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http://www.webmedcentral.com/images/Header_Logo.giftext/html2012-12-06T14:22:13+01:00http://www.webmedcentral.com/Dr. Avnish KumarImportance of Using DNA Microarray in Studying Medicinal Plant
http://www.webmedcentral.com/article_view/3876
Medicinal plants have promised a positive concept of health based upon certain basic principles of physical, chemical and biological sciences in present busy life. Drugs from these are said to be potentially safe casting no side effects over synthetic drugs like antibiotics, analgesics, antihelminitics etc., Pyrostegia venusta (Ker Gawl.) Miers, (Bignoniaceae), is native to the Brazilian Cerrado and popularly known as “cipó-de-são-joão” (Sandwith and Hunt, 1974; Scalon et al., 2008). Pyrostezia venusta have reported folklore used as medicinal plant for common diseases of disease of eye, nose, etc. The flowers of P. venusta are used as a general tonic and a treatment for diarrhea, vitiligo, cough, and common infections and inflammatory diseases of the respiratory system (Ferreira et al., 2000). The flower consist of gallo-tannic acid and glycoside bellericanin, which is bitter, pungent acrid, digestible, laxative, anti-helminthic, anti-bronchitis, sore throat, anti-inflammatory, anti-asthamic. The fruit in combination with other drugs is prescribed for snake bites (Ferreira et al., 2000, Scalon et al., 2008; Velosoa et al 2010, Roy et al 2012).Economical importance:Although herbs had been priced for their medicinal, flavouring and aromatic qualities for centuries, the synthetic products of the modern age surpassed their importance, for a while. However, after a boom of allopath now these days herbs are staging a comeback and herbal ‘renaissance’ is happening all over the globe (Huff & Toby, 2003;). The herbal products today symbolise safety in contrast to the synthetics that are regarded as unsafe to human and environment (Wealth of India Dictionary CSIR, 2007). Over 80% of the world population depends mainly on plants and plant extracts for health care (Joy et al 1989, WHO 2002, Jawla et al 2009). Thus business world estimated that plant derived drugs may account for about Rs. 2,00,000 crores. Presently, India contribution is less than Rs. 2000 crores which is growing steadily. The annual production of medicinal and aromatic plant’s raw material is worth about Rs. 200 crores and it supposed to be raised to US $5 trillion by 2050 (Joshi et al, 2004). Even developed countries such as United States constitute 25% of the total drugs as plant drugs, while in most populated as well as fast developing countries such as China and India utilizing about 80% plant based drugs (Jawla et al 2009). The World Health Organization (WHO) has estimated the present demand for medicinal plants is approximately US $14 billion per year (Sharma, 2004). Epidemiology: These developing countries provide two third of the plants used in modern system of medicine and the health care system of rural population depend on indigenous systems of medicine. At present it is reported that there are more than 80,000 are medicinal higher plant species on earth. Among plant based drug producer, India is one important biodiversity centres with the recognition of over 45000 different plant species. India’s biodiversity have 16 different agro-climatic zones, 10 vegetation zones, 25 biotic provinces and 426 biomes (habitats of specific species). This constitute, about 15000-20000 plants with good medicinal value. Of these 7000-7500 species are used for their folk medicinal values since ancient time (Kirtikar & Basu 1995). In India, drugs of herbal origin have been used in traditional systems of medicines such as Unani and Ayurveda since ancient times. The Ayurveda system of medicine uses about 700 species, Unani 700, Siddha 600, Amchi 600 and modern medicine around 30 species. About 8,000 herbal remedies have been codified in Ayurveda. The Rigveda (5000 BC) has recorded 67 medicinal plants, Yajurveda 81 species, Atharvaveda (4500-2500 BC) 290 species, Charak Samhita (700 BC) and Sushrut Samhita (200 BC) had described properties and uses of 1100 and 1270 species respectively, in compounding of drugs and these are still used in the classical formulations, in the Ayurvedic system of medicine (Astin, 1998; Barnes et al 2002; Christie, 1991; Engebretson & Wardell, 1993; Francis, 1994; Indian Herbal Pharmacopoeia 1999; Jugalbandhi of Ayurveda with Allopathy, 2006; Mohar, 1999; Maclennan et al.,1996; Mukherjee, 2002; Rangari, 2002; WHO 1993 Research Guidelines; WHO 2002 Policy Perspectives of Medicines; Traskie, 1997; Govt of India 2001 The