Hypofibrinogenemia due to novel 316 Asp --> Tyr substitution in the fibrinogen Bbeta chain.

We investigated the molecular basis of hypofibrinogenemia in a woman with a plasma fibrinogen of 1.0 mg/mL. After sequencing the coding region and intronic boundaries of all three fibrinogen genes a single heterozygous GAC-->TAC mutation was identified at codon 316 of the Bbeta gene. This Asp-->Tyr substitution segregated with the hypofibrinogenemia in the only other affected family member. Examination by SDS-PAGE, isoelectric focussing, reverse phase chromatography and electrospray ionisation (ESI) mass spectrometery, failed to detect expression of the new Bbeta chain in purified plasma fibrinogen. The absence of the variant chain was confirmed by ESI tryptic mapping; while the [M + 1 H] and [M + 2 H] ions of the affected peptide (MGPTELLIEMEDWK) were clearly visible at 1,692 and 847 m/z, there were no new signals (1,741 or 871 m/z) that would at indicate expression of the variant in plasma. Asp 316 and its gamma chain homologue (Asp 252) are conserved in all known species and this is the first report of a mutation at either of these. The residue appears to be critical in maintaining the structure of the five stranded sheet that forms the dominant structural feature of the D domains.

Gamma371 Thr-->Ile substitution in the fibrinogen gammaD domain causes hypofibrinogenaemia.

Six members of a family with hypofibrinogenaemia had fibrinogen concentrations ranging from 0.5 to 1.1 mg/ml and, after sequencing the entire coding region and the intron exon boundaries of all three fibrinogen genes, a single heterozygous ACT-->ATT mutation was identified in the gamma gene. This novel mutation was not detected in normal family members or unrelated controls. The gamma371 Thr-->Ile substitution occurs at a conserved threonine in the gammaD domain, but molecules containing the new isoleucine were not present in circulating fibrinogen. The evidence for this was that purified gamma chains had a normal mass of 48375 Da compared to a control of 48374 Da, and tryptic peptide maps were entirely normal. The mutation predicts a mass increase of 12 Da in peptide T-36, but on mass mapping only the normal [M+2H] ion was detected, at 948 m/z. There was no new signal at 954 m/z that would indicate expression of variant chains. Also the normal 948 m/z signal was at the same intensity in digests from the proposita and controls. Crystal structures show a hydrogen bond from the threonine hydroxyl to the main chain and this case suggests this bond is critical in maintaining the structure of the gammaD domain.

High sensitivity and specificity of a laboratory-based serological test, pylori DTect ELISA, for detection of Helicobacter pylori infection.

A number of commercial ELISA kits are now available for detection of Helicobacter pylori infection. Generally, whereas the manufacturers have claimed high sensitivity and specificity, independent studies have often failed to confirm the results. The aim of this study was to independently evaluate the pylori DTect ELISA, a commercial kit for detection of H. pylori infection, in Australian patients with dyspepsia and reflux symptoms. Two hundred and nine consecutive patients (102 males and 107 females, mean age 52.8 years) who were referred for endoscopy due to upper gastrointestinal symptoms, but had not received anti-H. pylori therapy were enrolled. A 10 mL blood sample was obtained from each subject and used to evaluate the kit. The absorbance index (AI) was calculated from the mean of two readings of optical density (OD) of each serum sample. Eight biopsies from the gastric antrum (x3), body (x2), fundus (x2), and incisura (x1) were obtained from each patient for CLO-testing (x1), culture (x3), and histological examination (x4) for H. pylori. Overall, 84 (40.2%) patients were infected with H. pylori as determined by the biopsy-based "gold standard." The AIs ranged from 0 to 1.86; 0.12 to 1.86 in H. pylori positive patients and 0 to 1.49 in negative patients. The pylori DTect ELISA obtained an accuracy of 94 to 95% under AI ranges between 0.20 to 0.40, with the highest accuracy being 95% under AIs of 0.25 and 0.35. An AI of 0.25 was recommended as the best cut-off AI, with a sensitivity of 96.4%, specificity of 93.6%, positive predictive value of 91% and negative predictive value of 97.5%. It is concluded that the pylori DTect ELISA is accurate for detecting H. pylori infection in patients with dyspepsia and reflux symptoms in Australia, when an AI of 0.25 is taken as the cut-off value.

Defective fibrinogen polymerization associated with a novel gamma279Ala-->Asp mutation.

