Aspirin dose dependently inhibits the interleukin-1 beta-stimulated increase in inducible nitric oxide synthase, nitric oxide, and prostaglandin E(2) production in rat ovarian dispersates cultured in vitro.

OBJECTIVE: Determine if aspirin inhibits the IL-1 beta-stimulated expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and prostaglandin E(2) (PGE(2)) in rat ovarian dispersates cultured in vitro. DESIGN: Prospective, controlled in vitro study. SETTING: Academic research laboratory. ANIMALS: Ovaries collected from immature rats. INTERVENTION(S): Ovaries were collected from immature rats and enzymatically dispersed. Ovarian dispersates were placed into plates containing media alone or media supplemented with IL-1 beta (100 U/mL) and varying concentrations of aspirin (0, 1, 3, 5 and 10 mM). Ovarian dispersates were cultured in a humidified environment of 5% CO(2) in air at 37 degrees C for 24 or 48 hours. MAIN OUTCOME MEASURE(S): Twenty-four- and 48-hour iNOS, nitrite (a stable metabolite of NO), and PGE(2) levels were determined from ovarian dispersates cultured in vitro. RESULT(S): Administration of IL-1 beta increased nitrite and PGE(2) levels over that observed in the control group after culture of ovarian dispersates for 24 and 48 hours. Aspirin dose dependently reduced the IL-1 beta-stimulated increase in nitrite production from ovarian dispersates after culture for 24 and 48 hours. Aspirin completely (24 hours) or dose dependently (48 hours) prevented the IL-beta-stimulated increase in PGE(2.) Coadministration of IL-1 beta and aspirin (10 mM) attenuates IL-1 beta-stimulated iNOS expression after culture for 24 and 48 hours. CONCLUSION(S): Aspirin significantly inhibits the IL-1 beta-stimulated expression of iNOS, NO, and PGE(2) in ovarian dispersates cultured in vitro.

Evaluation of two string tests for obtaining gastric juice for culture, nested-PCR detection, and combined single- and double-stranded conformational polymorphism discrimination of Helicobacter pylori.

We have compared two gastric string tests for obtaining gastric juice for culture of Helicobacter pylori and for nested-PCR detection and PCR-based combined single- and double-stranded conformational polymorphism (SDSCP) discrimination of infecting strains. String test specimens were obtained from one seropositive volunteer for 13 consecutive weeks. The distal 10 cm of each string was suspended in 1 ml saline and quantitatively cultured. An additional nine volunteers with histories of upper-gastrointestinal complaints were given a string test for culture and nested-PCR assay. H. pylori isolates and/or gastric juice from each volunteer were extracted for DNA and analyzed by PCR-based SDSCP. Quantitative culture showed that the Entero-test was four times as sensitive as the Gastro-test but was more prone to contamination by oral flora. However, the two string tests are equally sensitive by PCR assays. Thus, the Gastro-test is more suitable for culture detection of H. pylori, since it is less prone to oral contamination and its shorter length is better tolerated. SDSCP analysis of H. pylori DNA from four PCR-positive volunteers without requiring culture showed four distinct profiles, indicating different infecting strains. SDSCP analysis of strains isolated before and after treatment of one volunteer had the same SDSCP profile, suggesting endogenous reinfection by the same strain.

Inhibiting early activation of tissue nuclear factor-kappa B and nuclear factor interleukin 6 with (1-->3)-beta-D-glucan increases long-term survival in polymicrobial sepsis.

