Critical von Willebrand factor A1 domain residues influence type VI collagen binding.

BACKGROUND: von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen. OBJECTIVES: We report here on several mutations that exclusively alter binding to type VI collagen. PATIENTS/METHODS: Healthy controls and index cases from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen. VWF gene sequencing was performed for all subjects. RESULTS: Two healthy controls and one type 1 VWD subject were heterozygous for an A1 domain sequence variation, R1399H, and displayed a selective decreased binding to type VI collagen but not types I and III. Expression of recombinant 1399H VWF resulted in absent binding to type VI collagen. Two other VWF A1 domain mutations, S1387I and Q1402P, displayed diminished binding to type VI collagen. An 11 amino acid deletion in the A1 domain also abrogated binding to type VI collagen. CONCLUSIONS: VWF:CB may be useful in diagnosis of VWD, as a decreased VWF:CB/VWF:Ag ratio may reflect specific loss of collagen binding ability. Mutations that exclusively affect type VI collagen binding may be associated with bleeding, yet missed by current VWF testing.

Incidence of weak D in blood donors typed as D positive by the Olympus PK 7200.

The incidence of weak D has been reported to be between 0.23 and 0.5 percent in Europe and 3.0 percent in the United States. All studies were performed before the introduction of monoclonal anti-D reagents. Using current commercial reagents, this study evaluated D+ samples for the presence of weak D. D+ donors, typed by the Olympus PK 7200, using diluted monoclonal blend anti-D and diluted polyclonal anti-D, were selected by sampling batches of 100 to 200 samples from the previous day's collection. Anti-D reagents used on the Olympus PK 7200 are required to detect RBCs with the weak D phenotype which do not agglutinate at immediate spin (IS) when tested with polyclonal anti-D by manual tube methods. More than 95 percent of donors tested were Caucasian. Using tube tests with two different monoclonal blend anti-D reagents and one polyclonal anti-D typing reagent, the presence or absence of the D antigen was evaluated after the IS reading. Donors found negative or weakly positive (< 2+) at IS were further typed for weak D by the IAT. The weak D samples were RHD genotyped by allele-specific PCR. Of 1,005 donors tested, 4 (0.4%) were classified as weak D by one or more anti-D reagents. Polyclonal anti-D reagent demonstrated weaker reactions when compared with the monoclonal blends. All weak D samples were found positive for exon 4, intron 4, and exon 10, a finding consistent with most D+ samples. The incidence of weak D found in this study is not significantly different from that found in earlier studies using polyclonal anti-D reagents.

Prenatal Genotyping of the RhD Locus to Identify Fetuses at Risk for Hemolytic Disease of the Newborn.

Hemolytic disease of the newborn (HDN) can occur when there are fetomaternal incompatibilities within any number of different erythrocyte antigen systems, including the RhD, Cc, Ee, Kidd and Duffy, and Kell antigen systems. In these disorders, maternal antibodies are developed by alloimmunization of the mother to fetal red blood cells during pregnancy when the fetal cells carry an alloantigen inherited from the father. The maternal antibodies result in the destruction of fetal erythrocytes leading to severe hemolytic anemia and hyperbilirubinemia. Permanent neurologic damage can result from HDN, and in extreme cases loss of the fetus or death of the neonate may occur. In subsequent pregnancies, it is important to determine the status of the incompatible allele in the fetus. If the father is heterozygous or homozygous for the allele, the chance of the fetus inheriting the paternal alloallele to which the mother is immunologically sensitized is 50 or 100%, respectively. Fetuses that do not inherit the alloallele will not be at risk for HDN.

Quantitative evaluation of post-bone marrow transplant engraftment status using fluorescent-labeled variable number of tandem repeats.

BACKGROUND: The analysis of highly polymorphic variable number of tandem repeat (VNTR) loci is useful for the estimation of donor-host chimerism in bone marrow transplant recipients. METHODS AND RESULTS: A rapid and sensitive engraftment assay has been developed in which the VNTR loci, D1S80, D17S5, D1S111, and apoB, are amplified with fluorescent-labeled (Cy5.5) oligonucleotide primers, followed by analysis using the Visible Genetics, Inc, OpenGene System. The degree of chimerism is then calculated by determining the percentage of host contribution to the total informative allele peak area. Reconstitution experiments and analysis of 383 posttransplantation DNA samples, isolated from 71 different bone marrow transplant recipients, were evaluated as part of assay development. Reconstitution studies showed assay linearity and sensitivity of at least 1%. Patient results were compared with a previous analysis in which unlabeled PCR products were quantified on silver-stained polyacrylamide gels. High concordance was observed between fluorescent analysis and silver-staining method in all 71 patients. CONCLUSIONS: Fluorescent analysis offers many advantages over previous methods, including faster turnaround time, decreased DNA requirements, greater resolution and/or sensitivity, and objective interpretation.

The sensitivity of allele-specific polymerase chain reaction can obviate concern of maternal contamination when fetal samples are genotyped for immune cytopenic disorders.

