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Biomedical subjects

M So

Publications and source records attributed to M So.

At least 19 recordsLinked to original sources
PubMed2006

Homeobox genes are differentially expressed in macrovascular human umbilical vein endothelial cells and microvascular placental endothelial cells.

Angiogenesis is fundamental to normal placental development. Aberrant angiogenesis within the placental terminal villi is a characteristic of significant placental pathologies and includes structural and vascular abnormalities as well as altered endothelial cell function, which substantially impacts on maternal-fetal exchange. Homeobox gene transcription factors regulate vascular development in embryonic and adult tissues, but their role in the placental microvasculature is not well known. In this study, we isolated and enriched human placental microvascular endothelial cells (PLEC) by a perfusion-based method and compared homeobox gene expression between PLEC and macrovascular human umbilical vein endothelial cells (HUVEC). Reverse transcriptase PCR detected mRNA expression of homeobox genes DLX3, DLX4, MSX2, GAX and HLX1 in both PLEC and HUVEC. DLX4 and HLX1 have not been previously detected in PLEC and with the exception of GAX, none of these homeobox genes have been previously identified in HUVEC. There was lower expression of HLX1 mRNA in HUVEC compared with PLEC. Using real-time PCR analysis PLEC HLX1 mRNA expression relative to housekeeping gene GAPDH was 0.9+/-0.06 fold of the calibrator (n=6) versus 0.2+/-0.06 (n=6) for HUVEC, p<0.001. These data provided evidence of heterogeneity in homeobox gene expression between microvascular PLEC and macrovascular HUVEC that most likely reflects significant differences in endothelial cell function in the two different cellular environments.

Endothelial Cells↗
PubMed2006

Intra-operative monitoring of vagal nerve activity with wire electrodes.

A monitoring system for tracking the electromyogram (EMG) of the vocal cords with wire electrodes embedded in an endotracheal tube was designed to identify the recurrent laryngeal nerve during thyroidectomy. Our recent experience in two cases suggests that vagal nerve activity can be correctly detected by recording of the EMG of the vocal cords using a special endotracheal tube embedded with wire electrodes.

Adult↗
PubMed2001

The pilus-induced Ca2+ flux triggers lysosome exocytosis and increases the amount of Lamp1 accessible to Neisseria IgA1 protease.

The IgA1 protease secreted by the pathogenic Neisseriae cleaves Lamp1, a major integral membrane glycoprotein of lysosomes, and significantly reduces its steady-state levels in an infected cell. IgA1 protease hydrolysis of Lamp1 is inefficient at the low pH of lysosomes, strongly suggesting that the enzyme is unlikely to reduce Lamp1 levels within lysosomes to any appreciable extent. We therefore explored the possibility that the protease may reach Lamp1 through an alternative route. We demonstrate that Neisseria pili induce a transient increase in the levels of cytosolic free Ca2+ in A431 human epithelial cells, as demonstrated previously for ME180 cells. This Ca2+ flux triggers lysosome exocytosis, quickly altering the cellular distribution of Lamp1 and increasing surface Lamp1 levels. Finally, we demonstrate that surface Lamp1 is cleaved by IgA1 protease secreted by adherent bacteria. We conclude that the pilus-induced Ca2+ flux increases the amount of Lamp1 that is cleavable by the IgA1 protease.

Antigens, CD↗
PubMed2000

Pilus retraction powers bacterial twitching motility.

Twitching and social gliding motility allow many gram negative bacteria to crawl along surfaces, and are implicated in a wide range of biological functions. Type IV pili (Tfp) are required for twitching and social gliding, but the mechanism by which these filaments promote motility has remained enigmatic. Here we use laser tweezers to show that Tfp forcefully retract. Neisseria gonorrhoeae cells that produce Tfp actively crawl on a glass surface and form adherent microcolonies. When laser tweezers are used to place and hold cells near a microcolony, retractile forces pull the cells toward the microcolony. In quantitative experiments, the Tfp of immobilized bacteria bind to latex beads and retract, pulling beads from the tweezers at forces that can exceed 80 pN. Episodes of retraction terminate with release or breakage of the Tfp tether. Both motility and retraction mediated by Tfp occur at about 1 microm s(-1) and require protein synthesis and function of the PilT protein. Our experiments establish that Tfp filaments retract, generate substantial force and directly mediate cell movement.

