Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Thein vitro effect of theophylline on 17α, 20ß-dihydroxy-4-pregnen-3-one-induced germinal vesicle breakdown in the catfish,Clarias batrachus

The effects of theophylline (a phosphodiesterase inhibitor) and cAMP on 17α, 20ß-dihydroxy-4-pregnen-3-one-induced germinal vesicle breakdown was investigatedin vitro in catfish (Clarias batrachus) oocytes. Folliculated oocytes incubated with 17α, 20ß-dihydroxy-4-pregnen-3-one at the concentration of 1 μg/ml induced 93.2 ± 2.23% germinal vesicle breakdown. When the oocytes were prestimulated with 17α,20ß-dihydroxy-4-pregnen-3-one for 6 h and then treated with different concentrations of theophylline, there was a significant drop in the frequency of germinal vesicle breakdown at the concentrations 2.0, 1.5 and 1.0 mM. However, theophylline was found to be incapable of inhibiting germinal vesicle breakdown at its lowest concentration (0.5 inM). In the time course study, significant inhibition of germinal vesicle breakdown was recorded when 1 mM theophylline was added up to 30 h of 17α,20ß-dihydroxy-4-pregnen-3-one Stimulation but the inhibitory effect of theophylline gradually (time dependent manner) declined if the stimulatory time of 17α,20ß-dihydroxy-4-pregnen-3-one was increased. A similar inhibition of germinal vesicle breakdown was also recorded with various concentrations of cAMP. Except 0.5 mM, all the higher concentrations of cAMP significantly inhibited 17α,20ß-dihydroxy-4-pregnen-3-one induced germinal vesicle breakdown.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

A 12-Crown-4 Ether Containing Dipeptide Boc-12-Crown-4-<Emphasis Type="SmallCaps">l</Emphasis>-DOPA-Gly-OMe Induces Cell Cycle Arrest and Apoptosis in Rat Eggs Cultured In Vitro

The study was designed to investigate whether crown ether containing dipeptide Boc-12-crown-4-l-DOPA-Gly-OMe has potential to induce meiotic cell cycle arrest and apoptosis in rat eggs cultured in vitro. The immature female rats were subjected to superovulation induction protocol and ovulated eggs were collected from ampulla of the fallopian tube. Ovulated eggs arrested at metaphase-II (M-II) stage of meiotic cell cycle were cultured in media-199 with or without various concentrations (0.0, 0.025, 0.050, 0.10, and 0.20 mM) of dipeptide for 3 h in vitro. Morphological apoptotic changes, hydrogen peroxide (H₂O₂) concentration, cytochrome c level, caspase-3 level as well as activity and DNA fragmentation were analysed in eggs cultured in vitro. Culture of M-II arrested eggs in plain medium for 3 h in vitro induced meiotic exit from M-II arrest in majority of eggs as evidenced by initiation of extrusion of second polar body (II PB). The dipeptide induced maintenance of M-II arrest and morphological apoptotic features in a concentration-dependent manner prior to degeneration. The dipeptide-induced morphological features were associated with increased H₂O₂ and cytochrome c levels in treated eggs. The increased cytochrome c induced caspase-3 level and activity and thereby DNA fragmentation as evidenced by DAB positive staining in treated eggs. Our results suggest that dipeptide Boc-12-C-4-l-DOPA-Gly-OMe induces cell cycle arrest at M-II stage and apoptosis in rat eggs cultured in vitro.     

Reduction of nitric oxide level leads to spontaneous resumption of meiosis in diplotene-arrested rat oocytes cultured in?vitro

The present study was aimed to investigate whether a decrease of nitric oxide (NO) level is beneficial for sponateous resumptiom of meiosis in diplotene-arrested oocytes cultured in vitro. For this purpose, diplotene-arrested oocytes were collected from ovary of immature female rats after a single subcutaneous injection of 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48 h. In vitro effects of S-nitroso-l-acetyl penicillamine (SNAP; an NO donor) and aminoguanidine (AG; an inducible NOS [iNOS] inhibitor), intracellular NO, cyclic guanosine monophosphate (cGMP), Cdc25B, Thr-14/Tyr-15 and Thr-161 phosphorylated cyclin-dependent kinase-1 (CDK1), and cyclin B1 levels were analyzed. The SNAP inhibited spontaneous meiotic resumption form diplotene arrest in a concentration-dependent manner, while AG-induced meiotic resumption form diplotene in 0.1 mmol/L 3-isobutyl-1-methylxanthine (IBMX)-treated oocytes in a concentration-dependent manner. The intracellular NO as well as cGMP levels were decreased significantly during spontaneous meiotic resumption from diplotene arrest. The reduction of Cdc25B expression level was associated with the accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. However, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels were reduced significantly during meiotic resumption from diplotene arrest. Taken together, these data suggest that the inhibition of iNOS expression leads to a decrease of NO and cGMP levels thereby decreasing Cdc25B level. The reduced CDC25 B level leads to accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. As a result, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels are decreased leading to maturation-promoting factor (MPF) inactivation. The inactive MPF finally induced meiotic resumption from diplotene stage in rat oocytes cultured in vitro.     

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson''s disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser⁶⁵) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser¹¹¹) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser¹¹¹ of each of the Rabs, we demonstrate that Rab Ser¹¹¹ phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser¹¹¹ phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser¹¹¹ phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser⁶⁵. We further show mechanistically that phosphorylation at Ser¹¹¹ significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser¹¹¹ may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson''s disease.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.