Search

We have developed a novel vaccine against Shiga toxin (Stx)-producing Escherichia coli (STEC) infection using a recombinant Mycobacterium bovis BCG (rBCG) system. Two intraperitoneal vaccinations with rBCG expressing the Stx2 B subunit (Stx2B) resulted in an increase of protective serum IgG and mucosal IgA responses to Stx2B in BALB/c mice. When orally challenged with 10(3) CFU of STEC strain B2F1 (O91: H21), the immunized mice survived statistically significantly longer than the nonvaccinated mice. We suggest that intraperitoneal immunization with rBCG expressing Stx2B would be a potential vaccine strategy for STEC.

Improvement of a drug's binding activity using the conformational restriction approach with sp³ hybridized carbon is becoming a key strategy in drug discovery. We applied this approach to BACE1 inhibitors and designed four stereoisomeric cyclopropane compounds in which the ethylene linker of a known amidine-type inhibitor 2 was replaced with chiral cyclopropane rings. The synthesis and biologic evaluation of these compounds revealed that the cis-(1S,2R) isomer 6 exhibited the most potent BACE1 inhibitory activity among them. X-ray structure analysis of the complex of 6 and BACE1 revealed that its unique binding mode is due to the apparent CH-π interaction between the rigid cyclopropane ring and the Tyr71 side chain. A derivatization study using 6 as a lead molecule led to the development of highly potent inhibitors in which the structure-activity relationship as well as the binding mode of the compounds clearly differ from those of known amidine-type inhibitors.

Van-M-02, a novel glycopeptide, was revealed to exert potent activities against Gram-positive bacteria, including vancomycin-resistant enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA). A crude assay system was then used to study the mode of action of Van-M-02 as a peptidoglycan synthesis model of both vancomycin-susceptible and -resistant strains. The results suggested that Van-M-02 inhibits the synthesis of lipid intermediates irrespective of their termini. This inhibitory activity may contribute to the anti-VRE and anti-VRSA activities observed.

Betamethasone sodium phosphate (BSP) is usually used as a steroid therapy for human brain edema. High doses of BSP (36mg/kg) twice a day for two days statistically reduced the mortality rate and improved the survival period of Stx2 (1.4mug/kg; 1.6LD(50))-toxemic rabbits. We made evaluations on three kinds of magnetic resonance images (MRI) including T1-weighted, T2-weighted, and enhanced MRI using gadopentetate dimeglumine (Gd-DTPA) to detect brain lesion induced by an intravenous injection of Stx2 in rabbits. Enhanced T1-weighted MRI was the most sensitive tool to find leakage of Gd-DTPA suggesting impairment of the blood brain barrier caused by Stx2. Enhanced MRI revealed that BSP treatment inhibited the leakage of Gd-DTPA, as directly evidenced by the protective effect of BSP against brain edema induced by intravenous injection of Stx2. Interleukin 1beta was not induced after Stx2 treatment in brain primary mixed cell culture.

We have identified a series of novel non-peptide compounds that activate the thrombopoietin-dependent cell line Ba/F3-huMPL. The compounds stimulated proliferation of Ba/F3-huMPL in the absence of other growth factors, but did not promote proliferation of the thrombopoietin-independent parent cell line Ba/F3. The thrombopoietin-mimetic compounds elicited signal-transduction responses comparable with recombinant human thrombopoietin, such as tyrosine phosphorylation of the thrombopoietin receptor, JAK (Janus kinase) 2, Tyk2 (tyrosine kinase 2), STAT (signal transducer and activator of transcription) 3, STAT5, MAPKs (mitogen-activated protein kinases), PLCgamma (phospholipase Cgamma), Grb2 (growth-factor-receptor-bound protein 2), Shc (Src homology and collagen homology), Vav, Cbl and SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) and increased the number of CD41(+) cells (megakaryocyte lineage) in cultures of human CD34(+) bone-marrow cells (haematopoietic stem cells). These findings suggest that this series of compounds are novel agonists of the human thrombopoietin receptor and are possible lead compounds for the generation of anti-thrombocytopaenia drugs.