Fimbriae, Bacterial↗
PubMed2000

Alteration of epithelial cell transferrin-iron homeostasis by Neisseria meningitidis and Neisseria gonorrhoeae.

Iron is an essential element for nearly all organisms. In mammals, iron is transported to body tissues by the serum glycoprotein transferrin. Transferrin-iron is internalized by binding to specific receptors followed by endocytosis. In vitro, Neisseria meningitidis and Neisseria gonorrhoeae can use iron from a variety of iron-containing compounds, including human transferrin. In vivo, transferrin is an important source of iron for N. gonorrhoeae: a mutant that is unable to bind and use transferrin-iron is unable to colonize the urethra of men or initiate disease at this site. As pathogenic Neisseria and its human host derive much of their iron from transferrin, we reasoned that a competition may exist between microbe and host epithelial cells for transferrin-iron at certain stages of infection. We therefore tested the hypothesis that N. meningitidis and N. gonorrhoeae may actively interfere with host transferrin-iron metabolism. We report that Neisseria-infected human epithelial cells have reduced levels of transferrin receptor messenger RNA and cycling transferrin receptors. The ability of infected cells to internalize transferrin receptor is also reduced. Finally, the relative distribution of surface and cycling transferrin receptors is altered in an infected cell. We conclude that Neisseria infection alters epithelial cell transferrin-iron homeostasis at multiple levels.

Epithelial Cells↗
PubMed2000

Incentives and obstacles to the routine use of outcomes measures by clinicians.

OBJECTIVE: Unless clinicians are forced by funds providers to use outcomes measures, clinicians will choose to use them only if the value they provide is commensurate with the administration time required. This study was designed to determine which aspects of outcomes measures encouraged clinicians to use them and which aspects discouraged clinicians from using them. DESIGN: Clinicians who had used one or more outcomes measures were surveyed using a reasonably open-ended survey. Comments were analyzed by categorising them and counting the number of comments within each category. RESULTS: Aspects that were most valued included determining the clients' goals and expectations at the outset of the rehabilitation program, as well as measures of improvement and of residual hearing difficulties at the end of the program. Aspects that most discouraged the use of outcomes measures included collection or recording of information that the clinician would prefer to collect and record in other ways, and an excessive amount of time to administer. CONCLUSION: To maximize the adoption of outcomes measures by clinicians, the tools should provide information in addition to a simple quantification of benefit. For example, this might comprise information about clients' initial goals and/or expectations.

Attitude↗
PubMed2000

Isolation of Neisseria gonorrhoeae mutants that show enhanced trafficking across polarized T84 epithelial monolayers.

Initiation of a gonococcal infection involves attachment of Neisseria gonorrhoeae to the plasma membrane of an epithelial cell in the mucosal epithelium and its internalization, transepithelial trafficking, and exocytosis from the basal membrane. Piliation and expression of certain Opa proteins and the immunoglobulin A1 protease influence the transcytosis process. We are interested in identifying other genetic determinants of N. gonorrhoeae that play a role in transcellular trafficking. Using polarized T84 monolayers as a model epithelial barrier, we have assayed an N. gonorrhoeae FA1090 minitransposon (mTn) mutant bank for isolates that traverse the monolayer more quickly than the isogenic wild-type (WT) strain. From an initial screen, we isolated four mutants, defining three genetic loci, that traverse monolayers significantly more quickly than their WT parent strain. These mutants adhere to and invade cells normally and do not affect the integrity of the monolayer barrier. Backcrosses of the mutations into the WT FA1090 strain yielded mutants with a similar fast-trafficking phenotype. In two mutants, the mTns had inserted 370 bp apart into the same locus, which we have named fit, for fast intracellular trafficker. Backcrosses of one of these mutants into the MS11A genetic background also yielded a fast-trafficking mutant. The fit locus contains two overlapping open reading frames, fitA and fitB, whose deduced amino acid sequences have predicted molecular weights of 8.6 and 15.3, respectively. Neither protein contains a signal sequence. FitA has a potential helix-turn-helix motif, while the deduced sequence of FitB offers no clues to its function. fitA or fitB homologues are present in the genomes of Pseudomonas syringae and Rhizobium meliloti, but not Neisseria meningitidis. Replication of the MS11A fitA mutant in A431 and T84 cells is significantly accelerated compared to that of the isogenic WT strain. In contrast, growth of this mutant in liquid media is normal. Taken together, these results strongly suggest that traversal of N. gonorrhoeae across an epithelial barrier is linked to intracellular bacterial growth.