BACKGROUND: Recent data implicate the activation of nuclear factor-kappa B (NF-kappa B) and nuclear factor interleukin 6 (NF-IL6) as important steps in the pathophysiologic mechanisms of adult respiratory distress syndrome and systemic inflammatory response syndrome. METHODS: This study evaluated the effect of immunomodulating polysaccharides on transcription factor activation, cytokine expression, and mortality in a murine cecal ligation and puncture (CLP) model. ICR/HSD mice were treated with glucan (50 mg/kg) 1 hour before or 15 minutes after CLP. Liver and lung tissue were harvested at 3 hours and mortality trends were observed for 20 days. RESULTS: CLP increased liver and lung NF-kappa B and NF-IL6 nuclear binding activity as well as tumor necrosis factor-alpha and interleukin 6 messenger RNA levels at 3 hours. Pretreatment or posttreatment with glucans inhibited tissue NF-kappa B and NF-IL6 nuclear binding activity and tissue cytokine messenger RNA levels. Prophylaxis with glucan phosphate or scleroglucan increased (P < .001) long-term survival (20% CLP vs 65% glucan phosphate, 75% scleroglucan). Posttreatment with glucan phosphate also increased (P < .05) long-term survival (20% vs 65%). CONCLUSIONS: Pretreatment or posttreatment with biologic response modifiers decreased tissue transcription factor nuclear binding activity and cytokine message in liver and lung of septic mice. Inhibiting early transcription factor activation and cytokine message expression correlates with improved outcome in polymicrobial sepsis as denoted by increased long-term survival.

Identification of H. pylori in saliva by a nested PCR assay derived from a newly cloned DNA probe.

A novel probe was developed from genomic DNA of Helicobacter pylori ATCC type strain 43629. It hybridized with all 73 H. pylori clinical isolates tested but not with any of 183 non-H. pylori DNAs in dot blot hybridization. Typing tests revealed 41 different HaeIII-digestion patterns from 57 H. pylori strains tested. Based on the sequence of the probe, a nested PCR was developed that detected as little as 2 fg of H. pylori DNA or approximately equivalent to one cell. No PCR products were amplified from any of 21 non-H. pylori strains tested. Using this nested PCR, H. pylori DNA was detected in 33 of 45 (73%) saliva samples collected from patients with gastric H. pylori infection. These data suggest that the probe is useful for typing H. pylori and that the nested PCR is a valuable tool for detecting H. pylori DNA in saliva.

Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture.

Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori. Genomic DNA preparations from these gastric biopsy specimens and the corresponding H. pylori isolates were tested by our PCR-based RFLP assay. The 1,179-bp H. pylori DNA fragments amplified by the PCR assay were digested with the restriction enzymes HhaI, MboI, and AluI and analyzed by agarose gel electrophoresis. HhaI, MboI, and AluI digestion produced 11, 10, and 6 distinguishable digestion patterns, respectively, from the 19 H. pylori isolates tested and generated 13, 11, and 6 different patterns, respectively, from the 19 gastric biopsy specimens. The patterns from 13 of the 19 gastric biopsy specimens matched those of the H. pylori isolates from the corresponding patients. The patterns from the remaining six biopsy specimens appeared to represent infection by two strains of H. pylori; the pattern of one strain was identical to that of the isolate from the corresponding patient. By combining all the restriction enzyme digestion patterns obtained by using HhaI, MboI, and AluI, we observed 19 distinct RFLP patterns from the 19 specimens. The results suggest that the PCR-based RFLP analysis method may be useful as a primary technique to identify and distinguish H. pylori strains directly from gastric biopsy specimens without culture of the organisms.

A high molecular weight protein from Staphylococcus intermedius cross-reacts with Staphylococcus aureus enterotoxin antibodies.

Enterotoxin production by Staphylococcus species other than Staphylococcus aureus has been reported. Staphylococcus strains (104 in toto) representing twelve species and subspecies were examined for enterotoxins using a commercial staphylococcal enterotoxin ELISA immunoassay (TECRA, International Bioproducts). Staphylococcus intermedius (24 strains) and S. aureus (7 strains) were positive with this test. Western blots of S. aureus exoproteins demonstrated proteins of approximately 30 kD, consistent with known staphylococcal enterotoxins. The major antigen in all S. intermedius strains, a 75 kD protein, was not analogous to previously described staphylococcal enterotoxins. This protein was unique to S. intermedius. Gel filtration data indicate that the protein is a subunit of a larger protein in vivo. The 75 kD protein cross-reacts with several enterotoxin antibodies. It is unclear whether the protein is a toxin, but its homology with S. aureus enterotoxins may indicate a shared toxic region, or this protein may create false positive results in screening for enterotoxin.

