Functional analysis of the cag pathogenicity island in Helicobacter pylori isolates from patients with gastritis, peptic ulcer, and gastric cancer

Helicobacter pylori is the causative agent of a variety of gastric diseases, but the clinical relevance of bacterial virulence factors is still controversial. Virulent strains carrying the cag pathogenicity island (cagPAI) are thought to be key players in disease development. Here, we have compared cagPAI-dependent in vitro responses in H. pylori isolates obtained from 75 patients with gastritis, peptic ulcer, and gastric cancer (n = 25 in each group). AGS gastric epithelial cells were infected with each strain and assayed for (i) CagA expression, (ii) translocation and tyrosine phosphorylation of CagA, (iii) c-Src inactivation, (iv) cortactin dephosphorylation, (v) induction of actin cytoskeletal rearrangements associated with cell elongation, (vi) induction of cellular motility, and (vii) secretion of interleukin-8. Interestingly, we found high but similar prevalences of all of these cagPAI-dependent host cell responses (ranging from 56 to 80%) among the various groups of patients. This study revealed CagA proteins with unique features, CagA subspecies of various sizes, and new functional properties for the phenotypic outcomes. We further showed that induction of AGS cell motility and elongation are two independent processes. Our data corroborate epidemiological studies, which indicate a significant association of cagPAI presence and functionality with histopathological findings in gastritis, peptic ulcer, and gastric cancer patients, thus emphasizing the importance of the cagPAI for the pathogenicity of H. pylori. Nevertheless, we found no significant association of the specific H. pylori-induced responses with any particular patient group. This may indicate that the determination of disease development is highly complex and involves multiple bacterial and/or host factors.     

Mosaic genes and mosaic chromosomes: intra- and interspecies genomic variation of Streptococcus pneumoniae

Streptococcus pneumoniae remains a major causative agent of serious human diseases. The worldwide increase of antibiotic resistant strains revealed the importance of horizontal gene transfer in this pathogen, a scenario that results in the modulation of the species-specific gene pool. We investigated genomic variation in 20 S. pneumoniae isolates representing major antibiotic-resistant clones and 10 different capsular serotypes. Variation was scored as decreased hybridization signals visualized on a high-density oligonucleotide array representing 1,968 genes of the type 4 reference strain KNR.7/87. Up to 10% of the genes appeared altered between individual isolates and the reference strain; variability within clones was below 2.1%. Ten gene clusters covering 160 kb account for half of the variable genes. Most of them are associated with transposases and are assumed to be part of a flexible gene pool within the bacterial population; other variable loci include mosaic genes encoding antibiotic resistance determinants and gene clusters related to bacteriocin production. Genomic comparison between S. pneumoniae and commensal Streptococcus mitis and Streptococcus oralis strains indicates distinct antigenic profiles and suggests a smooth transition between these species, supporting the validity of the microarray system as an epidemiological and diagnostic tool.     

Molecular mechanism of temperature sensing by the circadian clock of Neurospora crassa

Expression levels and ratios of the long (l) and short (s) isoforms of the Neurospora circadian clock protein FREQUENCY (FRQ) are crucial for temperature compensation of circadian rhythms. We show that the ratio of l-FRQ versus s-FRQ is regulated by thermosensitive splicing of intron 6 of frq, a process removing the translation initiation site of l-FRQ. Thermosensitivity is due to inefficient recognition of nonconsensus splice sites at elevated temperature. The temperature-dependent accumulation of FRQ relative to bulk protein is controlled at the level of translation. The 5'-UTR of frq RNA contains six upstream open reading frames (uORFs) that are in nonconsensus context for translation initiation. Thermosensitive trapping of scanning ribosomes at the uORFs leads to reduced translation of the main ORF and allows adjustment of FRQ levels according to ambient temperature.     

Lipopolysaccharide-binding proteins of Limulus amebocyte lysate.

Limulus amebocyte lysate, obtained from horseshoe crab (Limulus polyphemus) blood cells, contains a coagulation system which is activated by bacterial lipopolysaccharide (LPS). A chromatographic fraction of Limulus lysate, containing the endotoxin-sensitive factor(s) which initiates the coagulation cascade, was studied. We utilized a photoreactive, cleavable, radiolabeled derivative of Salmonella minnesota LPS, LPS-(p-azidosalicylamido)-1,3'-dithiopropionamide (LPS-ASD), to identify LPS-binding proteins. The lysate fraction was incubated with LPS-ASD, and LPS-binding proteins were identified by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. An 82-kDa protein, a major protein component of this fraction from Limulus lysate, was identified as a LPS-binding protein in a majority of lysates. Incubation of whole Limulus lysate with antiserum to this protein resulted in enhanced sensitivity of the lysate to LPS, suggesting that this 82-kDa protein is a negative regulator of coagulation. A minor 50-kDa protein component of lysate also was identified as a LPS-binding protein and is a candidate for the LPS-sensitive coagulation protein in L. polyphemus.     