Shiga toxin 1 (Stx1) and Stx2 produced by Escherichia coli O157 are known to be cytotoxic to Vero and HeLa cells by inhibiting protein synthesis and by inducing apoptosis. In the present study, we have demonstrated that 10 ng/ml Stx2 induced DNA fragmentation in human brain microvascular endothelial cells (HBMEC), with cleavage activation of caspase-3, -6, -8, and -9. A microarray approach used to search for apoptotic potential signals in response to Stx2 revealed that Stx2 treatment induced a marked upregulation of C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible protein 153 (GADD153). Increased CHOP expression was dependent on enzymatically active Stx1. Knockdown of CHOP mRNA reduced the activation of caspase-3 and prevented apoptotic cell death. These results suggest that Stx2-induced apoptosis is mediated by CHOP in HBMEC and involves activation of both the intrinsic and extrinsic pathways of apoptosis.

UDP-N-acetylmuramic acid:L-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short L-alanine side chain. Purified H343Y and wild-type MurC had K(m) values for L-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their K(m) values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of L-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded L-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of L-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to L-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.

Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.

We constructed the expression vector pSK-SCP containing the streptococcal exotoxin B gene (spe b) which expressed protease activity. We showed that the recombinant streptococcal pyogenic exotoxin B/streptococcal cysteine protease (rSPE B/SCP) was secreted into the culture supernatant of the transformant and retained its SCP activity, which was equivalent to or greater than that of the naturally occurring molecule. The secreted rSPE B/SCP induced histamine release and degranulation of the human mast cell line HMC-1. This study may contribute to the understanding of the pathogenic role of SPE B/SCP in streptococcal infection and streptococcal toxic shock syndrome.

Objective: The aim of this study was to evaluate the toxicity of Shiga toxin (Stx) 1 and 2 on amniotic cells in vitro., Methods: WISH cells, which were derived from human amniotic cells, and Vero cells were cultured with or without Stxs. After 24 hours of culture, cell viability was measured by Cell Counting Kit-8, and extracted DNA was electrophoresed on a 1% agarose gel. The morphologic changes were observed by Papanicolaou staining, and the apoptotic index (percentage of apoptotic nuclei per total nuclei) was calculated. Quantification of apoptotic cells was also measured by an enzyme-linked immunosorbent assay., Results: The viability of WISH cells decreased in proportion to the concentrations of Stxs. Cellular ladder formation was observed by DNA electrophoresis of Stx-treated WISH cells, and the typical morphologic changes were observed by Papanicolaou staining. The proportion of apoptotic cells increased in response to Stxs., Conclusions: Stxs injured WISH cells directly and induced apoptosis in vitro. WISH cells were as sensitive as Vero cells to Stxs and cell death occurred by apoptosis.

The action of Shiga toxin (Stx) on the central nervous system was examined in rabbits. Intravenous Stx1 was 44 times more lethal than Stx2 and acted more rapidly than Stx2. However, Stx1 accumulated more slowly in the cerebrospinal fluid than did Stx2. Magnetic resonance imaging demonstrated a predominance of Stx1-dependent lesions in the spinal cord. Pretreatment of the animals with anti-Stx1 antiserum intravenously completely protected against both development of brain lesions and mortality.

Shiga toxin 2 (Stx2) is produced by enterohemorrhagic Escherichia coli (EHEC) and is known as the major virulence factor of EHEC. The aim of this study was to evaluate the effects of Stx2 on (i) maternal lethality, (ii) fetuses, (iii) delivery period, and (iv) maternal behavior after delivery. Timed pregnant ICR mice were injected intravenously with Stx2 on day 5 of pregnancy (early stage) or on day 15 (late stage). In early-stage experiments, the number of normal fetuses of mice injected with Stx2 was significantly lower than that of control mice. In late-stage experiments, mothers injected with Stx2 delivered normal numbers of neonates, but could not take care of them. The lethal doses of Stx2 were not different for pregnant and nonpregnant female mice at either stage. We conclude that Stx2 is toxic to the fetus in early pregnancy and affects maternal puerperal behavior in late pregnancy.

