Cobalt-dependent transcription of the nitrile hydratase gene in Rhodococcus rhodochrous M8

Abstract The effects of cobalt ions on the activities of Rhodococcus rhodochrous M8 enzymes for nitrile utilization, nitrile hydratase and amidase, were investigated. In contrast to amidase, synthesis of nitrile hydratase and its activity required cobalt ions in the growth medium. Northern blot analysis showed that in the presence of cobalt ions, the level of mRNA for nitrile hydratase genes was several times higher than that under cobalt-limited conditions. It was assumed that the low nitrile hydratase activity in cells grown in the absence of cobalt ions is connected either with the weak expression of nitrile hydratase genes or with the rapid degradation of nitrile hydratase mRNA.     

Virulence-associated characteristics and phage lysogenicity of two morphologically distinct colonies of Vibrio cholerae O139 serogroup

Abstract The presence of a temperate phage was demonstrated in a strain of Vibrio cholerae O139 isolated from a patient. Spontaneous variants with translucent colonies had lost this phage. The loss of the phage was associated with increased hydrophobicity, indicating the loss of the capsule. These clones were sensitive to serum bactericidal activity, showed decreased expression of such presumed virulence factors as proteases, motility and mannose-sensitive pili. Furthermore, excision of the phage made the strain dependent on purines for growth.     

Gm and Km immunoglobulin allotypes in Reindeer Chukchi and Siberian Eskimos

Summary Blood samples from 403 Reindeer Chukchi of inland Chukotka, and 100 samples from Chaplin Eskimos of the Chukot Peninsula were tested for G1m (z,a,x,f), G2m (n), G3m (g,b0,b1,b3,b5,s,t), and Km (1) allotypic determinants. An apparent affinity between the Chukchi and the Eskimos could be inferred from similar frequencies of the two common haplotypes, Gm^za;g and Gm^za;bst, and from very similar frequencies of the Km¹ allele. However, none of the Eskimos had Gm^zax;g, though it occurred at a low or moderate frequency in the five Chukchi populations studied. It is assumed that Chukchi can be distinguished from adjoining Eskimos by the same G1m (x) outlier, that has been considered as a taxonomic marker useful in differentiating between Eskimos and American Indians. Comparison of North Asian and North American populations with respect to the array and frequencies of Gm haplotypes and the Km¹ allele, supports the hypothesis of a nonrandom distribution of the Gm^za;bst and Km¹ on both sides of the Bering Strait.     

Nitrile metabolism in fungi: A review of its key enzymes nitrilases with focus on their biotechnological impact

Nitriles are abundant in the plant kingdom. The ability to detoxify them is beneficial for microbes living in the plant environment. Nitrilases (NLases; EC 3.5.5.-), which hydrolyze nitriles to carboxylic acids, have been well characterized in bacteria, and classified into various substrate-specificity subtypes (aromatic NLases, aliphatic NLases, arylacetoNLases). NLases also occur in filamentous fungi, mainly in Ascomycota (subdivision Pezizomycotina), as documented by genome mining. However, the investigation of NLases in fungi has been delayed compared to bacteria. Only a few NLases (aromatic NLases) were purified from native fungal strains (mainly Fusarium), which were grown under suitable induction conditions. Over a few past years, the spectrum of known fungal NLases was broadened by expressing fungal NLase genes in Escherichia coli. Thus functional NLases were reported for the first time in fungi of genera Auricularia, Macrophomina, Nectria, Neurospora, Pichia, Talaromyces, Trichoderma and Trichophyton. Two major substrate-specificity subtypes were identified in them, i.e. aromatic NLases and arylacetoNLases, apart from a few NLases with broad substrate specificities. The biotechnological impact of fungal arylacetoNLases was explored with a focus on the enantioselective hydrolysis of (R,S)-mandelonitrile, the selective hydrolysis of one cyano group in dinitriles and the hydrolysis of nitrile precursors of the taxol sidechain. Despite recent advances, the wealth of fungal NLases whose sequences have been deposited in databases has not yet been fully exploited. Overproduction in E. coli has the potential to bring these NLases to life. This will enable to estimate the natural roles of NLases in fungi and will also provide new catalysts for biotechnological uses.     

Interaction of piroxicam with bovine serum albumin investigated by spectroscopic,calorimetric and computational molecular methods

Abstract

The binding of drugs to serum proteins is governed by weak non-covalent forces. In this study, the nature and magnitude of the interactions between piroxicam (PRX) and bovine serum albumin (BSA) was assessed using spectroscopic, calorimetric and computational molecular methods. The fluorescence data revealed an atypical behavior during PRX and BSA interaction. The quenching process of tryptophan (Trp) by PRX is a dual one (approximately equal static and dynamic quenched components). The FRET results indicate that a non-radiative transfer of energy occurred. The association constant and the number of binding sites indicate moderate PRX and BSA binding. The competitive binding study indicates that PRX is bound to site I from the hydrophobic pocket of subdomain IIA of BSA. The synchronous spectra showed that the microenvironment around the BSA fluorophores and protein conformation do not change considerably. The Trp lifetimes revealed that PRX mainly quenches the fluorescence of Trp-213 situated in the hydrophobic domain. The CD and DSC investigation show that addition of PRX stabilizes the protein structure. ITC results revealed that BSA-PRX binding involves a combination of electrostatic, hydrophobic and hydrogen interactions. The analysis of the computational data is consistent with the experimental results. This thorough investigation of the PRX-BSA binding may provide support for other studies concerning moderate affinity drugs with serum protein.

Communicated by Ramaswamy H. Sarma     

Interaction of plasma membrane Ca-ATPase isoform 4 with calcineurin A: Implications for catecholamine secretion by PC12 cells

PMCA1–4 isoforms have been recently recognised as regulators of various signalling pathways in mammalian cells. PMCAs were found to interact with calcineurin A in an isoform specific manner. In this study we focus on the interaction of calcineurin A with PMCA4 and its effect on catecholamine secretion in PC12 cells with reduced PMCA2 or PMCA3 content. Reduction of synthesis of PMCA2 or PMCA3 led to upregulation of PMCA4 manifested by preferential interaction of PMCA4 with calcineurin A. On the other hand, we observed a significant reduction of dopamine secretion, which did not correspond with an increased [Ca²⁺]_c. This result indicates that the interaction of PMCA4 with calcineurin A plays a regulatory role in the signalling during catecholamine secretion.