Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties

Human protein S (HPS), a regulator of hemostasis, is a vitamin K- dependent plasma protein with potential clinical utility. We have obtained high-level expression of the cDNA for HPS in two mammalian cell lines. Both cell lines secreted single chain recombinant HPS (rHPS) in serum-free medium as determined by Western blot analysis. The ability of the rHPS from both cell lines to act as a cofactor for human protein C (HPC) was determined; the rHPS secreted from the human 293 cell line had an activity six times that of the rHPS from the AV12-664 Syrian hamster cell line. Furthermore, the relative specific cofactor activity of rHPS from the 293 cell line was actually 2.5-fold higher than that of single-chain human plasma-derived HPS. Essentially all of the rHPS secreted from the 293 cell line exhibited a calcium-dependent elution profile on anion exchange chromatography, whereas only 25% to 35% of the hamster cell-derived rHPS exhibited this profile. However, the calcium-eluted rHPS from the AV12 cell line had a high specific cofactor activity, equivalent to that of the 293-derived rHPS. A NaCl- elutable rHPS fraction (calcium nondependent) was isolated from the recombinant AV12-664 cell line, further purified, and found to have reduced activity, only 40% that of the calcium-dependent rHPS. The only observable difference in the calcium-dependent and nondependent rHPS molecules was in the content of gamma-carboxyglutamic acid (Gla); the calcium-dependent material contained approximately 10 mol Gla/mol protein whereas the calcium-nondependent material contained only approximately 8 mol Gla/mol of protein. In addition, the calcium- nondependent rHPS had reduced ability to interact with phospholipid vesicles as evidenced by an eightfold increase in the apparent kd. Our data demonstrate the isolation of rHPS with high specific activity, and show that a reduction in as few as two Gla residues dramatically decreases its functional cofactor activity for HPC, due to a reduction in ability to interact with the phospholipid bilayer.     

Enhancement of erythrocyte superoxide dismutase activity: effects on cellular oxidant defense

To delineate further the role of superoxide dismutase (SOD) in red blood cell (RBC) oxidant defense, normal human erythrocytes were osmotically lysed and resealed in the presence of varying concentrations of exogenous SOD. This resulted in a dose-dependent increase in SOD activity in the resealed erythrocytes while maintaining nearly normal RBC hemoglobin concentration (less than 10% decrease from the control value), cell volume, and cellular deformability. Surprisingly, a five- or ninefold increase in SOD activity yielded no additional protection against superoxide-generating drugs (phenazine methosulfate or menadione sodium bisulfite). No significant differences were observed between the control and SOD-loaded RBCs in O2-driven methemoglobin formation or generation of thiobarbituric acid-reactive substances. In contrast, RBCs with elevated SOD activity pretreated with sodium azide (to block catalase activity) or 1-chloro-2,4- dinitrobenzene (to deplete reduced glutathione, GSH) showed significantly enhanced methemoglobin generation in response to superoxide generating drugs. No differential response was noted between the control, control-resealed, and SOD-loaded RBCs to oxidants other than superoxide. Based on our results and other data, we conclude that elevated SOD activity may imbalance cellular oxidant defense, resulting in enhanced oxidation due to the accelerated generation of H2O2, the product of O2- dismutation. This effect is significantly exacerbated under conditions in which H2O2 catabolism is altered.     

