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.     

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.     

Common action of certain viruses,toxins, and activated complement: pore formation and its prevention by extracellular Ca2+

Haemolysis by Sendal virus, -toxin, and activated complement is inhibited by high concentrations of divalent cations. In Daudi cells, sublytic amounts of these agents induce the following changes: collapse of surface membrane potential, uptake of Na⁺ and loss of K⁺ from cells, and leakage of phosphorylated metabo-tites from cells. The changes induced by Sendal virus and complement are sensitive to physiological concentrations of extracellular Ca²⁺. It is concluded that fluctuations in plasma Ca²⁺ concentration may affect the damaging action of certain pore-forming agents on susceptible cells.     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery. Failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, 99mTc, and an aqueous marker, 125I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped 125I-labelled poly(vinyl pyrrolidone) were labelled with 99mTc by the SnCl2 method. 99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than 125I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, 99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than 125I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60-70% of 125I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of 99mTc-radioactivity was co-eluted with the liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all 99mTc-radioactivity does not represent the 60-70% of intact liposomes present in lymph nodes. Using the aqueous marker 125I-labelled poly(vinyl pyrrolidone), the lymph node localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that 125I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier, where the authors used 99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became 'leaky' to low-molecular-weight drugs, for example, methotrexate (Mr 454) to a much greater extent than with a large-molecular-weight substance, 125I-labelled poly(vinyl pyrrolidone) (Mr 30 000-40 000), when incubated with rat lymph at 37 degrees C. Using the two markers 99mTc and 125I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining lambda-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with 99mTc rather than using the SnCl2 technique, or using other radionuclides as markers for gamma-scan imaging.