Amino Acid Sequence↗
PubMed2000

Effects of the immunoglobulin A1 protease on Neisseria gonorrhoeae trafficking across polarized T84 epithelial monolayers.

We previously demonstrated that the Neisseria IgA1 protease cleaves LAMP1 (lysosome-associated membrane protein 1), a major integral membrane glycoprotein of lysosomes, thereby accelerating its degradation rate in infected A431 human epidermoid carcinoma cells and resulting in the alteration of lysosomes in these cells. In this study, we determined whether the IgA1 protease also affects the trafficking of Neisseria gonorrhoeae across polarized T84 epithelial monolayers. We report that N. gonorrhoeae infection of T84 monolayers, grown on a solid substrate or polarized on semiporous membranes, also results in IgA1 protease-mediated reduction of LAMP1. We demonstrate that iga mutants in two genetic backgrounds exited polarized T84 monolayers in fewer numbers than the corresponding wild-type strains. Finally, we present evidence that these mutants have a statistically significant and reproducible defect in their ability to traverse T84 monolayers. These results add to our previous data by showing that the IgA1 protease alters lysosomal content in polarized as well as unpolarized cells and by demonstrating a role for the protease in the traversal of epithelial barriers by N. gonorrhoeae.

Antigens, CD↗
PubMed2000

Interactions of pathogenic neisseriae with epithelial cell membranes.

The closely related bacterial pathogens Neisseria gonorrhoeae (gonococci, GC) and N. meningitidis (meningococci, MC) initiate infection at human mucosal epithelia. Colonization begins at apical epithelial surfaces with a multistep adhesion cascade, followed by invasion of the host cell, intracellular persistence, transcytosis, and exit. These activities are modulated by the interaction of a panoply of virulence factors with their cognate host cell receptors, and signals are sent from pathogen to host and host to pathogen at multiple stages of the adhesion cascade. Recent advances place us on the verge of understanding the colonization process at a molecular level of detail. In this review we describe the Neisseria virulence factors in the context of epithelial cell biology, placing special emphasis on the signaling functions of type IV pili, pilus-based twitching motility, and the Opa and Opc outermembrane adhesin/invasin proteins. We also summarize what is known about bacterial intracellular trafficking and growth. With the accelerated integration of tools from cell biology, biochemistry, biophysics, and genomics, experimentation in the next few years should bring unprecedented insights into the interactions of Neisseriae with their host.

Animals↗
PubMed1999

Recombinant, attenuated Salmonella typhimurium stimulate lymphoproliferative responses to SIV capsid antigen in rhesus macaques.

Recombinant bacteria are useful vectors for delivering foreign antigens to mucosal surfaces and may elicit immune protection against sexually-transmitted pathogens. Recombinant, attenuated Salmonella typhimurium expressing the Simian Immunodeficiency Virus capsid protein (p27) were given to rhesus macaques by intragastric intubation. This route of immunization was compared with intramuscular injection of soluble p27 in adjuvant, and with immunization protocols that combined intragastric and intramuscular antigen exposures. Recombinant Salmonella stimulated p27-specific lymphoproliferative responses that were present transiently in peripheral blood, and were recalled easily by booster immunizations. Intramuscular p27 injection elicited strong serum antibody responses, but only low level capsid-specific proliferative responses. Recombinant Salmonella immunization elicited low levels of p27-specific antibodies in serum and did not suppress subsequent responses to parenteral immunization. Intragastric immunization of macaques with recombinant Salmonella typhimurium was safe and induced immune responses specific for the expressed, foreign antigen.

Animals↗
PubMed1999

Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells.

