[A new mutation c.422C>G (p.S141C) in homo- and heterozygous forms of the human leptin gene].

Mutation g.15409C>G, c.422C>G (p.S141C) in homo- and heterozygous forms of the human LEP gene was identified among some patients of the high mountain village of Karaul, Ashkhabad oblast, Turkmenistan, some of which suffer from adiposity. It causes the substitution S120C in the secreted leptin. The mature leptin molecule (146 aa) has only two Cys residues (C96 and C146) forming an S-S bridge, which is important for the hormone function. A third mutation, C120, in the molecule might disturb the correct formation of the S-S bond and could alter the leptin activity.

[The interconnections of molecular mechanisms of hormone actions and their role in pathogenesis of obesity, insulin resistance, and diabetes mellitus].

The various hormones, proteins and other compounds related to developing obesity, insulin resistance and type 2 diabetes are analyzed in the paper. 1) Leptin, ciliary neurutrophic factor, adiponectin, glucagon-like peptide 1, peptide YY, neuromedin S, as well as the protein receptors of these hormones decrease the food consumption, increase the energy turnover, and prevent obesity, insulin resistance, and type 2 diabetes development. The mediators of these hormone and receptor actions are melanocyte stimulating hormone (MSH), corticotropin-releasing hormone (CRH), and the others. 2) Ghrelin, endogenose cannabinoides, galanin-like peptide and the mediators of their actions: neuropeptide Y (NPY) and Agouti gene related protein (AGRP) increase the appetite and food consumption. Peroxisome proliferation-activated receptor (PPAR) performs the similar action on food intake. The activation of the first group compound functioning decreases the obesity, increases the energy turnover, facilitates the insulin action and prevents the insulin resistance and type 2 diabetes. Increasing the activities of the second group, as well as, decreasing the actions of the first one of substances induce the opposite effects and facilitate obesity, insulin resistance, and type 2 diabetes developments. The interconnections of the molecular mechanisms of so many hormone actions make the very complicated tusk to study the various endocrine disorders including diabetes mellitus as well.

[Molecular and genetic study of the role of hormones, receptors, and enzymes in regulation of reproduction, lipid metabolism, and other human physiological functions].

Prevalence of uterine progesterone receptors over estrogen ones, high uterine cAMP level, and low uterine prostaglandin level are necessary conditions of normal pregnancy. In cases of spontaneous and antiprogestin RU486-induced abortions, estrogen receptors prevail over progesterone ones, cAMP level decreases, and prostaglandin concentration in decidual tissue increases. Porcine and bovine beta-lipotropines were the first proteins, whose correct amino acid sequence was first determined in Russia. Several research centers carried out collaborative studies of the nucleotide sequences of human and animal proopiomelanocortin (lipotropin precursor) and prolactin cDNA. Researchers constructed genetic engineering producers of human pre-proinsulin and somatostatin, identified structural genes expressed in pancreatic beta-cells, studied antigenic properties of glutamic acid decarboxylase (GAD), which determine insulin-dependent diabetes, and identified the cholesterase determinant. They revealed mutations in the genes of proopiomelanocortin and melanocortin receptors (MC4-P), which inhibit leptin regulation of appetite and are associated with human obesity.

[Screening of mutations in genes of pro-opiomelanocortin in patients with constitutional exogenous obesity]. / Skrining mutatsii v gene proopiomelanokortina u bol'nykh konstutsional'no-ékzogennym ozhireniem.

Proopiomelanocortin (POMC) is a precursor of ACTH, beta- and gamma-liportopins, alpha-, beta- and gamma-MSH, beta-endorphin. alpha-, beta- and gamma-MSH are synthesized by hypothalamus neurons, and leptin stimulates their synthesis. These hormones regulate food consumption and energy metabolism by via melanocortin receptors (MC3-R and MC4-R) in hypothalamus. Screening mutations in the coding region of human POMC has been carried out with PCR, SSCP and DNA sequencing and the association study of these mutations and human obesity has been performed. Group of patients with the exogenous obesity (BMI 37.8 +/- 6.8 kg/m2) consisted of 228 persons (173 women and 55 men). 145 blood donors (67 women and 78 men) without obesity (BMI J25 kg/m2, 23.1 +/- 2.2 kg/m2) and 170 women without apparent obesity at the beginning of the study were included in the control group. 8 polymorph sites: insertions; missense and silent mutations have been identified in the coding region of POMC. Among them 1) two heterozygous mutations: the insertion of 6 b.p. (GGGCCC) in codon 176 inducing the insertion of two amino acid residues (Arg-Ala) in POMC and nonsense mutation (G-7316-T) in codon 180 of gamma-LTH coding region of the same DNA chain were identified in 4 women (5.8%) out of 69 patients with morbid obesity (BMI 40-53 kg/m2). These mutations were not found in control (n = 315). 2) The new heterozygous mutation T-7130-C (Phe118Leu) in active site of alpha-MSH has been identified in POMC gene of a woman suffering with obesity since the early childhood. 3) Mutation A-7341-G (Glu188Gly) seemed to have a protective effect because it was revealed more frequently in control (3.9%) than in obese patients (0.66%). The results of genetic study of two pedigrees suggested the dominant influence of the first two mutations (1 and 2) on woman obesity.