Escherichia coli clones expressing recombinant Shiga toxin (Stx)1-A and recombinant Stx1-B subunits, were established. Culture supernatants of these clones were examined for inhibitory activity on in vitro protein synthesis using luciferase as a reporter enzyme. Culture supernatant of the clone expressing Stx1-A, but not Stx1-B, showed the inhibitory activity. Neither recombinant Stx1-A nor Stx1-B showed Vero cell cytotoxicity. For reconstitution of biologically active toxin, the culture supernatants of the Stx1-A clone and the Stx1-B clone were mixed. The reconstituted recombinant Stx1 showed both Vero cell cytotoxicity and inhibition of in vitro protein synthesis.

Acute encephalopathy is the major determinant of death in an early stage of Shiga toxin (Stx)-producing Escherichia coli infection. Rapid progress toward refractory hypotension and dysfunction of breathing implies autonomic center dysfunction of patients. To clarify whether autonomic dysfunction becomes an ultimate cause of death in Shiga toxemia, we injected purified Stx2 (20 microg/kg) intravenously into rabbits, and monitored changes in cardiovascular and respiratory function together with renal sympathetic nerve activity (RSNA) in the conscious state. After an approximately 24-hour silent (lag) period, all rabbits given Stx2 developed hemorrhagic diarrhea (25.7 +/- 1.1 hours) and limb paralysis (31.2 +/- 1.3 hours). This limb paralysis was observed initially in the hind legs, and then it gradually extended to the forelegs. After 23.2 +/- 2.3 hours, RSNA increased gradually, and arterial blood pressure was maintained within normal limits together with an increase in the maximum gain of baroreflex response. Severe hypotension developed within 34.8 +/- 2.2 hours, without any increase in heart rate; RSNA significantly increased by 39.5 +/- 0.9 hours. In the final stage, RSNA decreased concurrently with decreases in arterial blood pressure, heart rate, and baroreflex response, suggesting dysfunction of the baroreflex control system. Thereafter, all rabbits died within 47.8 +/- 1.2 hours after the intravenous Stx2 injection. Magnetic resonance imagings of the central nervous system (T2-weighted images) showed high-intensity areas in the dorsal two-thirds of the cervical spinal cord and brainstem 48 hours after Stx2 administration. These results show that the cause of death is circulatory failure caused by impairment of the cardiovascular center in the medulla. We believe that this animal model helps to clarify the mechanism of rapid progress to death of patients with Shiga toxin-producing E. coli infection.

The initial brain lesions in rabbits given intravenous Shiga toxin 2 (Stx2) were noted at 24 h in an area around the third ventricle (Fujii et al., Infect Immun 1996, 64: 5053-60). This result implied that Stx2 is present in the cerebrospinal fluid (CSF) despite the fact that the toxin was administered intravenously. We measured Stx2 activity in CSF by using a Vero cell cytotoxicity assay at various times after an intravenous injection of Stx2. Stx2 was detected from 2 h after the injection, and its concentration in CSF remained at a high level for a further 6 h. Fifty percent lethal doses (LD 50) of Stx2 were measured in rabbits after intravenous and intrathecal Stx2 injections; The LD 50 after an intrathecal injection of Stx2 was 0. 36 microg/kg, which was 9.2-fold lower than that of an intravenous injection of Stx2 (3.4 microg/kg). Magnetic resonance images obtained after an intrathecal Stx2 injection (5 microg/kg) were compared with those obtained after an intravenous Stx2 injection (5 microg/kg). At 48 h, the cerebellar lesions had spread from the area in contact with the CSF on a T2-weighted image, which suggests that the intrathecal Stx2 may invade the cerebellum directly. We then examined whether anti-Stx2 antiserum injected intrathecally protects rabbits against brain damage. Eighty percent of the rabbits infected with Stx2 at 5 microg/kg died within 8 days from brain damage. Rabbit anti-Stx2 sera (with titres of x16 and x64 by the Ouchterlony precipitation method) were administered into the CSF space through the cisterna magna. All the rabbits ( n=10) survived when they were given an intrathecal injection of rabbit anti-Stx2 antiserum 2 h before the intravenous injection of Stx2. Our results suggest that a leakage of Stx2 into the CSF from the choroid plexus causes brain damage, and that an intrathecal injection of anti-Stx2 antiserum could be a therapy for acute encephalopathy caused by Stx2-producing Escherichia coli., (Copyright 1998 Academic Press)