Psychiatric features of individuals with problematic internet use

BACKGROUND: Problematic internet use has been described in the psychological literature as 'internet addiction' and 'pathological internet use'. However, there are no studies using face-to-face standardized psychiatric evaluations to identify behavioral characteristics, psychiatric comorbidity or family psychiatric history of individuals with this behavior. METHODS: Twenty individuals with problematic internet use were evaluated. Problematic internet use was defined as (1) uncontrollable, (2) markedly distressing, time-consuming or resulting in social, occupational or financial difficulties and (3) not solely present during hypomanic or manic symptoms. Evaluations included a semistructured interview about subjects' internet use, the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders-IV (SCID-IV), family psychiatric history and the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) modified for internet use. RESULTS: All (100%) subjects' problematic internet use met DSM-IV criteria for an impulse control disorder (ICD) not otherwise specified (NOS). All 20 subjects had at least one lifetime DSM-IV Axis I diagnosis in addition to their problematic internet use (mean+/-SD=5.1+/-3.5 diagnoses); 14 (70.0%) had a lifetime diagnosis of bipolar disorder (with 12 having bipolar I disorder). LIMITATIONS: Methodological limitations of this study included its small sample size, evaluation of psychiatric diagnoses by unblinded investigators, and lack of a control group. CONCLUSIONS: Problematic internet use may be associated with subjective distress, functional impairment and Axis I psychiatric disorders.     

Specific characterization of isoelectrofocused immunoglobulins in polyacrylamide gel by reaction with 125 I-labeled protein antigens or antibodies

A method is described for the visualization and characterization of isoelectrofocused antibodies, immunoglobulins in general, and many other proteins in thin plates of polyacrylamide gel. The novel aspect of the method is that the protein bands after electrofocusing are immobilized in the gel by treatment with glutaraldehyde after soaking with 18 % sodium sulfate solution. Such treatment is shown not to affect the binding activity of antibodies. Antibody bands are located by treatment of the plates with ¹²⁵I-labeled specific antigen. Other proteins can be located by ¹²⁵I-labeled specific antibody. The bands thus located are visualized by radioautography. The immobilization of the focused proteins allows the excess radiolabeled reagent macromolecules to be washed out of the plates without disrupting the focused pattern. Under the conditions described, background radioactivity and nonspecific pickup by other serum proteins in the plates are negligible. Examples of the use of the method are given for anti-4-azonaphthalene-1-sulfonate and anti-4-hydroxy-5-iodo-3-nitro-phenacetyl antibodies, using the ¹²⁵I-labeled hapten-specific antigens, and for a mouse IgG myeloma protein, MOPC 195, using ¹²⁵I-labeled goat anti-mouse IgG antibody. Extension of the method would allow differences between proteins to be characterized by appropriate ¹²⁵I-labeled antibodies. Thus immunoglobulins could be characterized by ¹²⁵I-labeled antibodies specific for immunoglobulin markers (e.g. Ig class, L chain type, etc.). The advantages of this method over other methods include the ease and generality of using radioiodine labeled macromolecules – especially antibodies – as compared to the restriction imposed by other methods which require small labeled ligands. The method can be extended to the use of fluorescent-labeled proteins and radiolabeled polysaccharide antigens as locators.     

Risk factors for wheezing in a subtropical environment: role of respiratory viruses and allergen sensitization

BACKGROUND: Risk factors for acute wheezing among children in subtropical areas are largely unknown. OBJECTIVE: To investigate the role of viral infections, allergen sensitization, and exposure to indoor allergens as risk factors for acute wheezing in children 0 to 12 years old. METHODS: One hundred thirty-two children 0 to 12 years of age who sought emergency department care for wheezing and 65 children with no history of wheezing were enrolled in this case-control study. Detection of respiratory syncytial virus antigen, rhinovirus and coronavirus RNA, adenovirus, influenza, and parainfluenza antigens was performed in nasal washes. Total IgE and specific IgE to mites, cockroach, cat, and dog were measured with the CAP system. Major allergens from mites, cockroach, cat, and dog were quantified in dust samples by ELISA. Univariate and multivariate analyses were performed by logistic regression. RESULTS: In children under 2 years of age, infection with respiratory viruses and family history of allergy were independently associated with wheezing (odds ratio, 15.5 and 4.2; P = .0001 and P = .008, respectively). Among children 2 to 12 years old, sensitization to inhalant allergens was the major risk factor for wheezing (odds ratio, 2.7; P = .03). High-level allergen exposure, exposure to tobacco smoke, and lack of breast-feeding showed no association with wheezing. CONCLUSIONS: Some risk factors for wheezing previously identified in temperate climates were present in a subtropical area, including respiratory syncytial virus infection in infants and allergy in children older than 2 years. Rhinovirus was not associated with wheezing and did not appear to be a trigger for asthma exacerbations.     

The use of multistate life-table models for improving population health

We demonstrate how incidence, prevalence, remission, mortality(IPRM) models may be constructed on population life-tables,how the incidence of a condition may be calculated, and howthe consequences of demographic changes and public health interventionsmay be predicted. We illustrate the methodology by applyingit to the epidemiology of diabetes, physical inactivity andobesity in New Zealand.