Effects of pituitary adenylate cyclase activating polypeptide on small intestinal INT 407 cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the gastrointestinal tract. It has been shown to exert actions on intestinal functions, mainly affecting glandular secretion and motility. PACAP has several different effects on cell survival depending on the cell type and the applied stimulus. Its influences on small intestinal epithelial cells are not yet elucidated, therefore the aim of the present study was to investigate the effects of PACAP on intestinal epithelial cells having high turnover (INT 407) against different harmful stimuli, such as oxidative stress, in vitro hypoxia and gamma radiation. We tested the effect of PACAP on proliferation and cell survival using MTT assay. Moreover, various cancer-related factors were evaluated by oncology array. PACAP did not influence the proliferation rate of INT 407 cells. Its cell survival-enhancing effect could be detected against oxidative stress, but not against in vitro hypoxia or gamma irradiation. Clonogenic survival assay was performed to analyze the effect of PACAP on clonogenic potential of cells exposed to gamma radiation. Surprisingly, PACAP enhanced the clone-forming ability decrease induced by irradiation. Western blot analysis of ERK1/2 phosphorylation was performed in order to obtain further information on the molecular background. Our data showed phospho-ERK1/2 suppression of PACAP in irradiated cells. Furthermore, the role of endogenous PACAP against oxidative stress was also investigated performing ADCYAP1 small interfering RNA transfection. We found significant difference in the cell vulnerability between cells undergoing silencing and cells without transfection suggesting the protective role of the endogenously present PACAP against oxidative stress in INT 407 cells. In summary, PACAP seems to be able to exert contradictory effects in INT 407 cells depending on the applied stressor, suggesting its regulatory role in the cellular household.

O-GlcNAcylation of STAT5 controls tyrosine phosphorylation and oncogenic transcription in STAT5-dependent malignancies.

The signal transducer and activator of transcription 5 (STAT5) regulates differentiation, survival, proliferation and transformation of hematopoietic cells. Upon cytokine stimulation, STAT5 tyrosine phosphorylation (pYSTAT5) is transient, while in diverse neoplastic cells persistent overexpression and enhanced pYSTAT5 are frequently found. Post-translational modifications might contribute to enhanced STAT5 activation in the context of transformation, but the strength and duration of pYSTAT5 are incompletely understood. We found that O-GlcNAcylation and tyrosine phosphorylation act together to trigger pYSTAT5 levels and oncogenic transcription in neoplastic cells. The expression of a mutated hyperactive gain-of-function (GOF) STAT5 without O-GlcNAcylation resulted in decreased tyrosine phosphorylation, oligomerization and transactivation potential and complete loss of oncogenic transformation capacity. The lack of O-GlcNAcylation diminished phospho-ERK and phospho-AKT levels. Our data show that O-GlcNAcylation of STAT5 is an important process that contributes to oncogenic transcription through enhanced STAT5 tyrosine phosphorylation and oligomerization driving myeloid transformation. O-GlcNAcylation of STAT5 could be required for nutrient sensing and metabolism of cancer cells.

Impact of medications on bacterial growth in syringes.

Syringes used to administer intravenous medications in an intensive care unit were cultured, and the isolates were compared with those from positive blood cultures from the same patients. The overall contamination rate was 16%, and syringes used for drugs such as insulin, which support bacterial growth, had higher contamination rates. All syringes should be changed routinely after 6h.

Erythroid progenitor renewal versus differentiation: genetic evidence for cell autonomous, essential functions of EpoR, Stat5 and the GR.

The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.

Virulence factors of uropathogenic Escherichia coli.

Virulence factors of Escherichia coli are of two main types; those produced on the surface of the cell and those produced within the cell and then exported to the site of action. Those on the surface include different sorts of fimbriae that have a role in adhesion to the surface of host cells but may also have additional roles such as tissue invasion, biofilm formation or cytokine induction. The activities of cell wall components are discussed and several exported virulence factors are described that have anti host cell activities. Others virulence factors enable the bacteria to grow in an environment of iron restriction.

Enhancement of the antiparakeratotic potency of calcitriol and tacalcitol in liposomal preparations in the mouse tail test.

In order to test the advantage of vitamin D(3) preparations in liposomal form, calcitriol, the natural activated form of vitamin D(3), and tacalcitol, a vitamin D(3) analogue, were employed in various concentrations and using different vehicles in the mouse tail test, an animal model for testing the antiparakeratotic efficacy of topical medications. The optimal concentration in petrolatum turned out to be similar to that in commercial preparations. The liposomal preparations were superior to those in petrolatum and to those in nonliposomal phospholipids. The antiparakeratotic potency (drug activity) of liposomal tacalcitol in a concentration of 2 microg/g was twice that of the commercial preparation with a higher concentration of 4 microg/g. These results suggest that the use of liposomal vitamin D(3) preparations can achieve a given antipsoriatic effect with a reduced concentration of the active substance thereby reducing the risk of skin irritation and of hypercalcemia.