[Study of the association between constitutional exogenous obesity and polymorphism of the apolipoprotein B gene]. / Issledovanie sviazi konstitutsional'no-ékzogennogo ozhireniia s polimorfizmom v lokuse gena apolipoproteina B.

An attempt was made to associate the insertion-deletion (Ins/Del) polymorphism of the apolipoprotein B gene (apoB) with obesity and to identify alleles and genotypes predisposing to this disorder. The apoB Ins/Del allele frequencies observed in the Russian population were similar to those in West European populations and significantly differed from frequencies reported for Asian populations. Patients with obesity did not differ from healthy individuals in allele and genotype frequencies regardless of whether total or sex-stratified samples were compared. Estimation of relative risk for individuals with genotype Ins/Ins did not reveal a significant association between obesity and this genotype. Thus, constitutional exogenous obesity did not prove to be associated with the Ins/Del polymorphism of the apoB gene in the Russian population.

[Cloning, determination of primary structure, and expression of the C-terminal segment of human cholesterol-esterase/lipase, containing the antigenic determinant of the protein, in Escherichia coli]. / Klonirovanie, opredelenie pervichnoi struktury i ékspressiia v Excherichia coli C-kontsevogo uchastka chelovecheskoi kholesterol-ésterazy/lipazy, soderzhashchego antigennuiu determinantu belka.

Clone pHICE0.9 was selected from human insulinoma cDNA library by immunoscreening with antibodies against total human insulinoma proteins. This clone contains a 0.9 kb cDNA insert and expresses a fusion protein with beta-galactosidase. Nucleotide sequences of 5'- and 3'-terminal regions of this cDNA insert show that clone pHICE0.9 expresses a protein which is identical to the C-terminal fragment (amino acids 483 to 745) of human pancreatic cholesterol esterase and Homo sapiens bile-acid-salt-stimulated lipase from milk. It is concluded that the protein fragment contains the antigenic determinant of human cholesterol esterase/lipase, and can be used for lipase determination in blood.

[Study on structural gene expression in human insulinoma]. / Isslenovanie strukturnykh genov, ékspressiruiushchikhsia v insulinome cheloveka.

A human insulinoma cDNA library was constructed in the expression plasmid vector pUEX1. The clone pUEX1Ins12 was selected by means of hybridization with an insulin probe. It codes for full size amino acid sequence preproinsulin. The bacterial strain pUEX3Ins8 producing proinsulin as beta-galactosidase fusion protein was obtained for the use of recombinant protein as an antigen in an ELISA to detect serum antibodies in subjects with IDDM. Recombinant clones containing the middle, N- and C-terminal domains of the GAD65, the major autoantigen in IDDM, were constructed in pVEX1. These clones may become important tools to study the nature of GAD autoreactivity in IDDM. The clone pHICEO.9 was selected from the human insulinoma cDNA library by immunoscreening with total human insulinoma protein antibodies. This clone expresses the C-terminal fragment of human cholesterol esterase/lipase containing its antigenic determinant and can be used for blood lipase determination. Four clones containing cDNA inserts (0.47-1.42 kb) without any significant homologies to the known sequences in the Gene Bank were obtained by means of statistic selection.

[Cloning, primary structure determination and expression of preproinsulin cDNA from human insulinoma in Escherichia coli]. / Klorirovanie, opredelenie pervichnoi struktury i ékspressiia kDNK preproinsulina iz insulinomy cheloveka v Escherichia coli.

