Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli

The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP. Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing the cof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.     

Demonstration of cholera toxin-related factor in cultures of Aeromonas species by enzyme-linked immunosorbent assay.

The production of toxins by Aeromonas species was examined by the suckling mouse test, the hemolysin test, and the enzyme-linked immunosorbent assay with anticholera enterotoxin. A factor that was immunologically related to cholera enterotoxin was produced by 5 of 14 strains of Aeromonas hydrophila and 4 of 15 strains of Aeromonas sobria. Analysis by these assays and by a test for heat stability suggested that the factor differed from hemolysin and from toxin that was active in the suckling mouse test.     

Temporal features of the responses of primate spinothalamic neurons to noxious thermal stimulation of hairy and glabrous skin

Extracellular recordings were made from 81 primate spinothalamic (STT) neurons in the L7-S1 segments of the spinal cord. The majority of the sample was recorded from within laminae IV-V. The temporal features of the responses to noxious thermal stimulation of glabrous and hairy skin were studied in an attempt to determine whether natural groupings of STT neurons could be identified on the basis of response time course alone and whether these groups were skin type dependent. The relationship between these groups and those based on static response features (37) was also explored in an attempt to define more fully their potential functional roles. In most STT neurons, the thermally evoked responses typically appeared to have two response components, particularly at stimulus temperatures above 49 degrees C. The first response phase typically peaked within 1-12 s of stimulus onset and then adapted. The second phase slowly rose to a maximum, typically 15-30 s following stimulus onset. The existence of natural groupings of STT neurons based upon the characteristics of these two response components was assessed with a k-means cluster analysis. On the basis of the onset and early peak latencies, two well-defined short and long latency neuronal clusters were found in the responses evoked from both glabrous and hairy skin; these were referred to as the SP1 and LP1 classes, respectively. The glabrous and hairy skin SP1 classes did not differ significantly in either onset or early peak latency for stimuli of 47-55 degrees C. However, the hairy skin LP1 class had significantly shorter onset latencies than the glabrous skin LP1 class for stimuli of 49-53 degrees C, as well as shorter peak latencies for stimuli of 49 and 51 degrees C. The SP1 class constituted 62% of the hairy skin subset, whereas the LP1 class constituted 57% of the glabrous skin subset. A cluster analysis of the late-peak latencies also revealed two subgroups. In the responses evoked from both glabrous and hairy skin, the longer latency classes (LP2) constituted more than 80% of the samples. With one exception, no dependence upon the type of skin that was stimulated was found in the latencies of either the LP2 class or the shorter latency SP2 class. Prior conditioning of the skin with a 30-s thermal pulse of 51-55 degrees C led to a suppression of the early response phase and an enhancement of the late phase in nearly all cases examined (n = 11). This pattern was independent of skin type.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of a cell-associated hemagglutinin of Vibrio parahaemolyticus.

We found a positive correlation between cell-associated mannose-sensitive hemagglutination and adherence of Vibrio parahaemolyticus to rabbit enterocytes by investigating 35 strains of V. parahaemolyticus for cell-associated hemagglutinin (cHA) and for the ability to adhere to the enterocytes. We purified a mannose-sensitive cHA from a Kanagawa phenomenon-positive clinical strain of V. parahaemolyticus that exhibited a high level of mannose-sensitive hemagglutination and strongly adhered to the enterocytes. The purified cHA is a heat-labile, tetrameric protein consisting of four identical subunits of approximately 26 kDa each. The adherence to rabbit enterocytes was inhibited in a dose-dependent manner by pretreatment of the bacterial cells with D-mannose and with the Fab fraction of immunoglobulin G against the purified cHA. Furthermore, pretreatment of the enterocytes with the purified cHA inhibited the adherence of V. parahaemolyticus. Immunogold electron microscopy revealed that the cHA is located on the bacterial cell surface and is not associated with pili. These results suggest that cHA is involved in the adherence mechanisms of V. parahaemolyticus to the enterocytes and that the receptors for cHA on the enterocyte appear to be a D-mannose-containing compound.     

A mutant toxin of Vibrio parahaemolyticus thermostable direct hemolysin which has lost hemolytic activity but retains ability to bind to erythrocytes.