The gene encoding a mitogenic factor, termed MF, was cloned from Streptococcus pyogenes and the recombinant MF was overexpressed in Escherichia coli. Both the natural and recombinant MF had heat-resistant nuclease activity. The nuclease activity of MF was characterized using the recombinant protein. MF showed endonuclease activity, digesting ssDNA, dsDNA and tRNA. The optimal pH for the DNase activity of MF was 9.5. The DNase activity was enhanced approximately tenfold by the simultaneous presence of two divalent cations, Mg2+ and Ca2+, compared to either alone and was inhibited by EDTA or NaCl. The heat stability of MF was biphasic; the DNase activity was heat-stable from 0 to 50 degrees C over 80 degrees C but very unstable at around 60 degrees C. DNA digested by MF possessed 5'-phosphorylated and 3'-hydroxylated termini, identical to those obtained by digestion of DNA by pancreatic deoxyribonuclease I. A mutant clone revealed that His122 was a residue essential to the nuclease activity.

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology and is associated with marked activation of T cells and monocyte macrophages, leading to the assumption that superantigens are involved in its pathogenesis. To determine if an association exists between streptococcal superantigens and KD, we examined serum antibody responses to superantigens in sera from 50 paired acute and convalescent KD patients using purified recombinant streptococcal superantigens, such as SPEA, SPEC, SSA and MF. We found a very low frequency of detection of anti-superantigen antibodies by ELISA and no marked IgG seroconversion to each superantigen, indicating the absence of a serological relationship between toxin-producing streptococcal infection and the onset of KD.

Human T cell activation by recombinant mitogenic factor (rMF) was investigated in comparison with that by recombinant streptococcal pyrogenic exotoxins (rSPE) A, B, and C. Recombinant MF, rSPEA, and rSPEC were mitogenic for peripheral blood mononuclear cells (PBMC), whereas rSPEB was not. Recombinant MF required only HLA-DR for the stimulation of PBMC, as determined using monoclonal antibodies (mAb) to HLA class II molecules and the mouse L cells transfected with HLA class II molecules. Recombinant SPEA and rSPEC required HLA-DR or HLA-DQ molecule. Recombinant MF selectively stimulated V beta 2, V beta 7, V beta 8, V beta 18 and V beta 21-bearing T cells, whereas rSPEA and rSPEC activated V beta 2 and V beta 6-bearing T cells as evaluated by the quantitative T cell receptor (TCR) analytical method. No clonality was observed in the nucleotide sequences of complementarity determining region 3 of TCR V beta in T cells responding to rMF. The profiles of cytokine production by PBMC in response to rMF, rSPEA, and rSPEC were quite similar. In summary, these results demonstrate that both HLA class II molecules and the TCR V beta required for rMF-mediated T cell activation are distinct from those required for rSPEA or rSPEC-mediated activation. Therefore, the MF is a novel streptococcal super-antigen which is different from SPEA, SPEB, and SPEC.

We recently cloned a gene encoding a new mitogenic factor (MF) from Streptococcus pyogenes NY-5. In the present study, we determined the distribution of this MF gene (mf) by PCR based upon its sequence. Of 371 streptococcal group A strains isolated from clinical specimens, 370 (99.7%) were positive for mf. The strain that was negative for the MF gene was also negative for the streptolysin O gene (slo). Some streptococcal strains belonging to groups C and G were negative for mf but positive for slo. Group B strains were negative for both. Furthermore, we examined the presence of mf in 54 strains belonging to 28 families and found mf only in group A streptococci. These results indicate that mf is distributed specifically in group A streptococci and the presence of mf in clinical samples strongly suggests infection with group A streptococci.

