Lalit Singh

Identification of Autoantibodies against Transthyretin for the Screening and Diagnosis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic and inflammatory rheumatic disease that leads to inflammation of the joints and surrounding tissues. Identification of novel protein(s) associated with severity of RA is a prerequisite for better understanding of pathogenesis of this disease that may also have potential to serve as novel biomarkers in the diagnosis of RA. Present study was undertaken to compare the amount of autoantigens and autoantibodies in the plasma of RA patients in comparison to healthy controls. Plasma samples were collected from the patients suffering from RA, Osteoarthritis (OA), Systemic lupus erythematosus (SLE) and healthy volunteers. The screening of plasma proteins were carried out using 2-dimensional gel electrophoresis followed by identification of differentially expressed protein by MALDI-TOF MS/MS. Among several differentially expressed proteins, transthyretin (TTR) has been identified as one of the protein that showed significantly up regulated expression in the plasma of RA patients. The results were further validated by Western blot analysis and ELISA. In comparison to OA synovium, an exclusive significantly high expression of TTR in RA has been validated through IHC, Western blotting and IEM studies. Most importantly, the increase in expression of TTR with the progression of severity of RA condition has been observed. The autoantibodies against TTR present in the RA plasma were identified using immunoprecipitation-Western methods. The significant production of autoantibodies was validated by ELISA and Western blot analysis using recombinant pure protein of TTR. Hence, these novel observations on increase in TTR expression with the increase in severity of RA conditions and significant production of autoantibodies against TTR clearly suggest that a systematic studies on the role TTR in the pathogenesis of RA is immediately required and TTR may be used as a serum diagnostic marker together with other biochemical parameters and clinical symptoms for RA screening and diagnosis.

Mycobacterium tuberculosis Cyclophilin A Uses Novel Signal Sequence for Secretion and Mimics Eukaryotic Cyclophilins for Interaction with Host Protein Repertoire

Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial two-hybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins.

Zinc regulates the activity of kinase-phosphatase pair (BasPrkC/BasPrpC) in Bacillus anthracis

Bacillus anthracis Ser/Thr protein kinase PrkC (BasPrkC) is important for virulence of the bacterium within the host. Homologs of PrkC and its cognate phosphatase PrpC (BasPrpC) are the most conserved mediators of signaling events in diverse bacteria. BasPrkC homolog in Bacillus subtilis regulates critical processes like spore germination and BasPrpC modulates the activity of BasPrkC by dephosphorylation. So far, biochemical and genetic studies have provided important insights into the roles of BasPrkC and BasPrpC; however, regulation of their activities is not known. We studied the regulation of BasPrkC/BasPrpC pair and observed that Zn2+ metal ions can alter their activities. Zn2+ promotes BasPrkC kinase activity while inhibits the BasPrpC phosphatase activity. Concentration of Zn2+ in growing B. anthracis cells was found to vary with growth phase. Zn2+ was found to be lowest in log phase cells while it was highest in spores. This variation in Zn2+ concentration is significant for understanding the antagonistic activities of BasPrkC/BasPrpC pair. Our results also show that BasPrkC activity is modulated by temperature changes and kinase inhibitors. Additionally, we identified Elongation Factor Tu (BasEf-Tu) as a substrate of BasPrkC/BasPrpC pair and assessed the impact of their regulation on BasEf-Tu phosphorylation. Based on these results, we propose Zn2+ as an important regulator of BasPrkC/BasPrpC mediated phosphorylation cascades. Thus, this study reveals additional means by which BasPrkC can be activated leading to autophosphorylation and substrate phosphorylation.

Serine/threonine protein phosphatase PstP of Mycobacterium tuberculosis is necessary for accurate cell division and survival of pathogen

Protein phosphatases play vital roles in phosphorylation-mediated cellular signaling. Although there are 11 serine/threonine protein kinases in Mycobacterium tuberculosis, only one serine/threonine phosphatase, PstP, has been identified. Although PstP has been biochemically characterized and multiple in vitro substrates have been identified, its physiological role has not yet been elucidated. In this study, we have investigated the impact of PstP on cell growth and survival of the pathogen in the host. Overexpression of PstP led to elongated cells and partially compromised survival. We find that depletion of PstP is detrimental to cell survival, eventually leading to cell death. PstP depletion results in elongated multiseptate cells, suggesting a role for PstP in regulating cell division events. Complementation experiments performed with PstP deletion mutants revealed marginally compromised survival, suggesting that all of the domains, including the extracellular domain, are necessary for complete rescue. On the other hand, the catalytic activity of PstP is absolutely essential for the in vitro growth. Mice infection experiments establish a definitive role for PstP in pathogen survival within the host. Depletion of PstP from established infections causes pathogen clearance, indicating that the continued presence of PstP is necessary for pathogen survival. Taken together, our data suggest an important role for PstP in establishing and maintaining infection, possibly via the modulation of cell division events.