A mutant toxin, R7, of thermostable direct hemolysin (TDH) with a single amino acid substitution at glycine 62 was analyzed. The hemolytic activity of R7 decreased to less than 1/1,000 of that of wild-type TDH, and its mouse lethality was undetectable. This mutant toxin, however, showed a marked inhibitory effect on hemolysis by wild-type TDH. Enzyme immunoassay and flow cytometric analysis demonstrated that R7 retained approximately 50% of the ability to bind to erythrocytes compared with that of wild-type TDH, suggesting that its inhibition of hemolysis by wild-type TDH might be due to blocking the binding sites on the erythrocyte membrane. Wild-type TDH affected the erythrocyte membrane by causing an influx of calcium and propidium iodide, while R7 showed no detectable effects of these kinds. These results suggest that hemolysis by TDH consists of at least two steps, binding and postbinding, and that R7 is likely to be a postbinding activity-deficient mutant toxin of TDH.     

Monoclonal antibodies against synthesized short peptides corresponding to human AA amyloid protein

Monoclonal antibodies (McAbs) were raised against the synthesized short peptides corresponding to 37-47 residues in amino acid sequence of human AA protein. The McAbs reacted immunohistochemically to amyloid tissues from cow, mouse, swan, and human AA amyloidosis. We concluded that the McAbs were useful for identification of AA type amyloidosis of various species, and that the 37-47 residues were effective antigenic sites in AA protein.     

Cell lineage specificity of newly raised monoclonal antibodies against gastric mucins in normal, metaplastic, and neoplastic human tissues and their application to pathology diagnosis

The specificity of monoclonal antibodies against gastric mucins (designated as HIK1083, PGM 36, and PGM 37) was studied immunohistochemically in normal, metaplastic, and neoplastic human tissues. These antibodies labeled class III mucin-producing cells identified by paradoxical concanavalin A staining in normal stomach, duodenum (Brunner gland), biliary tract, and main pancreatic duct; in mucinous metaplasia of pancreas and gallbladder; and in adenocarcinomas of stomach (90%), bile duct (80%), gallbladder (100%), pancreas (80%), lung (100% of goblet cell type adenocarcinomas), ovary (67% of mucinous carcinomas), and uterine cervix (100% of adenoma malignum tumors). Normal and neoplastic cells of esophagus, colon, salivary gland, kidney, endometrium, breast, prostate, and liver, as well as normal small intestine, lung, and uterine cervix, were all negative. The antibodies used should be valuable for the detection of class III mucin and class III mucin-producing cells in normal, metaplastic, and neoplastic tissues.     

Mouse erythroblast formation is inhibited by anemia-inducing substance from the plasma of a patient with a malignant neoplasm

An anemia-inducing substance (AIS) was found as a protein, with a molecular weight of 50,000 on SDS electrophoresis gel, that decreased the osmotic resistance of erythrocytes. In this study, the plasma fraction containing AIS is shown to inhibit mouse erythroblast formation in vitro. The addition of the plasma fraction from a patient with an advanced malignant neoplasm to a liquid culture of mouse bone marrow cells with erythropoietin results in low numbers of erythroblast formation and low cellular yields of alpha- and beta-globin mRNAs. These inhibitions were not observed after immunoadsorption of the plasma fraction with an antiserum against AIS.     

Regulation of the type I IFN induction: a current view

The type I IFN-alpha/beta gene family was identified about a quarter of a century ago as a prototype of many cytokine gene families, which led to the subsequent burst of studies on molecular mechanisms underlying cytokine gene expression and signaling. Although originally discovered for their activity to confer an antiviral state on cells, more evidence has recently been emerging regarding IFN-alpha/beta actions on cell growth, differentiation and many immunoregulatory activities, which are of even greater fundamental biological significance. Indeed, much attention has recently been focused on the induction and function of the IFN-alpha/beta system regulated by Toll-like receptors (TLRs), which are critical for linking the innate and adaptive immunities. The understanding of the regulatory mechanisms of IFN-alpha/beta gene induction by TLRs and viruses is an emerging theme, for which much new insight has been gained over the past few years.     

CO2 dissociation curves of oxygenated whole blood obtained at rest and in exercise

Summary In vitro CO₂ dissociation curves for oxygenated whole blood were determined in 19 healthy male subjects at rest and during submaximal and maximal bicycle work. Hemoglobin concentration and blood lactate increased with increasing work load and accordingly buffer value of the whole blood increased while bicarbonate and Base Excess (BE) decreased, resulting in a downward shift of the CO₂ dissociation curve during exercise. Despite the marked increase in buffer values of the blood, the slopes of the CO₂ dissociation curves during exercise were found to be about the same as those obtained at rest. It was inferred that the increasing effect of increased buffer value, on the dissociation slope, was essentially compensated by the decreasing effect of diminished bicarbonate content. The advantages of this relatively constant CO₂ dissociation slope for the indirect measurement of cardiac output by the Fick principle are discussed.