D-lactate specific pyridine nucleotide lactate dehydrogenase in animals.

A survey of representative invertebrates has revealed the presence of pyridine nucleotide-linked (D)-lactate dehydrogenase in a number of groups. All species studied contained either D(D)-or (L)L-lactate dehydrogenase, but no species contained both enzymes. The (D)-lactate dehydrogenase from Limulus polyphemus has been purified and has a molecular weight of 65,000.

A genetic basis for the interrelation of coagulation factors.

BACKGROUND: Evidence found in the literature for a strong correlation between coagulation factors suggests that single genes might influence the plasma concentrations of multiple coagulation factors (i.e. pleiotropically acting genes). OBJECTIVE: To determine whether there is a genetic basis for the correlation among coagulation factors by assessing the heritability of interrelated coagulation factors. PATIENTS/METHODS: We performed principal components analysis, and subsequently variance components analysis, to estimate the heritability of principal components of coagulation factors in family members of a large French-Canadian kindred. RESULTS: Four clusters were identified by principal components analysis in 200 family members who did not carry the protein C 3363C mutation. Cluster 1 consisted of prothrombin, factor VII (FVII), FIX, FX and protein S; cluster 2 consisted of FV, FIX, protein C and tissue factor pathway inhibitor; cluster 3 consisted of FVIII and von Willebrand factor; and cluster 4 consisted of antithrombin, protein C and FVII. The heritability of the principal components estimated by variance components analysis was, respectively, 37%, 100%, 37%, and 37%. CONCLUSION: Our findings support the hypothesis that genes can influence plasma levels of interrelated coagulation factors.

Chronic venous abnormalities in symptomatic and asymptomatic protein C deficiency.

BACKGROUND: Thrombophilia is a frequent medical condition associated with symptomatic deep vein thrombosis (DVT). Unlike other clinical risk factors associated with DVT, such as surgery, thrombophilia has not been demonstrated to be associated with asymptomatic venous thrombotic events. Our aim was to search for asymptomatic sequelae of DVT in a protein C (PC)-deficient family. METHODS: We studied 228 individuals from a large kindred with PC deficiency and performed a systematic ultrasound examination. RESULTS: Among the 203 patients without a known history of venous thrombosis we found seven patients with abnormalities indicative of prior asymptomatic thrombosis: six (7.4%) in the PC-deficient group (n = 81) and only one (0.8%) in the non-deficient group (n = 122). The relative risk for these sequelae associated with PC deficiency was 9.0 (95% CI: 1.1-73.7). CONCLUSIONS: These data suggest that chronic venous abnormalities are frequently present and that thrombotic events in asymptomatic individuals with familial PC deficiency may be underestimated.

Cloning and expression in COS-1 cells of a full-length cDNA encoding human coagulation factor X.

A 1.5-kb cDNA (FX) encoding full-length human coagulation factor X was isolated from a human fetal liver cDNA library. The identity of the insert in a selected phage lambda clone was confirmed to be FX by nucleotide (nt) sequence analysis and restriction mapping. This FX cDNA clone contained 1467 bp of coding sequence, no 5'-untranslated sequence, a short 3'-untranslated sequence of 10 nt and a poly(A) tail at the 3'-end. The FX cDNA was inserted into a mammalian expression vector and transfected into COS-1 monkey kidney cells. Media from transfected cells showed evidence of factor X antigen and, following addition of Russel's viper venom factor X activator, enhanced amidolytic activity toward a synthetic peptide rho-nitroanilide substrate. Immunoprecipitation with an anti-factor X monoclonal antibody of [35S]methionine-labeled cell-conditioned media showed evidence of polypeptides of 74, 55, and 17 kDa, as determined by SDS-PAGE followed by autoradiography. Together, these results indicate that an active factor X can be successfully expressed in a recombinant DNA expression system. This approach will allow the systematic structure/function investigation of this important blood-clotting enzyme.

Genome scan of venous thrombosis in a pedigree with protein C deficiency.

Kindred Vermont II has a high frequency of venous thrombosis, occurring primarily in pedigree members with type I protein C deficiency due to a 3363 inserted (Ins) C mutation in exon 6 of the protein C gene. However, only a subset of 3363 InsC carriers have suffered thrombotic episodes, suggesting that the increased risk of thrombosis results upon the co-occurrence of 3363 InsC with a second, unknown, thrombophilic mutation that segregates independently within the pedigree. To test this hypothesis and to localize the co-occurring gene, we performed a genome scan of venous thrombosis in Kindred Vermont II. Non-parametric linkage statistics identified three potential gene locations, on chromosomes 11q23 (nominal P < 0.0001), 18p11.2-q11.2 (P < 0.0007), and 10p12 (P < 0.0003), supporting the presence of at least one additional thrombophilic mutation in the pedigree. Identification of the unknown mutation(s) promises to reveal a new genetic risk factor for thrombophilia, contribute to our understanding of the blood clotting mechanism, and expand our knowledge of the diversity of oligogenic disease.