On the basis of the known streptolysin O (SLO) genomic sequence of Streptococcus pyogenes group A, we identified the SLO genes in some strains of group C and group G streptococci by the polymerase chain reaction procedure (PCR). The entire open reading frame region of these genes was cloned and analyzed. Their nucleotide sequence data showed that the defined SLO genes in group C and group G are almost identical to that of group A.

A new type of mitogenic factor, termed MF, has been found in the culture supernatant of Streptococcus pyogenes and its N-terminal amino acid sequence has been determined. On the basis of this sequence, an S. pyogenes gene encoding MF was cloned and its nucleotide sequence was determined. The MF gene includes a long, open reading frame with 813 nucleotides capable of encoding the MF precursor protein with 271 amino acids. Removal of the putative 43 residues as a signal peptide results in the mature MF protein with 228 amino acids. The molecular mass of the mature MF is calculated as 25,363 which is consistent with the previously determined value of 25,370 for MF secreted from S. pyogenes. Neither nucleotide nor amino acid sequence homology was found between the mature MF and other streptococcal pyrogenic exotoxins, such as SPE A, SPE B and SPE C. The mature MF was recombinantly overexpressed as a fusion protein with glutathione S-transferase in Escherichia coli. The recombinant protein showed mitogenic activity in rabbit peripheral blood lymphocytes and immunoreactivity with the rabbit antiserum raised against the secreted MF from S. pyogenes. These data indicate that a unique gene encoding MF was cloned from S. pyogenes.

Objectives: This study was performed to assess the length and contractile performance of human left ventricular papillary muscles and to determine the relation between papillary muscle dysfunction and mitral regurgitation., Background: Assessment of human papillary muscle contractility remains a clinical challenge., Methods: Two-dimensional echocardiographic examinations were performed in 16 normal subjects and 31 patients with prior myocardial infarction. Apical echocardiograms were used to obtain long-axis views of the anterior and posterior papillary muscles. The end-systolic and end-diastolic lengths of the papillary muscles were measured and fractional shortening was calculated., Results: Fractional shortening in normal subjects was 27 +/- 8% for the anterior papillary muscle and 30 +/- 8% for the posterior papillary muscle. In patients with prior myocardial infarction, a significant decrease in fractional shortening was observed in proportion to the severity of left ventricular wall motion abnormalities at the site of papillary muscle implantation. Moderate or severe mitral regurgitation was significantly more frequent in patients with combined anterior and posterior papillary muscle dysfunction than in those with isolated anterior or posterior dysfunction or with normal function of both papillary muscles (p < 0.05)., Conclusions: Two-dimensional echocardiography is useful for demonstrating abnormal contractility of human left ventricular papillary muscles. Papillary muscle contractility should be analyzed in each case to elucidate the mechanism of mitral regurgitation in patients with papillary muscle dysfunction.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.

The aim of this study is, first, to analyze pulmonary venous flow velocity (PVFV) pattern in normal subjects and second, to compare it with the various diseased state. PVFV was recorded in eleven normal volunteers, five patients with lone atrial fibrillation, twenty eight patients with valvular heart diseases and six patients with cardiomyopathy using transesophageal color Doppler echocardiography by placing the sample volume at the junction of the left superior pulmonary vein and left atrium. PVFV in normal subjects demonstrated distinct four waveforms: due to atrial systole (AS wave) and diastole (AD wave), and due to ventricular systole (VS wave) and diastole (VD wave). PVFV changed with respiration in normal subjects. The peak velocity of VD wave was increased with inspiration (p less than 0.001). The ratio of velocity VS/VD was increased during expiration (p less than 0.01). The ratio of area AD + VS/VD was significantly decreased with inspiration (p less than 0.01). We feel that this is the normal variation in pulmonary venous return during respiration , influenced by changes in the venous return on the right side of the heart. In all patients with atrial fibrillation, AS and AD waves were disappeared. The negative deflection occasionally observed was due to mitral valve closure. In patients with mitral stenosis, the peak velocity of VD wave was significantly decreased compared to that of normal subjects, but it was not significantly different between the patients with mitral valve replacement and normal subjects. The peak velocity of VD wave was also correlated with pressure half time among the patients with mitral stenosis, mitral valve replacement and mitral commissurotomy. On the other hand, it was significantly increased in patients with mitral regurgitation and returned to normal level after the operation. The peak velocity of VS wave was correlated with the left atrial dimension among the patients with mitral valve diseases except these with mitral regurgitation. In patients with mitral regurgitation, the peak velocity was decreased compared to that of normal subjects and reversed flow was seen in half of the patients. Also, it was decreased in patients with dilated cardiomyopathy and increased in patients with hypertrophic cardiomyopathy. In conclusion, PVFV is influenced not only by changes of venous return with respiration but also by the left atrial size, presence or absence of MS or MR, left atrial or left ventricular systolic and diastolic function in the various diseased states.

