Factor XKetchikan: a variant molecule in which Gly replaces a Gla residue at position 14 in the light chain.

To seek the possible molecular defect in a patient with deficient factor X plasma procoagulant activity, factor X gene exosn and splice junctions were subjected to heteroduplex analyses and sequencing. A mutation in exon 2 was confirmed as substitution of A by G at nucleotide position 206, coding for Gly instead of a Glu which is a normal precursor for gamma-carboxylated glutamic acid (Gla) at amino acid position 14. An abolished TaqI restriction site was used to indicate homozygosity of the defect, but occurrence of a gene deletion with attendant heterozygosity could not be excluded.

Sulphation of L-tyrosine in mammalian cells: a comparative study.

Chang liver cells, Caco-2 human intestinal epithelial cells and Madin-Darby canine kidney (MDCK) cells, labelled with [35S]sulphate in the presence of different concentrations of cycloheximide, produced 87.7-95.3%, 35.

Quantitative comparison of asbestos and talc bodies in an individual with mixed exposure.

Tissue from an individual with a history of exposure to asbestos and other dust was referred for particulate analysis. The digested material was reviewed by light microscopy to establish the numbers of ferruginous bodies per gram of tissue. Typical asbestos bodies were found at levels consistent with occupational exposure.

Genetic differences between BCG substrains.

Setting: University-affiliated Mycobacteriology Reference Laboratory.

Objective: To determine the genetic differences of 25 BCG isolates representing 16 referenced substrains.

Design: Non-randomized, observational study based on the visual comparison of the large restriction fragment (LRF) patterns created by digesting each BCG isolate's DNA with an infrequent cutting restriction endonuclease (DraI, AsnI, XbaI or SpeI) and separating the resultant DNA fragments with pulsed field gel electrophoresis.

A role for neutrophil elastase in solar elastosis.

Hairless (SKH-1) mice were mated with beige (C57BL/bb) mice to produce a hairless mouse deficient in neutrophil elastase (hhbb). These mice were exposed to 0.09 J UVB radiation for 5 months to see if neutrophil elastase was an important factor in the development of solar elastosis.

Measurement of urinary desmosine as an indicator of acute pulmonary disease.

A modified radioimmunoassay (RIA) for desmosine in the urine was investigated as a tool for the rapid estimation of lung elastin catabolism. Cystic fibrosis (CF) and oxygen toxicity were chosen as conditions that might show altered elastin destruction. Using an antibody bound to magnetic particles the RIA was adapted to handle large numbers of samples requiring only 50 microliters or urine.

Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity.

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF.

PCR amplification and restriction endonuclease analysis of a 65-kilodalton heat shock protein gene sequence for taxonomic separation of rapidly growing mycobacteria.

A total of 129 reference and clinical strains of rapidly growing mycobacteria (RGM) belonging to 10 taxonomic groups were studied for restriction fragment length polymorphism patterns from a PCR-amplified 439-bp segment of the 65-kDa heat shock protein (HSP) gene. Of 24 endonucleases evaluated, restriction fragment length polymorphism patterns produced by HaeIII and BstEII and then by AciI and CfoI gave the best separation. Sixty percent of all RGM taxa studied were differentiated by HaeIII digests alone.

De novo sulfation of L-tyrosine in HepG2 human hepatoma cells and its possible functional implication.

HepG2 human hepatoma cells, labeled with [35S]sulfate in the presence of 10-30 micrograms/ml of cycloheximide, released up to 64% of the amount of free tyrosine-O-[35S]sulfate produced and released by cells labeled in the absence of cycloheximide. A time-course study revealed that, in cells incubated in medium containing [3H]tyrosine, free [3H]tyrosine-O-sulfate was produced within 5 min of incubation, whereas no [3H]tyrosine-sulfated proteins were detected until 20 min after the incubation had begun. Using 3'-phosphoadenosine, 5'-phospho[35S]sulfate as the sulfate donor, HepG2 cell homogenate was shown to contain enzymic activity catalyzing the sulfation of L-tyrosine with the formation of tyrosine-O-[35S]sulfate.

Differing effects of airway obstruction on physical work capacity and ventilation in men and women with COPD.

Background: Although the natural history of COPD is thought to be well known, studies assessing differences in the onset and course of the disease by gender are surprisingly lacking. This study is a cross-sectional analysis using progressive cycle ergometry exercise testing to assess male and female patients at specific levels of airway obstruction to see if they differ in their exercise capacity and decline in functional capacity.

Methods: The study group included 417 patients with COPD, 55 to 85 years of age, who were compared with 29 controls of similar age; all patients had COPD (FEV1/FVC < 75% predicted) without restrictive disease.

A case of recurrent typhoid fever in the United States: importance of the grandmother connection and the use of large restriction fragment pattern analysis of genomic DNA for strain comparison.

An 8-year old girl was infected for a second time with Salmonella typhi by contact with her grandmother, a known typhoid carrier. The S. typhi from both patient and grandmother had closely related genomic pulsed field gel electrophoresis patterns that differed from epidemiologically unrelated strains.

Identifying disagreement in ordinal ratings within a fixed panel of cytotechnologists.

In many clinical settings, a fixed number of raters screen specimens with more than two ordinal outcomes (for example, mild, moderate, severe). A design is proposed that facilitates the measurement of both interrater and intrarater agreement and associated trends. Design deficiencies are discussed, as are the propriety and interpretation of some common indices of reliability and reproducibility.

Recent changes in taxonomy and disease manifestations of the rapidly growing mycobacteria.