A novel dimorphism in the human SRY gene: usefulness in human migration studies

A nucleotide polymorphism of C or T was detected at position 465 in the sex-determining region Y (SRY) gene. To evaluate the utility of this dimorphism in human population studies, the frequency and the frequency of the haplotype combined with the two polymorphic loci YAP and M9 were examined in a total of 130 unrelated Japanese and 130 unrelated German males. The T nucleotide was found in 24.6% (32/130) of the Japanese but not in any of the 130 German males. Accordingly, four of the eight possible combination haplotypes of SRY/YAP/M9 were identified in the Japanese population, but one of the four haplotypes comprising SRY(T) was absent in the German samples. This suggests that the C to T transition may be more recent than the YAP insertion or the M9 transversion and the change might have occurred in an ancestral Asian population. These results imply that the dimorphism at the SRY gene is one of the Y-linked markers useful for human population studies and also for ethnic identification of forensic samples. Received: 9 March 1999 / Accepted: 9 September 1999     

Pulmonary capillary leak syndrome with intravenous cyclosporin A in pediatric renal transplantation

Despite frequent use of intravenous (i.v.) cyclosporin A (CsA) in the early post-operative course of transplant recipients, allergic reactions have been infrequently described. Of 134 transplants, we report four pediatric renal transplant recipients with severe reaction to i.v. CsA with pulmonary capillary leak syndrome. Pulmonary edema developed at a mean time of 3.5 h after commencement of i.v. CsA, with two patients requiring mechanical ventilation. Discontinuation of i.v. CsA and conversion to oral CsA was followed by rapid resolution of pulmonary edema, suggesting that cremaphor, the solubilizing agent in the i.v. formulation, is likely to be responsible for this adverse response. Skin prick testing with cremaphor was negative in all patients and alternative mechanisms for the cremaphor response are proposed. It is likely that inadequate mixing of the i.v. CsA solution triggered this reaction, by delivering a higher concentration of cremaphor at the start of the CsA infusion. Pulmonary edema in the early post-transplant course in the absence of obvious fluid overload should prompt the diagnosis of an i.v. CsA reaction. This life-threatening reaction is easily reversible if recognized, and can be managed easily without compromise to the allograft, by discontinuing i.v. CsA and switching early to an oral CsA formulation.     

Transepithelial paracellular leakiness induced by chronic phorbol ester exposure correlates with polyp-like foci and redistribution of protein kinase C-alpha

Although exposure of LLC-PK1 epithelial cell sheets to phorbol esters (TPA) causes a near immediate and total decrease of transepithelial electrical resistance (TER), continuation of exposure for 3 to 4 days results in a tachyphylactic response as TER begins to return to control levels. Recovery of TER is maximal by 5 to 6 days, but reaches only 70 to 80% of control level. A reciprocal change in the transepithelial flux of D-mannitol indicates that the TER decrease is indicative of an increase in tight junction permeability. Exposure of cell sheets to TPA for several days also results in the appearance of multilayered polyp- like foci (PLFs) across the otherwise one cell layer thick cell sheets. The pattern of penetration of the electron dense dye, ruthenium red, from the apical surface, across the tight junction and into the lateral intercellular space indicates that the tight junctions of the cell sheet become uniformly leaky after acute exposure to TPA. However, when exposure is continued for several days, only the junctions of cells in the PLFs manifest leakiness. The decrease in TER following acute TPA exposure correlates with the translocation of protein kinase C-alpha (PKC alpha) into a membrane-associated compartment. With exposure of several days, only a trace of PKC alpha is visible by Western immunoblot, and this is in the membrane-associated compartment. Immunofluorescent microscopy indicates that the trace of PKC alpha seen in the Western immunoblots is ascribable distinctly to cells of the PLFs. Monolayer areas between PLFs show no discernible immunofluorescent signal. The data therefore indicate that tight junction barrier function may be restored in certain areas by the down regulation of PKC alpha from the membrane-associated compartment. Failure to down regulate may result in the paracellular leakiness and abnormal cell architecture of the PLFs. Possible implications of this model for in vivo epithelial tumor promotion are discussed.      

