The frequencies of Gm(1), Gm(2), Gm(4), Gm(12), and Inv(1) in Hessen (Germany)

Summary The serum groups Gm(1) [Gm(a)], Gm(2) [Gm(x)], Gm(4) [Gm(f)]. Gm(12) [Gm(b)] and Inv(1) [Inv(1)] of 2000 sera of healthy blood donors from the land Hesse were examined. The results obtained were compared with those known until now. Three persons, not related to each other, possessed the extremely rare phenotype Gm(-1, 2, 4, 12) [Gm (a-x+b+f+)]. In 0.75% of the cases we found a discordant behaviour of the factors Gm(4) and Gm(12) [Gm(f) and Gm(b)].

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Zusammenfassung 2000 Seren von gesunden Blutspendern aus Hessen wurden bezüglich der Gamma-Globulin-Serumgruppen Gm(1) [Gm(a)], Gm(2) [Gm(x)], Gm(4) [Gm(f)]. Gm(12) [Gm(b)] und Inv(1) [Inv(1)] untersucht. Die gefundenen Resultate wurden mit den bisher bekannten verglichen. Drei miteinander nicht verwandte Personen wiesen den äußerst seltenen Phänotyp Gm(-1, 2, 4, 12) [Gm(a-x+b+f+)] auf. In 0.75% der Fälle fanden wir ein diskordantes Verhalten der Faktoren Gm(4) und Gm(12) [Gm(f) und Gm(b)].

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Director: Prof. Dr. W. Wachsmuth

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Director: Prof. Dr. W. Spielmann

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The nomenclature suggested by WHO at a round-table conference over genes, genotypes and allotypes of immunglobulins is used. The conference took place in Geneva on the 1965 31. 5. to the 5. 6. [5].

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With technical assistance of S. Mohs.     

Chromosomal assignment of four rat genes coding for the spermatid-specific proteins proacrosin (ACR), transition proteins 1 (TNP1) and 2 (TNP2), and protamine 1 (PRM1)

The genes for proacrosin, protamines, and transition proteins are exclusively expressed in haploid spermatogenic cells. From the analysis of mouse x rat cell hybrids which segregate rat chromosomes, the rat gene for proacrosin (ACR) was assigned to chromosome 7, that for transition protein 1 (TNP1) to chromosome 9, and the genes for transition protein 2 (TNP2) and protamine 1 (PRM1) to chromosome 10.     

Physicochemical and biological properties of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose (6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin)

A unique multibranched cyclomaltooligosaccharide (cyclodextrin, CD) of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose [6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin, (G(2))(3)-betaCD] was prepared. The physicochemical and biological properties of (G(2))(3)-betaCD were determined together with those of monobranched CDs (6-O-alpha-D-glucopyranosyl-alpha-cyclodextrin (G(1)-alphaCD), 6-O-alpha-D-glucopyranosyl-beta-cyclodextrin (G(1)-betaCD), and 6-O-alpha-maltosyl-beta-cyclodextrin (G(2)-betaCD)). NMR spectra of (G(2))(3)-betaCD were measured using various 2D NMR techniques. The solubility of (G(2))(3)-betaCD in water and MeOH-water solutions was extremely high in comparison with nonbranched betaCD and was about the same as that of the other monobranched betaCDs. The formation of an inclusion complex of (G(2))(3)-betaCD with stereoisomers (estradiol, retinoic acid, quinine, citral, and glycyrrhetinic acid) depends on the cis-trans isomers of guest compounds. The cis isomers of estradiol, retinoic acid, and glycyrrhetinic acid were included more than their trans isomers, while the trans isomers of citral and quinine fit more tightly than their cis isomers. (G(2))(3)-betaCD was the most effective host compound in the cis-trans resolution of glycyrrhetinic acid. Among the branched betaCDs, (G(2))(3)-betaCD exhibited the weakest hemolytic activity in human erythrocytes and showed negligible cytotoxicity in Caco-2 cells up to 200 microM. These results indicate unique characteristics of (G(2))(3)-betaCD in some biological responses of cultured cells.     

