The Menace of Radicals

We all know that we need oxygen to live. But research has now established an astounding fact. The very thing which promotes life is killing us. Shocked? Relax.. research has now proved that oxidation in the body cells ( the process by which we are continuously burning our calories to get energy) releases dangerous, very active molecules known as free radicals. These damaging fellows attack other (neighborhood) cells, the cells walls, and the genetic material (DNA) within the cells and over a long time period, such damage can become irreversible (like mutation) and cause disease (e.g. cancer). Even if it doesn’t lead to cancer, the old age symptoms (lack of energy, poor memory, loss of hearing, falling hair) are definitely associated with damaged or weak cells. In addition, free radicals contribute to alcohal-induced liver damage, perhaps more than alcohal itself. Radicals in cigarette smoke have been implicated in inactivation of alpha 1 trypsin in the lung, which promotes the development of emphysema and it is now proved that these free radicals are the main culprit. While nature has created this problem, it has provided the solution as well.

What happens in oxidation (burning)?

Burning is quite aptly associated with loss. Things loose their colour, taste or odour when they are burnt. How does an apple retain its fresh red colour even when there’s so much oxygen and sunlight in the nature? It’s the antioxidant. But if it is cut open, it turns brown after a while. That’s oxidation. the presence of every easily oxidisable compounds, called Antioxidants,in the system can “mop up” free radicals before they damage other essential molecules. Therefore, Antioxidants play a key role in these defense mechanisms. An Antioxidants is a chemical that prevents the oxidation of other chemicals. Consuming more antioxidants helps provide the body with tools to neutralise harmful free radicals. It’s estimated that there are more than 4,000 compounds in foods that acts as antioxidants. The most studied include Vitamin C & E, beta-carotene and the mineral selenium. Besides antioxidants, there are several enzyme systems within the body that scavenge free radicals, the principal micronutrient (vitamin) antioxidants are Vitamin E, beta carotene, & Vitamin C. Additionally, selenium, a trace metal that is required for proper function of one of the body’s antioxidant enzyme systems, is sometimes included in this category. The body cannot manufacture these micronutrient so they must be supplied in the diet.

The following vitamins have shown positive antioxidants effects:

Vitamin A or Retinol,or beta-carotene. It has been discovered that beta-carotene protects dark green, yellow and orange vegetables and fruits from solar radiation damage and it is thought that it plays a similar role in human body. Carrots, Squash, Sweet Potatoes, Tomatoes,Peaches and apricots are particularly rich sources of beta-carotene. (NOTE: Vitamin A has no antioxidant properties and can be quite toxic when taken in excess).

Vitamin C: also called Ascorbic acid, is a water-soluble vitamin found in all body fluids, so it may be one of our first lines of defense. This powerful antioxidant cannot be stored by the body, so it’s important to get some regularly-not a difficult task if fruits and vegetable are regularly consumed. Important sources include Citrus Fruits (like Oranges, sweet lime,etc.), green peppers, green leafy vegetables, strawberries, raw cabbage, tomatoes and potatoes.

Vitamin E: A fat soluble vitamin that can be stored with fat in the liver and other tissues, vitamin E is promoted for a range of purposes- from delaying aging to healing sunburn. While it’s not a miracle worker, it’s another powerful antioxidant. Important sources include wheat germ, nuts, seeds, whole grains, green leafy vegetable, vegetable oil and fish-liver oil.

Beta-Carotene: the most studied of more than 600 different carotenoids that have been discovered, beta-carotene protects dark green, yellow and orange vegetables and fruits from solar radiation damage. It is thought that it plays a similar role in the body. Carrots, Squash, Sweet potatoes, peaches and apricots are particularly rich sources of beta-carotene.

Beta Carotene rich foods

Selenium: This mineral is thought to help fight cell damage by oxygen-derived compounds and thus may help protect against cancer. It is best to get selenium through foods foods, as large doses of the supplement from can be toxic. Good food sources include fish, shellfish,red meat, grains, eggs chicken and garlic. Vegetables can also be a good source if grown in selenium rich soils.

