Association between insertion-deletion polymorphism of the angiotensin-converting enzyme gene and development of angiopathies in patients with non-insulin dependent diabetes mellitus from the Chuvash Republic

Insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) gene was analyzed in patients with non-insulin-dependent diabetes mellitus (NIDDM) and in the control group consisting of healthy subjects. The insertion allele (I) and genotype II were found to be associated with NIDDM. The frequencies of diabetic retinopathy and nephropathy in NIDDM patients were not associated with this polymorphism. However, an association was found between the DD genotype of the ACE gene and diabetic angiopathy in lower extremities.     

Catalytic properties of Glu-plasminogen in a complex with monoclonal antibody IV-1c

Earlier it was shown that anti-plasminogen monoclonal antibody IV-1c was able to induce a catalytic activity in plasminogen. IV-1c activates plasminogen by binding to plasminogen protease domain with antigen binding site and to lysine-binding sites by C-terminal lysines of gamma-chains. The effect of plasminogen and IV-1c concentration on rate of catalytic activity induce in Pg-IV-1c complex has been investigated. It was found that IV-1c inhibited an activation reaction at concentrations higher of equimolar to Glu-Pg. Glu-Pg did not inhibit reaction of activation in higher to IV-1c concentrations. Role of IV-1c gamma-chain C-terminal lysine concentration in Pg activation is discussed.     

Down-regulation of the expression of O-acetyl-GD3 by the O-acetylesterase cDNA in hamster melanoma cells: effects on cellular proliferation, differentiation, and melanogenesis

The composition of the gangliosides of hamster melanoma cells is closely related to their cellular growth and degree of differentiation, with slow-growing, highly differentiated melanotic melanoma MI cells expressing GM3 and fast-growing, undifferentiated amelanotic Ab melanoma cells having a preponderance of GD3 and O-acetyl-GD3. To study the putative function of O-acetyl-GD3, we established stably transfected AbC-1 amelanotic hamster melanoma cells with O-acetylesterase gene from influenza C virus to hydrolyze the O-acetyl group from O-acetyl-GD3. The content of O-acetyl-GD3 in the transfected cells expressing O-acetylesterase gene was reduced by >90%. These O-acetyl-GD3-depleted cells differed from the parental ones in their cellular morphology, growth behavior, and melanogenesis activity. The absence of O-acetyl-GD3 in the transfected cells was accompanied by increased thick dendrite formation with an enlarged cell body, which is in striking contrast to the control cells, which were rounded and flattened, with few processes. Their growth was significantly slower than that of the control cells. They also demonstrated significantly lower tyrosinase activity and melanogenic potential. We suggest that the enhanced expression of melanoma-associated O-acetyl-GD3 ganglioside may stimulate cellular growth and suppress certain differentiated phenotypes such as dendrite formation but not melanogenesis.     

Analysis of the allele polymorphism of (CTG)n and (GAG)n triplet repeats in DM,DRPLA, and SCA1 genes in various populations of Russia

Polymorphism of highly polymorphic triplet repeats CTG of the 3'-untranslated region of the myotonin protein kinase gene and CAG of the genes associated with dentatorobral-pallidoluysian atrophy (DRPLA, or Hew River syndrome) and spinocerebellar ataxia type 1 (SCA1) was analyzed in several ethnic populations of Russia. A difference in allele spectra of the three genes was demonstrated for populations differing in ethnic origin.     

Analysis of the 32 bp deletion in the CCR5 chemokine receptor gene in people from Moscow, infected with HIV-1

Chemokine receptors have recently been shown to mediate HIV-1 entry into cells. The chemokine receptor CCR5 plays a key role in this process. A 32-bp deletion within the coding region of the CCR5 gene generates a truncated nonfunctional receptor. In HIV-1-infected individuals homozygous for this mutation, disease progression is inhibited. We analyzed the frequencies of the deletion in HIV-1-infected seropositive individuals. No significant differences in allelic frequencies of the CCR5 gene between the control and general HIV-1-infected cohorts and within the latter group between the infected individuals and patients with AIDS symptoms were revealed.     