To ascertain the usefulness of continuous wave Doppler echocardiography in evaluating the severity of mitral regurgitation (MR), 29 patients with MR and 10 normal subjects were examined. The patients were categorized in three groups according to the angiographic evidence of severity of MR. To analyze the flow velocity patterns of MR, the time to peak velocity index (time from onset of MR signal to peak flow velocity/duration of MR signal), the A/B ratio (the ratio of the first and second half of the systolic MR signal area), systolic peak velocity, and diastolic peak velocity were measured using continuous wave Doppler echocardiograms. The velocity patterns of MR differed significantly among the three groups. With severer MR, the flow velocity pattern showed an earlier appearance of the peak in systole, a steeper decrease in systole and a greater increase in early diastole. The time to peak velocity index was 55 +/- 7% (mean +/- SD) in mild MR, 42 +/- 6% in moderate MR and 35 +/- 5% in severe MR. This index shortened significantly in accord with the severity of MR (mild vs moderate MR: p less than 0.001, moderate vs severe MR: p less than 0.05). The A/B ratio was 1.06 +/- 0.12 in mild MR, 1.23 +/- 0.10 in moderate MR and 1.41 +/- 0.07 in severe MR. This ratio increased significantly with the severity of MR (mild vs moderate MR: p less than 0.01, moderate vs severe MR: p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Purified Vero toxin 2 (VT2) was separated into A and B subunits by treatment with 6 M urea in 0.1 M propionic acid (pH 4.0). The isoelectric points of the isolated A and B subunits were determined to be 8.1 and 4.1, respectively. The A subunit of the purified VT2 was not nicked, but could be nicked in vitro by trypsin. Biologically active toxin was reconstituted from the isolated A and B subunits of VT2. Hybrid toxins with biological activity were obtained in vitro from the A subunit of Vero toxin 1 (VT1) and the B subunit of VT2, and from the A subunit of VT2 and the B subunit of VT1. The hybrid toxins showed similar cytotoxicity to native VT1 and VT2 on Vero cells. The in vitro formations of hybrid toxins were confirmed by polyacrylamide disc gel electrophoresis.

The A and B subunits of Shiga toxin were isolated by high performance liquid chromatography and their physicochemical properties were examined. The A subunit of Shiga toxin purified from culture supernatant was not nicked, but it could be nicked in vitro by trypsin. The isoelectric points of the A and B subunits were determined to be 8.2 and 5.8, respectively. Amino acid compositions of the two subunits were also determined. The isolated A and B subunits were reconstituted to form active holotoxin which showed lethal activity to mice which was similar to that of native Shiga toxin.

A cytotoxin to Vero cells (Shiga-like toxin), which was neutralized by antibody against purified Shiga toxin produced by Shigella dysenteriae 1, was purified from Escherichia coli O157:H7, isolated from a patient with hemorrhagic colitis. The purification procedure consisted of ammonium sulfate fractionation, DEAE-cellulose column chromatography, chromatofocusing column chromatography and high performance liquid chromatography. About 200 micrograms of purified Shiga-like toxin was obtained from cell extracts of 14 liters of culture with a yield of about 15%. The purified Shiga-like toxin showed identical physicochemical, biological and immunological properties to those of Shiga toxin. Purified Shiga-like toxin and Shiga toxin also had the same mobilities on polyacrylamide disc gel electrophoresis and polyacrylamide gel isoelectrofocusing. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, purified Shiga-like toxin migrated as two bands corresponding to the A and B subunits, and these migrated to the same positions as A and B subunits of Shiga toxin. The amino acid composition of the purified Shiga-like toxin was also similar to that of Shiga toxin. The purified Shiga-like toxin showed various biological activities: lethal toxicity to mice when injected intraperitoneally, the LD50 being 30 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 6 pg; and fluid accumulation in rabbit ileal loops at concentrations of more than 1.25 micrograms/loop. These values are comparable with those obtained with Shiga toxin. In an Ouchterlony double gel diffusion test, the lines formed by the purified Shiga-like toxin and Shiga toxin fused, indicating that the two toxins were immunologically identical.

A Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli O157:H7 was shown to inhibit protein synthesis in a rabbit reticulocyte lysate, but not in wheat germ or Ercherichia coli lysates. The toxin, VT2, inactivated 60S ribosomal subunits of rabbit reticulocytes. The site of inhibition of protein synthesis by VT2 was shown to be elongation factor 1-dependent aminoacyl-tRNA binding to ribosomes. VT2 did not affect Met-tRNAf binding to ribosomes, non-enzymatic binding of aminoacyl-tRNA to ribosomes, peptide bond formation or translocation.

The primary structures of the A and B subunits of Shiga toxin and of Shiga-like toxin I (VT1), isolated from the culture supernatants of Shigella dysenteriae 1 and Escherichia coli O157:H7, respectively, were analyzed by Edman degradation of intact proteins and peptides in their digests with trypsin or Achromobacter protease I and also by fast atom bombardment mass spectrometry of the digests. The results indicated that the A and B subunits of Shiga toxin and Shiga-like toxin I have the same primary structures. The identity of their primary structures was confirmed by determining the nucleotide sequence of the gene encoding Shiga-like toxin I cloned from a Shiga-like toxin I converting phage. This nucleotide sequence was different from that reported by Jackson et al. (Microbial Pathogenesis 1987; 2: 147-153), by Calderwood et al. (Proc Natl Acad Sci USA 1987; 84: 4364-8) and by Grandis et al. (J Bacteriol 1987; 169: 4313-9) in one base at position 231, which was found to be adenine instead of thymine, which they reported. The amino acid residue at position 45 from the N-terminus of the A subunit of Shiga-like toxin I deduced from the nucleotide sequence determined in this study is threonine, which corresponds with that found by amino acid sequencing, whereas from previous reports by other investigators it is serine. Edman degradation of the intact A subunit of Shiga toxin indicated that the A subunit was nicked between Ala253 and Ser254 to form A1 and A2 fragments linked by a disulfide bond.

A cytotoxin to Vero cells (Vero toxin) was purified from Escherichia coli O157:H7 isolated from a patient with hemorrhagic colitis by ammonium sulfate fractionation, DEAE-cellulose column chromatography, repeated chromatofocusing column chromatography and repeated high performance liquid chromatography. About 440 micrograms of purified Vero toxin was obtained from 12 liters of culture with a yield of about 22%. The purified Vero toxin showed similar cytotoxic activity to that of Shiga toxin to Vero cells and killed about 50% of the Vero cells at 1 pg. The activity was lost on heating the toxin at 80 degrees C for 10 minutes, but not at 60 degrees C for 10 minutes. The toxin also showed lethal toxicity to mice when injected intraperitoneally, the LD50 being 1 ng per mouse. The purified Vero toxin consisted of A and B subunits with molecular weights of about 35,000 and 10,700, respectively, which were slightly larger than those of Shiga toxin. On polyacrylamide gel disc electrophoresis, the mobility of the purified Vero toxin differed from that of Shiga toxin. The isoelectric point of the toxin was 4.1, which was also different from that of Shiga toxin (pI = 7.0). Furthermore, Vero toxin and Shiga toxin were found to be immunologically unrelated; anti-Vero toxin did not react with Shiga toxin, and similarly anti-Shiga toxin did not react with the Vero toxin in either the Ouchterlony double gel diffusion test or enzyme-linked immunosorbent assay. The Vero toxin purified in this work was found to be immunologically identical to VT2 and Shiga-like toxin II reported previously.

