Absract Archive January 2008 Research article Optimization of Thermostable Laccase Production From Pleurotus eous Using Rice Bran Abstract A laccase producing fungus Pleurotus eous was obtained from Tamil Nadu Agricultural University, which produced a thermostable laccase enzyme utilizing rice bran. The optimum conditions for laccase activity was 500C at pH 6.0 containing 1% rice bran in the Malt extract medium after 7 days of incubation. Peptone, tryptone and yeast extract proved as good nitrogen sources while glucose, starch, and sucrose as carbon sources for the production of laccase enzyme. The extracellular production of the enzyme, its thermostable nature and its compatibility suggest its application in the textile and paper mill industrial wastewater treatment. Key words: laccase, rice bran, thermostability, enzyme Introduction Laccase formation in a number of white-rot fungi is known to be influenced by various physiological factors (Mansur et al., 1998) and it can be enhanced by the addition of certain organic compounds and metal ions. Fungi are capable of accumulating high concentrations of heavy metals (Melgar et al., 1998; Cihangir and Saglam,1999). This may pose serious human health risks in the case of edible mushrooms, especially if they are grown on agro industrial wastes, which may contain toxic substances such as heavy metals. Heavy metals can affect the growth (Pointing et al., 2000), the extracellular enzyme production (Baldrian et al.,2000), and the dye-decolorizing ability (Pointing et al., 2000) of the white rot fungi. Industrial applications require enzymes that are able to withstand altered physical conditions like temperature, pH etc. For production of enzyme for industrial use, isolation and characterization of new promising strains using cheap carbon and nitrogen source is a continuous process. Rice bran, a by-product of the milling of rice is a good source of proteins at present underutilized as a food material. The potential of producing rice bran at the global level is 27.3 million ton. The present study was undertaken to examine the effectiveness of rice bran as potent alternative protein source in production medium and optimization of parameters using rice bran for maximizing enzyme production. Authors:S Shanmugam, Rajasekaran, T. Sathish Kumar Research article Heterologous expression of envelope Protein (Domain III) of Dengue Virus Type 2 for Serodiagnosis Abstract Dengue is re-emerging as one of the most important public health problems. Since there is no specific therapy available, the timely and rapid diagnosis plays a vital role in patient management. Thus, there is a need to develop an alternate antigen to replace the whole virus antigen in diagnostic tests, which has several drawbacks. In this study, domain III of envelope gene of dengue virus type 2 was cloned in pQE 30UA expression vector and expressed in E. coli strain M15 (pREP4). The recombinant dengue-2 domain III protein was purified from inclusion bodies by affinity chromatography. The reactivity of this protein was checked by Western blot using patient serum sample. This protein was evaluated by an in-house ELISA employing a panel of human serum samples and the results were compared with Pan-Bio IgM capture ELISA. These results revealed that the product could be used as diagnostic reagent in dengue diagnosis. Keywords: Dengue virus, ELISA, rD2 DIII protein, affinity purification, Western blot. Introduction Dengue virus infection is now recognized as one of the most important mosquito borne human infection of 21st century. The global incidences of the dengue infection have increased enormously and an estimated 50-100 million cases of dengue infections are now reported annually from more than 100 tropical and sub tropical countries of the world (WHO, 2002). Dengue is caused by four antigenically distinct viruses designated as dengue virus type 1 - 4, belonging to genus Flavivirus of family Flaviviridae. Dengue is endemic in India and dengue 2 & 3 serotypes have been reported in various outbreaks in the past (Dar et al., 1996; Parida et al., 2002; Dash et al., 2005-06). However, the large and severe outbreaks were caused by dengue virus type-2. All the four serotypes of dengue viruses are primarily transmitted by Stegomyia (Aedes) aegypti. Definitive diagnosis of dengue infection depends on the identification of the virus, virus-encoded antigens, viral genomic RNA or the virus-induced antibodies (Gubler, 1998). Viral RNA can be detected with a high degree of sensitivity, using reverse transcription coupled with polymerase chain reaction (RT-PCR). The shortcoming common to all these methods is the narrow window period (5~days), available for successful detection, which coincides with the febrile period during which patients are viremic (Vaughn et al., 2000). This precludes diagnostic tests based on the identification of the virus or its RNA genome because of the short duration of viremia. Thus, in a majority of cases the only feasible diagnostic test would have to be based on the identification of