Synthesis of 1-deoxy-L-gulonojirimycin (L-guloDNJ) and 1-deoxy-D-talonojirimycin (D-taloDNJ)

Carbohydrate based syntheses of azasugars with unusual configurations viz. 1,5-dideoxy-1,5-imino-L-gulitol (L-guloDNJ) and 1,5-dideoxy-1,5-imino-L-talitol (L-taloDNJ) are reported, from D-mannose and D-fructose, respectively. The key steps in both syntheses involved reductive aminative cyclizations. Thus, L-guloDNJ was obtained by reduction of 2,3;4,6-di-O-isopropylidene-5-O-p-toluenesulfonyl-D-mannononitrile with LiAlH(4) in DME to give the protected azasugar which upon hydrolysis with HCl afforded crystalline L-guloDNJ as the HCl salt in 29% overall yield. Reduction of 6-azido-1-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-beta-D-ribohexulofuranose obtained from D-fructose in six steps, followed by treatment with HCl, afforded L-taloDNJ as an HCl salt in approximately 10% overall yield.     

Distinct recognition of collagen subtypes by alpha(1)beta(1) and alpha(2)beta(1) integrins. Alpha(1)beta(1) mediates cell adhesion to type XIII collagen

Two integrin-type collagen receptors, alpha(1)beta(1) and alpha(2)beta(1), are structurally very similar. However, cells can concomitantly express the both receptors and they might have independent functions. Here, Chinese hamster ovary (CHO) cells, which lack endogenous collagen receptors, were transfected with either alpha(1) or alpha(2) integrin cDNA. Cells were allowed to adhere to various collagen types and their integrin function was tested by observing the progression of cell spreading. The cells expressing alpha(1)beta(1) integrin could spread on collagen types I, III, IV, and V but not on type II, while alpha(2)beta(1) integrin could mediate cell spreading on collagen types I-V. Type XIII is a transmembrane collagen and its interaction with the integrins has not been previously studied. CHO-alpha1beta1 cells could spread on human recombinant type XIII collagen, unlike CHO-alpha2beta1 cells. Integrins alpha(1)beta(1) and alpha(2)beta(1) recognize collagens with the specific alphaI domains. The alpha(1)I and alpha(2)I domains were produced as recombinant proteins, labeled with europium and used in a sensitive solid-phase binding assay based on time-resolved fluorescence. alpha(1)I domain, unlike the alpha(2)I domain, could attach to type XIII collagen. The results indicate, that alpha(1)beta(1) and alpha(2)beta(1) have different ligand binding specificity. Distinct recognition of different collagen subtypes by the alphaI domains can partially explain the differences seen in cell spreading. However, despite the fact that CHO-alpha1beta1 cells could not spread on type II collagen alpha(1)I domain could bind to this collagen type. Thus, the cell spreading on collagens may also be regulated by factors other than the integrins.     

Total synthesis of the mollu-series glycosyl ceramides alpha-D-Manp-(1----3)-beta-D-Manp-(1----4)-beta-D-Glcp-(1----1)-Cer and alpha-D-Manp-(1----3)-[beta-D-Xylp-(1----2)]-beta-D-Manp-(1----4)-beta- D-Glcp-(1----1)-Cer

The mollu-series glycosphingolipids, O-alpha-D-mannopyranosyl-(1----3)-O-beta-D-mannopyranosyl-(1----4)-O-bet a-D-glucopyranosyl-(1----1)-2-N-tetracosanoyl-(4E)-sphingeni ne and O-alpha-D-mannopyranosyl-(1----3)-O-[beta-D-xylopyranosyl-(1----2])-O- beta-D-mannopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-2-N- tetracosanoyl-(4E)-sphingenine, were synthesized for the first time by using 2,3,4-tri-O-acetyl-D-xylopyranosyl trichloroacetimidate, methyl 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranoside, benzyl O-(4,6-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside 9, and (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol 6 as the key intermediates. The hexa-O-benzyl disaccharide 9 was prepared by coupling two monosaccharide synthons, namely, 2,3-di-O-allyl-4,6-di-O-benzyl-alpha-D-mannopyranosyl bromide and benzyl 2,3,6-tri-O-benzyl-beta-D-glucopyranoside. It was demonstrated that azide 6 was highly efficient as a synthon for the ceramide part in the coupling with both glycotriaosyl and glycotetraosyl donors, particularly in the presence of trimethylsilyl triflate.     

Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.

The alpha(1)beta(1) and alpha(2)beta(1) integrins are cell surface collagen receptors. Cells expressing the alpha(1)beta(1) integrin preferentially adhere to collagen IV, whereas cells expressing the alpha(2)beta(1) integrin preferentially adhere to collagen I. Recombinant alpha(1) and alpha(2) integrin I domains exhibit the same collagen type preferences as the intact integrins. In addition, the alpha(2) integrin I domain binds echovirus 1; the alpha(1) I domain does not. To identify the structural components of the I domains responsible for the varying ligand specificities, we have engineered several alpha(1)/alpha(2) integrin I domain chimeras and evaluated their virus and collagen binding activities. Initially, large secondary structural components of the alpha(2) I domain were replaced with corresponding regions of the alpha(1) I domain. Following analysis in echovirus 1 and collagen binding assays, chimeras with successively smaller regions of alpha(1) I were constructed and analyzed. The chimeras were analyzed by ELISA with several different alpha(2) integrin monoclonal antibodies to assess their proper folding. Three different regions of the alpha(1) I domain, when present in the alpha(2) I domain, conferred enhanced collagen IV binding activity upon the alpha(2) I domain. These include the alpha3 and alpha5 helices and a portion of the alpha6 helix. Echovirus 1 binding was lost in a chimera containing the alphaC-alpha6 loop; higher resolution mapping identified Asn(289) as playing a critical role in echovirus 1 binding. Asn(289) had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of multiple determinants of ligand binding specificities within the alpha(1) and alpha(2) integrin I domains.     

fra(1) (p11), fra(1) (q22) and r(1) (p11q22) in a retarded girl.

A mentally retarded girl with a 46,XX/47, XX+r(1) (p11q22q22p11)/47, XX+r(1) (p11q22) fra(1) (p31) fra(1) (p11) fra(1) (q22) karyotype who inherited the fragile sites from the normal mother was studied. The conicidence of fra(1) (p11) and fra(1) (q22) with the ring chromosome breakpoints strongly suggests a cause-effect relationship. This finding agrees with other reported associations between fragile sites and structural chromosome abnormalities and constitutes the fourth reported of a de novo structurally abnormal chromosome as a consequence of presumed in vivo fragile sites instability. Although risk figures for chromosome anomalies and cancer associated with fragile sites are lacking, carriers of fra (1) (p11) may have a higher risk for abnormalities of chromosome 1 in somatic and gonadal cells than the general population.     

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1)

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.     

N(1)-Acetyl-N(1)-deoxymayfoline from Maytenus buxifolia

A new alkaloid, N(1)-acetyl-N(1)-deoxymayfoline was isolated from Maytenus buxifolia growing in the vicinity of Santiago de Cuba. Plants of     

Isolation and characterization of CNBr peptides of human (alpha 1 (III) )3 collagen and tissue distribution of (alpha 1 (I) )2 alpha 2 and (alpha 1 (III) )3 collagens.

