Formation of stress fibres in human endothelial cells infected with Bartonella bacilliformis is associated with altered morphology, impaired migration and defects in cell morphogenesis

Bartonella bacilliformis , a Gram-negative, flagellated bacterium, infects human erythrocytes (haematic phase) and endothelial cells (tissue phase), resulting in a biphasic disease. In the tissue phase of disease (verruga peruana), infection leads to infection of endothelial cells and a pronounced proliferation of these cells, resulting in characteristic skin eruptions of papules and nodules. We have studied the properties of endothelial cells infected in vitro . Extensive cytoskeletal remodelling of endothelial cells occurred after infection in vitro with B. bacilliformis. The cells became spindle shaped and contained arrays of actin stress fibres orientated parallel to the long axis of the cell. Cell–cell contacts were disrupted , along with the distribution of the plasma membrane marker protein, PECAM-1, which participates in cell–cell junctions. The prominent stress fibres terminated in an increased number of focal contacts, which were studied using immunofluorescent staining for paxillin, a cytoplasmic protein that localizes in the focal adhesions. These morphological changes are consistent with activation of intracellular Rho by B. bacilliformis . Formation of stress fibres and the increased number of focal adhesions could be prevented by preincubation of the endothelial cells with C3 exoenzyme, which inactivates intracellular Rho by ADP ribosylation. Endothelial cell motility was greatly diminished in infected cells and the cells did not respond effectively to a stimulus that would evoke motility. In addition, infection of endothelial cells interfered with their ability to form networks of capillary tubes when suspended within three-dimensional collagen matrices. If the properties of infected endothelial cells in vivo are similar, the infected cells will probably not participate effectively in angiogenesis.     

Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b

The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, A. (2002) J. Biol. Chem. 277, 6822-6829). Asp(1080) had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp(1080) to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp(1080) mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp(1077) had no effect on basal activity or calmodulin activation. We propose that the conserved Asp(1080), even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.     

Decolorization of synthetic textile dyes by lignin peroxidase of Phanerochaete chrysosporium

Neem hull waste (containing a high amount of lignin and other phenolic compounds) was used for lignin peroxidase production byPhanerochaete chrysosporum under solid-state fermentation conditions. Maximum decolorization achieved by partially purified lignin peroxidase was 80% for Porocion Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for Navidol Fast Black MSRL. The effects of different concentrations of veratryl alcohol, hydrogen peroxide, enzyme and dye on the efficiency of decolorization have been investigated. Maximum decolorization efficiency was observed at 0.2 and 0.4 mmol/L hydrogen peroxide, 2.5 mmol/L veratryl alcohol and pH 5.0 after a 1-h reaction, using 50 ppm of dyes and 9.96 mkat/L of enzyme.     

Degradation of predigested distillery effluent by isolated bacterial strains

Batch studies were conducted on degradation of anaerobically digested distillery wastewater by three bacterial strains, viz. Xanthomonas fragariae, Bacillus megaterium and Bacillus cereus in free and immobilized form, isolated from the activated sludge of a distillery wastewater treatment plant. The removal of COD and colour with all the three strains increased with time up to 48 hr and only marginal increase in COD and colour removal efficiency was observed beyond this period up to 72 hr. After this period removal efficiency remained fairly constant up to 120 hr. The maximum COD and colour removal efficiency varied from 66 to 81% and 65 to 75%, respectively for both free and immobilized cells of all the three strains. The strain Bacillus cereus showed the maximum efficiency of COD (81%) and colour (75%) removal out of the three strains. An interrelationship between the percent COD and colour removal was carried out by correlation and regression analysis and was justified by high values of coefficient of correlation (r = 0.99) for all the cases. The first order removal rate kinetics was also applied and rate constants were evaluated for COD and colour removal efficiencies.     