Complex compounds of Pd(II), Pt(II), Ni(II), Hg(II) and Cu(I) with 1-benzylidene-2-phenazinoylhydrazine

Synthesis and physical-chemistry characterization of five new genuine complex compounds of 1-Benzylidine-2-phenazinoylhydrazine with Pd(II), Pt(II), Ni(II), Hg(II) and Cu(I) are presented. The chemical structure for these complexes is suggested by the elemental chemical analysis, molecular mass measurements, electric conductivities as well as by IR and UV-VIS spectra. The metal:ligand molar ratio is found 1:1, the obtained complexes belonging to the square planar geometry D4h.     

Linkage of isozyme loci in the mouse: Phosphoglucomutase-2 (Pgm-2), mitochondrial NADP malate dehydrogenase (Mod-2), and dipeptidase-1 (Dip-1)

The linkages of the isozyme genes Mod-2, Pgm-2, and Dip-1 have been determined in tests with established linkage group markers among inbred strains of mice. Unique alleles for both Mod-2 and Pgm-2 have been observed in the strain of SM/J. Linkage was determined from backcross progeny of the matings C57BL/6J×(SM/J×C57BL/6J)F₁, (SM/J×SWR/J)F₁×SM/J, and (SM/J×SWR/J)F₁×SJL/J. The gene Mod-2 is on linkage group 1. In a three-point cross of the loci Gpi-1, c, and Mod-2, the c locus was determined to be the middle gene. No double crossovers were observed. Our combined data show the following linkages: Gpi-1 to c, 28.3±3.2%; Gpi-1 to Mod-2, 33.3±3.0%; and c to Mod-2, 4.1±2.8%. The proposed gene order for four markers on LG I is Gpi-1-p-c-Mod-2. The gene Pgm-2 was linked to Gpd-1 (27.0±4.2%) on LGVIII. Two backcrosses segregating for Pgm-2 and b, (SM/J×DBA/2J) F₁×DBA/2J and (SM/J×DBA/2J)F₁×C57BR/cdJ, showed 9.1±4.3% recombination. The proposed gene order on LG VIII is b-Pgm-2-Gpd-1. The genes Pgm-1 and Pgm-2 are not linked (53.4±4.4%). Linkage of the isozyme genes Dip-1 and Id-1 on LG XIII was observed in backcross progeny of the crosses (SJL/J×C57BL/6J)F₁×SJL/J and C57BL/6J×(SM/J×C57BL/6J)F₁. The combined recombination was 23.8±2.8%. Two cases are established where genes whose enzyme products share substrate affinities (Pgm-1 and Pgm-2; Mod-1 and Mod-2) are not linked. Our data generally support the conclusion that functionally or metabolically related isozyme genes are not contiguous on mouse linkage groups.This investigation was supported in part by Public Health Service General Research Support Grant GM-09966 and in part by Public Health Service Training Grant 5T01 HD-00032-07 from the National Institute of Child Health and Human Development, and by Atomic Energy Commission contract AT(30-1)-3671.     

Investigation of inclusion complexation of paclitaxel by cyclohenicosakis-(1-->2)-(beta-D-glucopyranosyl), by cyclic-(1-->2)-beta-D-glucans (cyclosophoraoses), and by cyclomaltoheptaoses (beta-cyclodextrins)

Inclusion complexation of the poorly soluble drug, paclitaxel, was investigated with various host cyclooligosaccharides such as a family of isolated neutral cyclohenicosakis-(1-->2)-(beta-D-glucopyranosyl) (cyclic-(1-->2)-beta-D-glucans, cyclosophoraoses), dimethyl cyclomaltoheptaose (cyclodextrins, DM-beta-CD) and hydroxypropyl cyclomaltoheptaose (cyclodextrins, HP-beta-CD). Quantitative analysis with high-performance liquid chromatography (HPLC) indicated that all three cyclic oligosaccharides could increase the solubility of paclitaxel, where DM-beta-CD gave the best results and a family of cyclosophoraoses and HP-beta-CD, both gave similar results. Complexation of host molecules with paclitaxel was studied by NMR and fluorescence spectroscopic analyses. NMR spectroscopic analysis showed that the aromatic regions of paclitaxel experienced noticeable changes of the chemical shifts or peak shapes upon interaction with host molecules. The relatively bulky cyclosophoraoses allowed favorable accessibility to either the B-ring or A-ring of paclitaxel, while DM-beta-CD and HP-beta-CD allowed accessibility to all the aromatic rings including the C ring. The interaction of DM-beta-CD with paclitaxel greatly increased the fluorescence intensity compared with other host molecules, suggesting the more effective partitioning of a moderate fluorophore into a hydrophobic cluster adjacent to the C-ring of paclitaxel.     