Ayurvedic Pharmacopoeia of India; Evans, 2006).Source of plant drugs:Plant drugs can be derived either from the whole plant or from different organs, like leaves, stem, bark, root, flower, seed, etc. and even from excretory plant product such as gum, resins and latex. Presently, Allopathic medicine (e.g Codeine – Anticough; Morphin- painkiller; Artemisinin- antimalarial, etc.) has also been derived from a number of plants (Papaver somniferum, Papaver somniferum, Artemesia annua, respectively etc.) which form an important segment of the modern pharmacopoeia (Kumar et al 1997, Yue-Zhong Shu, 1998). Some more examples of important drugs obtained from plants are digoxin from Digitalis spp., quinine and quinidine from Cinchona spp., vincristrine and vinblastine from Catharanthus roseus, atropine from Atropa belladonna and morphine and codeine from Papaver somniferum. It is estimated that 60% of anti-tumour and anti-infectious drugs already on the market or under clinical trial are of natural origin (Yue-Zhong Shu, 1998). Few important chemical intermediates needed for manufacturing the allopathic drugs are also from plants origin (e.g. diosgenin, solasodine, b-ionone) (Tal et al 1983). In addition, a large number of natural products, especially plant-derived drugs, continues to be discovered on the basis of traditional or empirical local medical practices (Farnsworth et al 1985). A single medicinal plant may contain hundreds of natural constituents, and a mixed herbal medicinal product may contain several times that number. About 25% of the drugs prescribed worldwide come from plants, 121 such active compounds being in current use. Of the 252 drugs considered as basic and essential by the World Health Organisation (Farnsworth et al 1985). These and more similar molecule can be searched by using modern scientific tools. For example, GC-MS study of Pyrostelgia venusta flower indicated that the phytochemicals myoinositol, hexadecanoic acid, linoleic acid, oleic acid, stigmasteryl tosylate, diazoprogesterone, arabipyranose, propanoic acid, pentamethyldisilanyl ester, acetophenone, trans-3-Hexenedioic acid, and 9-Octadecenoic acid (Z)-methyl ester were present in the flower (Roy et al 2012). These compounds exhibit diverse activities, including: antiseptic; bactericidal; fungicidal; antiviral; analgesic; anesthetic; antihistaminic; anti-inflammatory; expectorant; antitussive; spasmolytic, spasmogenic; sedative; antiedemic; antidiabetic; anticancer and cancer preventative; antiatherogenic; liver protection; and fever reduction (Johnson & Gitksan, 2006).text/html2013-03-02T08:29:01+01:00http://www.webmedcentral.com/Dr. Narotam Sharma Hepatitis B Virus DNA Quantification Using TaqMan Probe and its Significance
http://www.webmedcentral.com/article_view/4042
Introduction Hepatitis B virus (HBV) is a partially double stranded DNA containing virus of Hepadnaviridae family (1). Hepatitis B virus (HBV), a 3.2 kb Orthohepadnavirus, is a well-known agent of acute and chronic hepatitis, with an estimated 350 million chronic carriers around the world (2, 3). HBV cause the Hepatitis B which may be acute or chronic in nature. As an estimate more than 2 billion people are infected with HBV and 350 millions are chronically infected (2). Hepatitis is associated with high rate of morbidity and mortality. It is a fatal disorder due to complications of cirrhosis, hepatocellular carcinoma (HCC) and portal hypertension. It enters into the liver by blood circulation. The new DNA strand are generated by reverse transcription of mRNA intermediate which is longer than that of its genome and is about 3.5 kb in length, the mRNA is called as pre-genomic RNA or PgRNA. The HBV genome encodes four partially overlapped open reading frames (ORF).SerologyHBV contains three types of serological markers, Hepatitis B surface Antigen (HBsAg) which is a surface coat protein and is also called as decoy particle and it has four phenotypes (adw,adr,ayw,ayr). Hepatitis B ‘c’ Antigen (HBcAg) which is inner core protein (single serotype) and HBeAg which is secreted protein (3). These serological markers play a vital role in diagnosis of acute HBV infection. HBV-preS specific receptors are primarily expressed on hepatocytes, however, viral DNA and proteins have also been detected in extra hepatic sites, suggesting that cellular receptors for HBV may also exist on extra hepatic cells (Illustration 1) (4).TransmissionHepatitis B is largely transmitted through exposure to bodily fluids containing the virus. This includes unprotected sexual contact, blood transfusions, re-use of contaminated needles and syringes, vertical transmission