Quantitative determination of oleane derivatives in Terminalia arjuna by high performance thin layer chromatography

A simple, precise and rapid high performance thin layer chromatographic method has been developed for the simultaneous quantitative determination of five oleane derivatives, namely, arjunic acid, arjunolic acid, arjungenin, arjunetin and arjunglucoside I from stem bark extract of Terminalia arjuna. The isolation and separation of these compounds was carried out on 60F254 layers eluted with chloroform:methanol (90:10), and the analytes were visualised through colour development with vanillin in concentrated sulphuric acid:ethanol. Scanning and quantification of the spots at 640 nm showed good recoveries in the range 96.40-101.7%.     

Helicobacter pylori eradication in patients on long-term H2 receptor antagonists. Economic and symptomatic benefits. A large prospective study in primary care

Background. A large proportion of patients in primary care are still being maintained on long‐term acid suppression, without any attempts to identify Helicobacter pylori status and to treat those that test positive. Objectives. To assess the prevalence and economic and symptomatic benefits of H. pylori eradication in patients maintained on long‐term H₂ receptor antagonists (H₂RA) in primary care. Patients and Methods. Patients on long‐term (i.e. 6 months or longer) H₂RA were identified from the computerised records of six practices in north England. Helicobacter pylori status was identified using serology and H. pylori positive patients were then offered standard 7‐day proton pump based triple therapy, followed by a urea breath test (UBT) to confirm H. pylori eradication. Those who had a positive UBT were offered a second line course of H. pylori eradication therapy. Follow up period was 1 year. The main outcome measures were improvement in dyspepsia symptom scores, amount of H₂RA being consumed, and economic benefits after H. pylori eradication. Results. One thousand and seven patients (1.5%) were identified on long‐term H₂RA, of whom 471 (46%) ultimately had their H. pylori serology assessed. Sixty‐three (297) percent of the patients tested had a positive serology for H. pylori, the majority of whom (58%, 172) had prior evidence of peptic ulcer disease. The mean duration of therapy and mean time since endoscopy/barium studies was significantly longer in patients with peptic ulcer disease compared to their counterparts with nonulcer dyspepsia and gastro‐oesophageal reflux disease, p= .0002 and .0001, respectively. After successful H. pylori eradication (which was possible in 84% of the patients), at the end of the 1‐year study period, on an intention to treat basis 62% of the patients could either stop or significantly reduce dosage of their H₂RA. There was also significant reduction in the mean dose of H₂RA being consumed and severity of symptoms at the end of the study period (p < .00001). Conclusion. Almost two‐thirds of patients on long‐term H₂RA in primary care will have a positive serology for H. pylori; the majority of these will have peptic ulcer disease. In over 60% of cases H. pylori eradication led to significant improvement in symptom scores and reduction in dosage of H₂RA being consumed. Cessation or reduction in long‐term H₂RA prescribing is cost effective.     

SOX10 mutations in chronic intestinal pseudo-obstruction suggest a complex physiopathological mechanism

The type IV Waardenburg syndrome (WS4), also referred to as Shah-Waardenburg syndrome or Waardenburg-Hirschsprung disease, is characterised by the association of Waardenburg features (WS, depigmentation and deafness) and the absence of enteric ganglia in the distal part of the intestine (Hirschsprung disease). Mutations in the EDN3, EDNRB, and SOX10 genes have been reported in this syndrome. Recently, a new SOX10 mutation was observed in a girl with a neural crest disorder without evidence of depigmentation, but with severe constipation due to a chronic intestinal pseudo-obstruction and persistence of enteric ganglia. To refine the nosology of WS, we studied patients with typical WS4 (including Hirschsprung disease) or with WS and intestinal pseudo-obstruction. We found three SOX10 mutations, one EDNRB and one EDN3 mutations in patients presenting with the classical form of WS4, and two SOX10 mutations in patients displaying chronic intestinal pseudo-obstruction and WS features. These results show that chronic intestinal pseudo-obstruction may be a manifestation associated with WS, and indicate that aganglionosis is not the only mechanism underlying the intestinal dysfunction of patients with SOX10 mutations.     

The MUR3 gene of Arabidopsis encodes a xyloglucan galactosyltransferase that is evolutionarily related to animal exostosins

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl-(1-->2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.