A human insulinoma cDNA library was constructed in expression plasmid vector pUEX1. Clone pUEX1Ins12 was selected from human insulinoma cDNA library by means of hybridization with the insulin probe and a nucleotide sequence of the insertion was determined. It codes for full size amino acid sequence preproinsulin and furthermore, contains the entire 3'-end of noncoding mRNA region and 44 nucleotides from the 5'-untranslated region. The bacterial strain pUEX3Ins8 producing preproinsulin as beta-galactosidase fusion protein was constructed.

[Molecular biological aspects of the pathogenesis of diabetes mellitus]. / Molekuliarno-biologicheskie aspekty patogeneza sakharnogo diabeta.

The discovery of the key role played by the immune system in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) opens up new possibilities for its early diagnosis, at the stages preceding its clinical manifestation. Analysed are the markers of genetic susceptibility to IDDM associated with some major histocompatibility complex antigens (specifically with HLA-DR 3, HLA-DR4, and HLA-DQ), of the cellular and humoral anti-islet autoimmunity, as well as the origin of the islet-cell autoantigens. The markers' significance for the diagnosis and prognosis is discussed.

[Mechanism of abortifacient effect of prostaglandins: changes in the level of cyclic adenosine monophosphate in the decidual tissue of women during abortion induced with sulprostone]. / O mekhanizme abortnogo deistviia prostaglandinov: izmenenie urovnia tsiklicheskogo adenozinmonofosfata v detsidual'noi tkani zhenshchin pri aborte, vyzvannom sul'prostonom.

PIP: The role of cyclic AMP (cAMP) in the mechanism of abortifacient effect of sulprostone was studied in women with pregnancy of 2-3 weeks of gestation (group 1) or 4-5 weeks of gestation (group 2). Pregnancy was confirmed by determining the beta-subunit of chorionic gonadotropin. The patients received intramuscular injection of sulprostone at 0.5 mg, 2 times with an interval of 4 hours (group 1), or 3 times with an interval of 3 hours (group 2). The cAMP level was determined in the decidual tissue removed immediately after abortion. Sulprostone-induced abortion resulted in marked decrease in the cAMP levels in the decidual tissue (from 491.5+/-70.4 picamol per 1 mg of protein to 224+/55.6 in group 1 and from 377.7+/-55.9 to 95.7+/-18.4 in group 2). Decrease in cAMP level was also observed during spontaneous abortion (122.3+/-28.5, compared with 691.1+/-110.5 during surgical abortion). To determine whether the decrease in cAMP level was associated with direct action of sulprostone, the decidual tissue was incubated with PGE1 in vitro (10, 25 or 50 microg/ml for 15 min). In vitro addition of PGE1 resulted in marked increase in the cAMP level. These findings indicated that decrease in cAMP level during sulprostone-induced abortion was associated not with its direct action on the decidual tissue but rather with reduced blood supply of the decidual tissue caused by uterine contractions.^ieng

[The role of periodic changes in cyclase and protein kinase activity in the mechanism of the proliferative effect of ACTH]. / Rol' periodicheskikh izmenenii aktivnosti tsiklaz i proteinkinaz v mekhanizme proliferativnogo deistviia AKTG.

A prolonged effect of ACTH on the state of adenylate and guanylate cyclase systems in the adrenal glands of experimental animals was investigated. It was found that in guinea pigs injected with ACTH (4 units daily for 1-50 days) the weight of adrenal glands and the DNA content in these organs increased 2.0-2.5-fold by the end of experiment; the increase in both values was stepwise. The corticosteroid level in the blood varied throughout the experiment: the changes in the DNA content in adrenals and in the corticosteroid content in the blood were oppositely directed. This was accompanied by cyclic changes in the basal and stimulated activities of adenylate and guanylate cyclases and proteinases in the adrenal glands occurring with a periodicity of 6-15 days. The activity peaks for cyclases and protein kinases preceded the rise in the DNA content in the adrenals. A clearcut correlation between the changes in the enzyme activity and the hormone dose was observed. The changes in the basal and stimulated activities of guanylate cyclase seem to be due to the control of cAMP level in the cell (stimulation of cGMP-dependent cAMP phosphodiesterase). Apparently, the periodic changes in the activity of cAMP-dependent protein kinases in the cytoplasmic and nuclear fractions and a relatively high activation of nuclear protein kinases (by 30-60%) in comparison of cytoplasmic ones (8-10%) are related to stimulation of DNA synthesis. It is concluded that the changes in the activity of cyclases and protein kinases play a role in the mechanism of proliferative effect of ACTH.

[Species and organ differences in the activity and regulation of adenylate and guanylate cyclases]. / Vidovye i organnye razlichiia v aktivnosti i reguliatsii adenilat- i guanilattsiklaz.

