mutation, (2) a promotion stage in which the initiated cell is stimulated to proliferate, and (3) tumor progression, in which mutations and changes in gene expression create variant cells exhibiting enhanced growth rates or other aggressive properties that give certain cells a selective advantage. Such cells tend to outgrow their companions and become the predominant cell population in the tumor. During tumor progression, repeated cycles of this selection process create a population of cells whose properties gradually change over time.

Ionizing and Ultraviolet radiation also cause DNA mutations that lead to cancer.

X-rays and related forms of radiation emitted by radioactive elements are called ionizing radiation because they remove electrons from molecules, thereby generating highly reactive ions that create various types of DNA damage, including single- and double-stranded breaks. Ultraviolet radiation (UV) is another type of radiation that causes cancer by damaging DNA. UV radiation is absorbed mainly by the skin, where it imparts enough energy to trigger pyrimidine dimer formation. If the damage is not repaired, distortion of the double helix causes improper base pairing during DNA replication. For example, CC = TT mutation (conversion of two adjacent cytosine to thymine) is a unique product of UV exposure and can be used as a distinctive "signature" to identify mutations caused by sunlight.

Viruses and other infectious agents trigger the development of some cancers.

A virus that causes cancer is called oncogenic virus. Denis Burkitt, a British surgeon noted large outbreaks of lymphocytic cancers of the neck and jaw. Because of this, it is known as Burkitt lymphoma, which is transmitted by an infectious agent. Epstein- Barr virus (EBV) can play a role in Burkitt lymphoma : (1) EBV DNA and proteins are often found in tumor cells obtained from patients with Burkitt lymphoma but not in normal cells from the same individuals, (2) adding EBV to normal human lymphocytes in culture causes cells to acquire some of the properties of cancer cells. (3) injecting EBV into monkeys causes lymphomas to arise.

In addition to EBV, several other viruses have been linked to human cancers. Among these are the hepatitis B and hepatitis C viruses, which trigger some liver cancers; human T-cell lymphotropic virus-I (HTLV-I), which causes adult T-cell leukemia and lymphomas; and the sexually transmitted human papillomavirus (HPV), which is associated with uterine cervical cancer. Moreover, chronic infection with the bacterium Helicobacter pylori is a common cause of stomach ulcers, this can also trigger stomach cancer. Flatworm infections have been linked to a small number of bladder and bile duct cancers. 

The mechanisms of action by these infectious agents can be grouped into two categories: 

(1) These agent causes tissue destruction and chronic inflammation. Under these conditions, cells of the immune system infiltrate the tissue and attempt to kill the infectious agent. The mechanism used by immune cells to fight infections often produce mutagenic chemicals such as oxygen free radicals. The net result is an increased likelihood that cancer-causing mutations will arise when cells proliferate to repair the damaged tissue.

(2) The other mechanism used by infectious agents to cause cancer is based on the ability of certain viruses to stimulate the proliferation of infected cells. Some viruses trigger the cell proliferation through the direct action of viral genes, whereas other viruses alter the behavior of host cell genes. 

ENZYMES

• Reaction catalysts of biological systems are called enzymes. They are the most remarkable and highly specialized proteins. They have extraordinary catalytic power, often far greater than that of synthetic or inorganic catalysts.
• Almost every biochemical reaction is catalyzed by an enzyme. They have a high degree of specificity for their substrates, they accelerate chemical reactions tremendously and they function in aqueous solution under very mild conditions of temperature and pH=7 (37 C).
• They catalyze the hundreds of stepwise reactions that degrade nutrient molecules, conserve and transform chemical energy and make biological macromolecules from simple precursors.

INTRODUCTION:

• Biological catalysts was first recognized and described in the late 1700s, in studies on the digestion of meat by secretions of the stomach.
• In the 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by "ferments". He postulated that these ferments were inseparable from the structure of living yeast cells; this view called vitalism.
• Then in 1897, Edward Buchner discovered that yeast extracts could ferment sugar to alcohol, proving that fermentation was promoted by molecules that continued to function when removed from the cells.
• Frederick W. Kuhne  gave the name enzymes to the molecules detected by Buchner.
• The isolation and crystallization of Urease by James Sumner in 1926 was a breakthrough. He found that urease crystals consisted entirely of proteins, and he postulated that all enzymes are proteins.
• John Northrop and Moses Kunitz crystallized pepsin, trypsin and other digestive enzymes and found them also to be proteins.
• J.B.S Haldane wrote a treatise entitled "Enzymes".

MOST ENZYMES ARE PROTEINS:

With the exception of a small group of catalytic RNA molecules, all enzymes are proteins. Their catalytic activity depends on the integrity of their native protein conformation. If any enzyme is denatured or dissociated into its subunits, catalytic activity is usually lost. If any enzyme is broken down into its component amino acids, its catalytic activity is always destroyed. Thus primary, secondary, tertiary and quaternary structures of protein enzymes are essential to their catalytic activity.

PROPERTIES OF AN ENZYME:

• Enzymes, like other proteins, have mol. wt. ranging from about 12,000 to more than 1 million.
• Some enzymes require no chemical groups for activity other than their amino acids residues.
• Other requires an additional chemical components called a cofactor, either 1 or more inorganic ions, such as Fe2+, Mg2+, Mn2+ or Zn2+.
• Or they require a complex organic or metallo-organic molecule called a coenzyme. Coenzymes act as transient carriers of specific functional groups.
• Some inorganic ions that serve as cofactors for enzymes: E.g., Cu2+ in cytochrome oxidase, Fe2+ or Fe3+ in peroxidase, catalase. Ni2+ in Urease. Se in Glutathione peroxidase. K+ in Pyruvate kinase, Mg2+ in hexokinase, glucose-6-Phosphatase. Mo in Dinitogenase.
• Some coenzymes that serve as transient carriers of specific atoms or functional groups: 1) Biocytin transfer CO2 derived from biotin. 2) Thiamin pyrophosphate transfer aldehyde 3) Pyridoxal phosphate transfers amino acids.
• Some enzymes requires both coenzymes and one or more metal ions for activity.
• A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein is called a prosthetic group.
• A complete catalytically active enzyme together with its bound coenzyme and/or metal ions is called a holoenzyme.
• The protein part of such an enzyme is called the apoenzyme or apoprotein.
• Some enzyme proteins are modified covalently by phosphorylation, glycosylation and other processes.