from mother to child during childbirth, and so on. Without intervention, a mother who is positive for the hepatitis B surface antigen confers a 20% risk of passing the infection to her offspring at the time of birth. This risk is as high as 90% if the mother is also positive for the hepatitis B e antigen. Transmission from mother to child during childbirth without intervention, a mother who is positive for HBsAg conferes a 20% risk of passing the infection to her offspring at the time of birth. Roughly 16-40% of unimmunized sexual partners of individuals with hepatitis B will be infected through sexual contact. The risk of transmission is closely related to the rate of viral replication in the infected individual at the time of exposure. Other risk factors for developing HBV infection include working in a health care setting, transfusion, dialysis, acupuncture, tattooing, extended over ease travel and residence in an institution. However hepatitis B viruses cannot spread by casual contact, such as holding hands, sharing eating utensils or drinking glasses, breast feeding, kissing, hugging, coughing or sneezing (5).Molecular tools for HBV detectionVirology techniques developed over the past 20 years are being used to diagnose and monitor chronic viral infections, such as those caused by hepatitis B virus (HBV) and hepatitis C virus (HCV). Molecular biology tools can be used to detect and quantify viral genomes, sequence them, assign them to a phylogenic clade or subclade (genotype or subtype), and identify clinically relevant nucleotide or amino acid substitutions, such as those associated with resistance to antiviral drugs. Recent advances include real-time target amplification methods for detecting and quantifying viral genomes and next-generation sequencing (NGS) techniques. Other new assays detect and quantify viral antigens, whereas point-of-care tests and alternatives to biologic tests that require whole-blood samples have been developed.Target Amplification MethodsFirst-generation target amplification techniques have been widely used to diagnose HBV and HCV infections and to monitor responses to antiviral therapies. In PCR or Transcription Mediated Amplification (TMA) assays, amplicons are detected at the end of the amplification process by their specific hybridization to immobilized oligonucleotide probes; the amplicon–probe hybrids are detected by an enzymatic reaction. They are quantified based on competitive amplification of the viral template with a known amount of synthetic standard added to each reaction tube; the relative amounts of viral template and standard amplicons are measured and the results are interpreted with a standard curve established in parallel.Because amplification reactions are saturable, these methods have a narrow dynamic range of quantification. As a result, high levels of virus are not always accurately quantified and require reanalysis after samples are diluted, whereas low levels of virus (such as in patients receiving antiviral therapy) are often not detectable. This problem was solved by development of real-time target amplification techniques in which quantification takes place during the exponential phase of the amplification reaction. In addition, the reaction is run in a closed system, which prevents carryover contamination and improves specificity.Real-Time PCRPCR uses a number of temperatures and a thermostable DNA polymerase to create double-stranded DNA amplicons. In assays for HBV, nucleic acids are isolated from a sample and the HBV DNA is directly amplified by PCR. Each complete PCR cycle doubles the number of DNA copies. The principle of Real-Time PCR is to detect amplicon synthesis during the PCR reaction and there by deduce the starting amount of viral genome in a clinical sample. A fluorescent probe is linked to a quencher and annealed to the target sequence, between the sense and antisense PCR primers. During each PCR reaction, as the DNA polymerase extends the primer, its intrinsic nuclease activity degrades the probe, releasing the reporter fluorochrome (Illustration 2, 3). The amount of fluorescence released during the amplification cycle and detected by the system is proportional to the amount of amplicons generated in each PCR cycle. Software is used to calculate the threshold cycle in each reaction (Ct), which has a linear relationship with the initial amount of nucleic acid. In each PCR run, parallel processing of a panel of quantified standards is used to establish a standard curve for quantification. Commercially Available Real-Time Target Amplification Assays for HBV DNA Quantification is shown in illustration 4.