Heritability of plasma concentrations of clotting factors and measures of a prethrombotic state in a protein C-deficient family.

BACKGROUND: Earlier studies found strong support for a genetic basis for regulation of coagulation factor levels and measures of a prethrombotic state (d-dimer, prothrombin fragment 1.2). OBJECTIVES: Estimation of how much of the variation in the levels of coagulation factors and measures of a prethrombotic state, including measures of protein C activation and inactivation, could be attributed to heritability and household effect. PATIENTS AND METHODS: Blood samples were collected from 330 members of a large kindred of French-Canadian origin with type I protein C deficiency. Heritability and common household effect were estimated for plasma concentrations of prothrombin, factor (F)V, factor VIII, factor (F)IX, fibrinogen, von Willebrand factor (VWF), antithrombin, protein C, protein S, protein Z, protein Z-dependent protease inhibitor (ZPI), fibrinopeptide A (FPA), protein C activation peptide (PCP), activated protein C-protein C inhibitor complex (APC-PCI), activated protein C-alpha1-antitrypsin complex (APC-alpha1AT), prothrombin fragment 1.2 (F1.2) and d-dimer, using the variance component method in sequential oligo-genic linkage analysis routines (SOLAR). RESULTS: The highest heritability was found for measures of thrombin activity (PCP and FPA). High estimates were also found for prothrombin, FV, FIX, protein C, protein Z, ZPI, APC-PCI and APC-alpha1AT. An important influence of shared household effect on phenotypic variation was found for VWF, antithrombin, protein S and F1.2. CONCLUSIONS: We found strong evidence for the heritability of single coagulation factors and measures of a prethrombotic state. Hemostatic markers with statistically significant heritability constitute potential targets for the identification of novel genes involved in the control of quantitative trait loci.

The effect of N-linked glycosylation on molecular weight, thrombin cleavage, and functional activity of human protein S.

Human protein S (HPS) has three potential N-linked glycosylation sites at Asn458, 468, 489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn458-->Gln, Ser460-->Gly; Asn468-->Gln, Thr470-->Gly; Asn489-->Gln, Thr491-->Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors, and conditioned medium was collected for recombinant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showing that each of the three potential N-glycosylation sites contain a similar amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular mass of approximately 78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses for cleavage by alpha-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor specific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component system, glycosylation mutants of protein S showed a slightly enhanced ability to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn458, Ser480) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the same apparent Kd as wild-type rHPS.

Homozygous type I protein C deficiency in two unrelated families exhibiting thrombophilia related to Ala136-->Pro or Arg286-->His mutations.

Separate single nucleotide mutations have been identified in two unrelated homozygous type I protein C deficient individuals suffering from thrombophilia. Each mutation, initially established by direct DNA sequencing of polymerase chain reaction amplification products, results in an amino acid substitution. The first mutation (PCClamart) results in an Ala136 to Pro substitution in the protein's second epidermal growth factor-like domain. The second mutation (PCMünchen) results in an Arg286 to His substitution in the serine protease domain. Comparison of the location of these two mutations and the relative conservation of the two regions in homologous vitamin K-dependent plasma proteins is consistent with the difference in severity of protein C deficiency and disease in the two individuals. Both mutations result in the abolition of a naturally occurring restriction endonuclease site, thereby allowing independent confirmation of the mutations and rapid and unambiguous genetic analysis of protein C deficiency in family members. In both families, the genetic analysis has proven useful in cases where an assignment of the protein C status based upon clinical laboratory measurements was either ambiguous or incorrect.

Genetic screening of candidate genes for a prothrombotic interaction with type I protein C deficiency in a large kindred.

