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H E Smith

Publications and source records attributed to H E Smith.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor.

To study the role of the capsule of Streptococcus suis serotype 2 in virulence, we generated two isogenic mutants disturbed in capsule production. For that purpose, we first cloned and characterized a major part of the capsular polysaccharide biosynthesis (cps) locus of S. suis serotype 2. Based on the established sequence, 14 open reading frames (ORFs), designated Orf2Z, Orf2Y, Orf2X, and Cps2A to Cps2K, were identified. Twelve ORFs belonged to a single transcriptional unit. The gene products of 11 of these ORFs showed similarity to proteins involved in polysaccharide biosynthesis of other gram-positive microorganisms. Nonencapsulated isogenic mutants were generated in the cps2B and cps2EF genes by insertional mutagenesis. In contrast to the wild-type S. suis serotype 2 strain, the nonencapsulated strains were highly sensitive to ingestion by porcine alveolar lung macrophages in vitro. More importantly, the nonencapsulated mutant strains were completely avirulent in young germfree pigs after intranasal inoculation. These observations indicate that the capsule of S. suis serotype 2 plays an essential role in the pathogenesis of S. suis serotype 2 infections.

Animals↗
PubMed1999

The cps genes of Streptococcus suis serotypes 1, 2, and 9: development of rapid serotype-specific PCR assays.

We developed three type-specific PCR assays for the rapid and sensitive detection of Streptococcus suis serotype 1 (plus 14), serotype 2 (plus 1/2), and serotype 9 strains in tonsillar specimens from pigs. The PCR primers were based on the sequences of type-specific capsular genes of S. suis serotype 1, 2, and 9 strains. We recently characterized a major part of the capsular biosynthesis (cps) locus of S. suis serotype 2. Here we extended these studies and characterized major parts of the cps loci of S. suis serotypes 1 and 9. Type-specific genes were identified by cross-hybridization experiments between the individual cps genes and chromosomal DNAs from the 35 different serotypes. Four genes of S. suis serotype 1 specifically hybridized with serotype 1 and 14 strains only. Five genes of S. suis serotype 2 specifically hybridized with serotype 2 and 1/2 strains only, and two genes of S. suis serotype 9 specifically hybridized with serotype 9 strains. Until now rapid and sensitive diagnostic tests were available only for pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1. The serotype-specific PCR assays can therefore be useful tools for the identification of serotype 1, 14, 2, 1/2, and 9 strains both for diagnostic purposes and in epidemiological and transmission studies. Therefore, these tests may facilitate control and eradication programs.

Base Sequence↗
PubMed1998

Identification of protein-protein interactions of the major sperm protein (MSP) of Caenorhabditis elegans.

In nematodes, sperm are amoeboid cells that crawl via an extended pseudopod. Unlike those in other crawling cells, this pseudopod contains little or no actin; instead, it utilizes the major sperm protein (MSP). In vivo and in vitro studies of Ascaris suum MSP have demonstrated that motility occurs via the regulated assembly and disassembly of MSP filaments. Filaments composed of MSP dimers are thought to provide the motive force. We have employed the yeast two-hybrid system to investigate MSP-MSP interactions and provide insights into the process of MSP filament formation. Fusions of the Caenorhabditis elegans msp-142 gene to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) interact to drive expression of a lacZ reporter construct. A library of AD-MSP mutants was generated via mutagenic PCR and screened for clones that fail to interact with LEXA-MSP. Single missense mutations were identified and mapped to the crystal structure of A. suum MSP. Two classes of mutations predicted from the structure were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface. Multiple additional mutations were obtained in the two carboxy-terminal beta strands, a region not predicted to be involved in protein folding or dimer formation. Size fractionation of bacterially expressed MSPs indicates that mutations in this region do not abolish dimer formation. A number of compensating mutations that restore the interaction also map to this region. The data suggest that the carboxy-terminal beta strands are directly involved in interactions required for MSP filament assembly.

Amino Acid Sequence↗
PubMed1998

The TAPS Project 41: homes for life? Residential stability five years after hospital discharge. Team for the Assessment of Psychiatric Services.

