cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors

cagA, a gene that codes for an immunodominant antigen, is present only in Helicobacter pylori strains that are associated with severe forms of gastroduodenal disease (type I strains). We found that the genetic locus that contains cagA (cag) is part of a 40-kb DNA insertion that likely was acquired horizontally and integrated into the chromosomal glutamate racemase gene. This pathogenicity island is flanked by direct repeats of 31 bp. In some strains, cag is split into a right segment (cagI) and a left segment (cagII) by a novel insertion sequence (IS605). In a minority of H. pylori strains, cagI and cagII are separated by an intervening chromosomal sequence. Nucleotide sequencing of the 23,508 base pairs that form the cagI region and the extreme 3′ end of the cagII region reveals the presence of 19 ORFs that code for proteins predicted to be mostly membrane associated with one gene (cagE), which is similar to the toxin-secretion gene of Bordetella pertussis, ptlC, and the transport systems required for plasmid transfer, including the virB4 gene of Agrobacterium tumefaciens. Transposon inactivation of several of the cagI genes abolishes induction of IL-8 expression in gastric epithelial cell lines. Thus, we believe the cag region may encode a novel H. pylori secretion system for the export of virulence determinants.

γ-Aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes

γ-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of γ-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K+ (Kir3) conductance. These GABA type B (GABAB)-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABAB agonist baclofen. Recent studies of native GABAB receptors (GABABRs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABABRs assemble heteromeric complexes from the GABABR1 and GABABR2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABAB agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K+ current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABAB antagonists, and prevented by pertussis toxin pretreatment. Both GABABR1 and GABABR2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABABRs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

β subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line

Human epithelial kidney cells (HEK) were prepared to coexpress α1A, α2δ with different β calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney α1A-transfected cells showed small inactivating currents in 80 mM Ba2+ that were relatively insensitive to calcium blockers. Coexpression of α1A, βIb, and α2δ produced a robust inactivating current detected in 10 mM Ba2+, reversibly blockable with low concentration of ω-agatoxin IVA (ω-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by α1A, β2a, α2δ subunits, which demonstrated the slowest inactivation and were relatively insensitive to ω-Aga IVA and sFTX. Coexpression of β3 with the same combination as above produced inactivating currents also insensitive to low concentration of ω-Aga IVA and sFTX. These data indicate that the combination α1A, βIb, α2δ best resembles P-type channels given the rate of inactivation and the high sensitivity to ω-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the β subunit associated with the α1A subunit.

Direct Evidence for Rapid Degradation of Bacillus thuringiensis Toxin mRNA as a Cause of Poor Expression in Plants1

It is well established that the expression of Bacillus thuringiensis (B.t.) toxin genes in higher plants is severely limited at the mRNA level, but the cause remains controversial. Elucidating whether mRNA accumulation is limited transcriptionally or posttranscriptionally could contribute to effective gene design as well as provide insights about endogenous plant gene-expression mechanisms. To resolve this controversy, we compared the expression of an A/U-rich wild-type cryIA(c) gene and a G/C-rich synthetic cryIA(c) B.t.-toxin gene under the control of identical 5′ and 3′ flanking sequences. Transcriptional activities of the genes were equal as determined by nuclear run-on transcription assays. In contrast, mRNA half-life measurements demonstrated directly that the wild-type transcript was markedly less stable than that encoded by the synthetic gene. Sequences that limit mRNA accumulation were located at more than one site within the coding region, and some appeared to be recognized in Arabidopsis but not in tobacco (Nicotiana tabacum). These results support previous observations that some A/U-rich sequences can contribute to mRNA instability in plants. Our studies further indicate that some of these sequences may be differentially recognized in tobacco cells and Arabidopsis.

Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit.

