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S Hopper

Publications and source records attributed to S Hopper.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1998

Aspirin after myocardial infarction: the importance of over-the-counter use.

BACKGROUND: Regular aspirin is beneficial after myocardial infarction (MI). Community-based surveys appear to show unsatisfactory uptake of aspirin after MI. This could be due to over-the-counter (OTC) use not being recorded. In a pilot study in one practice, 39% of patients on aspirin after MI used OTC preparations. In addition, women and older patients were less likely to use aspirin. OBJECTIVES: We aimed to describe aspirin use following MI in general practices in West London, including regimens and OTC use, and to demonstrate any association between aspirin use and gender, age or social class. METHODS: We used a postal questionnaire survey. The setting was a random sample of six general practices in Ealing, Hammersmith and Hounslow Health Authority who use Egton Medical Information Systems (EMIS) to store morbidity data. The subjects were registered patients with a history of MI. The main outcome measures were questionnaire data on aspirin use, regimen, OTC use, prescription charge exemption, age, gender, ethnic group and social class. RESULTS: The prevalence of previously recorded MI in all age groups was 0.64%. The response rate was 89%. Regular aspirin was used by 80%. Of these, 22% use OTC aspirin. OTC use was significantly more common in those paying prescription charges. Aspirin use was not associated with gender, age or social class. CONCLUSION: This study demonstrated a high uptake of aspirin among patients with previous MI as recorded by computer. OTC aspirin use was significant, and should be inquired about and recorded.

Age Distribution↗
PubMed1996

The nucleotide concentration determines the specificity of in vitro transcription activation by the sigma 54-dependent activator FhlA.

An in vitro transcription system has been set up for formate- and FhlA-dependent transcription activation at the -12/-24 promoter of the fdhF gene from Escherichia coli by sigma 54-RNA polymerase. It requires the presence of the upstream activation sequence on supercoiled DNA. Transcription is independent from the effector formate at nucleoside triphosphate concentrations of 400 microM and above and completely dependent on the presence of the effector when the concentration is lowered to 300 microM. Inclusion of nucleoside diphosphates in the system raises the nucleoside triphosphate level at which specific induction by formate can take place. The threshold level of FhlA relative to that of template DNA required for transcription activation in the absence of formate was lowered at a high nucleoside triphosphate concentration. On the other hand, transcription activation at the fdhF promoter lacking the upstream activation sequence requires an increased ratio of FhlA to promoter plus the presence of formate; high ATP concentrations cannot bypass the effect of formate. These results are interpreted in terms of a model which implies that FhlA must undergo a change in its oligomeric state for transcription activation and that this oligomerization is favored by high nucleoside triphosphate concentrations, by the effector formate, and by the target DNA. In the absence of the target DNA, FhlA can line up at unspecific DNA and activate transcription; in this case, however, presence of formate and a higher FhlA concentration are required to stabilize and increase the amount of active oligomer.

Base Sequence↗
PubMed1995

Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli.

The FHLA protein is the transcriptional regulator of the genes of the formate regulon from Escherichia coli. The protein shares homology with the sigma 54-dependent regulators of the NTRC family in the central and C-terminal domains but differs in possessing an extended N terminus lacking the aspartate residue which is the site of phosphorylation. Purified FHLA displays intrinsic ATPase activity which is stimulated weakly by formate and DNA. The presence of both formate and DNA carrying the upstream regulatory sequence to which FHLA binds leads to a large increase in the rate of ATP hydrolysis. Hypophosphite, a structural analog of formate, and azide, a transition state analog of formate, also stimulate ATPase activity, supporting the conclusion that formate is a direct ligand of FHLA. Half-maximal saturation of FHLA with formate took place at around 5 mM, and half-maximal saturation with target DNA took place at around 50 nM. The stimulation of ATPase activity by formate was conferred by a decrease in the apparent Km for ATP, whereas the effect of the DNA binding site also affected the Kcat of the reaction. The other nucleoside triphosphates, GTP, UTP, and CTP, competed with ATP cleavage by FHLA, suggesting at least their binding to FHLA. The specific ATPase activity of FHLA was dependent on the concentration of FHLA in the assay, especially in the presence of DNA and formate. Direct liganding of the effector, therefore, leads to the same consequence as phosphorylation for the NTRC-type regulators, namely, stimulation of ATPase activity.

Adenosine Triphosphatases↗
PubMed1994

Regulated expression in vitro of genes coding for formate hydrogenlyase components of Escherichia coli.

Purified FHLA, the transcriptional activator of the formate regulon from Escherichia coli, is able to efficiently stimulate transcription from the sigma 54-dependent promoters of the fdhF, hyp, and hyc transcriptional units. Expression was dependent on the presence of sigma 54, of the upstream activatory sequence (UAS), and of formate. Hypophosphite, a formate analogue, could substitute for formate in vitro suggesting that formate per se was active in regulation. The integration host factor (IHF) had a direct effect on the expression (in vivo and in vitro) of the hyp and hyc genes but not of the fdhF gene. Binding of IHF within the region between the hyp and the hyc operon could be shown. A model is proposed for the transcriptional regulation of the inversely oriented hyp and hyc operons. It involves two upstream regulatory sequences, one between the hyp and the hyc operon (IR1), and the other between hycA and hycB (IR2). The UAS situated within IR1 is responsible for activation of the hyc operon, that within IR2 for activation of the hyp operon. A supramolecular transcription complex is proposed which involves the binding of IHF to a site located between the UAS and the promoter responsible for transcription of the hyc operon.

Bacterial Proteins↗
PubMed1989

Glutaredoxin from rabbit bone marrow. Purification, characterization, and amino acid sequence determined by tandem mass spectrometry.

A glutaredoxin was purified from rabbit bone marrow, and its amino acid sequence was determined by high performance tandem mass spectrometry. The sequences of peptides generated by digestion with trypsin alone or in combination with thermolysin were determined from their collision-induced dissociation (CID) mass spectra. Alignment of these sequences and additional sequence information were obtained from the collision-induced dissociation mass spectra of peptides obtained from digestion of the intact protein with Staphylococcus aureus V8 protease and alpha-chymotrypsin. The resulting sequence of 106 amino acids is as follows: Ac-Ala-Gln-Glu-Phe-Val-Asn-Ser-Lys-Ile-Gln-Pro-Gly-Lys-Val-Val-Val-Phe- Ile-Lys-Pro-Thr-Cys-Pro-Tyr-Cys-Arg-Lys-Thr-Gln-Glu-Ile-Leu-Ser-Glu-Leu- Pro-Phe - Lys-Gln-Gly-Leu-Leu-Glu-Phe- Val-Asp-Ile-Thr-Ala-Thr-Ser-Asp-Met-Ser-Glu-Ile- Gln-Asp-Tyr-Leu-Gln-Gln-Leu-Thr-Gly-Ala-Arg- Thr-Val-Pro-Arg-Val-Phe-Leu-Gly-Lys-Asp-Cys-Ile- Gly-Gly-Cys-Ser-Asp-Leu-Ile-Ala-Met-Gln-Glu-Lys- Gly-Glu-Leu-Leu-Ala-Arg-Leu-Lys-Glu-Met-Gly- Ala-Leu-Arg-Gln. This glutaredoxin strongly resembles the corresponding calf and pig proteins (known as glutaredoxin and thioltransferase, respectively) with respect to its primary structure and enzymatic activity as a GSH:disulfide thioltransferase, an activity also found for the glutaredoxin from Escherichia coli. However, rabbit glutaredoxin was not active as a hydrogen donor for the reduction of ribonucleotides in the presence of the ribonucleotide reductases from rabbit bone marrow, Lactobacillus leichmannii, and Corynebacterium nephridii.

Amino Acid Sequence↗
PubMed1989

Salmonella enteritidis phage type 4 from the contents of intact eggs: a study involving naturally infected hens.

Two small flocks of egg-laying hens, naturally infected with Salmonella enteritidis, were housed in individual cages so that their eggs could be identified. During a longitudinal study where the contents of 1,119 eggs were examined, 11 were positive for S. enteritidis. One isolate was phage type (PT) 33 the others were PT4. The production of infected eggs was clustered though intermittent. The positive eggs, which were produced by 10 of the 35 hens, were all found to contain fewer than 10 salmonellas. Some birds were also apparently carrying S. hadar PT14 as this organism was isolated from the contents of six cracked eggs.

Animals↗
PubMed1988

Amino acid sequence of thioredoxin isolated from rabbit bone marrow determined by tandem mass spectrometry.

The amino acid sequence of the thioredoxin isolated from rabbit bone marrow was determined chiefly by high performance tandem mass spectrometry and fast atom bombardment mass spectrometry combined with manual Edman degradation. The sequences of peptides generated by digestion with trypsin alone or in combination with Staphylococcus aureus protease V8 or thermolysin were determined from their collision-induced dissociation mass spectra. Alignment of these sequences and additional sequence information were obtained from the collision-induced dissociation mass spectra of peptides obtained from digestion of the intact protein with S. aureus protease V8 and alpha-chymotrypsin. The resulting sequence of 104 residues is as follows: Val-Lys-Gln-Ile-Glu-Ser-Lys-Ser-Ala-Phe-Gln- Glu-Val-Leu-Asp-Ser-Ala-Gly-Asp-Lys-Leu-Val-Val- Val-Asp-Phe-Ser-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys- Met-Ile-Lys-Pro-Phe-Phe-His-Ala-Leu-Ser-Glu-Lys- Phe-Asn-Asn-Val-Val-Phe-Ile-Glu-Val-Asp-Val-Asp- Asp-Cys-Lys-Asp-Ile-Ala-Ala-Glu-Cys-Glu-Val-Lys- Cys-Met-Pro-Thr-Phe-Gln-Phe-Phe-Lys-Lys- Gly-Gln-Lys-Val-Gly-Glu-Phe-Ser-Gly-Ala-Asn-Lys- Glu-Lys-Leu-Glu-Ala-Thr-Ile-Asn-Glu-Leu-Leu.