This paper describes pathways followed by 567 long-stay patients who were resettled into the community, as part of a program to close two psychiatric hospitals in London and replace them with community-based services. Sixty-one percent of the former patients remained in their original placement--mostly group homes--over the five-year follow-up. Transition from one house to another mostly took a direct course, with only 27 patients changing houses by way of long intermediate hospitalisation. Change of residence was at the same level of support, with only a slight trend towards less supervised facilities. Ten patients could not be traced and have possibly become homeless. Only 3 patients were in prison during the five-year follow-up. More than a third of the sample were readmitted at least once during that period. It is concluded that community residences established under the resettlement program served as relatively stable homes for the majority of patients.

Adult↗
PubMed1998

Parental and professional perception of need for emergency admission to hospital: prospective questionnaire based study.

AIM: To compare views of parents, consultants, and general practitioners on severity of acute illness and need for admission, and to explore views on alternative services. METHOD: Prospective questionnaire based study of 887 consecutive emergency paediatric admissions over two separate three week periods in summer and winter of five Yorkshire hospitals, combined with a further questionnaire on a subsample. OUTCOME MEASURES: Parental scores of need for admission and parent and consultant illness severity scores out of 10. Consultant judgment of need for admission. Alternatives to admission considered by consultants and, for a subsample, by parents and family GP. RESULTS: Ninety nine per cent of parents thought admission was needed. Parents scored need for admission more highly than severity of illness with no association observed between severity and presenting problem or diagnosis. High parental need score was associated with a fit, past illness, and length of stay. Consultant illness severity scores were skewed to the lower range. Consultants considered admission necessary in 71%, especially for children aged over 1 year, presentation with breathing difficulty or fit, and after a longer stay. More admissions in the evening were considered unnecessary as were admissions after longer preadmission illness, gastroenteritis, or upper respiratory tract infection. Of a subsample of parents, 81% preferred admission during the acute stage of illness even if home nursing had been available. Similar responses were obtained from GPs. Alternative services could have avoided admission for 19% of children, saving 15.6% of bed days used. CONCLUSIONS: Medical professionals and parents differ in their views about admission for acute illnesses. More information is needed on children not admitted. Alternative services should take account of patterns of illness and should be acceptable to parents and professionals; cost savings may be marginal.

Acute Disease↗
PubMed1998

Medical and social factors associated with the admission and discharge of acutely ill children.

AIM: To examine medical and sociodemographic factors involved in acute paediatric admission. To compare outcome of admission with factors present at time of admission. METHODS: Prospective questionnaire based study of 887 consecutive emergency general paediatric admissions to five Yorkshire hospitals during two separate three week periods in summer and winter. MAIN OUTCOME MEASURES: Discharge diagnosis, length of stay. RESULTS: Most admissions (53%) occurred "out of hours" with a peak during the evening. Two thirds (64%) of patients were under 3 years of age and clinical problems varied with age. Self referral via an accident and emergency department occurred in one third and was more likely after a fit in older children and in more socioeconomically deprived children. The most frequent presenting problems were breathing difficulty (24%), fit (16%), and feverish illness (15%). One quarter (24%) were discharged within 24 hours and 61% spent, at most, one night in hospital. Length of stay was shorter for night admissions and longer for children with a discharge diagnosis of asthma. Although most children had mild, self limiting illnesses, serious illness was subsequently found in 13% and could not be predicted from the presenting problems. CONCLUSIONS: Current demand on emergency paediatric admission is mainly from young children with mild self limiting illnesses who spend one night or less in hospital. Changes in delivery of care to acutely ill children must take account of the pattern and nature of presenting problems and be rigorously audited to ensure that improvements in the health of children continue.

Acute Disease↗
PubMed1997

Virulence of Streptococcus suis type 2 for mice and pigs appeared host-specific.