Tazarotene induces epidermal cell differentiation in the mouse tail test used as an animal model for psoriasis.

Disturbed epidermal proliferation and keratinization are major features of psoriatic skin lesions. The so-called mouse tail test is known as an animal model to evaluate the antipsoriatic efficacy of topical drugs with regard to the induction of orthokeratosis. The purpose of the present study was to investigate the effect of tazarotene, a novel, receptor-specific retinoid, by the mouse tail test in a direct comparison to dithranol representing a classical topical antipsoriatic compound. The tails of CFLP mice were treated with tazarotene gel (0.1%, 0.05%), dithranol ointment (1.0%), tretinoin cream (0.05%) and methylcellulose (5%) hydrogel (vehicle control) for 2 weeks. Longitudinal histological sections were prepared from the tail skin, and the degree of orthokeratosis was determined by measuring the horizontal length of the fully developed granular layer within an individual scale in relation to its total length according to a method originally described by Bosman and co-authors. The degree of orthokeratosis was significantly (p < or = 0.05) increased by 0.1% tazarotene (87+/-20%), 1.0% dithranol (75+/-26%), 0.05% tazarotene (59+/-27%), and 0.05% tretinoin (23+/-13%) as compared to untreated (11+/-6%) and methylcellulose hydrogel-treated (13+/-6%) controls. Under the conditions of the mouse tail test, tazarotene showed a strong potency to induce orthokeratosis. With regard to clinically relevant concentrations this effect was even more pronounced than that observed for dithranol.

Halothane decreases bacterial adherence in vitro.

BACKGROUND: Adherence of pathogenic bacteria to host epithelial cells is thought to be the initial step in infection, while the presence of the commensal flora is an important host defence mechanism. Anything altering bacterial adherence to human epithelial cells may contribute to bacterial infections. The impact of anaesthesia on this first step to infection is not known. In this study the effect of halothane on bacterial adherence was investigated. METHODS: Human epithelial cells (HEp-2) and two strains of Escherichia coli were exposed to halothane 2% for 2 h. Then HEp-2 cells were coincubated with bacteria for 3 h. Bacteria attached to the epithelial cells were evaluated by light microscopy. RESULTS: Compared to the control, bacterial adherence was reduced by 37% to 56% with the different strains when HEp-2 cells were exposed to halothane. No significant difference was found when only bacteria were treated with halothane. CONCLUSION: Our results show that halothane reduces bacterial adherence to human epithelial cells in vitro. Reduced number or function of epithelial cell surface receptors may be responsible for the reduced adherence as no changes were observed when only the bacteria were exposed to halothane.

Influence of RecA on in vivo virulence and Shiga toxin 2 production in Escherichia coli pathogens.

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains 933 and 86-24 as well as the uropathogenic E. coli (UPEC) strain 536 were compared with their isogenic rec A mutants and rec A trans -complemented strains in intravenous lethality and lung toxicity assays in mice. While the wild-type EHEC strains were fully virulent, the virulence of the rec A mutants was strongly reduced. Complementation of the EHEC rec A mutants with the cloned E. coli recA gene restored their virulence capacity. The stx2EHEC mutant TUV86-2 as well as its isogenic rec A mutant were completely avirulent in both assays. In contrast, RecA had no influence on the virulence of UPEC strain 536. We conclude that the lethality observed with EHEC is presumably mainly due to Shiga toxin, which is severely down-regulated in the rec A mutants as a result of lacking spontaneous phage induction. Therefore, the EHEC rec A+strains 933 and 86-24 were compared for their Shiga toxin 2 (Stx2) production with the respective rec A-counterparts. The rec A mutants of the EHEC strains were significantly reduced in toxin synthesis and were devoid of Stx2 specific phage production. Complementation of the EHEC rec A mutants with the cloned rec A gene enabled the rec A mutants to restore toxin and phage production. These results suggest that the higher level of Stx2 synthesis in the EHEC strains is the result of a higher level of spontaneous Stx2 specific phage induction, which is controlled by RecA.

Diversity among clinical isolates of Proteus penneri detected by random amplified polymorphic DNA analysis.

DNA of thirteen haemolytic Proteus penneri strains of clinical origin, all producing calcium dependent haemolysin and having been derived from four European countries was examined for plasmid profile, and outer membrane protein profile, by random amplified polymorphic DNA-PCR (RAPD-PCR) method, and digestions with restriction endonucleases were performed. All strains contained two large plasmids of approximately 60 and 70 kilobase pairs (kb). In addition, four strains contained a small plasmid of about 6 kb. These four strains produced cell-bound haemolysin only. Outer membrane protein analysis revealed subtle differences between strains. RAPD-PCR with primer I (CCGCAGCCAA) revealed 13 types, whereas primer II (AACGCGCAAC) yielded only two main types of different patterns. Results with primer I suggests a DNA sequence diversity within this species. The RAPD-PCR method provides a fast, economical and reproducible means for the typing of P. penneri. Digestion with restriction endonucleases indicated a high level of DNA methylation in this species.

