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.

HindIII-digested DNA fragments derived from an EcoRI-digested 6.5-kb fragment of chromosomal DNA prepared from Helicobacter pylori ATCC 43629 (type strain) were cloned into the pUC19 vector. A 0.86-kb insert was identified as a potential chromosomal DNA probe. The specificity of the probe was evaluated by testing 166 non-H. pylori bacterial strains representing 38 genera and 91 species which included aerobic, anaerobic, and microaerophilic flora of the upper and lower gastrointestinal tracts. None of the 166 non-H. pylori strains hybridized with this probe (100% specificity), and the sensitivity of this probe was also 100% when H. pylori isolates from 72 patients with gastritis and with the homologous ATCC type strain were tested by dot blot hybridization. The capability of this probe for differentiating between strains of H. pylori was evaluated by Southern blot hybridization of HaeIII-digested chromosomal DNA from 68 clinical isolates and the homologous ATCC type strain of H. pylori. Fifty-one unique hybridization patterns were seen among the 69 strains tested, demonstrating considerable genotypic variation among H. pylori clinical isolates. We propose that this probe would be of significant value for conducting epidemiologic studies.

Natural product complex carbohydrate polymers are currently being developed as prophylactic and/or therapeutic drugs. These water-soluble carbohydrate pharmaceuticals, which are primarily 0-1,3-D-glucan polymers, belong to the class of drugs known as biological response modifiers (BRMs). A major obstacle to the development, understanding and clinical utilization of carbohydrate BRMs has been the difficulty involved in accurately characterizing high molecular weight (MW) carbohydrate polymers. Recent advances in aqueous gel permeation chromatography (GPC), differential viscometry (DV) and multi.

1. 1. Plasma carnitine and triglycerides were measured in five male Macaca arctoides and one female Macaca nemistrina during the course of feeding a low-fat (5.2% w/w), high carbohydrate diet and a high-fat (15.9% w/w), low carbohydrate diet. 2. 2. For each individual monkey, an inverse relationship was observed between plasma carnitine and triglyceride levels when the low-fat diet was fed but not when the high-fat diet was fed. 3. 3. The mechanism of the different responses to diet was not investigated but may be related to the primary source of the plasma triglycerides (i.e. endogenous origin or exogenous origin) or to differing hormonal effects. 4. 4. A close coupling between carnitine and triglyceride metabolism may be part of a sensitive homeostatic control mechanism that responds to endogenously-synthesized triglyceride.

A major barrier to the development, preclinical and clinical application of natural carbohydrate biological response modifiers has been the difficulty involved in accurately characterizing carbohydrate polymers with molecular masses ranging from 104 to 107 g/mol. Herein, we employed size exclusion chromatography with multi-angle laser light scattering and differential viscometry to compare and contrast structural properties of the biological response modifiers Krestin, schizophyllan and glucan phosphate. Krestin, schizophyllan and glucan phosphate exhibit significant differences in molecular mass moments, molecular mass distribution, polymer sizes, intrinsic viscosity and perhaps their solution behaviour. This knowledge of precise physicochemical data is required for a better understanding of the properties and higher structure of complex carbohydrate biological response modifiers.

Adult male albino Sprague Dawley rats were fed diets containing 15 % of casein with a constant 0.62 % of supplemental methionine, and three levels of inorganic sulfate, 0.0002 %, 0.02 %, and 0.42 %. The polyamines (putrescine, spermidine, and spermine) and the controlling enzymes for their biosynthesis, ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SAMD) were evaluated in liver tissue homogenates following a 17 day dietary period. There was no increase in the activity of ODC or the tissue concentration of putrescine in the liver tissue of rats fed the diet low in sulfate. There was an increase in SAMD activity and the concentration of spermidine and spermine. The activity of both ODC and SAMD and the tissue concentration of putrescine, spermidine, and spermine increased in the liver tissue from rats fed the diet high in inorganic sulfate. These data coupled with previous observations suggest that the metabolic effects of diets low in inorganic sulfate are probably not mediated through the regulatory activity of the polyamines. However, these data allow the suggestion that the metabolic alterations observed in rats fed diets high in inorganic sulfate may be due to the regulatory action of the polyamines.