A murine model for Streptococcus suis infection in pigs was validated by inoculating groups of 5 BALB/c and 5 CF1 mice with 10(7) CFU/ml of 13 different S. suis serotype 2 strains. The pathogenicity of these strains had been established in a standardized pig model of S. suis infection using one-week-old gnotobiotic pigs. We inoculated groups of mice intraperitoneally with 4 strains that were highly virulent for pigs and belonged to the phenotype MRP+EF+, with 4 strains, that were weakly virulent for pigs and belonged to the phenotype MRP+EF+, and with 5 strains that were non-virulent for pigs and belonged to phenotype MRP-EF-. The S. suis strains that were highly virulent for pigs caused high morbidity and an intermediate mortality in mice, the S. suis strains that were weakly virulent for pigs caused high morbidity but low mortality, and the strains that were non-virulent for pigs, induced highest morbidity and mortality. These results were comparable in both breeds of mice. In contrast to the pathology of S. suis infection in pigs with specific lesions, lesions in mice were histologically often characterized as non-specific, i.e., necrotizing encephalitis and focal or diffuse hepatitis sometimes with abscesses. Irrespective of breed (BALB/c vs. CF1), the murine model used for S. suis infection was incompatible with the pig model. This indicates that virulence of S. suis type 2 for mice and pigs is host-specific. Therefore, we regard the presently available murine models unsuitable for studying S. suis infections in pigs.

Animals↗
PubMed1997

Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile.

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.

Bacterial Typing Techniques↗
PubMed1996

Characterization of virulence of the Streptococcus suis serotype 2 reference strain Henrichsen S 735 in newborn gnotobiotic pigs.

Strain Henrichsen S 735 (NCTC 10234) of Streptococcus suis serotype 2 reference and three other such strains (strains S 4005, S 3921 and T 141) were tested for virulence by inoculating pigs intranasally and intravenously. The taxonomical properties of each strain were determined. Phenotypes were determined by Western blotting based on MRP and EF protein expression and genotypes were determined by Southern hybridization analysis of the mrp and epf genes. Reference strain S 735 and strain S 3921 produced the 136 kDa MRPh and a 180 kDa form of EF, and hence these strains belong to the MRP + EF phenotype. In accordance with previous experiments with this phenotype, strains S 735 and S 3921 appeared to be only weakly virulent for newborn gnotobiotic pigs. Strain S 4005 produced the 136 kDa MRP and the 110 kDa form of EF, hence it belongs to the MRP + EF + phenotype. This strain was highly virulent for pigs. Strain T 141 did not produce MRP or EF, and hence belongs to the MRP-EF- phenotype. It was nonvirulent for pigs. The route of inoculation did not influence the frequency or severity of clinical signs of disease or lesions, which demonstrated that the 110 kDa EF is not essential during invasion. Southern blot analysis showed that all four S. suis type 2 strains contain sequences that are homologous to the epf and mrp genes. For studies on pathogenesis of S. suis type 2 infections in pigs, we recommend the use of strains that have been tested in a standardized pig model and that belong to the MRP + EF + phenotype, such as strain S 4005.

Administration, Intranasal↗
PubMed1996

Histopathological changes in the lymphoid tissues of sheep exposed to the bovine immunodeficiency-like virus.

Six yearling sheep were inoculated intraperitoneally with peripheral blood from two sheep infected with the bovine immunodeficiency-like virus (BIV) strain R29. An additional five sheep served as sham-inoculated controls. Of the six sheep given BIV, five seroconverted, one of them remaining seropositive for the duration of the study. The polymerase chain reaction demonstrated BIV provirus in three of the five serologically positive animals. At necropsy, 1 year after inoculation, histological changes were found only in the lymphoid tissues. In sheep exposed to BIV, mild though significant increases were seen in the (1) number of splenic periarteriolar lymphoid sheaths, (2) number of secondary follicles in hilar and prescapular or popliteal lymph nodes, and (3) medullary sinus cellularity in prescapular and popliteal lymph nodes.

Animals↗
PubMed1996

Radiation protection systems for the final focus test beam at SLAC.

