Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits.

The A-subunit polypeptides of Shiga toxin, the Shiga-like toxins (SLTs), and the plant lectin ricin inactivate eucaryotic ribosomes by enzymatically depurinating 28S rRNA. Comparison of the amino acid sequences of the members of the Shiga toxin family and ricin revealed two regions of significant homology that lie within a proposed active-site cleft of the ricin A chain. In previous studies, these conserved sequences of the SLT-I and ricin A subunits have been implicated as active sites.

Construction and analysis of TnphoA mutants of enteropathogenic Escherichia coli unable to invade HEp-2 cells.

Enteropathogenic Escherichia coli (EPEC) strains have recently been shown to invade tissue culture cells. We describe a set of 22 Tn5 IS50L::phoA (TnphoA) insertion mutants of EPEC strain E2348-69 that are unable to invade HEp-2 cells. Each mutant was tested for the ability to adhere to and to induce the polymerization of actin in HEp-2 cells.

DNA binding of heat shock factor to the heat shock element is insufficient for transcriptional activation in murine erythroleukemia cells.

The heat shock response is among the most highly conserved examples of regulated gene expression, being present in all cellular organisms. Transcriptional activation of heat shock genes by increased temperature or other cellular stresses is mediated by the binding of a heat shock factor (HSF) to a conserved nucleotide sequence (the heat shock element) present in the promoter of heat-inducible genes. Despite the high degree of conservation of this response, embryonic stages of development are characterized by the absence of a heat shock response.

Characterization and sequence of a mouse hsp70 gene and its expression in mouse cell lines.

A heat-inducible member of the mouse hsp70 gene family has been isolated and characterized. The gene, hsp70.1, has a primary transcript of 2.

Members of the 70-kilodalton heat shock protein family contain a highly conserved calmodulin-binding domain.

The 70-kilodalton heat shock protein (hsp70) family members appear to be essential components in a number cellular protein-protein interactions. We report here on the characterization of a new functional region in hsp70, a calmodulin-binding site. We have identified a 21-amino-acid sequence within the hsp70 protein that contains a calmodulin-binding domain.

Identification of an iron-regulated virulence determinant in Vibrio cholerae, using TnphoA mutagenesis.

Several virulence determinants of bacteria are regulated by the concentration of iron in the medium, with increased expression occurring under low-iron conditions. Iron-regulated virulence factors have not been previously described in Vibrio cholerae. We used the transposon vector Tn5 IS50L::phoA (TnphoA) to obtain insertion mutations in iron-regulated genes of V.

Heat shock stimulates the release of arachidonic acid and the synthesis of prostaglandins and leukotriene B4 in mammalian cells.

Heat shock has a profound influence on the metabolism and behavior of eukaryotic cells. We have examined the effects of heat shock on the release from cells of arachidonic acid and its bioactive eicosanoid metabolites, the prostaglandins and leukotrienes. Heat shock (42-45 degrees) increased the rate of arachidonic acid release from human, rat, murine, and hamster cells.

Effects of nitroimidazoles on neuronal cells in vitro.

Peripheral neuropathy is a significant dose-limiting side effect of the nitroimidazole drugs in vivo. We have thus undertaken a study on the mechanisms of nitroimidazole neurotoxicity in the cultured neuronal cell lines, PC-12 (rat pheochromocytoma) and NB4183 (mouse neuroblastoma). Cells were differentiated with either nerve growth factor or dibutyryl cAMP and then were exposed to misonidazole and SR 2508.

Bacteremia with Streptococcus bovis and Streptococcus salivarius: clinical correlates of more accurate identification of isolates.

Two biotypes of Streptococcus bovis can be identified by laboratory testing and can be distinguished from the phenotypically similar organism Streptococcus salivarius. We assessed the clinical relevance of careful identification of these organisms in 68 patients with streptococcal bacteremia caused by these similar species. S.

Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I.

Escherichia coli Shiga-like toxin I, a close relative of Shiga toxin and a distant relative of the ricin family of plant toxins, inhibits eukaryotic protein synthesis by catalyzing the depurination of adenosine 4324 in 28S rRNA. By comparing the crystallographic structure of ricin with amino acids conserved between the Shiga and ricin toxin families, we identified seven potential active-site residues of Shiga-like toxin I. The structural gene encoding Shiga-like toxin I A chain (Slt-IA), the enzymatically active subunit, was engineered for high expression in E.

Effects of heat on cell calcium and inositol lipid metabolism.

Hyperthermia causes a large (three-to fivefold) increase in intracellular free calcium ([Ca2+]i) in HA-1 fibroblasts. Increased [Ca2+]i appears initially to be due to release of Ca2+ from an internal store, probably located in the endoplasmic reticulum. A subsequent influx of Ca2+ from the extracellular medium is then observed.

Confirmation of the Fur operator site by insertion of a synthetic oligonucleotide into an operon fusion plasmid.

We constructed a synthetic oligonucleotide corresponding to the previously proposed consensus binding site for the Fur protein, a central iron-regulatory protein of Escherichia coli. When this oligonucleotide was introduced at the start of transcription of an operon fusion between the ompF promoter and the lacZ structural gene, beta-galactosidase activity became iron regulated. This consensus sequence is sufficient to function as an operator site for the binding of Fur protein in vivo.

Native valve endocarditis due to coagulase-negative staphylococci. Clinical and microbiologic features.

Twenty-one patients with native valve endocarditis caused by coagulase-negative staphylococci were studied; 14 had pre-existing valvular or congenital heart disease. Although commonly subacute in presentation, complications of endocarditis were frequent: arterial emboli in five patients, new electrocardiographic conduction system abnormalities in nine, congestive heart failure in eight, annular or myocardial abscesses in five, and disruption of valve leaflets in three. Cures were achieved in 10 of 12 patients treated medically and seven of nine treated surgically.

Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus.

Shiga-like toxin is an iron-regulated cytotoxin quite similar to Shiga toxin from Shigella dysenteriae 1. The structural genes for Shiga-like toxin in Escherichia coli (sltA and sltB) appear to be transcribed as an operon from a promoter upstream of sltA. We used a gene fusion between the promoter and proximal portion of sltA with the gene for bacterial alkaline phosphatase to assess the regulation of toxin expression.

Effect of hyperthermia (45 degrees C) on calcium flux in Chinese hamster ovary HA-1 fibroblasts and its potential role in cytotoxicity and heat resistance.

Hyperthermia caused a major increase in uptake of 45Ca2+ into Chinese hamster ovary HA-1 cells. Increased permeability to Ca2+ was observed with heating periods as brief as 45 degrees C for 4 min and reached a maximum at 45 degrees C for 30 min. In addition to elevation of Ca2+ influx, heat induced an increase in 45Ca2+ exchange with the extracellular Ca2+ pool.

Nucleotide sequence of the Shiga-like toxin genes of Escherichia coli.

We have determined the nucleotide sequence of the sltA and sltB genes that encode the Shiga-like toxin (SLT) produced by Escherichia coli phage H19B. The amino acid composition of the A and B subunits of SLT is very similar to that previously established for Shiga toxin from Shigella dysenteriae 1, and the deduced amino acid sequence of the B subunit of SLT is identical with that reported for the B subunit of Shiga toxin. The genes for the A and B subunits of SLT apparently constitute an operon, with only 12 nucleotides separating the coding regions.

Ampicillin versus cefamandole as initial therapy for community-acquired pneumonia.

One hundred seven patients with community-acquired pneumonia thought to be of bacterial etiology by the admitting physician but whose initial sputum Gram stain was inadequate to direct specific therapy were randomized to receive either intravenous ampicillin or cefamandole as empiric therapy. Patients were excluded if the initial sputum Gram stain was highly suggestive of infection with Streptococcus pneumoniae, Staphylococcus aureus, or an enteric gram-negative bacillus. The two study groups had comparable demographic and presenting clinical features.

Heat stress stimulates inositol trisphosphate release and phosphorylation of phosphoinositides in CHO and Balb C 3T3 cells.

Exposure of eukaryotic cells to elevated temperature leads to profound switches in cell metabolism and gene expression which may be involved in cellular homeostatic mechanisms. We have investigated the effect of heat shock (45 degrees C) on the metabolism of the phosphoinositides, a class of phospholipids involved in the function of Ca2+ -linked membrane receptors. Heat shock led to stimulation of phosphoinositide turnover in HA1-CHO and Balb C 3T3 cells, resulting in the rapid accumulation of inositol trisphosphate (IP3).

Role of energy in cellular responses to heat.

We have examined the effect of heat on energy-generating processes and on parameters of bioenergetic status in animal cells. Heat inactivates several processes involved in uptake and metabolism of nutrients. In particular, insulin-stimulated hexose transport in HA-1 fibroblasts and electron transport in blowfly sarcosomes (Bowler, 1981) exhibit thermal sensitivities that reflect the vulnerability to heat of the whole cell or organism.

Binding of polycation DEAE-dextran to Chinese hamster ovary cells induces reversible Ca2+ influx and inhibits capping of concanavalin A acceptor proteins.

Binding of the polycation DEAE-dextran to the cell surface of HA-1 CHO cells caused a marked increase in 45Ca2+ exchange influx. The effect was fairly selective for Ca2+, undirectional (efflux was not increased) and was rapidly reversed by treatment with polyanion dextran sulfate. 45Ca2+ influx could not be stimulated by treatment with multivalent lectins or fibronectin.

Cardiac conduction abnormalities complicating native valve active infective endocarditis.

Two hundred eleven episodes of native valve active infective endocarditis treated at the Massachusetts General Hospital between 1975 and 1983 were reviewed. The aortic (36%) and mitral (33%) valves were most frequently involved, but in 21% of the cases the site of infection could not be localized. Streptococcal (50%) and staphylococcal (35%) species were the most frequently isolated pathogens.

Prosthetic valve endocarditis. Analysis of factors affecting outcome of therapy.

We analyzed the outcome of 116 patients with prosthetic valve endocarditis treated between 1975 and 1983 and used multivariate analysis to identify risk factors for in-hospital mortality and bad outcome during follow-up. Complicated prosthetic valve endocarditis was defined as the presence of a new or changing heart murmur, new or worsening heart failure, new or progressive cardiac conduction abnormalities, or prolonged fever during therapy. Complicated prosthetic valve endocarditis was present in 64% of patients; factors associated with complicated prosthetic valve endocarditis included aortic valve infection (odds ratio 4.

Rapid increases in inositol trisphosphate and intracellular Ca++ after heat shock.

Heat shock (45 degrees C) caused a rapid (less than 1 min) release of inositol trisphosphate from the membranes of HA-1 CHO fibroblasts. The rise in inositol trisphosphate concentration was followed by an increase in intracellular free Ca++. In addition to the heat induced rise in intracellular free Ca++, we observed an increase in 45Ca++ influx following nonlethal heat shock (45 degrees C/10 min).

Susceptibility of human and porcine neutrophils to hypothermia in vitro.

Hypothermia may contribute to serious life-threatening infections. An experimental model has been established in pigs in order to study the effects of hypothermia on host bacterial defenses. The function of blood neutrophils from pigs and humans was examined in vitro at 37 and 29 degrees C.