anti-dengue antibodies. Numerous commercial kits are available using whole virus cell culture antigen (Groen et al., 2000) in enzyme-linked immunosorbent assay (ELISA) format. The most commonly used diagnostic test is IgM-capture enzyme-linked immunosorbent assay (Groen et al., 2000) performed with a single specimen. Viral antigen, commonly prepared in cell culture or in suckling mouse brain, is often the limiting reagent in developing countries. Utilization of recombinant proteins, expressed using baculovirus can be produced more easily, and present little or no health hazard, are an attractive alternative (Cuzzubbo et al, 2001). The simplified production of recombinant dengue virus antigen in E.coli avoids problems associated with the quality and standardization of conventional dengue viral antigen preparations (Huang et al 2001; AnandaRao et al, 2005; Tripathi et al, 2007). Author : A. M. Jana Review Hepatitis A - a worldwide illness Previous Designations Infectious hepatitis, Epidemic hepatitis, Epidemic jaundice, Catarrhal jaundice, Infectious icterus, Botkin's disease and MS-1 hepatitis. Introduction Hepatitis means inflammation of the liver which can be caused by exposure to alcohol, from infections, certain medications, chemicals, poisons or from a disorder of the immune system. Hepatitis A refers to liver inflammation caused by infection with HAV-Hepatitis A Virus. HAV is one of several virus that cause hepatitis. Prevalence Infection with HAV is known to occur throughout the world. Risk of infection is greatest in developing countries with poor sanitation or poor personal hygiene standards. According to Centers for Disease control and Prevention, it is estimated that 84,000 to 1,34,000 of the total infections show symptoms of HAV. Each year, approximately 100 people die because of HAV. History In 1972, a scientist, Mac Callum suggested that the virus of infectious hepatitis should be called as virus A. This virus was first isolated by Purcell in 1973. It is a Picornavirus, formerly classified in the genus Enterovirus. Genome studies (sequence homology) showed that it did not belong in Enterovirus and has been reclassified in a genus of its own, Hepatovirus. HAV Hepatitis A Virus Structure Figure 2 - A cluster of hepatitis viruses, as seen through a transmission electron microscope. HAV is non enveloped with a diameter of 27-32 nm, it is composed entirely of viral protein and RNA. Electron microscopy show icosahedral nucleocapsid and virus replicates in cytoplasm of the cell. (Www.wales.nhs.uk) The hepatitis A virion is spherical with a diameter of about 27nm.The virion consists of a genome of linear single stranded RNA of messenger sense polarity and of a protein shell made of three major proteins, VP1 to VP3. The presence of fourth protein VP4 has been repeatedly described, but the reported apparent molecular weights (7-14KD) contrast sharply with those predicted from nucleic acid sequence data (1.5KD or 2.3KD). Propagation Hepatitis A virus has been propagated in fetal Rhesus monkey kidney cell lines, in human diploid lung, embryo fibroblast and in a variety of transformed cell lines. In cell culture, HAV appears to grow slowly, fails to reach high titers and does not inhibit macromolecular synthesis. Replication Replication of HAV occurs in the cytoplasm of the hepatocyte, within membrane bound vesicles. Figure 3 - Hepadnavirus replication occurs within a newly formed core particle in the cytoplasm of the infected cell. In the nucleus, the partially double stranded DNA is 'repaired' by the cell to form a complete circle of DNA. It then undergoes transcription by the host cell RNA polymerase and the transcript is translated by host cell ribosomes. New virus particles are formed, which acquire lipid from the endoplasmic reticulum of the host cell, and the genome is packaged within these particles, which then bud off from the cell. (www.gsbs.utmb.edu) Epidemiology • HAV is excreted in feces of infected people and can produce clinical disease when susceptible individuals consume     contaminated water or foods. • Cold cuts and sandwiches, fruits and fruit juices, milk and milk products, vegetables, salads, shell fish and iced drinks are    commonly implicated in outbreaks. • Contamination of foods by infected workers in food processing and restaurants is common. • It can be transmitted primarily through oral fecal contact. This includes sexual contact, especially oral-anal sex. • Hepatitis A can easily spread among young children in day care settings because many are in diapers and cannot wash    their own hands and no one may know that they are subjected to infection since children normally do not have symptoms    or have anicteric illnesses. • Spread can occur from non human primates to humans • HAV can be transmitted through blood transfusion, use of blood products, sharing needles or other injecting equipments    contaminated with HAV infected blood. Transmission by blood is rare because the presence of virus in the blood occurs with    the onset of infection and is not thought to be