Natural Sources of Antioxidants

The colorful stuff: carrots, apples, oranges, tomatoes (cooked), beetroot, brinjal, dark green vegetables, olives, strawberries, honey (the darker the better) and so on. The tasty stuff: garlic, ginger, onion, nutmeg. The smelly stuff: tea,green tea, spinach, tulsi and other herbs etc. Research says multi vitamin pills may slow the advance of HIV, as it appears to cut the levels of the virus and boost the number of immune cells.

Even though a tomato is rich in antioxidants, most of them can’t be absorbed by humans because they are too complex. Slow cooking brakes them down into simpler compounds that are easily observed. This antioxidant value of a tomato is said to be increase 5 times when it is cooked.

Preventing Cancer and Heart disease – Do Antioxidants help?

Epidemiologic observations shows lower cancer rates in people whose diets are rich in fruits & vegetables. This has led to the theory that these diets contain substances, possibly antioxidants, which protect against the development of cancer. There is currently intense scientific investigation into this topic. thus far, none of the large, well designed studies have shown dietary supplementation with extra antioxidants reduces the risk of development of cancer. in fact one study demonstrated an increased risk of lung cancer in male smokers who took antioxidants vs,. male smokers who did not supplement. Whether this effect was from the antioxidants is unknown but it does raise the issue that antioxidants may be harmful under certain conditions.

Recommendations

Follow a balanced training program that emphasize regular exercise and eat 5 servings of fruits & Vegetables per day. This will ensure that you are developing your inherent antioxidant systems and that your diet is providing the necessary components.

POLYMERASE CHAIN REACTION: PRINCIPLE AND APPLICATIONS

BY DAKSHITA NAITHANI

The polymerase chain reaction (PCR) is an in-vitro (laboratory) technique used to produce huge amounts of DNA.

•             PCR is a cell-free amplification method that produces billions of identical copies of any DNA of interest. PCR, which was invented by Karry Mullis in 1984, is today regarded as a fundamental technique for molecular methods. It is the most widely used approach for multiplication of target nucleic acids.

•              The method usually combines complementary nucleic acid hybridization and nucleic acid replication principles, which are applied repeatedly over many cycles to amplify a single and original copy of a nucleic acid target, which is often undetectable by standard hybridization methods, and multiply to 107 or more copies in a short amount of time. In result, it gives a large number of targets which may be identified using a variety of ways.

ADVANTAGES:

•             Despite being simple it is a very powerful technique.

 •            It enables for massive amplification of any particular sequence of DNA given that short sequences on each side of it are known.

•             Improves sensitivity and specificity while allowing for speedier diagnosis and recognition.

PRINCIPLE OF PCR:

Double-stranded DNA in question is denatured, resulting in two independent strands and  each strand is allowed to hybridise using a primer (renaturation). The enzyme DNA polymerase is used to synthesise DNA from the primer-template duplex. To create various forms of target DNA, the three processes of denaturation, renaturation, and synthesis are performed numerous times.

ESSENTIAL REQUIREMENTS FOR PCR:

•             A target DNA which is around 100-35,000 bp in length.

•             Two primers (synthetic oligonucleotides of 17-25 nucleotides length ) that are complementary to regions flanking the target DNA.

•             Four deoxyriobonucleotides  are used(d ATP, d CTP, d GTP, d TTP)

•             MgCl2 (Magnesium Chloride)

•             Nuclease free water

•             Taq DNA polymerase buffer

•             A thermo-stable DNA polymerase is one that can tolerate temperatures up to 95 degrees Celsius.

The target DNA, two primers (in excess), a thermo-stable DNA polymerase (Taq DNA polymerase), and four deoxyribonucleotides are all included in the reaction mixture. It is a method that includes a series of cycles for DNA amplification.