The incomplete penetrance of thrombosis in familial protein C deficiency suggests disease occurs when this deficit is combined with additional abnormalities in the hemostatic system. The pattern of inherited thrombophilia in the Vermont II kindred, which is affected by a clinically dominant type I protein C deficiency, provides strong evidence for a second unidentified gene that segregates independently of protein C deficiency and increases susceptibility to thrombosis. To test the second gene hypothesis, thirty-four candidate genes for proteins involved in hemostasis or inflammation were tested as the unknown defect, using highly polymorphic short tandem repeat (STR) markers in an informative subset (n = 31) of the kindred. The genes considered are; alpha-fibrinogen, beta-fibrinogen, gamma-fibrinogen, prothrombin, tissue factor, factor V, protein S, complement component 4 binding protein, factor XI, factor XII, factor XIIIa, factor XIIIb, histidine rich glycoprotein, high molecular weight kininogen, kallikrein, von Willebrands factor, platelet factor 4, thrombospondin, antithrombin III, alpha-1-antitrypsin, thrombomodulin, plasminogen, tissue plasminogen activator, urokinase plasminogen activator, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, protein C inhibitor, alpha-2-plasmin inhibitor, kallistatin, lipoprotein a, interleukin 6, interleukin 1, cystathionine-beta-synthase, and methylenetetrahydrofolate reductase. Mutations in many of these genes have been previously established as independent risk factors for thrombosis. However, linkage analysis provided no evidence to implicate any of the candidate genes as the second inherited factor that promotes thrombophilia in this kindred.

The G20210A prothrombin polymorphism is not associated with increased thromboembolic risk in a large protein C deficient kindred.

Likelihood analysis was used to test the effect of the G20210A prothrombin mutation and the His107Pro protein C mutation (resulting from a C insertion) on thrombosis status and prothrombin level in a large kindred of French Canadian descent with type I protein C deficiency. Genotypes were available on 279 pedigree members or their spouses. Of this total, 36 pedigree members were heterozygous for the G20210A variant and one pedigree member was homozygous for G20210A, while 64 were heterozygous for the His107Pro protein C mutation. The factor V Leiden mutation (Arg506Gln) was observed in only one of 181 tested family members. Objectively verified thrombosis was present in 26 of the 279 pedigree members. Thrombosis was suspected in an additional 19 pedigree members. The transmission disequilibrium test of Spielman, 1996, as extended to pedigrees, was used to test for excess transmission of G20210A or His107Pro to thrombosis cases, with transmission of 0.5 specifying no effect. Although the His107Pro mutation was over transmitted (0.837 +/- 0.075 p <0.001) to thrombosis cases in this pedigree, the G20210A variant was not (0.491 +/- 0.130 NS). Measured genotype analysis was used to examine a total of 184 individuals for the relationship between prothrombin level and both the G20210A variant and thrombosis. The G20210A variant increased prothrombin level from 97 +/- 2% to 124 +/- 4% (p <0.0001), but thrombosis status was not associated with any additional increase in prothrombin level. Thus, in a large thrombophilic, protein C deficient kindred, with the G20210A variant present in a proportion (13%) far higher than the general Caucasian population (approximately 2%), neither the presence of the variant nor the plasma concentration of prothrombin were associated with increased risk for thrombosis. These findings contrast with those of others who have established the G20210A variant as a thrombophilic risk factor; and emphasize the complex nature of the multigenic pathogenesis of thrombophilia.

Evidence of a founder effect for the protein C gene 3363 inserted C mutation in thrombophilic pedigrees of French origin.

We have previously reported that the 3363 inserted (Ins) C mutation in exon 6 of the protein C gene was present in four unrelated French patients and in four French Canadian families with type I protein C deficiency as well as in a large Vermont protein C deficient kindred of French Canadian origin. The present study was designed to investigate the likelihood of the existence of a founder effect for this mutation in protein C deficient individuals of French origin living in France, Quebec and Vermont. In order to demonstrate a possible founder effect for the 3363 InsC mutation, we have previously constructed a high-resolution genetic map to locate several highly polymorphic markers close to the protein C locus. Thereafter, the markers D2S347, D2S2339, D2S383, D2S2271 and D2S2215 were genotyped in 117 heterozygotes from France (n = 7), Quebec (n = 36) or Vermont (n = 74). The allelic frequency distribution of these five markers was also determined in fifty control French Canadian subjects and thirty-two unaffected members of the Vermont kindred with normal protein C levels and compared with their frequency in our cohort of heterozygotes. Our data suggest that patients from Quebec and Vermont carry a common haplotype at the protein C locus. Moreover, in order to study the evolutionary history of the 3363 InsC mutation, we traced back the ascending genealogy of one proband in each of the families with this mutation. These results showed that the 3363 InsC mutation was most probably introduced in North America by a couple of French settlers who established themselves in 1669 on Isle d'Orleans located near Quebec City. All heterozygotes for the 3363 InsC mutation living in North America are related to these founders within 10 generations. Thus, these families afford a unique opportunity to evaluate the role of the protein C system in thrombophilia due to the high degree of linkage disequilibrium at the protein C gene, which in essence holds that variable more constant than in a more heterogeneous population.