Genetic diversity in Proteus penneri.

DNA of thirteen Proteus penneri strains derived from four European countries (nine strains from Germany, two strains from United Kingdom, one strain from Turkey, one strain from Hungary) was examined by random amplified polymorphic DNA-PCR (RAPD-PCR) method. RAPD with primer AACGCGCAAC gave different patterns, which suggests a DNA sequence microdiversity within this species. The method provides a fast, economical and reproducible means for typing P. penneri.

Effect of fumaric acid, its dimethylester, and topical antipsoriatic drugs on epidermal differentiation in the mouse tail model.

Fumaric acid, fumaric acid dimethylester, and the dithranol derivative C4-lactone were studied in the mouse tail test to evaluate their effects on epidermal cell differentiation compared with other topical antipsoriatic drugs, such as betamethasone, calcipotriol, and dithranol. Mouse tails were treated for 2 weeks and longitudinal histological sections prepared of the tail skin. The length of the orthokeratotic regions (stratum granulosum) was measured on 10 sequential scales per tail and expressed as percentage of the full length of the scale. In addition, epidermal thickness was measured and the efficacy of the various compounds evaluated. In comparison to 2% salicylic acid ointment, all tested compounds except fumaric acid significantly (p < or = 0.05) increased the proportion of the orthokeratotic region. C4-lactone and calcipotriol were less effective than dithranol, fumaric acid dimethylester only moderately influenced cell differentiation, and betamethasone showed the least potent effect. Dithranol was the most potent substance inducing orthokeratosis without increasing epidermal thickness.

tRNA genes and pathogenicity islands: influence on virulence and metabolic properties of uropathogenic Escherichia coli.

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries two unstable DNA regions on its chromosome which were termed pathogenicity islands (Pais). Both pathogenicity islands, Pai I and Pai II, are incorporated into tRNA specific loci: Pai I is located in the tRNA gene for selenocysteine (selC), and Pai II is integrated in the leucine-specific tRNA locus leuX. Mutant strain 536-21 has lost the two pathogenicity islands together with the intact tRNA genes. While 536 is a virulent strain, 536-21 has lost a number of properties, including in vivo virulence. In previous publications we reported that the genes coding for two haemolysins (hly I, hly II) and P-related fimbria (prf) are located on the Pais. In this paper, we demonstrate that the expression of other gene products influencing metabolic properties in addition to in vivo virulence are strongly dependent on the intact tRNA loci selC and leuX. In order to determine the influence of the two tRNAs on the expression of these properties, the genes selC and leuX were cloned from the genome of strain 536 and then introduced into the mutant 536-21. Our results clearly show that the seleno-cysteine-specific tRNA (tRNA(Sec)) directly influences the ability of the bacteria to grow under anaerobic conditions, because selenocysteine is part of the enzyme formate dehydrogenase (FDH) which is involved in mixed acid fermentation. The rare leucine-specific tRNA5(Leu), encoded by leuX, influences a number if properties including type 1 fimbria production, flagellation and motility, production of enterobactin and serum resistance, and is also necessary for full in vivo virulence. While the tRNA(Sec) is directly involved in the production of FDHs, the leuX specific tRNA5(Leu) appears to influence the expression of various factors through specific transcriptional or translational control mechanisms.

Genetic-epidemiologic study of haemophilia A and B in Hungary.

All known surviving haemophiliacs A and B and their relatives were reexamined by laboratory and clinical methods and evaluated by a genetic-epidemiologic approach in 4 north-western countries of Hungary. The prevalence of haemophilia A and B patients born in the fifties was 2.73 and 0.25 per 10,000 persons, respectively. The reproductive fitness was found to be 0.3 in haemophilia A, and 0.8 in haemophilia B patients. The mutation rates calculated by the indirect method were 6.3 x 10(-5) for haemophilia A and 0.2 x 10(-5) for haemophilia B.

Haematological reactions of rabbits infected intravenously with Listeria strains of different virulence.

Listeria strains of different virulence were injected intravenously into rabbits of both sexes (2-4 kg). The infectious dose was 10(8) cells/kg. Blood samples were taken from the ear wein one day and immediately before the infection, then 3 h, 1, 2, 3, 6 and 10 days after it. Total white blood cell counts and their changes were determined and the number of lysosomal granules in neutrophils were counted. A marked monocytic reaction was observed after the injection of virulent Listeria monocytogenes 18 and 87/5467, partly virulent Listeria ivanovii, non-pathogenic Listeria seeligeri 87/5626 and 87/5575, and Listeria murrayi G44 strains. A late slow growth was provoked by Listeria innocua C644 and a very slight reaction by Listeria welshimeri 1830 strains. There was no change after injection of L. innocua strain 10. The number of lysosomal granules decreased significantly and remained at a low level for 6 days after the infection of L. monocytogenes strain 18 and for 3 days after the injection of L. innocua strain 10.