A woman with menorrhagia was investigated for a suspected fibrinogen mutation when coagulation tests revealed prolonged thrombin (55 s) and reptilase (43 s) times together with a functional and an antigenic fibrinogen concentration of 0.7 and 2.8 mg/ml respectively. Heterozygosity for a gamma-chain mutation was suggested by a doublet gamma band on SDS-PAGE and an increased negative charge was observed on isoelectric focusing of HPLC-isolated gamma-chains. Electrospray ionization mass spectrometry revealed a gamma-chain mass of 48 411 Da, which was 20 Da more than the control value of 48 391 Da. Because the normal and variant gamma-chains were not resolved, this implied a 40-Da increase in 50% of the gamma-molecules. An increased negative charge and a 44-Da increase in mass was verified when DNA sequencing showed heterozygosity for an Ala (GCC)-->Asp (GAC) substitution at codon 279 of the gamma-gene. Fibrin polymerization curves indicated a delay in the onset, and a decrease in the rate, of polymerization. Examination of crystal structures showed that the adjacent Tyr-gamma280 side chain is involved in bonding across the D-D interface, and from the proximity of the gamma279Ala-->Asp mutation it would appear that this perturbs the end-to-end DD interactions between fibrin units of the growing polymer.

Antral-type mucosa in the gastric incisura, body, and fundus (antralization): a link between Helicobacter pylori infection and intestinal metaplasia?

OBJECTIVES: Helicobacter pylori is a carcinogen; gastric carcinoma involves a multistep process from chronic gastritis to atrophy, intestinal metaplasia, and dysplasia. The aims of this study were to determine the types of mucosa at different gastric sites in H. pylori-infected and uninfected patients, and whether the presence of antral-type mucosa in the incisura, body, and fundus is associated with gastric atrophy and intestinal metaplasia. METHODS: Two hundred and sixty-eight patients with dyspepsia were enrolled. Eight biopsies (i.e., antrum x3, body x2, fundus x2, and incisura x1) were obtained. One antral biopsy was used for the CLO-test. Three (each from the antrum, body, and fundus) were cultured. The remaining biopsies were examined histologically according to the updated Sydney System after staining with hematoxylin and eosin and Giemsa. A validated serological test was also applied. RESULTS: Overall, 113 (42%) patients were infected with H. pylori. At the incisura, antral-type mucosa was more prevalent in infected than in uninfected patients (84% vs. 18%; odds ratio [OR] = 23.9, 95% confidence interval [CI] 12.5-45.8; p<0.001). Atrophic gastritis and intestinal metaplasia at the incisura was present in 19.5% and 13.3%, respectively, of infected, and 4.5% and 3.2%, respectively, of uninfected patients (both p<0.01). Moreover, atrophic gastritis at the incisura was associated with the presence of antral-type mucosa at the site (termed antralization); the prevalence of atrophic gastritis was 19.5% (24/123) in the presence of antralization, whereas the rate was 2.1% (3/145) without antralization (OR = 11.4, 95% CI 3.4-39.2; p<0.001). Similarly, at the incisura, 16.3% (20/123) of "antralized" cases and 1.4% (2/145) of "unantralized" cases had intestinal metaplasia (OR = 13.8, 95% CI, 3.2-60.7; p<0.001). The association between antralization at gastric body and fundus also appeared to be associated with atrophic gastritis and intestinal metaplasia at these sites. CONCLUSIONS: Atrophic gastritis and intestinal metaplasia occurs predominantly at the gastric antrum and incisura with H. pylori infection. Antralization of the gastric incisura is a common event in H. pylori-infected patients, and appears to be associated with an increased risk of atrophic gastritis and intestinal metaplasia.

Nizatidine in combination with amoxycillin and clarithromycin in the treatment of Helicobacter pylori infection.

BACKGROUND: The efficacy of H2-antagonists in combination with antibiotics in curing Helicobacter pylori infection remains controversial, and it is uncertain whether double dose H2-antagonist therapy is superior to standard dose. AIM: To determine the efficacy of two doses of nizatidine in combination with two antibiotics in the treatment of H. pylori. METHODS: A randomized controlled trial was conducted in 160 patients comparing nizatidine 150 mg with 300 mg b.d. (standard vs. double dose), in combination with clarithromycin (500 mg) and amoxycillin (1000 mg) b.d. for 14 days, in Australia and Taiwan. Compliance was based on a clinical assessment and pill count. H. pylori status was determined by histology (antrum and corpus) and CLO-test. RESULTS: Baseline clinical and endoscopic findings were similar in both arms of the study. Based on an intention-to-treat analysis, cure of H. pylori was achieved in 78% (95% CI: 70.4-85.4%) in the standard nizatidine dose arm and 70% (95% CI: 61.6-78.2%) in the double dose arm (P=0.2). Similar cure rates were observed in ulcer and non-ulcer patient groups. Compliance was excellent in the single and double dose arms (85 and 91%, respectively). CONCLUSIONS: The combination of nizatidine in standard or double dose with clarithromycin and amoxycillin is similarly efficacious in curing H. pylori infection.

Lipomatous infiltration of the canine salivary gland.