Anti-resorptive activity and pharmacokinetic study of N(1),N(1)-diisopropyl-N(2)-(diphenylphosphoryl)-2-(4-nitrophenyl)acetamidine

In vitro anti-resorptive activity, mechanism of action, pharmacokinetic profile and in vivo anti-resorptive activity of N¹,N¹-diisopropyl-N²-(diphenylphosphoryl)-2-(4-nitrophenyl)acetamidine (1) were evaluated.     

Glycogen debranching enzyme 6 (AGL), enolase 1 (ENOSF1), ectonucleotide pyrophosphatase 2 (ENPP2_1), glutathione S-transferase 3 (GSTM3_3) and mannosidase (MAN2B2) metabolism computational network analysis between chimpanzee and human left cerebrum

We identified significantly higher expression of the genes glycogen debranching enzyme 6 (AGL), enolase 1 (ENOSF1), ectonucleotide pyrophosphatase 2 (ENPP2_1), glutathione S-transferase 3 (GSTM3_3) and mannosidase (MAN2B2) from human left cerebrums versus chimpanzees. Yet the distinct low- and high-expression AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism networks between chimpanzee and human left cerebrum remain to be elucidated. Here, we constructed low- and high-expression activated and inhibited upstream and downstream AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network between chimpanzee and human left cerebrum in GEO data set by gene regulatory network inference method based on linear programming and decomposition procedure, under covering AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 pathway and matching metabolism enrichment analysis by CapitalBio MAS 3.0 integration of public databases, including Gene Ontology, KEGG, BioCarta, GenMapp, Intact, UniGene, OMIM, etc. Our results show that the AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network has more activated and less inhibited molecules in chimpanzee, but less activated and more inhibited in the human left cerebrum. We inferred stronger carbohydrate, glutathione and proteoglycan metabolism, ATPase activity, but weaker base excision repair, arachidonic acid and drug metabolism as a result of inducing cell growth in low-expression AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network of chimpanzee left cerebrum; whereas stronger lipid metabolism, amino acid catabolism, DNA repair but weaker inflammatory response, cell proliferation, glutathione and carbohydrate metabolism as a result of inducing cell differentiation in high-expression AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network of human left cerebrum. Our inferences are consistent with recent reports and computational activation and inhibition gene number patterns, respectively.     

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.

     

Biochemical markers in rats: Linkage relationships of aconitase (Acon-1), aldehyde dehydrogenases (Ahd-2 and Ahd-c), alkaline phosphatase (Akp-1), and hydroxyacid oxidase (Hao-1)

We have examined the linkage relationships between five biochemical markers, Acon-1, Ahd-2, Ahd-c, Akp-1, and Hao-1, and 19 other genetic loci in five breeding combinations. The genetic locus that codes for a recently described aldehyde dehydrogenase in the liver (Ahd-c) has been assigned to linkage group X (LG X). Hydroxyacid oxidase is coded for by a locus (Hao-1) that is linked to genes that encode agouti coat color and seminal vesicle proteins in linkage group IV. Alkaline phosphatase (Akp-1) was linked to the locus that encodes the C6 component of complement and this association provisionally defines a new linkage group (LG XI) in the rat. The locus Acon-1 could not be positively assigned to a specific linkage group but the results from one breeding combination suggest that this locus may be included in linkage group II. No linkage relationship could be detected for the aldehyde dehydrogenase coded for by Ahd-2.This work was supported by Grant GM 32580 from the National Institutes of Health, United States Public Health Service.     