The pathogenic Neisseriae Neisseria meningitidis and Neisseria gonorrhoeae, initiate colonization by attaching to host cells using type IV pili. Subsequent adhesive interactions are mediated through the binding of other bacterial adhesins, in particular the Opa family of outer membrane proteins. Here, we have shown that pilus-mediated adhesion to host cells by either meningococci or gonococci triggers the rapid, localized formation of dramatic cortical plaques in host epithelial cells. Cortical plaques are enriched in both components of the cortical cytoskeleton and a subset of integral membrane proteins. These include: CD44v3, a heparan sulphate proteoglycan that may serve as an Opa receptor; EGFR, a receptor tyrosine kinase; CD44 and ICAM-1, adhesion molecules known to mediate inflammatory responses; f-actin; and ezrin, a component that tethers membrane components to the actin cytoskeleton. Genetic analyses reveal that cortical plaque formation is highly adhesin specific. Both pilE and pilC null mutants fail to induce cortical plaques, indicating that neisserial type IV pili are required for cortical plaque induction. Mutations in pilT, a gene required for pilus-mediated twitching motility, confer a partial defect in cortical plaque formation. In contrast to type IV pili, many other neisserial surface structures are not involved in cortical plaque induction, including Opa, Opc, glycolipid GgO4-binding adhesins, polysialic acid capsule or a particular lipooligosaccharide variant. Furthermore, it is shown that type IV pili allow gonococci to overcome the inhibitory effect of heparin, a soluble receptor analogue, on gonococcal invasion of Chang and A431 epithelial cells. These and other observations strongly suggest that type IV pili play an active role in initiating neisserial infection of the mucosal surface in vivo. The functions of type IV pili and other neisserial adhesins are discussed in the specific context of the mucosal microenvironment, and a multistep model for neisserial colonization of mucosal epithelia is proposed.

Animals↗
PubMed1999

Neisseria gonorrhoeae mutants altered in toxicity to human fallopian tubes and molecular characterization of the genetic locus involved.

In an effort to identify potential cytotoxins expressed by Neisseria gonorrhoeae, we have identified a locus that, when mutated in the gonococcus, results in a significant increase in toxicity of the strain to human fallopian tube organ cultures (HFTOC). This locus, gly1, contains two open reading frames (ORFs) which are likely cotranscribed. ORF1 encodes a polypeptide of 17.8 kDa with a signal sequence that is recognized and processed in Escherichia coli and N. gonorrhoeae. The 15.6-kDa processed polypeptide has been observed in membrane fractions and filtered spent media from cultures of E. coli expressing gly1 and in outer membrane preparations of wild-type N. gonorrhoeae. The gly1 locus is not essential for bacterial survival, and it does not play a detectable role in epithelial cell adhesion, invasion, or intracellular survival. However, a gly1 null mutant causes much more damage to fallopian tube tissues than its isogenic wild-type parent. A strain complemented in trans for the gly1 mutation showed a level of toxicity to HFTOC similar to the level elicited by the wild-type parent. Taken together, these results indicate an involvement of the gly1 locus in the toxicity of N. gonorrhoeae to human fallopian tubes.

Amino Acid Sequence↗
PubMed1999

Neisseria gonorrhoeae PilA is an FtsY homolog.

The pilA gene of Neisseria gonorrhoeae was initially identified in a screen for transcriptional regulators of pilE, the expression locus for pilin, the major structural component of gonococcal pili. The predicted protein sequence for PilA has significant homology to two GTPases of the mammalian signal recognition particle (SRP), SRP54 and SRalpha. Homologs of SRP54 and SRalpha were subsequently identified in bacteria (Ffh and FtsY, respectively) and appear to form an SRP-like apparatus in prokaryotes. Of the two proteins, PilA is the most similar to FtsY (47% identical and 67% similar at the amino acid level). Like FtsY, PilA is essential for viability and hydrolyzes GTP. The similarities between PilA and the bacterial FtsY led us to ask whether PilA might function as the gonococcal FtsY. In this work, we show that overproduction of PilA in Escherichia coli leads to an accumulation of pre-beta-lactamase, similar to previous observations with other bacterial SRP components. Low-level expression of pilA in an ftsY conditional mutant can complement the ftsY mutation and restore normal growth to this strain under nonpermissive conditions. In addition, purified PilA can replace FtsY in an in vitro translocation assay using purified E. coli SRP components. A PilA mutant that is severely affected in its GTPase activity cannot replace FtsY in vivo or in vitro. However, overexpression of the GTPase mutant leads to the accumulation of pre-beta-lactamase, suggesting that the mutant protein may interact with the SRP apparatus to affect protein maturation. Taken together, these results show that the gonococcal PilA is an FtsY homolog and that the GTPase activity is necessary for its function.

