gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function.

The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.     

Haploinsufficiency of cytosolic serine hydroxymethyltransferase in the Smith-Magenis syndrome.

Folate-dependent one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth, and serine hydroxymethyltransferase (SHMT) is central to this process. Our studies reveal that the gene for cytosolic SHMT (cSHMT) maps to the critical interval for Smith-Magenis syndrome (SMS) on chromosome 17p11.2. The basic organization of the cSHMT locus on chromosome 17 was determined and was found to be deleted in all 26 SMS patients examined by PCR, FISH, and/or Southern analysis. Furthermore, with respect to haploinsufficiency, cSHMT enzyme activity in patient lymphoblasts was determined to be approximately 50% that of unaffected parent lymphoblasts. Serine, glycine, and folate levels were also assessed in three SMS patients and were found to be within normal ranges. The possible effects of cSHMT hemizygosity on the SMS phenotype are discussed.     

Sublocalization of an Ataxia-Telangiectasia Gene Distal to D11S384 by Ancestral Haplotyping In Costa Rican Families

In an effort to localize a gene for ataxia-telangiectasia (A-T), we have genotyped 27 affected Costa Rican families, with 13 markers, in the chromosome 11q22-23 region. Significant linkage disequilibrium was detected for 9/13 markers between D11S1816 and D11S1391. Recombination events observed in these pedigrees places A-T between D11S1819 and D11S1960. One ancestral haplotype is common to 24/54 affected chromosomes and roughly two-thirds of the families. Inferred (ancestral) recombination events involving this common haplotype in earlier generations suggest that A-T is distal to D11S384 and proximal to D11S1960. Several other common haplotypes were identified, consistent with multiple mutations in a single gene. When considered together with all other evidence, this study further sublocalizes the major A-T locus to ≈200 kb, between markers S384 and S535.     

Thiosulfate Reduction, an Important Physiological Feature Shared by Members of the Order Thermotogales

Several members of the order Thermotogales in the domain Bacteria, viz., Thermotoga neapolitana, Thermotoga maritima, Thermosipho africanus, Fervidobacterium islandicum, and Thermotoga strain SEBR 2665, an isolate from an oil well, reduced thiosulfate to sulfide. This reductive process enhanced cellular yields and growth rates of all the members but was more significant with the two hyperthermophiles T. neapolitana and T. maritima. This is the first report of such an occurrence in this group of thermophilic and hyperthermophilic anaerobic bacteria. The results suggest that thiosulfate reduction is important in the geochemical cycling of sulfur in anaerobic thermal environments such as the slightly acidic and neutral-pH volcanic hot springs and oil reservoirs.     

Reevaluating the classification of Halobacteroides and Haloanaerobacter species based on sequence comparisons of the 16S ribosomal RNA gene

Abstract The 16S rRNA gene (rDNA) sequence analysis of four halophilic anaerobes: Halobacteroides halobius, H. lacunaris, Haloanaerobacter (Hb.) chitinovorans and H. acetoethylicus confirmed that they were all members of the family Haloanaerobiaceae. H. lacunaris and H. halobius were found to be more closely related to each other and were distantly related to Sporohalobacter lortetti and the members of the genera Haloanaerobium and Halothermothrix . These data are in agreement with their assignment to the genus Halobacteroides . Further analysis indicated that Hb. chitinovorans was closely affiliated to members of the genus Halobacteroides , and therefore we propose to transfer it to the genus Halobacteroides as H. chitinovorans comb. nov. This transfer would invalidate the genus Haloanaerobacter , as Hb. chitinovorans is the only member of this genus. The 16S rDNA sequence analysis of H. acetoethylicum indicated that it was very closely related to members of the genus Haloanaerobium , viz. Haloanaerobium (Ha.) praevalens, Ha. salsugo , and Ha. alcaliphilum , and hence we propose to transfer it to the genus Haloanaerobium as Ha. acetoethylicus comb. nov.     

Callus Cultures from Zygotic Embryos of Costus speciosus and Their Morphogenetic Responses

Soft, nodular and hard types of calli were initiated on mature zygotic embryo explants of two tetraploid clones of Costus speciosus, of which, only the hard calli were amenable to morphogenetic responses. The two clones differed in their growth regulator requirements both for the initiation of calli and for shoot regeneration. De novo formation of both shoot bud meristems and somatic embryoids were observed. Latter were encased partially or fUlly by coleoptilar sheath. Embryoids could be isolated as discrete units. On maturity, a stock like appendage developed from the base and finally embryoids got detached from the subtending tissue. Both shoot-bud meristems and somatic embryoids developed into complete plantlets, the former upon sequential transfer of calli on Schenk and Hildebrandt’s (SH) basal medium containing lower levels of growth hormones, while the latter only on basal medium. These culture regenerants were subsequently transferred to the field. The morphogenetic behaviour of these two tetraploid clones reflects their marked genotypic difference inspite of their same ploidy status.     

Solvation of platinum anti-cancer drugs in methanol-water mixtures

Solubilities and transfer chemical potentials of carboplatin, cisplatin, iproplatin, and several related platinum complexes have been determined in methanol-water mixtures. the range of solvation behaviour is discussed in relation to possible oral administration of complexes of this type.