[Synthesis of spyropyran derivatives of retinal and study of their interaction with bacterioopsin from Halobacterium salinarum].

The synthesis of retinal analogue series that contain a spyropyran moiety instead of a trimethylcyclohexene ring was proposed. The process of the retinal analogue interaction with bacterioopsin from apomembranes of Halobacterium salinarum and the spectral properties of the newly formed pigments were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

[The role of emotional pressure in the development of the early forms of chronic cerebrovascular insufficiency]. / Rol' émotsional'nogo napriazheniia v razvitii nachal'nykh form khronicheskoi tserebrovasculiarnoi nedostatochnosti.

The paper presents a comparative analysis of the results of biochemical, neuropsychological and neurophysiological examination of initial chronic cerebrovascular insufficiency in patients without significant psycho-emotional tension (the 1-st group--37 individuals) and with significant emotional tension (the 2-nd group--44 persons). Statistically significant differences between the groups concerned both cerebral hemodynamic parameters and EEG data. On the basis of the results obtained several possible pathogenetic variations of chronic cerebral circulatory insufficiency are suggested on the domination of either primary damages of the structures of the brain and its vessels or the secondary disorder of energetic metabolism and cerebral blood circulation. That, in turn, may be conditioned both by supertension of cerebral systems in chronic stress, and by somatic disturbances. More precise definition of the main pathogenetic factors of initial chronic cerebrovascular insufficiency may be useful for improving efficiency of rehabilitation in these patients.

Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta.

Marrow stromal cells from wild-type mice were infused into transgenic mice that had a phenotype of fragile bones resembling osteogenesis imperfecta because they expressed a human minigene for type I collagen. In mice that were irradiated with potentially lethal levels (700 cGy) or sublethal levels (350 cGy), DNA from the donor marrow stromal cells was detected consistently in marrow, bone, cartilage, and lung either 1 or 2.5 mo after the infusions. The DNA also was detected but less frequently in the spleen, brain, and skin. There was a small but statistically significant increase in both collagen content and mineral content of bone 1 mo after the infusion. Similar results were obtained with infusion of relatively large amounts of wild-type whole marrow cells into the transgenic mice. In experiments in which male marrow stromal cells were infused into a female osteogenesis imperfecta-transgenic mouse, fluorescense in situ hybridization assays for the Y chromosome indicated that, after 2.5 mo, donor male cells accounted for 4-19% of the fibroblasts or fibroblast-like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin. In a parallel experiment in which whole marrow cells from a male mouse were infused into a female immunodeficient rag-2 mouse, donor male cells accounted for 4-6% of the fibroblasts or fibroblast-like cells in primary cultures. The results support previous suggestions that marrow stromal cells or related cells in marrow serve as a source for continual renewal of cells in a number of nonhematopoietic tissues.

Specific inhibition of expression of a human collagen gene (COL1A1) with modified antisense oligonucleotides. The most effective target sites are clustered in double-stranded regions of the predicted secondary structure for the mRNA.

A series of antisense oligonucleotides (ASOs) were synthesized and tested to define the best target sites within an RNA transcript of collagen for effective inhibition of expression. The test system consisted of mouse NIH 3T3 fibroblasts that were stably transfected with a human minigene for procollagen I so that the cells simultaneously synthesized full-length mouse pro alpha 1 (I) chains and internally deleted human pro alpha 1 (I) chains. The sequences of the transcripts from both genes were compared, and a series of 28 ASOs were designed to target sites in which there were at least two base differences within a 20-nucleotide sequence between the human and mouse transcripts. Six of the ASOs specifically decreased the levels of pro alpha 1 (I) chain synthesized from the human gene without a decrease in the levels of pro alpha 1 (I) chains from the mouse endogenous gene. The most effective ASOs reduced the intracellular levels of human pro alpha 1 (I) chains relative to the mouse pro alpha 1 (I) chains to 37-67% of the control values. Combined addition of two effective ASOs or a second administration of the same effective ASO did not produce any additive effect. The results did not support previous suggestions that the best target sites for ASOs were sequences around initiation codons for translation, at intron-exon boundaries, or in single-stranded loops in hairpin structures. Also, the results did not support previous suggestions that the most effective ASOs are those with the highest affinities for their target sequences. Instead, the most consistent pattern in the data was that the most effective ASOs were those targeted to sequences that were predicted to form clustered double-stranded structures in RNA transcripts.