Oral administration of autoantigens can prevent and partially suppress autoimmune diseases in a number of experimental models, Depending on the dose of antigen fed, this approach appears to involve distinct yet reversible and short-lasting mechanisms (anergy/deletion and suppression) and usually requires repeated feeding of large (suppression) to massive (anergy/deletion) amounts of autoantigens to be effective. Most importantly, this approach is relatively less effective in animals already systemically sensitized to the fed antigen, such as in animals already harboring autoreactive T cells and, thus, presumably also in humans suffering from an autoimmune disorder. We have previously shown that feeding a single dose of minute amounts of antigens conjugated to cholera toxin B subunit (CTB) can effectively suppress delayed-type hypersensitivity reactions in systemically immune animals. We now report that feeding small amounts of myelin basic protein (MBP) conjugated to CTB either before or after disease induction protected rats from experimental autoimmune encephalomyelitis. Such treatment was as effective in suppressing interleukin 2 production and proliferative responses of lymph node cells to MBP as treatment involving repeated feeding with much larger (50- to 100-fold) doses of free MBP. Different from the latter treatment, which led to decreased production of interferon-gamma in lymph nodes, low-dose oral CTB-MBP treatment was associated with increased interferon-gamma production. Most importantly, low-dose oral CTB-MBP treatment greatly reduced the level of leukocyte infiltration into spinal cord tissue compared with treatment with repeated feeding of large doses of MBP. These results suggest that the protection from experimental autoimmune encephalomyelitis achieved by feeding CTB-conjugated myelin autoantigen involves immunomodulating mechanisms that are distinct from those implicated by conventional protocols of oral tolerance induction.

A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein.

A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels.

Differential involvement of N-type calcium channels in transmitter release from vasoconstrictor and vasodilator neurons

1 The effects of calcium channel blockers on co-transmission from different populations of autonomic vasomotor neurons were studied on isolated segments of uterine artery and vena cava from guinea-pigs. 2 Sympathetic, noradrenergic contractions of the uterine artery (produced by 200 pulses at 1 or 10 Hz; 600 pulses at 20 Hz) were abolished by the N-type calcium channel blocker omega-conotoxin (CTX) GVIA at 1-10 nM. 3 Biphasic sympathetic contractions of the vena cava (600 pulses at 20 Hz) mediated by noradrenaline and neuropeptide Y were abolished by 10 nM CTX GVIA. 4 Neurogenic relaxations of the uterine artery (200 pulses at 10 Hz) mediated by neuronal nitric oxide and neuropeptides were reduced < 50% by CTX GVIA 10-100 nM. 5 Capsaicin (3 muM) did not affect the CTX GVIA-sensitive or CTX GVIA-resistant neurogenic relaxations of the uterine artery. 6 The novel N-type blocker CTX CVID (100-300 nM), P/Q-type blockers agatoxin IVA (10-100 nM) or CTX CVIB (100 nM), the L-type blocker nifedipine (10 muM) or the 'R-type' blocker SNX-482 (100 nM), all failed to reduce CTX GVIA-resistant relaxations. The T-type channel blocker NiCl2 (100-300 muM) reduced but did not abolish the remaining neurogenic dilations. 7 Release of different neurotransmitters from the same autonomic vasomotor axon depends on similar subtypes of calcium channels. N-type channels are responsible for transmitter release from vasoconstrictor neurons innervating a muscular artery and capacitance vein, but only partly mediate release of nitric oxide and neuropeptides from pelvic vasodilator neurons.

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway.

Mucopolysaccharidosis Type Ii: Identification Of 30 Novel Mutations Among Latin American Patients.

In this study, 103 unrelated South-American patients with mucopolysaccharidosis type II (MPS II) were investigated aiming at the identification of iduronate-2-sulfatase (IDS) disease causing mutations and the possibility of some insights on the genotype-phenotype correlation The strategy used for genotyping involved the identification of the previously reported inversion/disruption of the IDS gene by PCR and screening for other mutations by PCR/SSCP. The exons with altered mobility on SSCP were sequenced, as well as all the exons of patients with no SSCP alteration. By using this strategy, we were able to find the pathogenic mutation in all patients. Alterations such as inversion/disruption and partial/total deletions of the IDS gene were found in 20/103 (19%) patients. Small insertions/deletions/indels (<22 bp) and point mutations were identified in 83/103 (88%) patients, including 30 novel mutations; except for a higher frequency of small duplications in relation to small deletions, the frequencies of major and minor alterations found in our sample are in accordance with those described in the literature.

Pyrimidine-5'-nucleotidase Campinas, A New Mutation (p.r56g) In The Nt5c3 Gene Associated With Pyrimidine-5'-nucleotidase Type I Deficiency And Influence Of Gilbert's Syndrome On Clinical Expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Elevated Micronucleus Frequency In Patients With Type 2 Diabetes, Dyslipidemia And Periodontitis.