Benign connective tumours of the canine salivary glands are rare. This report describes lipomatous infiltration of parotid or submandibular salivary glands in seven dogs in which the glands were enlarged as a result of infiltration by fat cells; they appeared to have been successfully treated by local excision. The precise cause of the lipomatous infiltration in the dogs is unclear but different causes of similar lesions in humans are discussed.

The development of a novel strategy for the microbial treatment of acrylonitrile effluents.

Effluent from the manufacture of acrylonitrile is difficult to biodegrade. It contains nine major organic components: acetic acid, acrylonitrile, acrylamide, acrylic acid, acrolein, cyanopyridine, fumaronitrile, succinonitrile, and maleimide. A range of bacteria have been isolated that can grow on, or convert all of the organic components of effluent from the manufacture of acrylonitrile. These bacteria can be used as the basis of a mixed culture system to treat the effluent. The bacteria were utilised in batch and continuous cultures to degrade a synthetic wastewater containing acrylonitrile, acrylamide, acrylic acid, cyanopyridine and succinonitrile. The mixed microbial population was adapted by varying the growth rate and switching from continuous to batch and back to continuous growth, to degrade these five compounds as well as acrolein, fumaronitrile and maleimide.

Feline spongiform encephalopathy: fibril and PrP studies.

The brains from 18 cats were examined for the presence of the fibrils and modified PrP protein which are molecular diagnostic markers for scrapie-like diseases. Thirteen cats were referred with clinical neurological signs potentially indicative of feline spongiform encephalopathy (FSE). Of these, five had histopathological changes of FSE, five had other lesions of the central nervous system, and in three the brain was normal. The remaining five cats had no clinical neurological signs and were selected as controls. Fibrils and modified PrP protein were found in the brains of the five cats with FSE and in one of the cats with neurological signs but no histopathological changes in the central nervous system. Fibrils were present in the absence of modified PrP in the brains of two cats, one with neurological signs and a histologically confirmed meningioma, and one with no neurological signs and a histologically normal brain.

Naturally occurring scrapie-like spongiform encephalopathy in five domestic cats.

Naturally occurring transmissible spongiform encephalopathies have been recognised in sheep, man, mink, captive deer and cattle. Recently a similar disease was reported in a domestic cat. This paper describes the clinical and pathological findings in five cats with similar signs, including further observations on the original case. All the cats had a progressive, neurological disease involving locomotor disturbances, abnormal behaviour and, in most cases, altered sensory responses. Histopathological examination of the central nervous system revealed changes pathognomonic of the scrapie-like encephalopathies, including widespread vacuolation of the grey matter neuropil, vacuolation of neuronal perikarya and an astrocytic reaction.

Biotechnological treatment of industrial waste water.

The production of toxic or recalcitrant waste effluents by the chemical industry is leading to major problems of their disposal. New biotechnological approaches are now being used which will enable biological treatment of these wastes and will, in future, replace existing methods of effluent treatment.

The industrial potential of microbial nitrile biochemistry.

Nitrile compounds are manufactured widely by man as well as in Nature. The industrial potential of using the microbial metabolism of nitriles and related compounds for the manufacture of a variety of chemicals has started to be realised. The microbial metabolism of nitrile-using microorganisms can also be harnessed to biodegrade toxic waste effluent streams that contain nitriles and are formed during the chemical synthesis of nitrile compounds.

Genetics of lipid deposition in the Japanese quail.

The research presented here was designed to investigate the mode of inheritance of fat and lean tissue deposition, and the relationship between them and body weight in Japanese quail. Heterotic effects were found for weight, size, and number of adipocytes in the abdominal fat depots, weight of the sartorial fat depot and percentage carcass fat with means for the hybrids being lower than those for the parental lines. General inferences concerning the importance of nonadditive genetic variation for lean and body weight were precluded due to inconsistencies observed among mating combinations. Thus, although heterosis and recombination effects were general for characteristics associated with fat deposition, the situation for body weight and lean was unique to the populations involved. It may be hypothesized that heterosis in the efficiency of feed utilization is reflected by the heterosis for fat deposition which explains why hybrids utilize feed better than their parental lines.

Relation of R factor and chromosomal beta-lactamase with the periplasmic space.

The release of several R factor and chromosomal beta-lactamases by osmotic shock treatment was studied. It was found that those beta-lactamases with a molecular weight of about 20,000 were released, but those with a molecular weight of about 30,000 to 44,000 were not released during osmotic shock. This differential release did not depend on whether the structural genes were on the chromosome or on the genome of an R factor. The release or retention of the beta-lactamases appeared to be a characteristic of the enzyme rather than the host cell since the same results were obtained when the R factors were harbored by a variety of host bacteria. Studies with bacteria which produced more than one beta-lactamase showed that each enzyme reacted independently to the presence of other beta-lactamases produced by the host bacterium.