Molecular analysis of PKU in Ireland

Classical phenylketonuria (PKU: McKusick No. 261600) is caused by mutations occurring at the phenylalanine hydroxylase (PAH) locus on chromosome 12 and has a prevalence in Ireland of 1 in 4500. We examined 304 independent alleles from 350 patients for the presence of six mutations and have characterized VNTR alleles within the minisatellite region 3' to the PAH gene in patients carrying the most prevalent mutation. R408W was the most common mutation found, with a relative frequency of 42%. All other mutations had relative frequencies of <10%. VNTR analysis showed that the R408W mutation is associated with the VNTR-8 allele in the Irish population, indicating that R408W is associated with RFLP haplotype 1. This differs from that reported from eastern Europe where R408W is associated with RFLP haplotype 2/VNTR-3; an observation which has led several groups to propose a Balto-Slavic origin for this mutation. These results support the hypothesis of a second, independent founding event for the R408W mutation on an RFLP haplotype 1 VNTR-8 chromsome background in the Irish/Celtic population.     

Effect of 1-methyl-2-mercapto-5-(3-pyridyl)-imidazole (KC-6141) on rabbit platelet aggregation in vitro and rat platelet retention

Effect of 1-methyl-2-mercapto-5-(3-pyridyl)-imidazole (KC-6141) on rabbit platelet aggregation in vitro and rat platelet retention investigated. In the in vitro study, KC-6141 inhibited ADP-induced aggregation by 27% at 5 X 10(-4)M, being more active than dipyridamole but much less than adenosine. Inhibition of arachidonic acid- and collagen-induced aggregation by KC-6141 was more effective than that of ADP-induced one and its ED50 was 2.1 X 10(-5) and 8 X 10(-5)M, respectively. KC-6141 was 10 and 4 times more potent than aspirin in arachiodonic acid- and collagen-induced aggregation, respectively. The dose-response curve of KC-6141 was parallel to that of aspirin, suggesting it is an aspirin-like compound. In the platelet retenion study, a method for determining platelet retintion in rats was devised so that platelet retention can be measured with a volume of blood as small as possible. By use of the method, effects of KC-6141, aspirin and dipyridamole were compared. When deministered intraperitoneally at 100 mg/kg, KC-6141 indicated 54.8% inhibition of platelet retention, whereas aspirin and dipyridamole showed only 23.5 and 5.2% inhibition, respectively. On the oral administration at 200 mg/kg KC-6141 inhibited by 60.8% and its ED50 was 125 mg/kg. The activity lasted over 32 hr. The above results demonstrated that KC-6141 is a compound with more potent action on the platelet aggregation, as well as on the platelet retention than aspirin and dipyridamole-a known antithrombotic drug.     

Esterase D phenotypes in north-eastern Japan

Summary Esterase D (EsD) was typed in 2,367 unrelated healthy blood donors from Yamagata and Miyagi districts, north-eastern area of Japan. The gene frequencies observed were: EsD¹=0.642, EsD²=0.358. In these blood donors two rare EsD phenotypes, EsD 3-2 and a new variant, were found. As for this new variant, the mode of hereditary transmission was discussed by family study.     

Recent progress in mitochondrial DNA analysis

In this review, we describe the current state of knowledge of mitochondrial genetics of East Asian populations and its application to forensic science. Recent advances in mitochondrial DNA (mtDNA) phylogeny have identified haplogroup-specific single nucleotide polymorphisms (SNPs) and the control region motifs of haplogroups. By analyzing haplogroup-specific SNPs, we can rapidly and accurately connect the mtDNA under study to the relevant haplogroup. Haplogroups are fairly continent- and/or region-specific; therefore, we can infer the ethnic background of that mtDNA. In addition, errors in hypervariable region sequences can be detected by means of haplogroup motif analysis.     

Fabrication of a Fixed Retrievable Implant‐supported Prosthesis Based on Electroforming: A Technical Report

This article describes a method of fabricating a fixed retrievable implant‐retained prosthesis based on electroforming. This method combines the advantages of both the cement‐ and screw‐retained prostheses, including passive fit, ease of fabrication, and retrievability. The absence of visible occlusal screw‐canals adds to its increased esthetic appeal.