Shiga toxin was purified from the culture supernatant of Shigella dysenteriae 1 by ammonium sulfate fractionation, DEAE-cellulose column chromatography and repeated chromatofocusing column chromatography. About 1.6 mg of purified Shiga toxin was obtained from 15 liters of culture with a yield of about 27%. The molecular weight of purified Shiga toxin was estimated to be 62,000. The toxin consisted of A and B subunits with molecular weights of about 30,000 and 5,000-6,000, respectively. The isoelectric point of purified Shiga toxin was 7.0. Purified Shiga toxin showed the following biological activities: lethal toxicity to mice when injected intraperitoneally with an LD50 of 28 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 1 pg and all of the cells at 10 pg; and fluid accumulation in rabbit ileal loops at a concentration of more than 1 microgram.

An outbreak of acute enteritis in children aged one to thirty-three months occurred from June 10th to 23rd, 1986, at a private orphanage in Matsuyama City. Twenty-two out of 23 children suffered from diarrhea. Nine of the 22 children excreted bloody stool. Fever and vomiting were observed with a few patients. One of them, a 33-month-old girl, developed hemolytic uremic syndrome, and died twelve days after the admission. Escherichia coli O111:H- was isolated from fecal specimens of 7 out of 15 patients. The culture filtrate of the isolate caused fluid accumulation in ligated ileal loops in a rabbit, and was lethal to mice. It was found that all isolates produced two kinds of Vero toxins (VT1 and VT2, or shiga-like toxin I and II). The amount of VT2 produced in vitro was about 10 times more than that of VT1.

A cytotoxin to Vero cells (Vero toxin), which was immunologically related to Shiga-like toxin II (SLT-II) (or VT2), was purified from a stain of Escherichia coli isolated from a patient with hemolytic uremic syndrome. The toxin was active on Vero cells but much less active on HeLa cells, a property similar to that of the recently identified SLT-II variant from E. coli strains that caused edema disease of swine. Thus the toxin purified in this report was tentatively named Shiga-like toxin II variant (Vero toxin 2 variant). The purification procedures consisted of ammonium sulfate fractionation, DEAE-Sepharose CL-6B column chromatography, chromatofocusing column chromatography, and repeated high performance liquid chromatography (HPLC) on TSK-gel G-2000SW column and on TSK-gel DEAE-5PW columns. About 90 micrograms of purified toxin was obtained from 451 of the culture supernatant with a yield of about 16%. The purified toxin consisted of A and B subunits of molecular sizes similar to those of SLT-II (VT2). The isoelectric point of the purified toxin was 6.1, which was different from that of SLT-II (VT2) (pI = 4.1). In an Ouchterlony double gel diffusion test, purified toxin and SLT-II (VT2) formed precipitin lines with spur formation against anti-purified toxin and anti-SLT-II (anti-VT2), respectively. The purified toxin was cytotoxic to Vero cells, about 6 pg of the toxin killing 50% of the Vero cells, and showed lethal toxicity to mice when injected intraperitoneally, the LD50 being about 2.7 ng per mouse.

The site of action of a Vero toxin (VT2 or Shiga-like toxin II) from enterohemorrhagic Escherichia coli and Shiga toxin from Shigella dysenteriae 1 on eukaryotic ribosomes was studied. Treatment of eukaryotic ribosomes with either toxin caused the release of a fragment of 400 nucleotides from 28S ribosomal RNA when the isolated ribosomal RNA was treated with aniline. Release of this fragment with aniline treatment was accompanied by inhibition of protein synthesis and of elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes. Analysis of the nucleotide sequence of the 3'-terminal fragment of 553 nucleotides of 28S rRNA of rat liver 60S ribosomal subunits suggested that an adenine base at position 4324 (A-4324) was absent in toxin-treated 28S rRNA. Further analysis by thin-layer chromatography demonstrated quantitative release of adenine from rat liver ribosomes on treatment with the toxins. These results indicate that both VT2 and Shiga toxin inactivate 60S ribosomal subunits by cleaving the N-glycosidic bond at A-4324 in 28S ribosomal RNA.