The activity of adenylate and guanylate cyclases was determined in adrenal, heart, liver and fat tissues of guinea pigs, mice, rabbits and monkeys. The enzymes activities varied markedly depending both on the species and organs. The highest basal activities of adenylate cyclase was observed in all organs of guinea pigs. It was found that organs with low basal level of adenylate cyclase possess high guanylate cyclase. Species variations of the basal and stimulated adenylate cyclase activity may determine the functional activity of an organ: the higher the adenylate cyclase activity, the more intensive steroidogenesis in adrenals, lipolysis in the fat tissue, muscle contraction and nerve impulse conduction in heart.

[Synthesis, cloning and primary structure of DNA complementary to mRNA for human pituitary pro-opiomelanocortin]. / Sintez, klonirovanie i opredelenie pervichnoi struktury DNK, komplementarnoi mRNK proopiomelanokortina iz gipofiza cheloveka.

cDNA coding for the human pro-opiomelanocortin (POMC) has been cloned and sequenced. It codes for full size amino acid sequence of POMC and furthermore, contains most part of the 3'-terminal noncoding mRNA region and 60 nucleotides coding for signal peptide.

[Adenylate- and guanylate cyclase systems in pathology of the adrenal glands]. / Adenilat- i guanilattsiklaznye sistemy pri patologii nadpochechnikov.

The authors have summed up the results of a study of the activity of adenylate and guanylate cyclase system in the adrenals of patients with the Icenko-Cushing disease and syndrome and in the adrenals of experimental animals after unilateral adrenalectomy and in ACTH administration. It has been established that the biochemical mechanisms of compensatory hypertrophy of the adrenals in the normal and pathological tissue differ significantly. It has been shown for the first time that an increase in the basal activity of adenylate cyclase, resulting in the restoration of the initial hormonal level, is an obligatory condition for the compensation of organ function. Disorder in hormonal regulation of adenylate cyclase and diverse changes of the cyclase activity may indicate to the transition of tissue into a pathological state. Cyclic changes of the basal and stimulated activity of cyclases and protein kinases have been established for the first time in the adrenals of animals during administration of 2.5 units of ACTH for 5-40 days. The importance of detected changes in the mechanism of ACTH proliferative action is discussed.

[Adenylate cyclase and cAMP-dependent protein kinase activity in the adrenal glands of animals after chronic administration of ACTH]. / Izuchenie aktivnosti adenilattsiklazy i cAMP-zavisimykh proteinkinaz v nadpochechnikakh zhivotnykh pri khronicheskom vvedenii AKTG.

ACTH, a prolonged action hormone, in a dose of 2.5 mu. was injected into guinea pigs daily for 5-35 days. The adenylate cyclase activity of the crude adrenal membrane fraction and the activity of cAMP-dependent protein kinases in the cytoplasmic fraction were determined. Cyclic changes in the basal and stimulated adenylate cyclase activities occurring with 15-20-day intervals have been established for the first time. The sensitivity of adenylate cyclase to ACTH, NaF and GTP did not change in the course of two cycles. The activity of cAMP-dependent protein kinases increased during the first few days after ACTH administration and decreased after further injections of the hormone. The role of cyclic changes of the enzyme activity in the mechanism of proliferative effect of ACTH is discussed.

[Effect of ACTH on protein phosphorylation in rat adrenal ribosomes]. / Vliianie AKTG na fosforilirovanie belkov ribosom nadpochechnikov krys.

The authors studied the influence of ACTH on the steroidogenesis and 32P-labeled KH2PO4 incorporation into the ribosome proteins under conditions of incubation of the rat adrenal glands in vitro. 30 minutes before the incubation ACTH added into the medium stimulated corticosteroid biosynthesis by 116 +/- 26% and increased the radioactive phosphorus incorporation into the sum total ribosome proteins by 38 +/- 7%. Ribosome proteins were separated by electrophoresis in polyacrylamide gel at pH 4.5 into 30 fractions. 5--6 protein fractions proved to be phosphorylated during the incubation. ACTH altered the phosphorylation of individual protein fractions differently: there was stimulation in 2 fractions and inhibition of phosphorylation--in 3. It was revealed that, along with steroidogenesis, ACTH altered the phosphorylation of ribosome proteins. The change of the rate of ribosome phosphorylation was possibly one of the mechanisms with the aid of which the steroidogenic action of ACTH is realized.