The over-production of reactive oxygen species (ROS) can cause oxidative damage to a large number of molecules, including DNA, and has been associated with the pathogenesis of several disorders, such as diabetes mellitus (DM), dyslipidemia and periodontitis (PD). We hypothesise that the presence of these diseases could proportionally increase the DNA damage. The aim of this study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 DM, dyslipidemia and PD. One hundred and fifty patients were divided into five groups based upon diabetic, dyslipidemic and periodontal status (Group 1 - poor controlled DM with dyslipidemia and PD; Group 2 - well-controlled DM with dyslipidemia and PD; Group 3 - without DM with dyslipidemia and PD; Group 4 - without DM, without dyslipidemia and with PD; and Group 5 - without DM, dyslipidemia and PD). Blood analyses were carried out for fasting plasma glucose, HbA1c and lipid profile. Periodontal examinations were performed, and venous blood was collected and processed for micronucleus (MN) assay. The frequency of micronuclei was evaluated by cell culture cytokinesis-block MN assay. The general characteristics of each group were described by the mean and standard deviation and the data were submitted to the Mann-Whitney, Kruskal-Wallis, Multiple Logistic Regression and Spearman tests. The Groups 1, 2 and 3 were similarly dyslipidemic presenting increased levels of total cholesterol, low density lipoprotein cholesterol and triglycerides. Periodontal tissue destruction and local inflammation were significantly more severe in diabetics, particularly in Group 1. Frequency of bi-nucleated cells with MN and MNF, as well as nucleoplasmic bridges, were significantly higher for poor controlled diabetics with dyslipidemia and PD in comparison with those systemically healthy, even after adjusting for age, and considering Bonferroni's correction. Elevated frequency of micronuclei was found in patients affected by type 2 diabetes, dyslipidemia and PD. This result suggests that these three pathologies occurring simultaneously promote an additional role to produce DNA impairment. In addition, the micronuclei assay was useful as a biomarker for DNA damage in individuals with chronic degenerative diseases.

Antioxidant And Anti-diabetic Potential Of Passiflora Alata Curtis Aqueous Leaves Extract In Type 1 Diabetes Mellitus (nod-mice).

Leaves of Passiflora alata Curtis were characterized for their antioxidant capacity. Antioxidant analyses of DPPH, FRAP, ABTS, ORAC and phenolic compounds were made in three different extracts: aqueous, methanol/acetone and ethanol. Aqueous extract was found to be the best solvent for recovery of phenolic compounds and antioxidant activity, when compared with methanol/acetone and ethanol. To study the anti-inflammatory properties of this extract in experimental type 1 diabetes, NOD mice were divided into two groups: the P. alata group, treated with aqueous extract of P. alata Curtis, and a non-treated control group, followed by diabetes expression analysis. The consumption of aqueous extract and water ad libitum lasted 28 weeks. The treated-group presented a decrease in diabetes incidence, a low quantity of infiltrative cells in pancreatic islets and increased glutathione in the kidney and liver (p<0.05), when compared with the diabetic and non-diabetic control-groups. In conclusion, our results suggest that the consumption of aqueous extract of P. alata may be considered a good source of natural antioxidants and compounds found in its composition can act as anti-inflammatory agents, helping in the control of diabetes.

Undulatory Physical Resistance Training Program Increases Maximal Strength In Elderly Type 2 Diabetics.

To investigate the effects of a specific protocol of undulatory physical resistance training on maximal strength gains in elderly type 2 diabetics. The study included 48 subjects, aged between 60 and 85 years, of both genders. They were divided into two groups: Untrained Diabetic Elderly (n=19) with those who were not subjected to physical training and Trained Diabetic Elderly (n=29), with those who were subjected to undulatory physical resistance training. The participants were evaluated with several types of resistance training's equipment before and after training protocol, by test of one maximal repetition. The subjects were trained on undulatory resistance three times per week for a period of 16 weeks. The overload used in undulatory resistance training was equivalent to 50% of one maximal repetition and 70% of one maximal repetition, alternating weekly. Statistical analysis revealed significant differences (p<0.05) between pre-test and post-test over a period of 16 weeks. The average gains in strength were 43.20% (knee extension), 65.00% (knee flexion), 27.80% (supine sitting machine), 31.00% (rowing sitting), 43.90% (biceps pulley), and 21.10% (triceps pulley). Undulatory resistance training used with weekly different overloads was effective to provide significant gains in maximum strength in elderly type 2 diabetic individuals.

Brooke-spiegler Syndrome Clinically Misdiagnosed As Neurofibromatosis Type 1.

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