Hepatitis-B viral antigen and antibodies detectable in the blood follows acute infection. Numerous tests are used for the detection of Hepatitis B viral infection which involves serum or blood tests that detect either viral antigen (protein produce by the virus) or antibodies produce by the host against the Hepatitis B viral protein. HBV testing begins with serological blood tests used to detect antibodies to HBV (6). Hepatitis B surface antigen (HBsAg) is a most frequently used marker to screen for the presence of this infection. It is the first detectable viral antigen, appearing during the infection. However in early infection, this antigen may not be present and it may be undetectable later in the infection stage as it is being cleared by the host, which is disadvantageous for HBV detection. Molecular tests help to determine the activity of HBV infection, the selection of patients for the treatment, and the efficacy of antiviral therapy, identifying the development of HBV drug resistant strains (7). Sample preparation is the major weakness in molecular tests, and improvement is constantly introduced to decrease the variability of techniques and the risk of contamination, such as ready-to-use reagents and automation of the extraction procedure. Overall HBV antibody test have a strong positive predictive value for exposure to HBV, but may miss patient who have not yet develop antibody, called seroconversion or have an insufficient levels antibodies to detect (8). Immunocompromised individuals infected with HBV may never develop antibodies to virus and never test positive using HBV antibody screening. Anti-HBV antibody indicate exposure to the virus, but cannot be determine if on going infection is present but initial stage of infection is very low amount of antibodies which are not detectable by serological methods. During HBV infection body immune system take a span of time for generation of antibodies against HBV infection (protein produced by Hepatitis B virions), this period of time is called ‘Window Period’(9). During this ‘window’ in which the host remains infected but is successfully clearing the virus, IgM antibodies to Hepatitis-B core antigen (HBc IgM) may be the only serological evidence of disease. After the appearance of HBsAg another Ag named as the HBeAg will appear. Traditionally, the presence of HBeAg in the host serum is associated with much higher rates of viral replication and enhanced infectivity. During the natural course of an infection, the HBeAg may be cleared an Ab to the ‘e’ Ag (Anti-HBe) will arise immediately afterwards (10). This conversion is usually associated with a dramatic decline in viral replication. If the host is able to clear the infection, eventually the HBsAg will become undetectable and will be followed by IgG antibody to the Hepatitis-B surface antigen and core antigen (Anti-HBs and Anti-HBc IgG). Individual who remains HBsAg positive for at least 6 months are considered to be Hepatitis-B carrier. Carriers of virus may have chronic Hepatitis-B, which would be reflected by elevated serum alanine aminotransferase (ALT) level and inflammation of liver as revealed by biopsy (11). Particularly those who acquired the infection as adults have very little viral multiplication and hence may be at little risk of long term complications or transmitting infection to others. Various advanced PCR based detection techniques have been developed for qualitative and quantitative detection of HBV infection (12).HBV Quantification by Real Time Utilizing TaqMan Probe AssayThe HBV viral load can be monitored utilizing COBAS TaqMan 48 Real Time PCR from Roche. This technique uses thermostable recombinant enzyme DNA polymerase (ZO5) for reverse transcription and as well as PCR amplification (13). Under the appropriate, conditions utilizing the Mn2+, ZO5 DNA polymerase involve the reverse transcription and PCR amplification together with real time detection of amplicon from processed specimen. In this technique dual labeled fluorescent probe called TaqMan is being utilized for real time detection of amplicon accumulation, which will be monitored by the emission intensity of fluorescent reporter