[Alpha 1(III)]3 collagen was solubilized by pepsin digestion of normal human placental membranes and was purified by differential salt precipitation and carboxymethylcellulose chromatography. This collagen was digested with CNBr, and the resultant nine peptides were isolated and characterized. The chains are cross-linked by cysteinyl residues in the COOH-terminal peptide. Isolation of peptides derived from CNBr digestion of insoluble tissues was used as an assay for the presence of [alpha 1(I)]2alpha 2 and [alpha 1(III)]3 collagens. Both types are present in human skin, intestine, liver, spleen, kidney, lung, aorta, umbilical cord, placental membranes, and myocardium. Bone and tendon contain [alpha 1(I)]2alpha 2 collagen but, unlike the other tissues, lack [alpha 1(III)]3 collagen. Both [alpha 1(I)]2alpha 2 and[alpha 1(III)]3 collagens are present in scars of human skin, myocardium, tendon, and liver and of rabbit skin. The degree of hydroxylation of proline was 4 to 5% lower in the same peptides in skin, bone, and tendon than in the other tissues. The degree of hydroxylation of lysine in the same peptides derived from different tissues varied more widely.     

Antioxidant activity and low toxicity of (E)-1-(1-(methylthio)-1-(selenopheny) hept-1-en-2-yl) pyrrolidin-2-one

The aim of the present study was to evaluate the potential pharmacological and toxicological properties of (E)-1-(1-(methylthio)-1-(selenopheny) hept-1-en-2-yl) pyrrolidin-2-one (compound 1), an organoselenium compound. In vitro experiments showed that compound 1 presented a reduction in the lipid peroxidation induced by Fe2? in thiobarbituric acid-reactive species (TBARS) production, and in the generation of reactive species caused by Fe2?/malonate in DCFH-DA oxidation. The high dose (500 mg/kg) induced an increase on ALT but not on AST activity. Hepatic, but not cerebral, δ-ALA-D activity from mice treated with 500 mg/kg presented a significant inhibition. Brain catalase activity was significantly inhibited by 100 mg/kg whereas hepatic catalase activity showed a significant increase at all doses. Hepatic lipid peroxidation was diminished only at lowest dose (100 mg/kg) whereas for brain tissue, all doses induced a significant reduction in TBARS levels. Brain and liver ascorbic acid contents were increased only at highest dose of compound 1. Urea and creatinine levels were not significantly altered by treatments. This is a promising compound with antioxidant activity and low toxicity, suggesting the potential beneficial activity of compound 1 against oxidative damage in many parameters studied in rats and mice.     

Glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) polymorphisms in a Brazilian mixed population

The GSTM1 and GSTT1 null genotype frequencies were significantly different between 658 nonblack and black healthy blood donors from a Brazilian mixed population (Rio de Janeiro). The GSTM1 phenotype distribution was not in Hardy-Weinberg equilibrium in either group, mainly because of an excess of the GSTM1*A/*B genotype.     

Expression of histone 1 (H1) and testis-specific histone 1 (H1t) genes during stallion spermatogenesis

In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged expression up to elongating spermatids (Sd1) at stage V. A fragment of the equine H1t gene was cloned (GenBank Accession No. AJ865320). The mRNA expression of H1t was found at the level in spermatogonia and in primary spermatocytes up to mid-pachytene at stage VIII/I, whereas H1t protein was found to be expressed up to round spermatides (Sa/Sb1) at stage VIII/I. In peripubertal animals, the H1t protein expression was detected up to elongating spermatids (Sb2) at stage II. Analysis of testes of different ages (< or =2 years) and (> or =3 years) by real-time RT-PCR revealed an increase of H1t mRNA expression, with a wide range of individual variety between 2 and 4 years old animals indicating a stable expression in animals older than 4 years old. This is the first study to show the testis-specific H1t in the stallion and gives evidence that the well-known peripubertal infertility in the stallion may be related to an insufficient histone to protamine exchange. The pattern of protamine gene expression, however, has still to be elucidated.     

G1m(1) and G1m(2) allotypes in Albanian towns of Calabria

The G1m(1) and G1m(2) allotype distribution was analyzed in a population sample from 11 Albanian towns of Calabria. The unusually high frequency of the G1m(1) marker already observed in Calabria as well as the presence of the Gm(2) phenotype were shown. The Calabrian and Albanian populations were similar, but significantly different from other Italian populations.     

Chromosomal mapping of calmodulin 1 (CALM1) and alpha-globin 1 genes (HBA1) in the bovine.