Physical distance between the site of type II DNA binding to the membrane and oriC on the Bacillus subtilis 168 chromosome.

The precise physical locations of the oriC region and the region for type II DNA binding to the membrane on the Bacillus subtilis 168 chromosome were determined. The DNA regions were physically mapped by creating new restriction sites (NotI and SfiI) within these regions. The physical distance between oriC and the type II DNA-binding region was verified with the creation of a novel sequence cleaved by endonuclease I-SceI in each of the above regions. Complete removal of the defined type II membrane-binding region produced no noticeable phenotype.

Characterization of a multienzyme complex derived from a Bacillus subtilis DNA-membrane extract that synthesizes RNA and DNA precursors.

The activity of a variety of enzymes involved in the synthesis of RNA and DNA precursors was found to copurify with initiation of DNA replication activity. These enzymes included ribo- and deoxyribonucleoside kinases, kinases for their phosphorylated intermediates, and ribonucleoside diphosphate reductase. This precursor-synthesizing complex is part of a Bacillus subtilis DNA-membrane extract originally shown to contain all of the enzymes and template necessary for initiation of DNA replication (J. Laffan and W. Firshein, J. Bacteriol. 169:2819-2827, 1987). Although the complex incorporated deoxyribonucleoside triphosphates into DNA, deoxyribonucleosides were incorporated even faster, suggesting catalytic facilitation. Both ribonucleosides and deoxyribonucleosides were found by thin-layer chromatography separation to be converted by the complex into their mono-, di-, and triphosphate derivatives. Ribonucleotides were incorporated into DNA via the action of ribonucleoside diphosphate reductase. Some regulatory mechanisms of the kinase system may also be retained by the complex. Electron microscope studies revealed that the precursor-synthesizing-initiation subcomplex is contained within a particulate fraction consisting of different-size vesicles resembling liposomes and that these particles may be structurally important in maintaining the synthetic activity of the subcomplex.

Origin-specific DNA-binding membrane-associated protein may be involved in repression of initiation of DNA replication in Bacillus subtilis.

Previous binding studies with labeled double-stranded Bacillus subtilis DNA fragments to a protein blot of renatured Bacillus membrane proteins showed selective binding of two adjacent origin fragments to a 64-kDa protein. The selective binding of the 64-kDa protein could be blocked by prior incubation of the blots with a specific polyclonal antibody. An in vitro replication system derived from a B. subtilis DNA-membrane complex showed initiation activity without addition of exogenous enzymes or template. When the complex was first incubated with the 64-kDa antibody or with its Fab fragments, initiation activity was enhanced. Antibodies to several other Bacillus membrane proteins as well as nonspecific antibodies did not show any significant stimulatory effect. A heavy-density-label experiment indicated that the complex initiated multiple rounds of replication in the presence of the 64-kDa antibody but not in its absence. The 64-kDa antibody plus an initiation inhibitor (streptovaricin) showed only repair and elongation activity. The 64-kDa protein may act in vivo as a repressor/regulator of initiation activity.

Effects of relaxin and insulin on reproductive tract size and early fetal loss in Squalus acanthias.

These experiments demonstrate that both porcine relaxin and bovine insulin can increase the cervical cross-sectional area in Squalus acanthias. When given after estradiol priming, the magnitude of this response is greater. This effect is limited to Stage C females in which pregnancy is over 75% complete. The overall result is premature loss of developing fetuses. The effect of relaxin or insulin on cervical cross-sectional area and fetal loss is unrelated to an effect on blood sugar. Cervical weights are affected little by hormone treatment. Relaxin and insulin do not increase the cross-sectional area of the anterior uterine constriction. These results suggest the involvement of a relaxin-like molecule during normal parturition in the spiny dogfish.