Regional localization of three convertases, PC1 (Nec-1), PC2 (Nec-2), and furin (Fur), on mouse chromosomes.

The genes for three convertases, PC1 (Nec-1), PC2 (Nec-2), and furin (Fur), have been regionally localized on chromosomes 13, 2, and 7, respectively, by interspecific backcross analysis. These results refine previous localizations by in situ hybridization as well as confirm and extend known regions of homology between mouse and human chromosomes.     

Localization of Rh1(D), 2(C), 3(E), 4(c), 5(e) and 25(LW) antigens of human Rh blood groups in fetal erythrocyte membranes

The fetal erythrocyte membranes were partially solubilized with Triton X-100 at the low concentration (0.5%). The localizations of Rh1(D), 2(C), 3(E), 4(c), 5(e) and 25(LW) were investigated. Using hemagglutination inhibition assay, Rh1(D) antigen activity was observed in the Triton-treated membrane (Triton shell) containing mainly band 1, 2 (spectrin), band 5 (actin), band 4.1 and a part of band 3, while Rh2(C), 3(E), 4(c), 5(e) and 25(LW) antigens were detected in the supernatant containing band 3, 6, 2.2, 2.3 and 4.2. It is suggested that: Rh1(D) antigen would associate with cytoskeleton matrix of fetal erythrocyte membranes; Rh1(D) and Rh25(LW) antigens might be integral membrane proteins, while Rh2(C), 3(E), 4(c) and 5(e) antigens would be surface membrane proteins which are easily released from membranes by EDTA, mercaptoethanol and alkaline treatments.     

Expression of PCSK1 (PC1/3), PCSK2 (PC2) and PCSK3 (furin) in mouse small intestine

The family of serine proteases known as the proprotein convertases subtilisin/kexin type (PCSK) is responsible for the cleavage and maturation of many precursor hormones. Over its three successive regions, the duodenum, the jejunum and the ileum, the small intestine (SI) expresses over 40 peptide hormones necessary for normal intestinal physiology. Most of these hormones derive from proteolytic cleavage of their cognate inactive polypeptide precursors. Members of the PCSK family of proteases have been implicated in this process, although details of enzyme-substrate interactions are largely lacking. As a first step towards elucidating these interactions, we have analyzed by immunohistochemistry the regional distribution of PCSK1, PCSK2 and PCSK3 in mouse SI as well as their cellular co-localization with substance P (SP), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP) and somatostatin (SS), 4 peptide hormones known to result from PCSK-mediated processing. Results indicate that PCSK1 is found in all three regions of the SI while PCSK2 and PCSK3 are primarily expressed in the upper two, the duodenum and the jejunum. In these proximal regions, PCSK1 was detectable in 100% of SP-positive (+) cells, 85% of CCK+ cells and 50% of GIP+ cells; PCSK2 was detectable in 40% of SS+ cells and 35% of SP+ cells; PCSK3 was detectable in 75% of GIP+ cells and 60% of SP+ cells. These histological data suggest that the 3 PCSKs may play differential and overlapping roles in prohormone processing in the three regions of the SI.     

Distribution of G1m(1), G1m(2) and G3m(5) allotypes in Aragon.

The distribution of G1m(1), G1m(2) and G3m(5) allotypes was studied in 700 unrelated individuals from Aragon (North-East Spain). The Gm haplotype frequencies were similar to those reported in French areas next to Aragon.     

P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors are coupled to Rho and Rho kinase activation in vascular myocytes

In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.     

Genetic interaction between AINTEGUMENTA (ANT) and the ovule identity genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2)

AINTEGUMENTA (ANT) promotes initiation and growth of ovule integuments which cell fate is specified by ovule identity factors, such as SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2). To study the genetic interaction between ANT and the ovule identity genes, we have obtained a stk shp1 shp2 ant quadruple mutant. The molecular and morphological characterization of the quadruple mutant and its comparison with the stk shp1 shp2 triple mutant, the shp1 shp2 ant triple mutant and the stk ant double mutant are here presented.     

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3)

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G(i) proteins linearly and not catalytically.