An autoantibody to C1-inhibitor recognizes the reactive center of the inhibitor

Immunohistochemistry was applied to AMeX-fixed sections of twelve cases of gastric carcinoma obtained at surgical resection to explore the occurrence and distribution of fibrin deposits in situ. Fibrinogen was distributed in abundance throughout perivascular zones and in the connective tissue of the tumor stroma. Fibrin II (des-fibrinopeptide B-type fibrin) was easily identified in a direct apposition to the surface membranes of viable carcinoma cells, predominantly at the host-tumor interface and in the regions immediately adjacent to the zones of angiogenesis. Further studies are required to identify the triggers of the coagulation reactions as well as fibrinolytic system components in the gastric cancer tissue.     

Mannolipid donor specificity of glycosylphosphatidylinositol mannosyltransferase-I (GPIMT-I) determined with an assay system utilizing mutant CHO-K1 cells

The microsomal enzyme glycosylphosphatidylinositol mannosyltransferase I (GPIMT-I) catalyses the transfer of a mannosyl residue from beta-mannosylphosphoryldolichol (beta-Man-P-Dol) to glucosamine-alpha(1,6)(acyl)phosphatidylinositol (GlcN-aPI) to form Man alpha(1,4)GlcN-aPI (ManGlcN-aPI), an intermediate in glycosylphosphatidylinositol (GPI) synthesis. While the transfer of [3H]mannosyl units to endogenous GlcN-aPI was not seen when membrane fractions from normal Chinese hamster ovary (CHO) K1 cells were incubated with exogenous [3H]Man-P-Dol, GPIMT-I activity could be characterized with an in vitro enzyme assay system employing membrane fractions from Lec15 or Lec35 cells. These CHO cell mutants apparently contain elevated levels of endogenous GlcN-aPI due to the inability to synthesize (Lec15) or utilize (Lec35) beta-Man-P-Dol in vivo. The presence of a saturated alpha-isoprene unit in the dolichyl moiety is required for optimal GPIMT-I activity since beta-mannosylphosphorylpolyprenol (beta-Man-P-Poly), which contains a fully unsaturated polyisoprenyl chain, was only 50% as effective as beta-[3H]Man-P-Dol as a mannosyl donor. When beta-[3H]-Man-P-Dol and alpha-[3H]Man-P-Dol were compared as substrates, GPIMT-I exhibited a strict stereospecificity for the mannolipid containing the beta-mannosyl-phosphoryl linkage. beta-[3H]Man-P-dolichols containing 11 or 19 isoprenyl units were equally effective substrates for GPIMT-I. Membrane fractions from Lec 9, a CHO mutant that apparently lacks polyprenol reductase activity and synthesizes very little beta-Man-P-Dol, but accumulates beta-Man-P-Poly, synthesized no detectable Man-GlcN-aPI when incubated with beta-[3H]Man-P-Dol in vitro. This indirect assay suggests that GlcN-aPI does not accumulate in Lec 9 cells, possibly because it is mannosylated via beta-Man-P-Poly, or perhaps the small amount of Man-P-Dol formed by the mutant in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enterocolitis associated with Clostridium perfringens infection in neonatal foals: 54 cases (1988-1997)

OBJECTIVE: To identify clinical signs, physical examination findings, results of diagnostic tests, treatments administered, and clinical outcome of neonatal foals with enterocolitis associated with Clostridium perfringens infection. DESIGN: Retrospective study. ANIMALS: 54 neonatal foals. RESULTS: Most foals had acute onset of obtunded mentation, colic, or diarrhea and developed leukopenia, neutropenia, an abnormally high number of band neutrophils, toxic WBC, and hypoproteinemia within 24 hours after admission, despite high serum IgG concentrations (> 800 mg/dl). Abdominocentesis and abdominal radiography of some foals revealed exudative peritonitis and gaseous distention of the small and large intestine, respectively. Cytologic examination of feces revealed spores or gram-positive rods in 8 of 10 foals. The most common genotypes of C perfringens isolates were type A and C, alone or in combination. Treatment did not alter mortality rate for most foals that had a positive culture for C perfringens type C. Of 54 foals, 29 (54%) that had C perfringens-associated enterocolitis died. Foals that had a culture that yielded C perfringens had higher sepsis scores, IgG concentrations, and mortality rates, compared with the overall hospital population of neonatal foals. CLINICAL IMPLICATIONS: Foals less than 7 days old that have enterocolitis associated with C perfringens infections, especially type C, have a guarded prognosis. Cytologic examination of feces to determine spore counts and detect rods may be a means for early identification of C perfringens infections. Polymerase chain reaction assays to determine genotype are important for designing preventive treatment regimens.     

Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry

Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism. Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme. These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da. Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate. The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme. Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme. Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70. A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130. A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde. Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.     

Sequence diversity in the NIb coding region of eight sugarcane mosaic potyvirus isolates infecting sugarcane in Australia

We have sequenced the NIb coding region of sugarcane mosaic potyvirus strain SC (SCMV-SC) and eight field isolates of SCMV from Australia. This region comprised 1563 nucleotides and encoded a putative protein of 521 amino acids containing the consensus motif GDD. The protease cleavage sites between the NIa/NIb and the NIb/coat protein were found to be Q/C and Q/A, respectively. The SCMV sequences were most similar to sorghum mosaic potyvirus with identities of 70% and 78% at the nucleotide and amino acid levels, respectively. When the sequences were compared to each other, there was a maximum of 3.3% variation between isolates at the nucleotide level and a maximum of 0.8% at the amino acid level. Phylogenetic analysis of the sequences indicated the field isolates were grouped according to their geographical location. The SCMV sequence with most homology to all other isolates has been selected to generate constructs for replicase-mediated resistance.     

A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in the brain

The Shc adaptor protein, hereafter referred to as ShcA, possesses two distinct phosphotyrosine-recognition modules, a C-terminal Src homology 2 (SH2) domain and an N-terminal phosphotyrosine-binding (PTB) domain, and is itself phosphorylated on tyrosine in response to many extracellular signals. Phosphorylation of human ShcA at Tyr-317 within its central (CH1) region induces binding to the Grb2 SH2 domain and is thereby implicated in activation of the Ras pathway. Two shc-related genes (shcB and shcC) have been identified in the mouse. shcB is closely related to human SCK, while shcC has not yet been found in other organisms. The ShcC protein is predicted to have a C-terminal SH2 domain, a CH1 region with a putative Grb2-binding site, and an N-terminal PTB domain. The ShcC and ShcB SH2 domains bind phosphotyrosine-containing peptides and receptors with a specificity related to, but distinct from, that of the ShcA SH2 domain. The ShcC PTB domain specifically associates in vitro with the autophosphorylated receptors for nerve growth factor and epidermal growth factor. These results indicate that ShcC has functional SH2 and PTB; domains. In contrast to shcA, which is widely expressed, shcC RNA and proteins are predominantly expressed in the adult brain. These results suggest that ShcC may mediate signaling from tyrosine kinases in the nervous system, such as receptors for neurotrophins.     

Variable distributions of Ca(2+)-permeable and Ca(2+)-impermeable AMPA receptors on embryonic rat dorsal horn neurons