Bacterial Proteins↗
PubMed1998

Infection of epithelial cells by pathogenic neisseriae reduces the levels of multiple lysosomal constituents.

Members of our group reported recently that neisseria infection of human epithelial cells results in accelerated degradation of the major lysosomal integral membrane protein LAMP1 and that this is due to hydrolysis of this glycoprotein at its immunoglobulin A1 (IgA1)-like hinge by the neisseria type 2 IgA1 protease (L. Lin et al., Mol. Microbiol. 24:1083-1094, 1997). We also reported that the IgA1 protease plays a major role in the ability of the pathogenic neisseriae to survive within epithelial cells and hypothesized that this is due to alteration of lysosomes as a result of protease-mediated LAMP1 degradation. In this study, we tested the hypothesis that neisseria infection leads to multiple changes in lysosomes. Here, we report that neisseria infection also reduces the levels of three other lysosomal markers: LAMP2, lysosomal acid phosphatase (LAP), and CD63. In contrast, neither the epidermal growth factor receptor level nor the beta-tubulin level is affected. A detailed examination of LAMP2 indicated that the reduced LAMP2 levels are not the result of an altered biosynthetic rate or of cleavage by the IgA1 protease. Nevertheless, the protease plays a role in reducing LAMP2 and LAP activity levels, as these are partially restored in cells infected with an iga mutant. We conclude that neisseria infection results in multiple changes to the lysosomes of infected epithelial cells and that these changes are likely an indirect result of IgA1 protease-mediated cleavage of LAMP1.

Acid Phosphatase↗
PubMed1997

Isolation and biochemical characterization of the PilA protein of Neisseria meningitidis.

PilA is the response regulator of a two-component regulatory system that controls a number of genes in the pathogenic Neisseria. Previous work has shown that Neisseria gonorrhoeae (GC) PilA binds DNA and also hydrolyzes GTP. Here, we report the cloning, sequencing, purification, and biochemical characterization of PilA from N. meningitidis (MC) strain 8013. MC pilA is 94% identical to GC pilA at the nucleotide level. Of the 78 nucleotide changes, 52 are silent, while 26 result in a total of 20 amino acid changes. Additionally, the MC homolog has a 4-amino acid insertion between the putative DNA-binding and GTP-binding domains. Purified MC PilA binds to the same DNA fragment we have previously shown to be bound by GC PilA specifically and also hydrolyzes GTP. The K(m) of MC PilA for GTP is 8.6 microM, similar to that determined for the GC protein. However, the maximum velocity (Vmax) is approximately 35-fold greater than the GC PilA activity. Additionally, the nucleotide specificity of MC PilA differs from that of GC PilA. While GC PilA hydrolyzes only GTP, MC PilA hydrolyzes GTP and ATP equally well, and CTP and UTP also compete for this activity.

Adenosine Triphosphatases↗
PubMed1997

Cloning and characterization of the Neisseria meningitidis rfaC gene encoding alpha-1,5 heptosyltransferase I.

We cloned and characterized the Neisseria meningitidis rfaC gene which encodes an enzyme, alpha-1,5 heptosyltransferase I, involved in the synthesis of the deep-core of the lipooligosaccharide. The N. meningitidis rfaC mutant, obtained by an allelic exchange, produced lipooligosaccharide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the lipooligosaccharide isolated from the wild-type N. meningitidis. The N. meningitidis rfaC mutant was not affected by growth in a rich microbiological medium and did not show any defect in adhesion to epithelial cell lines. Conversely, the rfaC mutant was attenuated in the infant rat model of meningococcemia, thus confirming the importance of intact lipooligosaccharide in the virulence of N. meningitidis.

Amino Acid Sequence↗