present long. • Infected persons can start spreading the infection about 1 week after their own exposure. People who do not have    symptoms can still spread the virus. Author : N.Vijayalakshmi. Review Dietary Phytochemicals: a Promise to Chemoprevention Abstract Cancer is a growing health problem around the world. Cervical cancer is commonest among women, followed by breast, esophagus, ovary, and stomach cancer. Among men most common are lung, bronchus, stomach oral cavity, pharynx, larynx, prostate, and rectum cancer. Synthetic anticancer drugs evoke severe side effects and in many cases patient may recover cancer but may die due side effects and severe immunosuppression. Several dietary phytochemicals are an alternative due to their various degree of antiproliferetive and immunostimulatory effects on various types of human cancers. This review will discuss the potentiality and promise of few plant derived secondary metabolites and there mode of actions in different human cancers. Facts and Figures WHO reported more than 10 million cases of cancer per year worldwide. In 2003, approximately 1,300,000 new cases were diagnosed, and more than 550,000 people died from cancer in US alone. As per National Cancer Registry Program of ICMR (1997), Chennai is the pocket of highest incidence of different cancers (stomach, cervix, ovary, prostate, and lung) in India (Chaudhry and Luthra). According to Mudur (2005), some Indian cities shows world’s highest cancer incidence. For example, New Delhi for gall bladder cancer in women, Wordha for mouth cancer, Pondicherry for tong cancer in men, and Kohima for nasopharyngeal cancer. Why Phytochemicals Cancer is a multistage carcinogenic process where there is a net accumulation of atypical cells arising from excess proliferation, an insufficient apoptosis or a combination of the two (Hetts, 1998). The conventional radiotherapy and chemotherapy with synthetic drugs evoke severe side effects including severe immunosuppression and in majority of cases patient may recover cancer but ultimately die due to infectious diseases and organ failure. Thus from therapeutic point of view, the best strategy is “induced apoptosis in the neoplastic cell line without affecting the normal cells of the body”(Fan et al., 1998). In this context, diatary phytochemicals are a potential alternative source of safer chemicals which are not only anticancerous but are also antioxidants, antidiabetic, antimutagenic and of other physiological benefits. Some of these common dietary bioactive secondary metabolites namely Curcumin, Resveratrol, Sulforaphane, Styrylpyrone derivatives, Theasinensin-A, Theaflavin, Acacetin etc. show various degree of their individual antiproliferetive effect and induction of apoptosis in various type of human cancers in various cytotoxic pathways. This review will deal with these phytochemicals and their mode of actions. Killing of Cancer cells Phyochemicals generally acts on apoptotic pathways. Apoptosis is a genetically controlled cellular suicide method by which unwanted cells are removed from the system with out necrosis. Generally there are two pathways: Extrinsic pathway (caspase-8 and 12) and Intrinsic pathway (caspase-9). The extrinsic pathway can be upregulated by treatment with drug like Cisplatin (Fulda et al, 1997), Doxorubicin and Methothrexate (Friesen et al., 1997). Once activated, caspase –8 activates downstream caspases by proteolytic cleavage of their zymogen forms (Muzio et al., 1997) thus amplifying the caspase signal to bring apoptosis. The intrinsic pathway is induced by ionizing radiation and also by chemotherapeutic drugs (Renatus et al., 2001) In intrinsic pathway, cytochrome-C is released from the mitochondria to the cytosol by he action of external stimuli (Kluck et al., 1997). Released Cyt-C acts as a co factor and interacts with Apaf-1 and procaspase –9 to form apoptosome. Apoptosome activates caspase-9 (Zou et al., 1997). Active casp-9 activates down stream (executioner) caspases like caspase-3,7 and 6 by limited proteolysis to facilitate cell death (Earnshaw et al., 1999). Most of these phytochemicals work on this pathway. Author:D. Barh Mini Review Integrating the Science of Pharmacology and Bio Informatics Phyllanthus "The wonder plant" Abstract Family            : Euphorbiaceae Genus            : Phyllanthus Synonyms      : Phyllanthus amarus, Phyllanthus carolinianus, P. sellowianus, P. fraternus,                         P. kirganella, P. lathyroides, P. lonphali,                         P. niruri, P. polyphyllus etc.                         