The Final Focus Test Beam (FFTB) is a new beam line at the Stanford Linear Accelerator Center designed to test new beam optics concepts, hardware, and techniques necessary to achieve and measure the small spot sizes required for future generations of high-energy e+e- linear colliders. The FFTB takes a 47 GeVc-1, 1 kW electron beam at the end of the Stanford Linear Accelerator Center linear accelerator and transports it to the FFTB beam dump. A radiation protection system was designed and installed for the FFTB with the primary goal that the integrated dose equivalent outside the shielding resulting from beam loss would not exceed 10 mSv y-1. This system is comprised of shielding, a beam containment system and a personnel protection system. This paper presents various aspects of radiation safety at Stanford Linear Accelerator Center that were considered in the design of the FFTB radiation protection system. Beam tests were conducted in which the performance of various beam containment devices and the shielding effectiveness were evaluated. Preliminary results from these tests are presented.

California↗
PubMed1996

Mutants of Streptococcus suis types 1 and 2 impaired in expression of muramidase-released protein and extracellular protein induce disease in newborn germfree pigs.

The contribution of muramidase-released protein (MRP) and extracellular factor (EF) to the virulence of Streptococcus suis type 1 and 2 infections was studied. For that aim, we constructed mutants of S. suis types 1 and 2 by inactivating the genes encoding MRP and EF. Moreover, we changed a type 2 strain producing the 110-kDa EF protein into a strain producing a modified protein (EF*) of increased molecular mass. The chromosomally located mrp and epf genes were inactivated by replacement recombination by using nonreplicative plasmids. Newborn germfree pigs were inoculated with pathogenic type 1 and 2 strains and with the isogenic mutant strains. Wild-type as well as mutant strains induced fever, specific signs of disease, and lesions. Moreover, all mutant strains could be reisolated from the central nervous system of infected pigs. These results showed that inactivation or alteration of the mrp and epj genes had no measurable effect on the pathogenicity of S. suis types 1 and 2.

Animals↗
PubMed1995

Binding of 5H-dibenzo[a,d]cycloheptene and dibenz[b,f]oxepin analogues of clozapine to dopamine and serotonin receptors.

Series of 5,11-dicarbo- and 11-carbo-5-oxy-10-(1-alkyl-1,2,3,6-tetrahydro-4 pyridinyl) analogues and a 11-carbo-5-oxy-10-(1-methyl-4-piperidinyl) analogue of the atypical antipsychotic agent clozapine were prepared and tested for binding to the dopamine D-2L and D-4 and serotonin S-2A and S-2C receptors. Some of these analogues were found to have dopamine D-2L and D-4 and serotonin S-2A and S-2C receptor binding activities as high as or higher than those of clozapine, indicating that neither the diazepine structure nor the piperazine ring present in clozapine is essential for high antidopamine activity and or for high dopamine D-4 selectivity (Ki for the dopamine D-2L receptor/Ki for the dopamine D-4 receptor). Increasing in the effective size of the alkyl substituent at the tertiary amine nitrogen atom in the 1,2,3,6-tetrahydro-4-pyridinyl moiety in the 5H-dibenzo[a,d]cycloheptene series reduces the affinity for the dopamine D-4 receptor, but in the dibenz[b,f]oxepin series, no significant change in binding affinity to the dopamine D-4 receptor was observed. Equal or slightly higher affinity for the serotonin S-2A and S-2C receptors was observed for the 10-(1-ethyl-1,2,3,6-tetrahydro-4- pyridinyl) analogues in both series, but for the 10-[1,2,3,6-tetrahydro-1-(2-propenyl)-4- pyridinyl] analogues, any favourable steric factor is overshadowed by an unfavorable electronic effect as a result of change in the basicity of the tertiary amino group in the pyridinyl moiety. Replacement of three of the four nitrogen atoms in clozapine with three carbon or two carbon atoms and an oxygen atom and removal of the chlorine atoms gives 10-(1,2,3,6-tetrahydro-1- methyl-4-pyridinyl)dibenzo[a,d]cycloheptene and 10-(1-methyl-4-piperidinyl)dibenz[b,f]oxepin, each having twice the binding activity to the dopamine D-4 receptor as does clozapine and a dopamine D-4 selectivity equal to that of clozapine.

Binding Sites↗
PubMed1995

Active-site-directed photoaffinity radioiodination of androgen-binding proteins.

Androgen-binding protein (ABP) in rat epididymal cytosol and sex hormone-binding globulin (SHBG) in rabbit serum and SHBG purified from human serum were active-site-directed photoaffinity radiolabeled with 17 alpha-[(E)-2-[125I]iodoethenyl]androstan-4,6-dien-17 beta-ol-3-one ([125I]1). The interaction of this compound with binding components in epididymal cytosol was dependent on exposure of the mixture to ultraviolet light and on the duration of exposure. Photolysis in the presence of [125I]1 and 5 alpha-dihydrotestosterone (5 alpha-DHT) resulted in a 40% inhibition of binding of [125I]1 to cytosolic components. These result indicate that, while [125I]1 interacted with 5 alpha-DHT binding sites, it also formed adducts with other sites. To characterize the labeled species, the photolysis mixture was subjected to electrophoresis under denaturing and reducing conditions. Autoradiography of the gel revealed that ABP and SHBG were labeled with [125I]1, but in cytosol and serum, higher and lower molecular weight components were also labeled. Purified SHBG was labeled, but no labeled contaminating protein was detected. The presence of 5 alpha-DHT completely inhibited [125I]1 photolabeling of human and rabbit SHBG and of ABP. However, in cytosol, the presence of 5 alpha-DHT also eliminated photolabeling to a component that may be albumin, but 5 alpha-DHT did not affect [125I]1 photolabeling of other contaminating proteins in cytosol. Thus, while [125I]1 is an effective photoaffinity radiolabel for ABP and SHBG, the observation that it also photolabels other proteins limits its practical use to the radiolabeling of purified ABP and SHBG preparations.

Affinity Labels↗
PubMed1995

High-efficiency transformation and gene inactivation in Streptococcus suis type 2.

An efficient electrotransformation system for Streptococcus suis type 2 is described. It is demonstrated that vectors based on the broad-host-range plasmid pWVO1 replicate in S. suis type 2. Transformation efficiencies of about 10(7) transformants per micrograms of plasmid DNA could be obtained. Derivatives of plasmid pBR322 containing S. suis chromosomal DNA did not replicate but integrated into the chromosome. Southern hybridization analysis revealed that double as well as single cross-over integration events had occurred. Double cross-over events occurred at a frequency of about 15%. With these transformation and integration systems, recombinant DNA technology can now be applied to this important pathogenic species.

Blotting, Southern↗
PubMed1995

Knockout mutants of Actinobacillus pleuropneumoniae serotype 1 that are devoid of RTX toxins do not activate or kill porcine neutrophils.

The Actinobacillus pleuropneumoniae RTX-toxins ApxI, ApxII, and ApxIII are important virulence factors of this swine pathogen. It is hypothesized that the Apx toxins are deleterious to defense cells of the host, enabling the bacterium to infect the host. To confirm this, we studied the effect on porcine polymorphonuclear neutrophils of mutant strains of A. pleuropneumoniae that were devoid of Apx toxins. For this purpose, we developed a system for targeted mutagenesis of A. pleuropneumoniae based on the conditionally replicating plasmid pVE6063 and insertional mutagenesis by homologous recombination. Employing this system on the reference strain of serotype 1, a strain that secretes ApxI and ApxII, we generated mutant strains that were devoid of ApxI and/or ApxII. We compared the ability of the parent strain and the mutant strains to provoke an oxidative burst in porcine neutrophils and to kill these cells. The parent strain and mutants that secreted either ApxI or ApxII provoked an oxidative burst and killed the neutrophils, whereas mutant strains that were devoid of ApxI and ApxII did not. These experiments indicate the importance of ApxI and ApxII to these profound effects on neutrophils and emphasize the importance of ApxI and ApxII in pathogenesis.

Actinobacillus pleuropneumoniae↗