KEY FACTORS OPTIMAL FOR PCR:

•             PRIMERS:

When it comes to determining PCR, these are crucial. Primers with no secondary structure and no complementarity amongst themselves (17-30 nucleotides) are excellent. In PCR, complementary primers can combine to produce a primer dimer, which can be amplified. The replication of target DNA is prevented as a result of this action.

•             DNA POLYMERASE:

Because it can resist high temperatures, Taq DNA polymerase is chosen. After the heat denaturation stage of the first cycle, DNA polymerase is introduced in the hot start procedure. This prevents the misaligned primers from extending, which is common at low temperatures.

Verification or proof reading of exonuclease (3′-5′) activity is absent in Taq polymerase, which might lead to mistakes in PCR products. Tma DNA polymerase from Thermotogamaritama and Pfu DNA polymerase from Pyrococcusfuriosus are examples of thermostable DNA polymerases with proof reading activity.

•             TARGET DNA:

In general, the smaller the target DNA sequence, the greater the PCR efficiency. A mplification of DNA fragments up to 10 kb has been documented in recent years. In PCR, the sequence of the target DNA is also crucial. As a result, CC-rich strand sections obstruct PCR.

•             PROMOTERS AND INHIBITORS:

 Addition of Bovine serum albumin (BSA) improve PCR by shielding DNA polymerase, humic acids which are commonly present in ancient samples of target DNA, hinder PCR.

EACH CYCLE HAS THREE STAGES:

1.            DENATURATION:

The DNA is denatured and the two strands split when the temperature is raised to around 95 degree celsius for about one minute.

2.            RENATURATION OR ANNEALING:

The primers base pair with the complementary regions flanking target DNA strands as the temperature of the mixture is gradually lowered to around 55 degree celsius.  Annealing seems to be the term for this procedure. Due to the high concentration of primer, annealing occurs between each DNA strand and the primer rather than between the two strands.

3.            EXTENSION OR SYNTHESIS:

The 3′-hydroxyl end of each primer is where DNA synthesis begins. By connecting the nucleotides that are complementary to DNA strands, the primers are expanded. The PCR synthesis process is quite similar to the leading strand DNA replication process.  The optimal temperature for Taq DNA polymerase is about 75 degree celsius. (For E.Coli DNA Polymerase is used). By increasing the temperature, the process can be halted (about 95 degree celsius).

Each cycle lasts around 3-5 minutes and in most cases, it is performed on computerised equipment. The corresponding sequence of the second primer lies beyond the new DNA strand linked to each primer. Long templates allude to these additional strands, which will be utilised in the second cycle.

The strands are denatured, annealed with primers, and exposed to DNA synthesis in the second cycle of PCR. Long and short templates are produced at the end of the second round.

The original DNA strands, as well as the short and long templates, are the starting materials for the third cycle of PCR. For each cycle, the procedures are used again and again. About a million-fold target DNA is produced by the conclusion of the 32nd cycle of PCR, according to estimates. As double-stranded molecules build, the small templates containing precisely the target DNA increase.

TYPES OF PCR:

1.            Real-time PCR

2.            Quantitative real time PCR (Q-RT PCR)

3.            Reverse Transcriptase PCR (RT-PCR)

4.            Multiplex PCR

5.            Nested PCR

6.            Long-range PCR

7.            Single-cell PCR

8.            Fast-cycling PCR

9.            Methylation-specific PCR (MSP)

10.          Hot start PCR

11.          High-fidelity PCR

12.          In situ PCR

13.          Variable Number of Tandem Repeats (VNTR) PCR

14.          Asymmetric PCR

15.          Repetitive sequence-based PCR

16.          Overlap extension PCR

17.          Assemble PCR

18.          Intersequence-specific PCR(ISSR)

19.          Ligation-mediated PCR

20.          Methylation –specifin PCR

21.          Miniprimer PCR

22.          Solid phase PCR

23.          Touch down PCR, etc

APPLICATIONS OF PCR:

1.            PCR IN CLINICAL DIAGNOSIS:

PCR’s specificity and sensitivity make it ideal for diagnosing a variety of human illnesses. RFLP is not involved in the development of many genetic diseases (restriction fragment length poly-morphism). For all of these problems, PCR is a godsend since it delivers straight DNA information it is accomplished by amplifying DNA from the appropriate area and then analysing the PCR results directly.

o             PRENATAL DIAGNOSIS OF INHERITED DISEASES:

It is used to diagnose hereditary disorders in the womb utilising chorionic villus samples or amniocentesis cells various c onditions such as sickle cell anaemia, p-thalassemia, and phenylketonuria can thus be identified in these specimens using PCR.

o             DIAGNOSIS OF RETROVIRAL INFECTIONS:

                PCR from cDNA is a useful technique for detecting and maintaining retroviral infections, such as HIV.

o             DIAGNOSIS OF BACTERIAL INFECTIONS:

o             PCR is used for the detection of bacterial infections such as tuberculosis which is caused by Mycobacterium tuberculosis.

o             DIAGNOSIS OF CANCERS:

PCR can identify some virally-induced malignancies, such as cervical cancer caused by the human papillomavirus it can also identify malignancies caused by chromosomal translocations (chromosome 14 and 18 in follicular lymphoma) containing known genes.

o             PCR IN SEX DETERMINATION OF EMBROYS:

The sex of human and animal eggs fertilised in vitro may be identified using PCR using sex chromosome-specific primers and DNA probes. This method can also be used to identify sex-related abnormalities in fertilised eggs.

2.            PCR IN DNA SEQUENCING:

 The process is useful for sequencing since it is considerably easier and faster to amplify DNA. Single strands of DNA are required for this function. Asymmetric PCR involves preferred amplification of a single strand. Strand removal can also be accomplished by digesting one strand.

3.            PCR IN FORENSIC MEDICINE:

For amplification, a single molecule from any source (blood strains, hair, semen, etc.) of a person is sufficient. As a result, PCR is critical for crime detection.

4.            PCR IN COMPARISON WITH GENE CLONING:

In comparison to traditional gene cloning procedures, PCR offers a variety of benefits. Improved efficiency, small amounts of beginning material (DNA), cost-effectiveness, low technical expertise, and the time frame are only a few of them. In the long run, PCR may be able to replace most gene cloning applications.

5.            PCR IN GENE MANIPULATION AND EXPRESSION STUDIES:

The benefit of PCR is that the primers do not need to be complementary to the target DNA. As a result, it may alter and amplify the nucleotide sequence in a portion of the gene (target DNA). The coding sequence of a protein of interest can be changed using this approach. Gene manipulations are also crucial for studying the impact of factors on gene expression.

The study of mRNAs, which are the results of gene expression, requires the use of PCR. Reverse transcription-PCR is used to accomplish this.

6.            PCR IN COMPARITIVE STUDIES OF GENOMES:

PCR using random primers can be used to assess the differences in the genomes of two species. Electrophoresis is used for separation of products for their comparative identification and it is predicted that two genomes from closely related species will produce more comparable bands.

The study of evolutionary biology, more especially phylogenetic biology, relies heavily on PCR. It has transformed palaeontology and archaeological research since it can amplify even minute amounts of DNA from any source (hair, mummified tissues, bone, or any fossilised material).

CANCER

WHAT IS CANCER?

The abnormal growth of cells anywhere in the body causes cancer. These cells are called cancer cells. These cells destroy the normal tissues in the body. Normal body cells grow, divide and die in an orderly way whereas cancer cells continue to grow and divide in a disorderly fashion and these cells do not die. These cells form a tumor. But, not all cancer cells form tumors. Cancer can occur at any age but 67% of cancer deaths occur in people who are 65 years. There are two types of tumors. Malignant tumors and benign tumors. Malignant tumors spread to other parts of the body at a rapid speed. It spreads to other parts through the bloodstream and lymphatic system. Whereas benign tumors stay in one place and start to grow slowly and expand in the same tissue. Although cancer is common, 5-10% of it can be hereditary, i.e they are inherited from their parents. The inherited cells undergo mutation and cause cancer.