dyes released during amplification process (14).TaqMan Probe Molecular ChemistryTaqMan probe consists of oligonucleotide sequence labeled with reporter and quencher dye. The TaqMan probes are designed in such a way, which contains complimentary sequence for target. The reporter region of the TaqMan probe binds to the 5’ end of the target. When these dual labeled fluorescent probes are present in bound state the quencher region emits the fluorescence which is absorbed by the reporter region. This type of emission of fluorescence of quencher region and absorbance based on the ‘forster type energy transfer effect’ (FRET) (as shown in illustration 2 and 3). During the amplification reaction the probes hybridized to a target sequences at 5’ and is cleaved by the 5’-3’ exonuclease activity of the thermostable ZO5 DNA polymerase. When the thermostable ZO5 DNA polymerase come in contact with hybridized probe during polymerization, reporter & quencher dyes are released and separated. Quenching no longer occurs and the fluorescent activity of quencher dye increases (15).Hepatitis B Virus DNA QuantificationAmplification of HBV DNA & HBV quantitation standard DNA is measured independently at different wavelengths. Emission intensity of individual reporter dye effectively increases in each cycle that allows independent identification of HBV DNA & HBV quantitation standard DNA. This intensity of signal is related to the amount of starting material at the beginning of PCR. Real Time PCR accumulates continuous collection of fluorescent signals from one or more polymerase chain reaction over a range of cycles. Quantitative Real Time PCR is the conversion of fluorescent signal from each reaction into numerical value for each sample by means of graph. As the concentration of the virus increases, the growth curves shift to earlier cycles. Therefore the leftmost growth curve corresponds to the highest viral titer level whereas the rightmost growth curve corresponds to the lowest viral titer level. Graphical representation of fluorescent signal from an ideal v/s actual reaction over 40 cycles of real time PCR in an ideal PCR, there are two phases, a baseline where the signal is below the level of instrument detection followed by the persistent geometric increases in fluorescence that continue over the remaining cycle of the experiment. However, is an actual PCR, there are four phases as in ideal reaction, there is a baseline followed by a geometric phase (16). However, the amplification becomes less than ideal leading to a linear phase & finally a plateau where no further increase in signal occur over the remaining cycle. The COBAS TaqMan 48 Analyzer automatically determines the HBV DNA titer for the specimen or control. The HBV DNA titer is expressed in International Units (IU)/mL.text/html2010-09-30T10:22:34+01:00http://www.webmedcentral.com/Prof. Krupanidhi SreeramaMolecular Interplay
http://www.webmedcentral.com/article_view/825
Molecules play important role at various levels in the life of organisms1. They are not only involved in the biological and physiological functions but also percolated in the field of behaviour and business2,3. The same has been highlighted in a poetic style to facilitate the students of Biology to know the scope of discipline of Biosciences.Molecules make lifeOrganisms studded with moleculesMolecules facilitate cells to communicate.Cells talk each other through signal moleculesMolecules recognize self and non-selfMolecules barricade us as immunological herds.Molecules either down or up regulate metabolismCascade of STAT is through moleculesMolecules fix axesMolecules determine organ systems.Molecules bring curiosity among biochemists and crystallographersMolecules keep up the healthMolecules provide livelihoodMolecules make marketInformatics revolves around molecules.Contemplation is through moleculesMemorisation is owing to moleculesTranquility and turmoil are due to moleculesMolecular interplay brings beauty to the bearer.Ultimately, molecules regulate gene expressionVia., DNA-Protein complexationForever, epigenome dictates life processes.text/html2010-10-30T22:27:56+01:00http://www.webmedcentral.com/Mr. Ahmad H IbrahimAmplification, Cloning And Characterization Of Rna Helicase Gene From Aedes Aegypti