Chromosomal mapping of the bovine calmodulin 1 and alpha-globin 1 genes was performed by analyzing bovine/murine somatic cell hybrid DNAs with PCR using primers specific for 3'-untranslated regions of those bovine genes. The calmodulin 1 and alpha-globin 1 genes were assigned to bovine chromosomes 25 and 29, respectively. Results from the present study should contribute to improvement in map resolution of bovine chromosomes and increase comparative information available on bovine chromosomes.     

Genotyping of the porcine ryanodine receptor 1 (RYR1) and estrogen receptor 1 (ESR1) genes by high resolution melting (HRM) approach

During the last decade, DNA mutations in the porcine ryanodine receptor 1 gene (RYR1, C1843T) and the estrogen receptor 1 gene (ESR1, T1665G), have been widely used in marker-assisted selection (MAS) for the pig industry. These 2 well-characterized SNPs in RYR1 and ESR1 are responsible for porcine stress syndrome (PSS) and litter size, respectively. Here, we describe a reliable, high-efficiency method for the genotyping of these 2 genes using the high-resolution melting (HRM) method. The HRM approach exhibited high-accuracy and repeatability, comparable with the classic PCR-restriction fragment length polymorphism (PCR-RFLP) approach, and is potentially suitable for large-scale genotyping in commercial pig farms.     

2(S)-(Cycloalk-1-enecarbonyl)-1-(4-phenyl-butanoyl)pyrrolidines and 2(S)-(aroyl)-1-(4-phenylbutanoyl)pyrrolidines as prolyl oligopeptidase inhibitors

In order to replace the P2-P1 amide group, different 1-cycloalkenyls and 2-aryls were studied in the place of the P1 pyrrolidine group of a 4-phenylbutanoyl-L-Pro-pyrrolidine structure, which is a well-known prolyl oligopeptidase inhibitor SUAM-1221. The 1-cyclopentenyl and the 2-thienyl groups gave novel compounds, which were equipotent with the corresponding pyrrolidine-analog SUAM-1221. It was shown that the P2-P1 amide group of POP inhibitors can be replaced by an alpha,beta-unsaturated carbonyl group or the aryl conjugated carbonyl group.     

GATA-binding Protein 4 (GATA-4) and T-cell Acute Leukemia 1 (TAL1) Regulate Myogenic Differentiation and Erythropoietin Response via Cross-talk with Sirtuin1 (Sirt1)

Erythropoietin (EPO), the cytokine required for erythrocyte production, contributes to muscle progenitor cell proliferation and delay myogenic differentiation. However, the underlying mechanism is not yet fully understood. Here, we report that EPO changes the skeletal myogenic regulatory factor expression program and delays differentiation via induction of GATA-4 and the basic helix-loop-helix TAL1 and that knockdown of both factors promotes differentiation. EPO increases the Sirt1 level, a NAD(+)-dependent deacetylase, and also induces the NAD(+)/NADH ratio that further increases Sirt1 activity. Sirt1 knockdown reduced GATA-4 and TAL1 expression, impaired EPO effect on delayed myogenic differentiation, and the Sirt1 knockdown effect was abrogated when combined with overexpression of GATA-4 or TAL1. GATA-4 interacts with Sirt1 and targets Sirt1 to the myogenin promoter and represses myogenin expression, whereas TAL1 inhibits myogenin expression by decreasing MyoD binding to and activation of the myogenin promoter. Sirt1 was found to bind to the GATA-4 promoter to directly regulate GATA-4 expression and GATA-4 binds to the TAL1 promoter to regulate TAL1 expression positively. These data suggest that GATA-4, TAL1, and Sirt1 cross-talk each other to regulate myogenic differentiation and mediate EPO activity during myogenic differentiation with Sirt1 playing a role upstream of GATA-4 and TAL1. Taken together, our findings reveal a novel role for GATA-4 and TAL1 to affect skeletal myogenic differentiation and EPO response via cross-talk with Sirt1.     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.