CAUSES OF CANCER:

There are several causes of cancer. The substance that causes cancer is known as a carcinogen. This carcinogen affects the normal growth of cells.

Lifestyle factors: Smoking, drinking alcohol and drugs high fat-diet, and working with some toxic chemicals are the risk factors that may cause cancer in adults.

Inheritance: Cancer cells can also be inherited from our past generation to the present generation in some cases. It may be caused by a genetic mutation, exposure to chemicals in the living residence, or a combination of these factors. 

Genetic disorders: Some syndromes like Wiskott-Aldrich and Beckwith-Wiedemann syndromes are responsible for altering the immune system. The immune system helps to fight back against diseases and infections. Sometimes the stem cells in the bone marrow become damaged and when they reproduce, it results in the growth of cancer cells. The stem cells are damaged by some inherited genetic defects.

Environmental exposure: The use of pesticides and fertilizers in the growth of food plants have a direct link to childhood cancers. There is also evidence of cancer occurring in a child who lives in a polluted environment.

 TYPES OF CANCER:

Cancers are classified into four types based on where it is found.

  1. Carcinomas: This type of cancer appears in the epithelial tissues of the skin or some inner tissues of the internal organs. Carcinomas may spread to other parts of the body or it may develop in the same tissues where it was provided. The risk of carcinoma increases with age, alcohol consumption, tobacco use, exposure to ultraviolet rays. Examples of carcinomas include breast cancer, lung cancer, liver cancer, colorectal cancer, and prostate cancer.
  2. Sarcomas: Sarcomas appears in the tissues that support and connect the body. Sarcomas can develop in fat, joints, blood vessels, muscles, nerves, lymph vessels, bones, cartilages. Example: Bone cancer (osteosarcoma), liposarcoma in fat, and rhabdomyosarcoma in muscles. Sarcoma occurs in both children and adults.
  3. Leukemia: Cancer that occurs in the blood is known as leukemia. Leukemia involves white blood cells (WBCs). WBCs are responsible for fighting against infection and diseases. Normally they grow in an orderly fashion. But in the case of leukemia, the bone marrow produces more amount of WBCs, and those cells do not function properly. Example: Blood cancer.
  4. Lymphomas: It begins from the lymphatic system. The lymphatic system consists of vessels and glands that help in fighting against infection. The most common types of lymphomas are Hodgkin lymphoma and non-Hodgkin lymphoma.

TREATMENT:

Some of the cancers will have only one treatment. But most of the cancers have combinations of treatment such as radiotherapy, chemotherapy, and surgery.

Chemotherapy: In this treatment, drugs are used to kill the cancer cells. The disadvantage is that it may also affect the normal cells and bone marrow and causes loss of hair and loss of weight and it reduces the immune responses.

Radiotherapy: In this treatment, high-energy rays are used to kill the cancer cells. These rays stop the growth and division of cancer cells. Some of the side effects are skin rashes, lowers WBCS count, and loss of appetite.

Surgery: In this treatment, the person has to undergo the surgery to remove the entire mass of cancer cells i.e, tumors in a particular area. In some cases, the only way to know if the person has cancer or what kind of cancer he has is by removing small tissue from the tumor and testing it.  

PREVENTION:

  • Always eat healthy foods. 
  • Consume more vegetables and fruits. 
  • Exercise regularly to keep your body fit and free from diseases. 
  • Don’t smoke and don’t drink alcohol. 
  • Minimize your exposure to harmful rays and chemicals. 
  • Avoid usage of tobacco. Reduce your exposure to the sun.
  • Consume nuts reduces the risk of cancer

We should follow preventive measures to avoid cancer. If already cancer has occurred, one has to undergo proper treatment.