Normal polymorphism of the (CTG)n repeat in the myotonin protein kinase (DM) gene on chromosome 19q13.3 in Western European populations

Polymorphism of a highly polymorphic CTG repeat in the 3'-untranslated region of the myotonin protein kinase gene was analyzed in healthy people from several Eastern European populations (Russians, Moldovans, Belarussians, Komis, Chuvashes, Udmurts, Bashkirs, Tatars, Maris, and Mordovians). In total, 26 alleles of the CTG repeat were found, the repeat number ranging from 5 to 33 (alleles with six and seven repeats were not detected). The heterozygosity of individual populations varied from 61 to 91%. In the total sample combining all populations, the observed and expected heterozygosities did not differ (fixation index -0.0022) suggesting selective neutrality of the normal polymorphism of the CTG repeat in the myotonin protein kinase gene.     

Molecular cloning and initial characterization of African green monkey (Cercopithecus aethiops) corticotropin releasing factor receptor type 1 (CRF1) from COS-7 cells

We report the expression of endogenous CRF1 in COS-7 cells (African green monkey origin). Cloning of the coding region of CRF1 gene identified three alternatively spliced isoforms with nucleotide and predicted amino acid sequences corresponding to the membrane bound alpha and c and soluble e isoforms. DNA sequencing of the main isoform CRF1alpha showed homologies of 99%, 97% and 91% with the rhesus monkey, human and rodent genes, respectively; the deduced protein sequence differed in only one amino acid with rhesus monkey and human. Western blot analysis with antibodies against human CRF1 demonstrated immunoreactive proteins with MW of 37, 52, 70 and 80-85 in crude membrane or cytoplasm preparation; two additional species of 40 and 60 kDa were detected only in the cytoplasmic fraction. On immunocytochemistry CRF1 was localized to both the cell surface and intracellularly. The receptor was functional, e.g., addition of CRF to COS-7 cells inhibited cell proliferation and stimulated release of arachidonic acid; nevertheless, it was poorly coupled to cAMP production (its stimulation was minimal in native cells). In conclusion, COS cells that are routinely used for the study of transfected CRF receptors do express endogenous CRF1 mRNA with splicing behavior similar to that reported in human and rodent cells, and translated into functional CRF1 receptors.     

Sequential Metabolism of 7-Dehydrocholesterol to Steroidal 5,7-Dienes in Adrenal Glands and Its Biological Implication in the Skin

Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3βHSD for 7DHP (V_m/K_m) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC→22(OH)7DHC→20,22(OH)₂7DHC→7DHP, with potential further metabolism of 7DHP mediated by 3βHSD or CYP17, depending on mammalian species. The 5–7 dienal intermediates of the pathway can be a source of biologically active vitamin D3 derivatives after delivery to or production in the skin, an organ intermittently exposed to solar radiation.     

CRH functions as a growth factor/cytokine in the skin

We tested the effect of CRH and related peptides in a large panel of human skin cells for growth factor/cytokine activities. In skin cells CRH action is mediated by CRH-R1, a subject to posttranslational modification with expression of alternatively spliced isoforms. Activation of CRH-R1 induced generation of both cAMP and IP3 in the majority of epidermal and dermal cells (except for normal keratinocytes and one melanoma line), indicating cell type-dependent coupling to signal transduction pathways. Phenotypic effects on cell proliferation were however dependent on both cell type and nutrition conditions. Specifically, CRH stimulated dermal fibroblasts proliferation, by increasing transition from G1/0 to the S phase, while in keratinocytes CRH inhibited cell proliferation. In normal and immortalized melanocytes CRH effect showed dichotomy and thus, it inhibited melanocyte proliferation in serum-containing medium CRH through G2 arrest, while serum free media led instead to CRH enhanced DNA synthesis (through increased transition from G1/G0 to S phase and decreased subG1 signal, indicating DNA degradation). CRH also induced inhibition of early and late apoptosis in the same cells, demonstrated by analysis with the annexin V stains. Thus, CRH acts on epidermal melanocytes as a survival factor under the stress of starvation (anti-apoptotic) as well as inhibitor of growth factors induced cell proliferation. In conclusion, CRH and related peptides can couple CRH-R1 to any of diverse signal transduction pathways; they also regulate cell viability and proliferation in cell type and growth condition-dependent manners.