Heart failure, the only cardiovascular disorder increasing in prevalence, afflicts 4.7 million Americans and costs more than 17.8 billion each year. This has been attributed to the fact that adult mammalian ventricular muscle cells are terminally differentiated cells which have lost their ability to multiply by cell division. This study is to use myogenic stem cells (satellite cells) from skeletal muscle to generate new heart muscles in the damaged heart for functional improvement. Autologous satellite cells were isolated from mongrel dogs before cultured, labeled (lac Z), and implanted into the injured myocardium (2 hrs coronary occlusion). Multiple necrotic sites were observed initially at the ischemic zone with satellite cells (X-Gal positive) and survived the early healing process. The implanted satellite cells gradually surrounded and infiltrated into the injured myocardium and eventually formed the new muscle tissue expressing β-galactosidase activity. For the control hearts (given culture medium), scar tissue was observed in the injured areas of myocardium. Change in wall thickness during each cardiac cycle was determined using the sonomicrometer. The regional wall thickening was not different from a normal heart at the regenerated myocardium. However, thinning and bulging during systole were observed for the control hearts at the injury sites. This result suggested that autologous satellite cells implanted into injured heart not only formed muscle cells similar to cardiomyocytes, but also restored the function of infarcted myocardium.

Glucans are fungal and bacterial derived glucose polymers. Glucans modulate immune function via macrophage modulation. A specific (1 → 3)-β-D-glucan receptor has been reported on monocytes/macrophages and their precursors. We studied the effect of different reagents on binding of a water soluble glucan to the human macrophage receptor. We investigated the effect of EGTA, EDTA, dexamethasone, colchicine, cytochalasin D and 2,4-dinitrophenol, on glucan binding to U937 cells, a human promonocytic cell line. Cells were incubated for 90 min with 3H-(1-3)-β-D-glucan phosphate (5-30 μg/well) in the presence of media or dinitrophenol ( 1 mM), EGTA (5 mM, 9.58 mM), EDTA (1, 5, 10 mM) dexamethasone (20, 200, 1000, 2000 nM), colchicine (50 mM) or cytochalasin D. We found that dinitrophenol, dexamethasone, EGTA and cytochalasin D did not affect binding. A 60-70% reduction in binding was seen at 5 and 10 mM EDTA. Colchicine, a microtubule inhibitor, suppressed binding by ∼50 %. We conclude that binding of (1-3)-β-D-glucan phosphate to its receptor is not dependent upon calcium. However, other divalent cations (Mg++, Mn++) may affect binding. The colchicine and cytochalasin D data indicate that microtubules, but not microfilaments, are involved in the binding of glucan to the human macrophage glucan receptor.

Adenosine prevents ischemic injury, induces preconditioning, and minimizes reperfasional damage. However, the beneficial mechanisms of adenosine in the cardiovascular system are not completely clear. The eukaryotic transcription factor NF-κB is activated by multiple stimuli and regulates many immediate-early gene expression involved in immune, inflammatory and defensive reactions. Ischemia rapidly induced NF-κB binding activity in rat heart was observed in our laboratory. The effect of adenosine (0.2 mM for 5 min) on NF-κB activation was studied in ischemic myocardium (0, 1, 2, 3, 4, 5, 7.5, 10, 15, and 30 min). Cytoplasmic and nuclear proteins were extracted for measuring IκBα levels and NF-κB binding activity. Western blot showed significant decrease of cytoplasmic IκBα during 3 to 7.5 min of ischemia while pretreating the hearts with adenosine prevented the loss of IκBα protein. Similarly, the activation and translocation of NF-κB due to myocardium ischemia (3 to 30 min) were blocked by adenosine. Northern blot hybridization also showed that adenosine maintained the normal levels of IκBα mRNA during the entire period of ischemia. The protective effect of adenosine and ischemia preconditioning may mediate through modulation of NF-κB activation which induces immediate-early gene expression that resulted in ischemic reperfusional injury.

Expression of immediate-early genes in response to ischemia/reperfusion has been reported, but little is known about the role of transcription factors that may regulate the gene expression. NF-κB is a critical regulator of many immediate-early gene-expression involved in immune responses, inflammatory reaction, and defense mechanisms. Isolated perfused rat hearts subjected to 0, 1, 2, 3, 4, 5, 7.5, 10, 15, and 30 min of global normothermic ischemia were used to study the activation and translocation of NF-κB. Western blot shows that cytoplasmic IκBα levels were reduced at 3 min of ischemia and markedly diminished by ischemia for 5 min, but were rapidly returned at 15 min after ischemia. Electrophoretic mobility shift assay using nuclear extract indicated NF-κB binding activity began at 3 min, peaked around 5 min, and persisted up to 30 min of myocardial ischemia. Concurrent to the disappearance of cytoplasmic IκBα protein, the NF-κB was activated and translocated to the nucleus. Pyrrolidine dithiocarbamate, an antioxidant, significantly inhibited the cytoplasmic IκBα disappearance and prevented the activation of NF-κB. The results suggest that ischemia rapidly induced NF-κB activation by dissociating its inhibitor IκBα. The activation of NF-κB which can be prevented by antioxidant indicates the reactive oxygen intermediates may serve as messengers.