Are L-tyrosine and L-dopa hormone-like bioregulators?

Some amino acids have bioregulatory functions, which far exceed those of precursors for proteins or of substrates for specific enzymes. Two of these amino acids, L-tyrosine and L-dopa, are precursors to melanin and catecholamines. In vertebrates, they can act as inducers and regulators of the melanogenic apparatus and of MSH receptors--two quite complex functions that could hardly be performed by mere substrates. Focussing on the pigmentary system as a study model, we therefore explore the hypothesis that L-tyrosine and L-dopa act as hormone-like bioregulators in mammals, with melanocytes regulating tyrosine and dopa activity via their metabolic consumption.     

Characterization of the serotoninergic system in the C57BL/6 mouse skin.

We showed expression of the tryptophan hydroxylase gene and of tryptophan hydroxylase protein immunoreactivity in mouse skin and skin cells. Extracts from skin and melanocyte samples acetylated serotonin to N-acetylserotonin and tryptamine to N-acetyltryptamine. A different enzyme from arylalkylamine N-acetyltransferase mediated this reaction, as this gene was defective in the C57BL6 mouse, coding predominantly for a protein without enzymatic activity. Serotonin (but not tryptamine) acetylation varied according to hair cycle phase and anatomic location. Serotonin was also metabolized to 5-hydroxytryptophol and 5-hydroxyindole acetic acid, probably through stepwise transformation catalyzed by monoamine oxidase, aldehyde dehydrogenase and aldehyde reductase. Activity of the melatonin-forming enzyme hydroxyindole-O-methyltransferase was notably below detectable levels in all samples of mouse corporal skin, although it was detectable at low levels in the ears and in Cloudman melanoma (derived from the DBA/2 J mouse strain). In conclusion, mouse skin has the molecular and biochemical apparatus necessary to produce and metabolize serotonin and N-acetylserotonin, and its activity is determined by topography, physiological status of the skin, cell type and mouse strain.     

Melanotropic activity of gamma MSH peptides in melanoma cells.

We studied the pigmentary activity of the peptides gamma 1, gamma 2 and gamma 3 melanocyte stimulating hormone (MSH), which differ in the structure of their C-termini, using hamster and mouse melanoma cell lines responsive to beta-MSH by increasing tyrosinase activity. Gamma 1-MSH alone or in combination with beta-MSH had no effect on either cell line. Gamma 2-MSH alone was biologically inactive but potentiated beta-MSH stimulation of tyrosinase activity. Gamma 3-MSH at high concentration (10 microM) induced tyrosinase activity and dendrite formation in the hamster melanoma line. When added together with beta-MSH, gamma 3-MSH partially inhibited the tyrosinase activity response to beta-MSH. Thus, gamma-MSH peptides have low intrinsic melanotropic activity in mammalian melanoma cells; the specific pigmentary responses appear to be affected by the structure of the C-terminal portion.     

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.