Molecular detection of carriers of hereditary amyloidosis in a Swedish-American family.

Autosomal dominant amyloidosis, also known as familial amyloidotic polyneuropathy (FAP), is a late-onset disorder associated with variants of the protein prealbumin. In FAP Type I, the variant contains a single amino acid substitution at position 30 in the subunit. This substitution corresponds to a single base change in the gene, coincidentally creating a new site for the restriction enzyme NsiI. This change is detectable in the DNA of gene carriers with restriction fragment length polymorphism (RFLP) methods. A well-characterized American family of Swedish origin was studied by this method using a prealbumin cDNA. The RFLP data were found to correlate with previous biochemical characterization of the prealbumin in this family, indicating that this test represents a reliable way to directly detect the DNA mutation responsible for the condition. This test can be used for preclinical diagnosis of gene carriers, including prenatal diagnosis.

Structure of mouse protein S as determined by PCR amplification and DNA sequencing of cDNA.

The cDNA sequence of mouse protein S was derived by conventional PCR amplification from liver mRNA, initially using primers derived from the human cDNA sequence, followed by direct DNA sequencing. Seven overlapping PCR fragments covering all of the mature protein, part of the propeptide, and the 3' noncoding region were generated and sequenced. In some cases primers based upon the human cDNA sequence were ineffective. Subsequent successful amplification with mouse-derived primers to the same regions and comparison of the mouse and human sequences in these regions suggest that the failure of the human primers was due to insufficient degree of heterospecies identity. The mouse protein S cDNA sequence of the coding region shares 82% identity to human. The 3' noncoding region of mouse protein S cDNA has several small deletions and insertions compared to human protein S cDNA. Mature mouse protein S consists of 634 amino acids in a single polypeptide chain and displays domain organization similar to that for other species. The amino acid sequence of mouse protein S is about 80% identical to that of other species. Eleven glutamic acid residues were found in the amino terminal region and are predicted to be sites of gamma-carboxylation. Amino acid residues #80-244 are defined as four cysteine-rich repeat sequences homologous to epidermal growth factor. The remainder of the molecule is homologous to plasma sex steroid binding protein. The mouse protein S contains two potential N-glycosylation sites at positions #458 and 468 and is lacking the putative glycosylation site at #490 found in human protein S.

Genetic analysis of a large kindred exhibiting type I protein C deficiency and associated thrombosis.

A previously described large Vermont kindred possessing a high incidence of venous thromboembolism with associated Type I protein C deficiency (1) has been genetically analyzed. All nine exons of the protein C gene, including both coding and non-coding regions, have been amplified from blood cell genomic DNA using the Tag DNA polymerase chain reaction (PCR) and primers corresponding to flanking intronic regions, and the products directly sequenced. An initial mutation (C-->T) resulting in Thr298-->Met was observed in one arm of the family exhibiting a history of thrombosis and protein C deficiency and was designated protein CVERMONT IIa. However, examination of the kindred member parent (male) of this arm and members of other arms of the kindred demonstrated that the mutation entered the arm via the genetically unrelated spouse. Further analysis of the father and members of other arms of the kindred revealed a different mutation (C insertion: CAT-->CCAT), resulting in a frameshift beginning at amino acid #107 (His-->Pro) and truncation of the protein at codon #119 of the mature protein. This mutation, called protein CVERMONT IIb, is associated with protein C deficiency and thrombosis throughout the kindred.

Factor X Stockton: a mild bleeding diathesis associated with an active site mutation in factor X.

A unique blood coagulation factor X variant has been identified in a family with a history of bleeding. Plasma from affected family members had prolonged prothrombin times and activated partial thromboplastin times, low to below normal factor X coagulant activity, and normal factor X antigen levels. Sequencing of DNA from the propositus revealed a single G to A substitution in one allele of factor X at base 964 resulting in an amino acid substitution of Asn for Asp at residue 282. This residue corresponds with the active site Asp102 of chymotrypsin. The substitution eliminates a TaqI restriction site and provided the basis for a screening assay to detect the mutation in polymerase chain reaction (PCR) amplified factor X exon VIII DNA. Fourteen additional family members were identified as having the mutation at base 964. Plasma factor X purified from the proposita using an anti-factor X monoclonal antibody immunoadsorbent exhibited an approximately 50% decrease in specific activity compared with factor X purified from a normal individual in a similar manner. Bleeding in family members with the mutation, termed factor X Stockton, appears to be due to disruption of normal hemostasis by the presence in plasma of circulating abnormal factor X. Factor X Stockton is the first naturally occurring substitution at the active site Asp of a serine protease and underscores the importance of this amino acid residue in factor Xa coagulant activity.