OBJECTIVE: Fetuses at risk for immune cytopenic disorders can be identified by molecular genotyping assays. To better understand the impact of maternal contamination on genotyping results, the levels of contamination that are routinely encountered during prenatal testing of fetal samples and the sensitivity of allele-specific polymerase chain reaction in detecting paternal alloalleles were examined. STUDY DESIGN: Reconstitution experiments were performed to define the sensitivity of allele-specific polymerase chain reaction assays. The sensitivities of allele-specific polymerase chain reactions and polymerase chain reaction-restriction fragment length polymorphism were compared for detection of the factor V Leiden mutation. RESULTS: A quantitative analysis of variable-number tandem repeat loci revealed maternal contamination in 4 of 56 fetal samples. Contaminating deoxyribonucleic acid compromised genotyping results when it comprised between 94% and 99% of the total deoxyribonucleic acid. Allele-specific polymerase chain reaction was found to be the more sensitive technique (0.8% sensitivity vs 13% sensitivity). CONCLUSION: These results illustrate that allele-specific polymerase chain reaction is well suited for reliable prenatal identification of fetuses at risk of immune cytopenic disorders.

Expression of spinach nitrite reductase in Escherichia coli: site-directed mutagenesis of predicted active site amino acids.

Spinach ferredoxin-nitrite reductase is a chloroplast enzyme that contains a coupled [Fe4S4]-siroheme-active site and catalyzes the six-electron reduction of nitrite to ammonia. An expression system which produced enzymatically active spinach nitrite reductase (NiR) in Escherichia coli was developed in order to study the structure-function relationships of the coupled active site using site-directed mutagenesis. The spinach NiR cDNA, without the sequences encoding the chloroplast transit peptide, was expressed as a beta-galactosidase fusion containing five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive. After optimizing growth conditions, active NiR represented 0.5-1.0% of the total protein. E. coli-expressed NiR was purified approximately 200-fold to homogeneity as indicated by SDS-polyacrylamide gel electrophoresis. The expressed NiR enzyme was recognized by rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E. coli-expressed NiR was identical to authentic spinach NiR with a Soret and alpha band at 386 and 573 nm, respectively, and a A278/A386 = 1.9. The addition of nitrite to the oxidized enzyme preparation produced the characteristic shifts in the spectrum. The specific activity for the methyl viologen-dependent reduction of nitrite of E. coli-expressed NiR was 100 U/mg and the Km determined for nitrite was 0.3 mM, which are in agreement with reported values for this enzyme. These results indicate that the E. coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity, and physical state.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of human liver sulfite oxidase.

A 2.4 kilobase cDNA clone of human sulfite oxidase was isolated from a human liver cDNA library in lambda gt10. Comparison of three sulfite oxidase sequences to several plant and fungal nitrate reductase sequences reveals a single conserved cysteine with highly conserved flanking sequences. The conserved cysteine is postulated to be a ligand of molybdenum in sulfite oxidase and nitrate reductase.

Rapid determination of platelet alloantigen genotypes by polymerase chain reaction using allele-specific primers.

BACKGROUND: The technique of allele-specific polymerase chain reaction (PCR) amplification has been adapted for DNA-based human platelet alloantigen typing. STUDY DESIGN AND METHODS: Sequence-specific primers were used to discriminate between the alleles encoding the six major human platelet alloantigens in a series of patients and normal blood donors. RESULTS: This technique allows the direct determination of platelet antigen genotypes from genomic DNA after PCR amplification and agarose gel electrophoresis. It offers significant advantages over previously described techniques for alloantigen identification, as the additional analytical steps of restriction enzyme digestion or dot blot hybridization are not required. The results obtained with this technique correlated precisely with those derived from serologic typing and allele-specific oligonucleotide hybridization. CONCLUSION: The use of allele-specific PCR for platelet alloantigen typing should facilitate the development of DNA-based typing in other regional blood centers and clinical laboratories.

Rat sulfite oxidase antibodies cross-react with two gene family-related proteins: albumin and vitamin D-binding protein.

Screening lambda cDNA libraries from rat liver with antibody to native rat liver sulfite oxidase (RLSO) showed cross-reaction with two proteins that belong to the same gene family: serum albumin and vitamin D-binding protein. Antibodies raised against native RLSO or sodium dodecyl sulfate-denatured protein cross-reacted with these proteins by Western blot analysis. The relative effectiveness of RLSO antibody binding was estimated to be 1/5 for rat serum albumin and 1/10 for rat vitamin D-binding protein. This result was not caused by contaminating proteins in the RLSO used for immunization as the RLSO preparation did not react with rat serum albumin antibody. RLSO antibodies, selected for their ability to bind rat serum albumin immobilized on nitrocellulose, recognized both rat serum albumin and RLSO. RLSO antibody, with albumin-reactive antibody removed, still recognized vitamin D-binding protein, suggesting that multiple determinants specific to each protein are involved in the cross-reaction. Comparison of RLSO antibody binding to the rat and human proteins indicated that the determinants were species-specific. cDNA clones identified by screening cDNA libraries with RLSO antibody demonstrated that these determinants reside in the C-terminal domain of these proteins. These results suggest that these proteins contain some common immunological features and may be evolutionarily related.

L-tryptophan injection fails to alter nutrient selection by rats.

Nutritional studies on rats given a choice between two diets differing in protein content have led to the proposal that brain 5-HT content regulates protein intake [2]. Pharmacologic studies under similar conditions of dietary self-selection suggest that brain 5-HT controls carbohydrate intake [41]. We tested the effect of elevating brain 5-HT via tryptophan injection (100 mg/kg) on short-term food intake and selection by rats choosing between two diets differing in protein and carbohydrate content. Under these conditions neither total food intake nor protein and carbohydrate selection were affected despite increases of 50% in brain concentrations of 5-HT and 5-HIAA. The effect of Trp administration was selective to serotonin metabolism as brain concentrations of NE, DA and DOPAC were not affected. These results suggest that alterations in brain 5-HT content which may occur following meal ingestion may not be of physiological importance in regulating nutrient intake and selection.