[Search for new products of gene expression in human cardiac muscle. Microsequencing of proteins after two-dimensional electrophoresis]. / Poisk novykh produktov gennoi ékspressii v serdechnoi myshtse cheloveka. Mikrosekvenirovanie belkov posle dvumernogo élektroforeza.

Proteins of the human heart muscle were studied using modified two-dimensional electrophoresis. After separation, proteins were electroblotted onto Immobilon P membranes and several protein spots were used for microsequencing analysis. In most cases the proteins analyzed have blocked N-terminal amino acids. In order to study the primary structure of these proteins, hydrolysis in situ by trypsin followed by reversed-phase HPLC and microsequencing of the resulting peptides were performed. Four protein were identified in 8 analyzed fractions, specifically myosin light chain 1 (MLCl-V/sB), fatty-acid binding protein (heart isoform), alpha (B)-crystallin and alpha-tropomyosin. Amino acid sequences of two proteins were not found among human amino acid sequences collected in SWISSPROT bank (v. 21).

Identification of an allelic variant of isoform MLC1-V/sB (human myosin light chain).

A study of 250 specimens of human myocardium by two-dimensional gel electrophoresis revealed an allelic variant of isoform MLC1-V/sB, which was identified by immunoblotting with monoclonal antibody against MLC1-V/sB and peptide mapping after in situ tryptic digestion of electroblotted proteins. The substitution Asn-144 for His-144 was found in this new allelic variant of MLC1-V/sB.

Membrane-bound phenylalanine hydroxylase of human liver.

Phenylalanine hydroxylase (EC 1.14.16.1) antigen and activity have been identified among proteins extracted with a buffer containing 0.4% Triton X-100 from adult human liver bioptate fraction, which was sedimented at 105,000 x g (n = 4). This enzyme fraction was designated as a 'membrane-bound form of phenylalanine hydroxylase'. It amounted to 5-15% of phenylalanine hydroxylase activity and 15-25% of phenylalanine hydroxylase antigen content. After immunoblotting two-dimensional gels, the soluble (cytoplasmic) form of phenylalanine hydroxylase antigen displayed three spots: one spot corresponded to the L-subunit with a molecular weight of 55,000, the two other spots corresponded to the H-subunit with a molecular weight of 57,000. Only the L-subunit was revealed in the membrane-bound enzyme form. Both phenylalanine hydroxylase activity and antigen were also demonstrated in extracts from human embryonic livers (n = 7). However, in this case the membrane-bound phenylalanine hydroxylase amounted to 85% of the antigen content. Subunit compositions of the enzymes were similar in adult and embryonic livers. The differences in the subunit compositions and enzyme activities of membrane-bound and cytoplasmic forms of phenylalanine hydroxylase in adults and embryos may be due to other functions of this enzyme in the hepatocyte membrane.

[Identification of alpha-tropomyosin of the human myocardium by two-dimensional electrophoresis, and sequencing]. / Identifikatsiia alpha-tropomiozina serdechnoi myshtsy cheloveka dvumernym élektroforezom i sekvenirovaniem.

Two-dimensional electrophoresis of total cardiac muscle extracts allows the detection of about 200 protein fractions. In preliminary studies the fraction D-10 protein was characterized in terms of relative molecular mass, isoelectric point and quantitative composition as alpha-tropomyosin. The similarity of the protein to human alpha-tropomyosin was confirmed by the results of analysis of the N-terminal sequence of the D-10 protein eluate in a gas-phase sequencer.

[The dynamics of the properties of liver phenylalanine hydroxylase in human embryogenesis]. / Dinamika svoistv fenilalaningidroksilazy pecheni v émbriogeneze cheloveka.