Characterization of the human prostaglandin H synthase 1 gene (PTGS1): exclusion by genetic linkage analysis as a second modifier gene in familial thrombosis.

Genetic evidence from a large Vermont kindred indicates that an unknown gene promotes thrombosis when inherited in conjunction with type I protein C deficiency. Cyclooxygenase-1 [prostaglandin H synthase 1 gene (PTGS1)] was tested as a plausible candidate for the unknown gene because of its role in primary hemostasis. The complete sequence of PTGS1 (25 638 nucleotides) was determined from a 37 kb human genomic cosmid clone to characterize intronic regions and subsequently to allow the search for mutations by direct sequencing of genomic DNA. Northern blot analysis confirms usage of a newly described distal poly-adenylation signal. Short tandem repeat (STR) sequences found in intron 2 and the 3' flanking region were developed as new genetic markers for PTGS1. The position of PTGS1 was refined on the CHLC chromosome 9 linkage map using the new markers scored in four Centre d'Etude du Polymorphisme Humain families and multipoint linkage analysis. Direct sequencing of DNA from members of the Vermont kindred led to the discovery of two new single nucleotide polymorphisms (SNPs) that give rise to non-conservative amino acid changes in the signal peptide (Arg(8) to Trp and Pro(17) to Leu) of cyclooxygenase-1. Linkage analysis of the SNP and STR markers indicated that PTGS1 is not the interacting gene associated with an increased incidence of thrombosis in the Vermont kindred.

Faculty and student cross-cultural learning through teaching health promotion in the community.

Cross-cultural learning for nursing students and faculty increased through a collaborative venture of teaching health promotion classes in a Hmong community. Through the learning process, nursing students and faculty learned about Hmong culture, and Hmong participants learned about health. The purpose of this article is to describe the process and evaluation of health promotion classes in a Hmong community.

Approaches to studying in deaf and hearing students in higher education.

We conducted a survey to compare the responses of 149 deaf students and 121 hearing students taking the same courses to a shortened and adapted version of the Approaches to Studying Inventory. In general, the impact of deafness on approaches to studying was relatively slight, and deaf students appeared to be at least as capable as hearing students of engaging with the underlying meaning of the materials to be learned. We used factor analysis to identify eight scales, and differences between the two groups were statistically significant on four of these scales. Discriminant analysis indicated that deaf students found it more difficult to relate ideas on different topics and that this was more marked in those who preferred to communicate using sign. However, deaf students were more likely than hearing students to adopt a critical approach and to analyze the internal structure of the topics studied.

Relationships among spatial skills, communication skills, and field independence in deaf students.

The hypotheses that deaf students would be more field-dependent than hearing students and that their competence in communication skills would be positively related to field-independence were supported for a group of 77 male and 67 female deaf students. Step-wise multiple regression analyses of the data showed that for females spatial skills followed by communication skills were significant predictors of field-independence; for males spatial skills followed by the extent of hearing loss were significant predictors of field-independence. Sex differences found on tests of field-independence and spatial relations were consistent with those obtained from the hearing population. It was suggested that socialization experiences and competence in communication skills may influence development of field-independence in deaf students.

[Clinical application of thoracoscopic surgery: a report of 19 cases].

Nineteen cases (14 males and 5 females) had been operated with thoracoscopic surgery. Ten cases of spontaneous pneumothorax and 1 spontaneous hemopneumothorax had been underwent thoracoscopic bullous or bleb resections by Endo-GIA 30 or 60. Four cases of plural effusion including primary and secondary malignancies were performed to be pleurodesis by talc sclerosis. Among 4 cases of lung mass, 3 were operated by wedge resection. Lobectomy was performed by VATS in one case with primary lung cancer (T1N0). VATS were satisfactory by using the 3 small incisions giving the advantage of rapid and complete recovery without any obvious complications. Even no any secondary pneumonia. So far the VATS is not fully accepted in China due to the cost of the disposable instruments.