Effect of pH on the conformation and stability of the plant toxin ricin

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.     

The structure of toxic protein, the mistletoe lectin, at different pH: the study using intrinsic fluorescence method

The effects of pH on the conformation of mistletoe lectin I and its isolated A- and B-subunits has been investigated by using the methods of intrinsic fluorescence. By the denaturating action of guanidine hydrochloride and the influence of the quenchers (I-, Cs+, acrylamide) the structural stability of the native protein and its isolated subunits was estimated. Treatment of the protein with the denaturant and quenchers revealed its different structure at pH 7.0 and 4.0. At pH 4.0 tryptophan residues become more accessible to quenchers, positive charge of the surrounding area increases and the protein becomes more stable to the action of denaturant. The structure of the isolated A- and B-chains of mistletoe lectin I differs considerably from that of the whole protein: a) its stability to the action of guanidine hydrochloride is lower; b) it depends on the ionic strength of the solvent; c) it is characterized by increased accessibility of tryptophan residues to quenchers (for B-chain). Differences between the conformations of the isolated chains at pH 7.0 and 4.0 are marked more strongly; moreover, at pH 4.5 the B-chain undergoes structural transition. The possible relationship between structural peculiarities of mistletoe lectin I and the mechanism of its transmembrane transfer is discussed.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using Mycobacterium neoaurum VKPM As-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM An-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI3 in situ.     

Protein splicing

Inteins are internal polypeptide sequences that are posttranslationally excised from a protein precursor by a self-catalyzed protein-splicing reaction. Most of inteins consist of N- and C-terminal protein splicing domain and central endonuclease domain. The endonuclease domain can initiate mobility of the intein gene, this process being named intein homing. This review is focused on the recent data about the structure and function of inteins. Main intein-mediated protein-engineering applications, such as protein purification, ligation and cyclization, new forms of biosensors, are presented.     

Profiles of the utilization of 20 amino acids as the only source of nitrogen and carbon in bacteria of the genera Klebsiella, Enterobacter, Serratia, Escherichia

The profiles of the utilization of 20 protein amino acids in 118 Klebsiella pneumoniae sub- sp. pneumoniae, K. oxytoca, K. planticola, K. mobilis, Enterobacter cloacae, Serratia marscescens, S. liquefaciens, Escherichia coli strains isolated from clinical material were studied. The utilization of amino acids was determined on minimal saline agar containing amino acid as the only source of nitrogen and carbon; the results were evaluated after 72-hour incubation at 37 degrees C. 17 profiles of amino-acid utilization were thus determined, most of them genus-specific in enterobacteria: Klebsiella (profiles No. 1--6, 9, 10), Enterobacter (No. 11--13), Serratia (No. 14--16), Escherichia (No. 17). The full coincidence of amino-acid utilization profiles in bacteria of K. mobilis (No. 1, 6) and K. pneumoniae subsp. pneumoniae with out of such profiles in bacteria of the genera Enterobacter, Serratia, Escherichia was established, which confirmed that K. mobilis (formerly Enterobacter aerogenes) belonged to the genus Klebsiella.     

Activity of the climbing fiber innervating various Purkinje cells

Low-amplitude potentials (10-130 microV) related to the action of a distant branch of the climbing fiber, which elicits complex spikes of the reference Purkinje cell were revealed by means of potential averaging synchronously with complex spikes of Purkinje cells in 10 out of 255 paired records of cerebellar Purkinje cells activity and extracellular field potentials at interelectrode distances of 200-1500 microns. These potential waves had a stable form in independent sets of data. In 3 out of 10 cases, the low-amplitude potentials included a slow (about 100 ms in duration) component. In one case, both test and reference electrodes recorded both simple and complex spikes of different Purkinje cells so that complex spikes of both cells were practically synchronous (conditional probability of complex spikes p = 0.97, onset time difference 0.54 ms). Thus for the first time in cerebellar physiology both simple and complex spikes activity of two Purkinje cells controlled by the same climbing fiber was recorded.     

Use of molecular-biological methods for identifying brucella in a comparative analysis of strains, isolated from sick dogs

Ten strains isolated from sick dogs in 1998 in St. Petersburg were studied by traditional and molecular biological methods of Brucella identification. PCR study confirmed that the isolated cultures were Brucellae, and comparative study of the traditional phenotypical characteristics and protein and antigenic composition allowed referring all the isolated strains to B. canis. Traditional identification showed similarity of 7 strains with the reference B. canis strain RM6/66, and 3 strains were similar to B. canis Mex 51 strain. These results confirmed the division of B. canis into two biovars. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate demonstrated the identity of protein profiles of 10 strains isolated from dogs to the reference B. canis RM6/66 strain. Immunoblotting analysis with S- and R-specific rabbit antisera also demonstrated the identity of antigens binding IgG antibodies in the strains isolated from dogs to the reference B. canis RM6/66 strain.     

Substrate phase state and time factor in variability of human fecal microbiota

Effects of substrate phase state and time factor on variability of human fecal microbiota were studied. It was shown that microecological system of native feces was characterized by marked time-dependent variability. It is unstable and begins to destruct after 24 hours of cultivation. The most sensitive elements of the system were bifidobacteria and Escherichia coli. Change of phase state of biotope eliminated the effect of factor limiting the microecosystem development, which allowed species of obligate and transitory microflora to freely colonize the growth substrate and interact with each other. The mentioned facts demonstrate that fecal microbiota exists in the environment of excess of growth substrate, which colonization is limited by cluster structure of biotope of native feces. It was concluded that phase state of growth substrate and duration of cultivation are important factors determining the population variability of fecal microbiota.     

Abnormal methylation of p16/CDKN2A AND p14/ARF genes GpG Islands in non-small cell lung cancer and in acute lymphoblastic leukemia

Multiplex methylation-sensitive PCR and methylation-specific PCR were employed in studying the methylation of CpG islands in the p16/CDKN2A and p14/ARF promoter and the first exon regions in non-small cell lung cancer (54 samples) and acute B-cell lymphoblastic leukemia (61 samples). Differences in methylation were detected between types of neoplasia as well as between CpG islands studied within the same types of tumors. High level of the p16/CDKN2A first exon CpC island methylation was revealed in non-small cell lung cancer (68%) and in acute B-cell lymphoblastic leukemia (55%) and the CpG island of p14/ARF first exon was nonmethylated in these types of tumors. The methylation of CpG-rich fragments of genes p16/CDKN2A and p14/ARF promoters was analysed. As was found out, CpG islands located in 5' areas of one and the same gene can differ in methylation frequencies. The comparison of sensitivity between methylation-specific PCR and methylation-sensitive PCR used in the methylations studies was carried out.     

Transport systems for carbonate in the extremely natronophilic cyanobacterium Euhalothece sp

The effect of carbonate concentration, pH of the medium, and illumination intensity on the major physiological characteristics (growth rate and the intensities of CO2 assimilation and oxygen photoproduction) of the natronophilic cyanobacterium Euhalothece sp. Z-M001 have been studied. It was established that the investigated microorganism has at least two transport systems (TS) for CO2, which differ in both the pH optimum and substrate affinity: TS I has a pH, 9.4-9.5 and a K(S) 0.5 of 13-17 mM, whereas TS II has a pH(opt) 9.9-10.2 and a K(S) 0.5 of 600-800 mM. The substrate affinity of these transport systems is several orders of magnitude lower than the substrate affinity of the transport systems of freshwater cyanobacteria. It is suggested that they are unique for extremely alkaliphilic cyanobacteria and reflect their adaptation to the seasonal cycles of the lake hydrochemistry.