Content, subunit composition and activity of phenylalanine hydroxylase's (PH) (EC 1.14.16.1) in cytoplasmic and membrane proteins extract of embryonic liver on week 6-11 of pregnancy were studied. PH enzymatic and antigenic activities were detected starting from the week 6 of pregnancy. Liver cytoplasmic PH antigen content increased gradually during development while its enzymatic activity remained practically unchanged. Concomitantly, relative content of L-subunit increased. Content of liver membrane PH antigen was constant during development. Samples of liver with relatively low specific PH activity were characterized by high content of PH in cytoplasm and vice versa. Since PH activity in extracts prepared from mixture of these samples decreased, an unknown PH inhibitor must be present in cytoplasmic protein extracts with relatively low specific PH activity.

[Immunochemical detection and characteristics of the subunit composition of phenylalanine hydroxylase in the brain of man]. / Immunokhimicheskoe vyiavlenie i kharakteristika sub"edinichnogo sostava fenilalaningidroksilazy v mozge cheloveka.

Immunochemical properties and subunit structure of an antigen were characterized in autopsy specimens of human liver and brain, using antiserum against human phenylalanine hydroxylase. An identical antigen was revealed in extracts of organs by immunoelectrophoresis. Its content was 1.5-2.0 mg/g tissue in the liver and 20-40 micrograms/g tissue in the brain. One L enzyme subunit and two H subunits were identified in the liver extracts after two-dimensional electrophoresis followed by immunoblotting. Subunit structure of phenylalanine hydroxylase in the brain was similar to that in the liver. The molecular weight of L subunit was 55,000 and it was located in the same area as albumin isoforms. The molecular weight of H subunits was 57,000 and they differed from L subunits in pI. The antigen was purified from crude extracts of biopsy liver by affinity chromatography on immunoadsorbent to phenylalanine hydroxylase and showed phenylalanine hydroxylase activity. An antigen with similar molecular weight was also purified from the brain extract by the same method. These data suggest that phenylalanine hydroxylase can be present in the human brain.

[Phenylalanine hydroxylase of human embryos: its homology with the enzyme from the liver of adults]. / Fenilalaningidroksilaza émbrionov cheloveka: gomologiia s fermentom iz pecheni vzroslykh liudei.

Phenylalanine hydroxylase (PH) activity was discovered in the liver of 7-12 week old human embryos. Embryonic and adult PHs were identical, as shown by immunoelectrophoresis. Unlike the adult liver PH, the PH content of the extract of cytoplasmic proteins of embryonic liver was reduced but the specific activity was increased more than by one order of magnitude. H (57,000 D) and L (55,000 D) subunits were detected by immunoblotting. The L subunit predominates in the extract of membrane proteins of embryonic liver. Hence, the major part of phenylalanine oxidizing activity in the embryonic liver is related to the enzyme immunochemically identical with the PH of adult liver but differing from it in some structural and functional properties.

[Immunochemical detection of phenylalanine hydroxylase in extracts of various human organs]. / Immunokhimicheskoe vyiavlenie fenilalaningidroksilazy v ékstraktakh nekotorykh organov cheloveka.

Phenylalanine hydroxylase was found in extracts from autoptates of human liver, kidney, myocardium, small intestine, lung and spleen. In all the tissues studied, except of spleen, the antigen was detected by immunoelectrophoresis with monospecific antiserum against human liver phenylalanine hydroxylase. As shown by immunoblotting carried out after electrophoresis under denaturating conditions, the antigen, observed in various tissues, exhibited similar electrophoretic mobility which was very close to that of the enzyme purified from human liver tissue. Molecular mass of revealed antigen was estimated 55-57 kDa. Coincidence of immunochemical and chemical properties of the protein suggested that the antigen, detected in the tissue extracts, was a product of phenylalanine hydroxylase gene expression. The antigen concentration did not correlate with the content of albumin in tissue extracts, thus demonstrating that the revealed antigen did not occur in these preparations with blood contaminations. Content of the antigen in the tissue extracts studied was (ug per g): liver - 1500-1900, kidney - 300-575, brain - 20-40, myocardium - 85-105, lung - 40-125, small intestine - 45-70, spleen - 0-12.

[Identification of the membrane form of phenylalanine hydroxylase from the human liver and characteristics of its subunit composition]. / Vyiavlenie membrannoi formy fenilalaningidroksilazy pecheni cheloveka i kharakteristika ee sub''edinichnogo sostava.

