Detection of spores of Bacillus anthracis from environment using polymerase chain reaction

A sensitive PCR based detection of Bacillus anthracis spores from environnment was standardized. Specific 1247bp amplicon could be detected with template concentration as low as 13 pg. Sensitivity was enhanced to 10 fold by nesting with second set of primers, forming 208bp amplicon. Extraction of DNA from spores purified from soil samples by aqueous polymer two-phase system followed by partial germination and freeze-thaw treatment yielded best results. Soil sample spiked with spores (8x10(2)/g of sample) could be detected with this method.     

Modulation by phenolic compounds of ABA-induced inhibition of mustard (Brassica juncea L. cv. RLM 198) seed germination

Exogenous abscisic acid (ABA) inhibited the germination of B. juncea seeds in a concentration dependent manner. As revealed in a time-course study, the ABA-induced inhibition got progressively alleviated with the lapse of time following ABA treatment possibly due to metabolic conversion of applied ABA in the seed tissue. A simultaneous application of certain phenolic compounds namely, p-coumaric-, vanillic-, gallic-, and chlorogenic acid (but not caffeic acid) also caused an alleviation of ABA effect. Of the above two patterns of recovery, the phenolic-dependent alleviation of ABA effect was apparent much earlier (24-48 hr treatment) than the time-dependent one (72 hr). It is likely that phenolics could accelerate ABA metabolism in the seed tissue leading to an early recovery from ABA-induced inhibition.     

Dose and time-related in vitro effects of glucocorticoid on phagocytosis and nitrite release by splenic macrophages of wall lizard Hemidactylus flaviviridis

Glucocorticoids (GC) are usually considered to be immunosuppressive and anti-inflammatory. However, recent studies in mammals have demonstrated the diverse effects of GC on non-specific host-defense mechanism, depending on dose or duration of treatment. Hence, in the present study in vitro dose and time-related effects of glucocorticoid, i.e. hydrocortisone (HC) on phagocytosis and nitrite production by LPS-induced splenic macrophages in wall lizard Hemidactylus flaviviridis has been investigated. Hydrocortisone suppressed percentage phagocytosis, phagocytic index and nitrite production by splenic macrophages even at the lowest concentration (10(-13) M) for a short-term exposure (4 h). Hydrocortisone-induced suppression enhanced with the increase of concentration or duration of exposure time. The suppressive effect of hydrocortisone on phagocytic and cytotoxic activities of splenic macrophages was further corroborated since the pre-exposure of macrophages to glucocorticoid-receptor blocker (RU 486) considerably reduced the hydrocortisone-induced suppression of phagocytosis and nitrite production. The present study suggests that GC instead of diverse effects, has dose- and time-dependent immunosuppressive effect on non-specific host-defense immune responses in wall lizard H. flaviviridis.     

Molecular mechanisms of Bartter syndrome caused by mutations in the BSND gene

Barttin, a gene product of BSND, was identified as a fourth gene responsible for Bartter syndrome. The co-expression of barttin with CLC-K chloride channels has been demonstrated to dramatically induce the expression of CLC-K current. However, it remains unknown how barttin interacts with CLC-K channels in mammalian cells and how the mutations of barttin lead to Bartter syndrome. In an attempt to clarify the effect of barttin expression on CLC-K2 cellular localization, we examined the expression of the CLC-K2 chloride channel and barttin, solely and in combination, in transient and stable expression systems in mammalian cells. In addition, we generated several stable cell lines expressing mutant barttins to clarify the consequence of the previously reported barttin mutations in Bartter syndrome. In immunocytochemistry, CLC-K2 was retained in the Golgi in the absence of barttin expression, but delivered to the plasma membrane when barttin was present. Barttin was coprecipitated with CLC-K2, suggesting a protein-protein interaction. Disease-causing mutant barttins, especially R8L, were retained intracellularly, but their binding ability to CLC-K2 was preserved. This led to a retention of CLC-K2 in intracellular organelles with barttin, and a loss of plasma membrane localization. The stability of the CLC-K2 protein was also markedly increased by coexpression with barttin. These results clarified that barttin determined cellular localization of CLC-K2 by protein-protein interaction. Thus, the mislocalization of CLC-K2 was identified as the molecular pathogenesis of Bartter syndrome by mutant barttins.     

Modulation of antigenicity of mycelial antigens during developmental cycle of Karnal bunt (Tilletia indica) of wheat

Indirect enzyme linked immunosorbent assays (ELISA) were developed using polyclonal antibodies against soluble cytoplasmic (SCA) and insoluble cell wall antigens (ICWA) for monitoring modulation of mycelial antigens during growth cycle of T. indica. With SCA, continuous decrease in ELISA reactivity was observed in maturing fungus cultures, suggesting that SCA were expressed predominantly during early vegetative phase and their decreasing role was apparent as the fungus matures possibly towards sporogenous mycelium. In case of ICWA, the reaction profile showed an increase up to exponential phase of growth probably due to increase in the cell division and branching of mycelium. But later, ICWA antibody reactivity was decreased which may be due to conversion of mycelial phase to sporogenous phase, a quiescent stage of growth. Characterization of changes in antigenic configuration during developmental cycle of Tilletia indica by these antibodies could prove to be useful in identification of developmentally related and virulence marker(s).     

Antifungal potential of some higher plants against Fusarium udum causing wilt disease of Cajanus cajan

The fungitoxic effects of different plant extracts on Fusarium udum, which causes wilt disease of Cajanus cajan in vitro and in vivo, were examined. The complete arrest of the radial growth of the pathogen occurred at a 10% concentration of leaf extract from Adenocallyma alliaceum. A leaf extract of Citrus medica, a root extract of Asparagus adscendens, rhizome extracts of Curcuma longa and Zingiber officinale, and a bulb extract of Allium sativum inhibited up to 100% growth at higher concentrations. A. alliaceum controlled the disease up to 100% by amending its 4% powder in unsterilized soil and 2% in sterilized soil. The population of F. udum was found to be markedly reduced following treatments with plant powders.     

Production of d-amino acids using immobilized d-hydantoinase from lentil, Lens esculenta, seeds

d-Hydantoinase from the lentil, Lens esculenta, seed is quite unstable, and has been immobilized on Diethyl amino ethyl (DEAE) cellulose by an adsorption and cross-linking method. The immboilized d-hydantoinase exhibited 80% enzyme activity and contained 86% protein. The immobilization of the enzyme preparation does not change its optimum pH, temperature or affinity constant, but increases its shelf-life, thermostability and stability in various organic solvents. This immobilized d-hydantoinase can be used effectively for the production of d-amino acids from the corresponding hydantoins and may therefore be of use in the chemical and pharmaceutical industries. Received: 28 April 1998 / Received last revision: 10 July 1998 / Accepted: 10 July 1998