1. By measuring the apparent reversal potential (aErev) of kainate- and alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA)-evoked currents associated with changes in extracellular Ca2+ concentration ([Ca2+]e), we have been able to identify embryonic dorsal horn neurons grown in tissue culture that express Ca(2+)-permeable non-N-methyl-D-aspartic acid (NMDA) receptors. 2. The relative expression of Ca(2+)-permeable and Ca(2+)-impermeable non-NMDA receptors varies from cell to cell. This was evident from the range of a ErevS observed for kainate-evoked currents in a 0 mM [Na+]e, 10 mM [Ca2+]e bath. Under these conditions, aErev ranged from -96 to -21 mV, suggesting that the percentage of the non-NMDA receptors on each neuron that are Ca(2+)-permeable is variable. 3. To determine the extent to which the variability in aErev is due to variable receptor expression rather than experimental variability, we compared the effects of changes in [Ca2+]e on kainate-evoked currents and NMDA-evoked currents on the same cells. Assuming that all of the NMDA receptors on each neuron have a similar Ca2+ permeability, this approach provides an index of the sensitivity of our assay system. The reversal potential of NMDA-evoked currents in 10 mM [Ca2+]e ranged from -30 to -7 mV, whereas on the same population of neurons, the aErev of kainate-evoked currents ranged from -92 to -40 mV. 4. The rectification properties of the non-NMDA currents were generally linear or outwardly rectifying in normal bath solution. When the PCa/PCs ratio in 0 mM [Na+]e, 10 mM [Ca2+]e bath solution was assessed as a function of the rectification index in standard bath, a poor correlation was found between Ca2+ permeability and the rectification index. 5. The aErev of kainate-evoked currents was similar to that of cyclothiazide-enhanced AMPA-evoked currents observed on the same cells (-66.5 +/- 18.4 and -64.0 +/- 13.9 mV, mean +/- SD, respectively). This suggests that kainate is primarily activating the AMPA receptor and that the majority of non-NMDA receptors on embryonic dorsal horn neurons in culture are high-affinity AMPA receptors. 6. Immunocytochemical evidence suggests that the AMPA receptor subunits GluR1-4 are expressed to a variable degree from cell to cell in our cultures. We found evidence for low levels of expression of the kainate receptor subunits GluR5-7. The immunocytochemical observations support the physiological data indicating that much of the kainate-evoked current recorded in our experiments can be accounted for by kainate activation of AMPA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

The pattern of inflammation in rat sepsis due to enterotoxin-producing Staphylococcus aureus: a comparison with ischemia-reperfusion injury

Sepsis and trauma have similarities in their immunopathologic profiles. Both conditions can result in multi-system organ failure which is sometimes associated with cytokine generation and inflammatory cell activation. Furthermore, decreases in peripheral blood monocyte expression of HLA-DR have been noted in both human sepsis and trauma. However, the magnitude, onset, and time course of such stimuli are often difficult to ascertain in human studies. Thus, to study a more detailed in vivo immunologic profile in these conditions, rat models were employed. Our aim was to describe and analyze cytokine and peripheral blood immunophenotype patterns in bacterially induced rat sepsis and to compare this to rat ischemia-reperfusion injury. Sprague-Dawley rats underwent either bacterial injection with enterotoxin producing Staphylococcus aureus or hind limb ischemia/ reperfusion. Two bacterial doses which were either lethal or sublethal at 24-48 hours were utilized. Peripheral blood neutrophils and B-lymphocytes were studied for expression of beta-integrins (CD11b and CD11b/c) and I-A, respectively, using flow cytometry. Corresponding plasma levels of TNF alpha and interferon gamma were measured by ELISA. At 24 hr, a lethal bacterial lethal bacterial dose injection resulted in significantly higher levels of neutrophil CD11b/c expression (p < 0.005) compared with ischemia-reperfusion treatment. B-cell I-A expression was also higher in lethal sepsis. Gamma interferon levels were significantly higher in lethal sepsis compared with ischemia-reperfusion (p = 0.005). Studies over time showed that CD11b expression and interferon gamma were both more marked at 6 hr than at 24 hr in lethal sepsis. This pattern was not observed in sublethal sepsis or in ischemia-reperfusion. CD11b/c expression on the other hand remained elevated at comparable levels at 6 and 24 hr in lethal sepsis. B-cell I-A expression in ischemia-reperfusion and sublethal sepsis decreased at 24 hr compared with baseline. Lethal sepsis in rats injected with enterotoxin producing staphylococcus results in phasic alterations in neutrophil CD11b and plasma interferon levels prior to death. In analogy to the findings of monocyte decreases in DR expression observed in human trauma and sepsis, rat B-cell I-A expression showed decreases in sublethal sepsis as well as in ischemia-reperfusion injury. However, this was not observed in lethal sepsis. These findings have implications in understanding the immunologic/inflammatory changes observed in human sepsis and trauma.