Phenylalanine hydroxylase was detected among human liver bioptats and autoptats extracted with 0.4% Triton X-100 from the 105,000 g homogenate fraction. In contrast to the membrane form of the rat liver enzyme, human liver phenylalanine hydroxylase is detected both by its enzymatic activity and immunochemically under non-denaturating conditions. The enzymatic activity of phenylalanine hydroxylase makes 5-15% of that of the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction as can be evidenced from rocket immunoelectrophoresis data. Immunoblotting of proteins performed after denaturating electrophoresis of the membrane and cytoplasmic fractions revealed an antigen band with a similar electrophoretic mobility. The subunit composition of the enzyme in both fractions was characterized by two-dimensional electrophoresis with subsequent immunoblotting. It was found that the membrane fraction of the enzyme is represented only by the L-subunit with Mr of 55 kD, whereas the cytoplasmic fraction, besides the predominant L-subunit, also contains 2H-subunits of the enzyme with Mr = 57 kD. These 2H-subunits differ between themselves as well as from the L-subunit by the pI value.

[Detection and characteristics of membrane form of phenylalanine hydroxylase (EC 1.14.16.1) from the rat liver]. / Vyiavlenie i kharakteristika membrannoi formy fenilalaningidroksilazy (KF 1.14.16.1) pecheni krys.

The proteins extracted with 0.4% Triton X-100 from the 105000 g homogenate fraction were shown to possess the phenylalanine hydroxylase (EC 1.14.16.1) activity. This phenylalanine hydroxylase fraction was designated as the membrane form of the enzyme. However, immunochemical methods of the antigen analysis performed under non-denaturating conditions and employing monospecific antisera to phenylalanine hydroxylase (double immunodiffusion in agar, racket immunoelectrophoresis, enzyme purification on immunoadsorbents) failed to reveal the antigen among the membrane fraction proteins of the liver. In this fraction the antigen was identified only by immunoblotting performed after electrophoresis of the proteins under denaturating conditions. The molecular mass of the cytoplasmic and membrane forms of the enzyme subunits is identical (52 kD). The Km value of phenylalanine for the cytoplasmic form of phenylalanine hydroxylase is 0.32.10(-3) M, that for the membrane form is 1.66.10(-3) M. Both enzyme forms can bind to phenyl-Sepharose after their activation by the substrate, and they dissociate from the carrier after phenylalanine removal from the incubation mixture, which points to the intactness of the phenylalanine binding allosteric center in the membrane form of the enzyme. This finding allowed for the purification of the membrane form of phenylalanine hydroxylase by affinity chromatography on phenyl-Sepharose.

[Isolation of inactive phenylalanine hydroxylase from autopsy specimens of human liver]. / Poluchenie neaktivnogo belka fenilalaningidroksilazy iz autoptatov pecheni cheloveka.

An electrophoretically homogeneous protein has been isolated from human liver autoptats, using a procedure employed for the isolation of phenylalanine hydroxylase from rat liver. The procedure includes chromatography of liver extracts on phenyl-Sepharose and subsequent purification on DEAE-Toyopearl. The activity of phenylalanine hydroxylase in the autoptats was markedly decreased in comparison with that in bioptats. The isolated protein possessed no enzymatic activity. However, the subunit composition of the protein, the molecular masses of protein subunits (55 and 57 kD) and the amino acid composition were close to those of the human enzyme. Antibodies to the protein inhibited the phenylalanine hydroxylase activity in human liver bioptats and weakly inhibited the rat enzyme. The experimental results suggest that the structural organization of phenylalanine hydroxylase does not alter as a result of the loss of enzymatic activity in cadaverous human liver.

[Clinico-rheoencephalographic characteristics and criteria for different initial forms of atherosclerotic brain lesions]. / Kliniko-reoéntsefalograficheskaia kharakteristika i kriterii razlichiia nachal'nykh form ateroskleroticheskikh porazhenii mozga.

The author analyzes the clinical and rheoencephalographic characteristics of the cerebral circulation in 252 patients with initial signs of cerebral circulation insufficiency, 198 patients with the initial stage of dyscirculatory encephalopathy due to atherosclerosis, and in 149 healthy subjects of a young age. Distinct criteria for differentiating the above groups have been defined. These criteria are necessary for developing an automated system of prophylactic examinations of population.