http://www.webmedcentral.com/article_view/1094
The genomic study of hazardous human parasites for instant, Aedes aegypti can improve our understanding of the disease vectors and help us to control the lethal diseases such as dengue fever and other transmitted diseases. The present study reported the amplification, cloning and determination of nucleotide sequences of the gene that encodes for DEAD box ATP-dependent RNA helicase, from Ae. aegypti. Totally, 1415 bp length of DNA segment from Ae. aegypti was amplified and cloned, which was found complementary to sequences at the 5´ end of the putative RNA helicase mRNA. Upstream sequences of the PCR primer at the 3´ end of our amplified DNA fragment matches exactly with the first 346 nt of the 5´ end of putative mRNA. The matched sequences consist of 297 bp exon and 49 bp untranslated region. Upstream of the matching sequences were comprised of another untranslated region (1048 bp), which may presumably be the promoter of the gene having nt sequences at -419 position, complementary to the TATA box.
text/html2010-11-15T18:09:29+01:00http://www.webmedcentral.com/Dr. Natalia NagradovaPeptidyl-prolyl Cis/trans Isomerase Activity In The Functioning Of Native Folded Proteins
http://www.webmedcentral.com/article_view/965
The finding that cis/trans isomerization of proline peptide bonds can provide the basis for conformational changes in native folded proteins, ensuring transition between functionally different states, has led to marked progress in this field of research. Presently, there are reasons to assume that the proline switch can serve as a precise regulator of biological function in native folded proteins. This review aims to summarize the available information on proline switch mechanisms in different proteins, focusing the attention on features pointing to similarities or distinctions between these mechanisms. Two groups of proteins are considered. The first one includes proteins which require the presence of an external factor accelerating the process of isomerization. These proteins are interleukin- 2 tyrosine kinase SH2 domain and the Crk adaptor protein. Each of them is able to catalyze an intrinsic intramolecular switch afforded by prolyl cis/trans isomerization, but this process is very slow; it can, however, be significantly accelerated by cyclophilin A (CypA), a peptidyl-prolyl cis/trans isomerase. The second group, comprising 5HT3 receptor of neurotransmitter-gated ion channel, cytochrome P450cam, and molecular chaperone Hsp70, can do without external catalysts; these proteins are able to accelerate cis/trans isomerization due to intrinsic structural features that facilitate this process. In each case, the likely mechanism of proline switch is discussed. Special attention is given to the possibility that a conformational switch may result from a shift of equilibrium between coexisting cis and trans conformers.text/html2013-10-07T07:06:12+01:00http://www.webmedcentral.com/Dr. Binod KumarDNAzyme mediated post-transcriptional gene silencing: A novel therapeutic approach
http://www.webmedcentral.com/article_view/4415
The increasing understanding of the regulatory mechanisms involved in the progression of diseases is opening up opportunities for new therapeutic intervention. DNAzymes, derived by in vitro selection processes, is one such discovery that has potential for selective gene silencing. The first report on DNAzymes came nearly eighteen years ago and since then hundreds of DNA sequences have been isolated in many research laboratories around the world to facilitate many chemical transformations of biological importance. These catalytic molecules have been successfully used against several targets including viruses, cardiovascular diseases, cancers and brain disorders with commendable results. The recent years have witnessed the use of these catalytic molecules for various innovation driven applications ranging from biosensing to gene regulation. This review will discuss the discovery, synthesis and applications of DNAzymes as potential therapeutic molecules.