Rapidly growing mycobacteria are a complex group of environmental organisms that cause human disease. Eight taxonomic groups of pathogens are recognized of which three, the Mycobacterium chelonae-like organism (MCLO) and the two Mycobacterium fortuitum third biovariant groups, have yet to receive names pending DNA-DNA homology studies. Clinical disease due to all eight taxons usually consists of skin or soft tissue infections following trauma.

Back-propagation and counter-propagation neural networks for phylogenetic classification of ribosomal RNA sequences.

A neural network system has been developed for rapid and accurate classification of ribosomal RNA sequences according to phylogenetic relationship. The molecular sequences are encoded into neural input vectors using an n-gram hashing method. A SVD (singular value decomposition) method is used to compress and reduce the size of long and sparse n-gram input vectors.

Evidence for conformational dynamics and molecular aggregation in cytochrome P450 102 (BM-3).

The native molecular weight of affinity-purified cytochrome P450 102 from barbiturate-induced Bacillus megaterium has been studied by sedimentation methods and HPLC size-exclusion chromatography. Sedimentation velocity experiments yielded an s020,w = 9.244 S for the holocytochrome, but the diffusion coefficient was unexpectedly large and varied widely with centrifugal field, ionic strength, and protein concentration.

Prothrombin Padua I: incomplete activation due to an amino acid substitution at a factor Xa cleavage site.

An individual and an affected brother previously identified as having the variant prothrombin Padua I were studied in order to identify underlying genetic defects. A heterozygous mutation in the prothrombin gene exon 8 was identified as substitution of A for G at nucleotide position 7,312 (Arg271 (CGT) to His (CAT)). An abolished RsaI restriction site was used to confirm heterozygosity for the defect.

On the domain structure of cytochrome P450 102 (BM-3): isolation and properties of a 45-kDa FAD/NADP domain.

Cytochrome P450 102 is a catalytically self-sufficient monooxygenase isolated from barbiturate-induced Bacillus megaterium. The enzyme contains FAD, FMN, and heme in a single polypeptide chain of 1048 residues, and each of the cofactors is believed to be located in a separate domain. In the present study we have used exhaustive endogenous proteolysis to produce a 45 kDa fragment of the cytochrome.

Detergent effects in rabbit liver microsomal UDP-glucuronosyltransferase studied by means of a continuous spectrophotometric assay with p-nitrophenol.

UDP-glucuronosyltransferase (UDPGT) has been studied with a continuous spectrophotometric assay employing UDP-glucuronic acid and p-nitrophenol as substrates. Activity is linearly dependent on the microsomal protein concentration. Male rabbit liver phenobarbital-induced microsomes exhibited a rate of 7.

Affinity isolation and characterization of cytochrome P450 102 (BM-3) from barbiturate-induced Bacillus megaterium.

Cytochrome P450 102 (BM-3) is a catalytically self-sufficient enzyme from Bacillus megaterium that is presently accepted as an important model of the mammalian microsomal P450 monooxygenase system. We have developed a novel affinity approach to purify P450 102 in a single chromatographic step and have studied the spectroscopic, catalytic, nucleotide binding, and crystallization properties of the highly purified enzyme. B.

Biological and kinetic characterization of recombinant human macrophage inflammatory peptides 2 alpha and beta and comparison with the neutrophil activating peptide 2 and interleukin 8.

We examined the biological and kinetic characteristics of two new members of the intercrine family of cytokines. Human macrophage inflammatory peptides 2 alpha and beta (huMIP-2 alpha and beta) were compared to human interleukin 8 (huIL-8), neutrophil activating peptide 2 (huNAP-2), and N-formyl-L-methionyl-L-phenylalanine (fMLP). The huMIP-2 peptides were the least potent cytokine tested in triggering neutrophil degranulation.

Changing strategies for treatment of chronic bronchitis.

There has been a recent re-examination of the therapeutic approach to chronic bronchitis, with or without airflow obstruction, partly as a result of intense interest in asthma therapy. Although the mainstay of therapy in chronic bronchitis is still smoking cessation, there has been a shift in importance and use of therapeutic interventions based on pathophysiological considerations. The presence of airway inflammation and bronchial hyperreactivity in chronic bronchitis, analogous to asthma, has spurred interest in the use of anti-inflammatory agents such as inhaled steroids, with the hope that these drugs will have the same favorable effects on airflow obstruction as in asthma.

Bronchiectasis.

Bronchiectasis, a condition characterized by abnormal and permanent dilatation of pulmonary airways, is an old disease, well recognized in the era before widespread immunization for childhood diseases and the development of antibiotics. After that time, interest in this disease waned. The recognition of bronchiectasis in association with congenital diseases awakened interest in the disease.

Chronic bronchitis in children.

Chronic bronchitis in childhood is a diverse group of illnesses with pathophysiology differing markedly from the adult condition. Airway disease in children is very common and results in significant and increasing morbidity and mortality. Chronic abnormalities in the bronchioles secondary to infection and morbidity of the bronchi secondary to neonatal problems are unique to children.

Identification and characterization of a major bovine serum tyrosine-O-sulfate-binding protein as a complement factor H.

A major tyrosine-O-sulfate (TyrS)-binding protein present in bovine serum was purified to electrophoretic homogeneity using a combination of TyrS-Affi-Gel 10 affinity chromatography, DEAE-Bio-Gel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified TyrS-binding protein migrated as doublet protein bands with apparent molecular weights of ca. 160,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions.