Biomedical subjects

G M Young

Publications and source records attributed to G M Young.

At least 19 recordsLinked to original sources

PubMed2001

Identification of a locus involved in systemic dissemination of Yersinia enterocolitica.

A putative LysR-type transcriptional activator, Hre20, was identified previously in an in vivo expression technology screen designed to identify factors which are expressed early during infection by Yersinia enterocolitica (G. M. Young and V. L. Miller, Mol. Microbiol. 25:319-328, 1997). An insertion in hre20, now designated rscR, resulted in increased splenic dissemination of bacteria during infection in a BALB/c mouse model. A nonpolar mutation was generated in rscR, and examination of this strain in the BALB/c mouse model demonstrated that the mutation in rscR was responsible for the increased dissemination to the spleen that was seen in the original experiments. RscR is homologous to the LysR family of transcriptional regulators; thus, a screen was undertaken to identify genes regulated by RscR. A strain containing an insertion in the chromosomal rscR gene and carrying rscR on a plasmid under the control of the inducible araBAD promoter was mutagenized with an mTn5Km-2 transposon containing a promoterless lacZY. Eighteen insertions were identified which appeared to respond to levels of RscR, and these were classified into four allelic groups based on Southern blot hybridization analysis. Representative members were sequenced from three allelic groups. Sequencing revealed insertions in an ORF with no known homologues, a homologue of OmpF of Serratia marcescens, and a locus (designated rscBAC) with similarity to the hmwABC locus of Haemophilus influenzae. The hmwABC locus promotes adherence of H. influenzae to host cells (S. J. Barenkamp and J. W. St. Geme III, Infect. Immun. 62:3320-3328, 1994; J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875-2879, 1993). A strain containing a deletion mutant of rscA, the hmwA homologue, exhibits increased splenic dissemination of bacteria during infection in a BALB/c mouse model, similar to the rscR mutant. This suggests that the phenotype of an rscR mutant is due to the loss of RscA.

Animals↗

PubMed2001

HreP, an in vivo-expressed protease of Yersinia enterocolitica, is a new member of the family of subtilisin/kexin-like proteases.

The role of proteases in pathogenesis is well established for several microorganisms but has not been described for Yersinia enterocolitica. Previously, we identified a gene, hreP, which showed significant similarity to proteases in a screen for chromosomal genes of Y. enterocolitica that were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like inv, the gene encoding the invasin protein of Y. enterocolitica, hreP is located in a cluster of flagellum biosynthesis and chemotaxis genes. The genomic organization of this chromosomal region is different in Escherichia coli, Salmonella, and Yersinia pestis than in Y. enterocolitica. Analysis of the distribution of hreP between different Yersinia isolates and the relatively low G+C content of the gene suggests acquisition by horizontal gene transfer. Sequence analysis also revealed that HreP belongs to a family of eukaryotic subtilisin/kexin-like proteases. Together with the calcium-dependent protease PrcA of Anabaena variabilis, HreP forms a new subfamily of bacterial subtilisin/kexin-like proteases which might have originated from a common eukaryotic ancestor. Like other proteases of this family, HreP is expressed with an N-terminal prosequence. Expression of an HreP-His(6) tag fusion protein in E. coli revealed that HreP undergoes autocatalytic processing at a consensus cleavage site of subtilisin/kexin-like proteases, thereby releasing the proprotein.

Amino Acid Sequence↗

PubMed2000

Motility is required to initiate host cell invasion by Yersinia enterocolitica.

Invasin-mediated invasion of host cells by the pathogen Yersinia enterocolitica was shown to be affected by flagellar-dependent motility. Motility appears to be required to ensure the bacterium migrates to and contacts the host cell. Nonmotile strains of Y. enterocolitica were less invasive than motile strains, but the reduction in invasion could be overcome by artificially bringing the bacteria into host cell contact by centrifugation. Mutations in known regulatory genes of the flagellar regulon, flhDC and fliA, resulted in less inv expression but did not have a significant effect on invasin levels. However, invasin levels were reduced for strains that harbored flhDC on a multicopy plasmid, apparently as a result of increased proteolysis of invasin.

Adhesins, Bacterial↗

PubMed2000

The Yersinia enterocolitica phospholipase gene yplA is part of the flagellar regulon.

Yersinia enterocolitica yplA encodes a phospholipase required for virulence. Virulence genes are often regulated in response to environmental signals; therefore, yplA expression was examined using a yplA::lacZY transcriptional fusion. Maximal yplA expression occurred between pH 6.5 and pH 7.5 and was induced in the mid-logarithmic growth phase. Potential Fnr, cyclic AMP (cAMP)-cAMP receptor protein (Crp), and sigma(F) regulatory sites were identified in the nucleotide sequence. Reduction of yplA expression by aeration, addition of glucose and sucrose, and application of high temperature and salt is consistent with Fnr-, cAMP-Crp-, and sigma(F)-mediated regulation, respectively. Expression of yplA was reduced in flhDC and fliA null strains, indicating that yplA is part of the flagellar regulon.

Bacterial Proteins↗

PubMed2000

Selective apoptosis within the rat subependymal zone: a plausible mechanism for determining which lineages develop from neural stem cells.

During development, the output of the subventricular zone (SZ) becomes increasingly restricted, yet it still harbors multipotential progenitors. The output of the SZ could be gated by selectively eliminating inappropriately specified progenitors. Using in situ end-labeling (ISEL) to identify apoptotic cells, nearly 60% of the ISEL(+) cells in the juvenile forebrain were localized to the SZ. Of these dying cells, at least 9% could be identified as neurons, 4% as astrocytes, and 12% as oligodendrocytes. The remainder were negative for the stem cell marker nestin, as well as other markers evaluated. To test the hypothesis that committed progenitors were under selective pressures, neural stem/progenitor cells were allowed to differentiate in vitro in the presence or absence of the caspase 3 inhibitor z-DEVD-fmk. DEVD increased neuronal production 10-fold over control cultures. By contrast, the development of astrocytes and oligodendrocytes was not affected. Altogether, these data support the hypothesis that selective forces within the postnatal rat forebrain control the types of precursors that emerge from the germinal matrix. Furthermore, they suggest that different mechanisms control neuronal versus glial cell numbers.

Animals↗

PubMed1999

Cycling cells in the adult rat neocortex preferentially generate oligodendroglia.

Gliogenesis in the mammalian central nervous system does not cease abruptly like neurogenesis. Instead, glia accumulate over a time period that extends into adulthood. To determine whether new glial cells in the adult cortex arise from resident progenitors and to determine the glial types to which these progenitors give rise to, cells in the perinatal subventricular zone (SVZ) were labeled with replication-deficient retroviral vectors, and clonal clusters of glia in the neocortex were examined from 1 week to 8 months of age. The average clonal cluster size increased during the first month of life. Interestingly, clusters containing oligodendrocyte lineage cells preferentially expanded with age, on average doubling every 3 months. Unexpectedly, the number of cells in astrocyte clusters decreased over time. In heterogeneous clusters, the numbers of oligodendroglia increased, whereas the number of astrocytes did not. Moreover, clonal clusters containing mature glia also contained less mature cells, indicating that clonally related progenitors do not differentiate synchronously in vivo. Thus, progenitors from the SVZ continue to cycle, resulting in an accumulation of oligodendroglia in the neocortex. These slowly cycling cells likely express the NG2 proteoglycan because a subset of the clonal clusters contained NG2(+) cells and these NG2(+) cells accumulated with time.

Animals↗

PubMed1999

A new pathway for the secretion of virulence factors by bacteria: the flagellar export apparatus functions as a protein-secretion system.

Biogenesis of the flagellum, a motive organelle of many bacterial species, is best understood for members of the Enterobacteriaceae. The flagellum is a heterooligomeric structure that protrudes from the surface of the cell. Its assembly initially involves the synthesis of a dedicated protein export apparatus that subsequently transports other flagellar proteins by a type III mechanism from the cytoplasm to the outer surface of the cell, where oligomerization occurs. In this study, the flagellum export apparatus was shown to function also as a secretion system for the transport of several extracellular proteins in the pathogenic bacterium Yersinia enterocolitica. One of the proteins exported by the flagellar secretion system was the virulence-associated phospholipase, YplA. These results suggest type III protein secretion by the flagellar system may be a general mechanism for the transport of proteins that influence bacterial-host interactions.

Anti-Bacterial Agents↗

PubMed1999

The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility.

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.

Bacterial Proteins↗

PubMed1997

An improved method for propagating oligodendrocyte progenitors in vitro.

Many investigators studying oligodendrocytes in vitro have sought out cell lines because it has been difficult to obtain sufficient numbers of primary oligodendrocytes for study. This paper describes three methodological improvements that facilitate culturing oligodendrocytes. We show that by detaching progenitor cells using papain instead of trypsin the total yield of oligodendrocyte progenitors can be doubled. We also show that papain can be used to subculture differentiated oligodendrocytes. Finally we report that primary O-2A progenitors can be cryo-preserved, reducing the demand upon laboratory personnel to produce and propagate them.

Animals↗

PubMed1997

Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis.

Pathogenic species of the genus Yersinia have a marked tropism for lymphoid tissue during the early stages of infection. Bacterial survival at this site determines whether the disease is localized or progresses systemically, leading to a high rate of mortality. Several plasmid-encoded virulence genes are known to be required for survival and pathogenesis, but the contribution of chromosomal genes has been largely unexplored. This study represents the first intensive effort to characterize and determine the function of Yersinia chromosomal genes expressed in lymphoid tissue after intragastric infection. Strains harbouring cat fusions expressed in the host were isolated from Peyer's patch tissue of mice intragastrically infected and treated with chloramphenicol (Cm); genes identified in this manner were designated hre for host responsive element. The hre::cat strains that were Cm resistant in vivo (in mouse tissue) and Cm sensitive in vitro (on laboratory media at 26 degrees C) were identified and shown to consist of 61 different allelic groups. The hre::cat fusions from 48 of the allelic groups were cloned and characterized by DNA sequence analysis. The results identified genes necessary for iron acquisition, protection from environmental stresses, biosynthesis of cell envelope components and other diverse metabolic activities. However, the DNA sequence of many clones had no homology to other known genes. Insertion mutations were constructed for four hre genes and the resulting Y. enterocolitica mutants were tested in the mouse model for effects on pathogenesis. All of the mutant strains were affected for virulence when assayed for survival in host tissues and LD50 analysis.

Animals↗

PubMed1997

A prospective, randomized study comparing intramuscular midazolam with intravenous diazepam for the treatment of seizures in children.

OBJECTIVE: To compare treatment of ongoing seizures using intramuscular (IM) midazolam versus intravenous (IV) diazepam. DESIGN: Controlled clinical trial. PATIENTS: Children with motor seizures of at least 10 minutes' duration. MAIN OUTCOME MEASURES: Time to cessation of seizures. RESULTS: Twenty-four patients were enrolled (13 midazolam, 11 diazepam). Initial treatment with either midazolam or diazepam was successful in 22 of the 24 patients. One patient in each group failed therapy and eventually required endotracheal intubation and general anesthesia for convulsive status epilepticus lasting more than one hour. Patients in the midazolam group received medication sooner (3.3 +/- 2.0 vs 7.8 +/- 3.2 minutes, P = 0.001) and had more rapid cessation of their seizures (7.8 +/- 4.1 vs 11.2 +/- 3.6, P = 0.047) than patients randomized to receive diazepam. CONCLUSIONS: IM midazolam is an effective anticonvulsant for children with motor seizures. Compared to IV diazepam, IM midazolam results in more rapid cessation of seizures because of more rapid administration. The IM route of administration may be particularly useful in physicians' offices, in the prehospital setting, and for children with difficult IV access.

Anti-Anxiety Agents↗

PubMed1996

Acute exposure to CNTF in vivo induces multiple components of reactive gliosis.

CNS trauma or disease induces a constellation of changes in the glia comprising the condition known as reactive gliosis. At present, little is known regarding the nature of the injury signals and the specific consequences of their actions. Ciliary neurotrophic factor (CNTF) induces acute phase proteins in liver and increases astrocytic glial fibrillary acidic protein (GFAP) both in vitro and in vivo. The purpose of the present study was to establish whether CNTF induces other aspects of gliosis. Between 10 and 72 h after 100 ng of recombinant human CNTF was administered into the adult rat neocortex, alterations were observed in a region extending several millimeters in circumference from the injection site. Microglia in this region were more apparent and astrocytes were hypertrophic. By in situ hybridization, mRNAs for GFAP, vimentin, and clusterin were upregulated when compared to the control hemisphere (which received heat-inactivated CNTF). By immunocytochemistry, GFAP, vimentin, glutathione-S-transferase mu, S-100, and OX-42 were elevated by 48 h. By contrast, the oligodendroglial marker GSTYp, the neuronal markers MAP-2 and NSE, the intermediate filament nestin, and the stress protein alpha B-crystallin were unchanged. In addition, a greater than twofold increase in the number of proliferating cells was observed. Since CNTF induces swelling and multiple "gliotic" genes in astrocytes, increases microglial number, and stimulates cell proliferation, we conclude that CNTF is sufficient to induce multiple aspects of gliosis. These data are consistent with a model whereby CNTF (which is synthesized by astrocytes) would be released when the integrity of the astrocyte membrane is compromised, whereupon it would elicit an inflammatory response.

Animals↗

PubMed1996

A bifunctional urease enhances survival of pathogenic Yersinia enterocolitica and Morganella morganii at low pH.

To infect a susceptible host, the gastrointestinal pathogen Yersinia enterocolitica must survive passage through the acid environment of the stomach. In this study, we showed that Y. enterocolitica serotype O8 survives buffered acidic conditions as low as pH 1.5 for long periods of time provided urea is available. Acid tolerance required an unusual cytoplasmically located urease that was activated 780-fold by low-pH conditions. Acid tolerance of Helicobacter species has also been attributed to urease activity, but in that case urease was not specifically activated by low-pH conditions. A ure mutant strain of Y. enterocolitica was constructed which was hypersensitive to acidic conditions when urea was available and, unlike the parental strain, was unable to grow when urea was the sole nitrogen source. Examination of other urease-producing gram-negative bacteria indicated that Morganella morganii survives in acidic conditions but Escherichia coli 1021, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii, and Pseudomonas aeruginosa do not. Consistent with these results, biochemical evidence demonstrated that Y. enterocolitica and M. morganii ureases were activated in vitro by low pH with an unusually low activity optimum of pH 5.5. In whole cells activation occurred as medium values decreased below pH 3.0 for Y. enterocolitica and pH 5.5 for M. morganii, suggesting that in vivo activation occurs as a result of cytoplasmic acidification. DNA sequence analysis of portions of the M. morganii ure locus showed that the predicted primary structure of the enzyme structural subunits is most similar to those of Y. enterocolitica urease. One region of similarity between these two ureases located near the active site is distinct from most other ureases but is present in the urease of Lactobacillus fermentum. This region of similarity may be responsible for the unique properties of the Y. enterocolitica and M. morganii ureases since the L. fermentum urease also has been shown to have a low pH optimum for activity.

Acids↗

PubMed1996

Persistence of multipotential progenitors in the juvenile rat subventricular zone.

The juvenile subventricular zone (SVZ) normally produces oligodendrocytes. To determine whether juvenile SVZ cells are lineage-restricted or whether they remain multipotential, we labeled SVZ cells and characterized their progeny after 1 week in vitro using cell morphology and antigen expression. Heterogeneous clones comprised of retrovirally labeled neurons and astrocytes or astrocytes and oligodendrocytes were observed, as well as homogeneous clones of neurons, astrocytes or oligodendrocytes. Large type-1 astrocyte clones were most common, and mixed oligodendrocyte/type-1-astrocyte clusters represented 15% of the total clusters. Twenty five percent of the total clusters contained at least 1 immature neuron. Of 128 clones, 6-10% contained neurons, astrocytes and oligodendrocytes. From these results we conclude that the juvenile SVZ is a mixture of lineage-restricted, bipotential and multipotential neural progenitors.

Aging↗

PubMed1994

Repression of tonB transcription during anaerobic growth requires Fur binding at the promoter and a second factor binding upstream.

Although iron is an essential nutrient, its toxicity at high levels necessitates regulated transport. In Gram-negative bacteria a central target for regulation is the TonB protein, an energy transducer that couples the cytoplasmic membrane proton motive force to active transport of (FeIII)-siderophore complexes across the outer membrane. We have previously demonstrated the threefold repression of tonB transcription by excess iron in the presence of Fur repressor protein under aerobic conditions. In this report, we examine tonB regulation under anaerobic conditions where the solubility of iron is not a limiting factor and, presumably, siderophore-mediated transport is not required. Under these conditions, tonB transcription is repressed at least 10-fold by excess iron in the presence of Fur, but can be fully derepressed in the absence of Fur. Based on several lines of evidence, this anaerobic repression is not due to increased negative supercoiling as previously postulated. Our results rule out both supercoiling-mediated decreased promoter function and increased Fur binding as mediators of anaerobic repression. Under iron-limiting anaerobic conditions tonB expression is as high or higher than under iron-limiting aerobic conditions, suggesting that promoter function has not decreased anaerobically. Furthermore, under anaerobic conditions in tonB+ strains, tonB promoter function is insensitive to the gyrase inhibitor novobiocin and to changes in medium osmolarity and temperature, three conditions known to change levels of supercoiling. We also rule out effects of mutations in arcA or fnr as mediators of anaerobic repression. Results from in vivo dimethyl sulphate protection foot-printing indicate that Fur binds to an operator site between the -10 and -35 regions of the promoter, but not to a less homologous operator site centered at +26. The binding is, if anything, weaker under anaerobic conditions, indicating that anaerobic repression is not mediated through Fur. Additional changes in the in vivo footprint upstream from the promoter implicate a second factor in tonB anaerobic repression. Together, these results suggest that the mechanism responsible for this regulation (and, by analogy, that of other anaerobically repressed, iron-regulated genes such as cir, exbB, and fhuA) is a novel one.