Phyllanthus urinaria Ref:www.easttennesseewildflowers.com/ gallery/view Introduction Phyllanthus, so aptly called the wonder plant perhaps has more useful properties to offer than mankind could ever use. The 10 sub-genera of Phyllanthus have more than 700 species. It was first identified in central and southern India in 18th century but is now found in many countries including Philippine, Cuba, Nigeria among others. Biliary and urinary conditions including kidney and gallbladder stones are areas where Phyllanthus plays an important role. It is also used for hepatitis, colds, flu, tuberculosis, and other viral infections; liver diseases and disorders including anemia, jaundice and liver cancer; and for bacterial infections such as cystitis, venereal diseases and urinary tract infections. Diabetes and hypertension are other disorders on which the wonder plant has profound effect on. Furthermore, extracts from P. amarus such as tannins and phenol have been associated with some medical importance. The tannins have antimicrobial activity. Even though this miracle herb has many therapeutic uses, here we will focus on its healing powers on: • Hepatitis • Kidney stones and • Tumors • Hepatitis Hepatitis is a disorder that primarily affects the liver. The disorder causes swelling of the liver that makes it stop working well. It can lead to scarring, called cirrhosis, or to cancer. Action of Phyllanthus on hepatitis virus Phyllanthus attacks DNA polymerase, which is essential for Hepatitis virus to reproduce. It also inhibits reverse transcriptase. Phyllanthin and Hypophyllanthin are two plant chemicals found in Phyllanthus, which are responsible for its anti-viral activity. Phyllanthin is soluble in chloroform, has a melting point of 196 - 198°C and a molecular weight of 418.53. They have been isolated from leaves along with others like Lignansniranthin, nirtetralin, and phyltetralin. They are major component of many popular liver tonics. Hypophyllanthin is soluble in ethanol, has a melting point of 128°C - 129°C and a molecular weight of 430.40. Phyllanthin and Hypophyllanthin are said to protect hepatocytes against carbon tetrachloride and galactosamine induced toxicity. They also reduce blood cholesterol and both LDL (low density lipoprotein) and VLDL (very low density lipoprotein), the two forms of cholesterol susceptible to oxidation. They act by reducing the ability of the liver to synthesize cholesterol and by increasing faecal bile acids excretion. The role that this plant plays in liver disorders is due to its febrifuge, antiseptic, astringent, stomachic, deobstruent and diuretic actions. Authors:Rakhee.R.Nair,Regin Susan Abraham. Short Communication Characterization of two Monoclonal Antibodies 10F3 and 15E5 directed against Mitotic Cytosolic Proteins of Chinese Hamster Ovary (CHO) Cells. Abstract Mitosis being a dynamic process is controlled by several factors of which certain protein complexes collectively called Mitosis Promoting Factors and Cell Cycle Control Factors are of importance and identified. Antibodies are powerful reagents for a variety of applications due to their specificity to a target antigen. Polyclonal and Monoclonal antibodies were generated for cytosolic protein extracts of CHO cells in the mitotic stage. Two hybridoma supernatants that showed the highest secretory activity from the panel of hybridomas obtained were characterized in this study. As there can be many common cytosolic proteins in the Interphase and the mitotic stages of a cell type, a sandwich ELISA was used which had the previously characterized Polyclonal antibodies as the primary antibody, the cytosolic protein extracts of cells block in G0/G1, S, G2 and M stages as the antigen and the two hybridoma supernatants 10F3 and 15E5 as the secondary antibodies. The study showed the specificity of the MAbs generated and also showed a trend of the level variation of Mitotic Factors through the various stages of the cell cycle studied. With finer characterization, it would be possible to employ the described two MAbs for a variety of applications making them useful biological reagents especially in mitosis and cell cycle related studies. Key Words: Cytosolic Proteins, Antibodies, ELISA, Mitotic Factors. Introduction Proliferating cells go through regular cycle of events taking place in an ordered progression, the mitotic cell-cycle, in which the genetic material is duplicated and divided equally between two daughter cells. Many of the genes and proteins involved in the mitotic cell cycle have been identified. The interphase of the cell cycle is generally divided into three phases G1 (Gap1), S (DNA synthetic), and G2 (Gap2). The mitotic part of the cycle (M) is divided into five phases: prophase, prometaphase, metaphase, anaphase and telophase. Mitosis is followed by cytokinesis, the division of the cytoplasm and cell membrane required to complete the formation of two daughter cells. All phases of the cell cycle are marked by an orderly progression of metabolic processes. Progression of the cell cycle is regulated at several checkpoints, which ensure that all cellular components are present and in good working order before the cell proceeds to the next stage. Both the G1/S and the G2/M checkpoints are regulated by a mechanism in which two proteins interact. The concentration of the first protein, cyclin, oscillates during the cell cycle. The second protein, cyclin dependent kinase (CDK) cannot function unless it is bound to cyclin. (1) Authors:Sukanya. S, Govind. P, Preetha ,B, Kavya. K, Solomon F D Paul, Ravi.M