Pharmacological evaluation of aqueous extract of syzigium cumini for its antihyperglycemic and antidyslipidemic properties in diabetic rats fed a high cholesterol diet—Role of PPARγ and PPARα

In India syzygium cumini (Myrtaceae) is commonly used traditional medicine to treat diabetes. The present study was undertaken to assess an investigation of antihyperglycemic and antidyslipidemic properties of aqueous extract of Syzigium Cumini (SC) in diabetic rats fed a high cholesterol diet. The aqueous extract of SC was screened for antihyperglycemic and antidyslipidemic activity by streptozotocin induced diabetes in rats. Animals were treated with 100, 200 and 400mg/kg body weight of aqueous extract of SC. Metformin were used as reference antihyperglycemic drugs for comparison. Administration of aqueous extract of SC or metformin for 21days resulted in a significant (P<0.05) reduction in serum glucose, insulin and Homeostasis model assessment of insulin resistance (HOMA-IR) compared with diabetic controls. Treatment with 100mg/kg/day aqueous extract of SC did not result in a significant reduction in serum insulin levels, but 200mg/kg/day and 400mg/kg/day, aqueous extract of SC and metformin showed significant reductions 17.89%, 19.60% and 24.40%, respectively. Furthermore, administration of 100, 200 and 400mg/kg/day, aqueous extract of SC and metformin resulted in significant decrease in insulin resistance of 19.20%, 41.59%, 51.55% and 68.68%, respectively. In high fat diet- streptozotocin (HFD - STZ) treated rats β-cells function (HOMA-B) were markedly reduced (5.8-fold), however observed a significant (P<0.01) improvement of β-cell function with aqueous extract of SC (400mg/kg/day) and metformin. The aqueous extract of SC seeds exhibits significant insulin-sensitizing, antidyslipidemic, antioxidant, anti-inflammatory and β-cell salvaging activity in HFD-STZ-induced type 2 diabetic rats via overexpression of PPARγ and PPARα activity, affirming its potential to be used in the prevention and treatment of type 2 diabetes mellitus (T2DM). Further isolation and characterization of active components in SC seed extract are needed to explore the other possible mechanisms and pathways that are involved in its anti-diabetic effect.

clpC operon regulates cell architecture and sporulation in Bacillus anthracis

The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.

BIPOLAR DISORDER IN ADULTS

Bipolar disorder is a chronic illness, which may require life-long treatment. Patients will spend 3-5 times more days in the depressed episode then in the manic phase. Due to this variability in episodes, polypharmacy is used quite frequently in practice, though the evidence to do this remains quite limited. Many positive and negative outcomes can occur from this practice. Bipolar disorder is the 6th leading cause of disability in the developed world among those between the ages 15 and 44 years age groups. Serotonin is one of the neurotransmitter in the brain, and one of that strongly affects the person mood. Clozapine (clozaril), olanzapine (zyperexa), risperidone (Risperdal), and ziprasidone (zeldox) and the clozapine may be helpful as mood stabilizer for people who do not respond to lithium and anticonvulsant.

“The Nutraceutical Amino Acids”- Nature’s Fortification for Robust Health

Nutraceuticals i.e. the product positioned at the interface between food and drugs, are an increasing group of products gaining importance; patients/consumers of drug always have a wish to have no or less side/ toxic effect and also complementary or alternative benefit of the drug products and that’s why nowadays they are using nutraceuticals. In recent years, because of health benefits and an alternative to modern medicine, there is a growing interest in nutraceuticals. Nutrients, functional foods, medicinal food, herbals and dietary supplements are major constituents of nutraceuticals these play an artistic instrumental role in health maintenance, they also show various acts against various disease conditions and thus promote and sustain the quality of life. The article aims to explore and discuss the ability of nutraceuticals to treat or prevent underlying causes of disease; the article outlines nutraceuticals with their therapeutic applications, adverse effects and interaction. Various researches on nutraceutical product are under process which will be integrating and assessing information further.

REVIEW ON RECENT ADVANCES IN A MODERN DAY TREATMENT: DIURETIC THERAPY

The choice of drugs to initiate therapy for the management of hypertension remains contentious and diu retics are central to this controversy. Because most of the major trials involve complex treatment algorithms and allow diverse background treatments, one of the greatest challenges lies in separating out true class specific effects – for example, separati ng true class - specific effects of diuretics from those of beta blockers . Thiazide diuretics were the first tolerated efficient antihypertensive drugs that significantly reduced cardiovascular morbidity and mortality in placebo - controlled clinical studies. Although these drugs today still are considered a fundamental therapeutic tool for the treatment of hypertensive patients. A description of successful use of diuretics in specific edematous states, such as congestive heart failure, chronic renal failure, nephrotic syndrome, and liver disease, is followed by a brief discussion of the management of resistant edema and the use of diuretics in non edematous states, including essential hypertension and other conditions. The elements required to successfully ach ieve adequate natriuresis under such conditions are analyzed. Because achieving diuresis may result in significant hypokalemi a, hyponatremia, metabolic alkalosis, and worsening prerenal azotemia, the prevention and management of these complications of diur etic therapy are also reviewed.

ROLE OF XANTHAN GUM ( XANTHOMONAS COMPESTRIS ) IN GASTRORETENTIVE DRUG DEL IVERY SYSTEM: AN OVERVIEW

Floating drug delivery system is the form of gastro - retentive drug delive ry system. That controls kinetic release rate of drug to a specific site for its pharmacological action. These are achieved by use of various polymeric substances including natural polymer such as xanthan g um. This delivery system prolongs the retention ti me of the drug in the stomach as compared to conventional dosage form. The present article highlights the use of xanthan gum for the formulation of the gastro - retentive drug delivery system especially with natural polymer (xanthan gum). The main goal of an y drug delivery system is to achieve desired concentration of the drug in blood or tissue, which is therapeutically effective and non toxic for a prolonge d period . Oral delivery of drugs is by far the most preferable route of drug delivery due to the ease of administration, patient compliance and flexibility in formulat ion etc. From immediate release to cite specific delivery, oral dosage forms have really progressed.