Anaerobiosis↗

PubMed1993

Management of children with epiglottitis during transport: analysis of a survey.

Because nationally accepted guidelines for the management of children with epiglottitis during transport have not been published, we surveyed physicians attending the 1990 Pediatric Critical Care Transport Leadership Conference in order to delineate current practices and to test for correlations between complications and methods of management. A 22-item questionnaire was distributed, addressing demographics, availability and composition of a designated transport team, methods of airway management, use of medications for sedation or paralysis, monitoring techniques, and complications encountered during transport. Forty-three of the 49 attendees completed the questionnaire (87.8%). Almost all were attending physicians (60.9% pediatric intensivists, 29.3% pediatric emergency physicians) practicing in tertiary care facilities (58.5% in children's hospitals, 41.5% in general hospitals). Eighty-three percent of centers had designated transport teams. For transfer of a child with suspected epiglottitis from a physician's office, virtually all respondents recommended transport by ambulance, 64% to the nearest facility and 36% directly to a tertiary care center. Regarding interhospital transfers, 49% recommended intubation prior to transport in all cases, whereas 49% considered it on an individual basis. The majority of respondents preferred nasal intubation. To prevent dislocation of the endotracheal tube, 79.1% recommended taping it to the face only (as opposed to around the skull), 70.7% administered paralytic agents, but only 35.2% used additional mechanical restraints.(ABSTRACT TRUNCATED AT 250 WORDS)

Ambulances↗

PubMed1993

Contrast sensitivity to patch stimuli: effects of spatial bandwidth and temporal presentation.

Models of the spatial response of human vision are important for applied work, but the available contrast sensitivity function (CSF) data vary widely due to the diverse spatiotemporal stimuli used over the years. To assist selection, this paper: (1) reports measurements of the effects on the CSF of varying the spatial and temporal windows of grating patches; (2) demonstrates that the widely discrepant CSFs from previous studies can be accounted for by using these results; and (3) discusses simple criteria for choosing CSFs for practical applications. CSFs were measured for several combinations of spatial and temporal waveforms, using the same subjects under otherwise identical conditions. The CSF was measured over the range of 0.5-10 c/deg using Gabor-type patches of 1.0-, 0.5-, 0.25-, and 0.125-octave spatial bandwidths using both abrupt and gradual temporal presentations. The results were compared with the CSF obtained with a fixed aperture (4 deg x 4 deg) grating pattern. Increasing the number of cycles resulted in increased sensitivity at intermediate frequencies, changing the CSF to a narrower bandpass shape. For each patch bandwidth, the gradual presentation CSF had a narrower spatial pass band than with the abrupt presentation. The relevance of the large differences in the CSFs obtained with different stimuli to our understanding of visual performance is discussed.

Contrast Sensitivity↗

PubMed1993

The Avianca plane crash: an emergency medical system's response to pediatric survivors of the disaster.

OBJECTIVE: On January 25, 1990, a jetliner crashed on Long Island, New York. Twenty-two children survived the crash. The purpose of this study was to evaluate the emergency medical system's response to these pediatric survivors. METHODS: A questionnaire was sent to all local, acute care hospitals to determine their specific pediatric capabilities and to rank them as level I, II, or III pediatric centers; level I centers are tertiary care facilities. A second questionnaire was sent to all hospitals that received pediatric survivors to collect specific clinical information for each patient. Based on this clinical information a Pediatric Trauma Score (PTS) was assigned to each patient. Children with a PTS < or = 8 are considered to be at increased risk of trauma-related mortality. The assigned PTS was compared to the level of the pediatric center to which each patient was transported. RESULTS: Of 25 children on board the plane, 22 (88%) survived the crash; of 135 adults on board, 70 (52%) survived (chi 2 = 9.9, P = .002). Seven children had a PTs < or = 8; only 1 of these high-risk patients was transported directly to a level I pediatric center, and only 2 of the 5 high-risk children initially transported to level III facilities were transferred to higher level pediatric centers. CONCLUSIONS: Pediatric survivors were neither adequately triaged nor transported to appropriate facilities which could optimize their care. Possible explanations for this include: (1) unique features of the rescue operation, (2) limited pediatric training of prehospital personnel, and (3) deficiencies of the regional disaster plan. Emergency medical services systems and disaster plans can be made more responsive to children's needs by: (1) acknowledging that children have special needs requiring referral, (2) improving the training of prehospital personnel in pediatric emergency care, (3) classifying ill and injured children according to appropriate triage criteria, (4) recognizing existing tertiary care pediatric centers as the optimal location for the treatment of critically ill and injured children, and (5) designating these centers as the appropriate transport destination for critically ill and injured children.

Accidents, Aviation↗