Amino Acid Sequence↗
PubMed1986

Twenty years of renal transplantation in children.

Two-hundred three children 1 to 16 years of age received kidney transplants during a 20-year period, 100 from living donors and 103 from cadaver donors. The overall survival rate was 79%. Actuarial patient and kidney survival rates at 15 years were 79% and 52%, respectively, for recipients of living donor kidneys and 57% and 19%, respectively, for recipients of cadaver donor kidneys. One of two children who received transplants in 1964 was alive 20 1/2 years later. Twenty-nine children had kidneys that had functioned more than 10 years; their mean serum creatinine concentration was 1.7 mg/dL and 24 were fully rehabilitated. Eighteen were more than 2 SD below the mean height of normal children, however. Comparison of survival rates during successive 5-year intervals showed significant improvement in patient survival during the 20-year period and smaller improvements in kidney survival after 1979. Patient survival after living donor transplants during the last 10 years was 100%, and kidney survival during the last 5 years was 92%. Improvement was attributed to the effect of experience, as well as to changes in immunosuppressive therapy in 1972 and the introduction of donor-specific transfusions in 1978.

Adolescent↗
PubMed1985

A seven-year experience with donor-specific blood transfusions. Results and considerations for maximum efficacy.

Two hundred thirty-nine transplants have been performed following donor-specific blood transfusions (DSTs) since 1978. Graft and patient survival in 1- and 0-haplotype-matched transplants with DST pretreatment is comparable to HLA-identical results through 4 years. Graft survival in 174 consecutive nondiabetic, non-HLA-identical DST recipients shows that the transfusion effect persists for at least 4 years, with graft survival of 88 +/- 3% at that time, compared with 83 +/- 4% in the concurrent HLA-identical group. Graft function, as determined by serum creatinine, was the same in both groups. Graft and patient survival in 20 0-haplotype matched pairs with DST pretreatment is 100% at 2 years. Low-dose Imuran coverage during DST administration (n = 91) was compared with a concurrent group with no Imuran (n = 93). Imuran had its maximum effect in patients undergoing their first transplant and with a pre-DST PRA less than 10% (12% vs. 21% sensitization rate in the no-Imuran group). Imuran did not appear to confer any beneficial effect in primary transplants with high PRAs and in patients undergoing a second or third transplant. The majority of patients formally excluded from transplantation because of a post-DST positive B-warm crossmatch can now be successfully transplanted with the use of flow cytometry analysis to rule out previously undetectable low levels of anti-T-lymphocyte antibodies. Of 62 patients with a positive B-warm crossmatch alone since 1982, 73% had a subsequent negative fluorescence-activated cell sorter (FACS) crossmatch permitting transplantation. Preliminary results of a DST and cyclosporine treatment study are described. In conclusion, a long-term immunologic effect of DST has been confirmed and the indications and considerations for optimum use of the DST protocol have been more clearly defined.

Adolescent↗
PubMed1985

Effect of donor-specific transfusions on renal transplantation in children.

Most family members who are evaluated as kidney donors for children have high reactivity in a mixed lymphocyte culture test and are thus excluded from donation. Fifty children, most of whom had highly reactive mixed lymphocyte cultures with their donors, were challenged with three blood transfusions from their donors before transplantation and were tested for the development of lymphocytotoxic antibodies. Ten children (20%) became sensitized and had a positive T-cell or B-cell crossmatch. Sensitization occurred less frequently in children treated with azathioprine during donor-specific transfusions (11%) than in those not treated (26%), but the difference was not significant. Thirty-seven children received renal transplants from their blood donors after the donor-specific transfusions. There were no deaths, and only two patients had kidney failure. Actuarial kidney survival was 93% after 6 years. The use of donor-specific transfusion has increased the number of related-donor transplants performed and the results have been highly successful.

Actuarial Analysis↗
PubMed1984

Dramatic improvement in the success rate for renal transplants in diabetic recipients with donor-specific transfusions.

The chance of achieving successful kidney transplants in diabetic patients was previously limited because few of them had optimally-matched (2-haplotype) related donors. Hence, transplants were usually not carried out until renal failure had already occurred. The application of donor-specific transfusions (DSTs) prior to transplantation to poorly matched donor-recipient pairs (1-haplotype) has been associated with a high success rate for type-I diabetic recipients in our center. The rate of graft survival for 35 consecutive transplants in this category was 88%, 80%, and 73% at 1, 2, and 5 years, respectively. Furthermore, the rate of patient survival was 94%, 90%, and 90% at 1, 2, and 5 years. These patient and graft survival data were without significant difference when compared with the corresponding data for 142 optimally-matched (2-haplotype) related transplants performed without DSTs for nondiabetic recipients, and also when compared with the corresponding data for 130 poorly matched (1 or 0-haplotype) related transplants involving nondiabetic recipients who were prepared for transplantation with DSTs. These good results with DSTs in diabetic recipients emphasize that earlier transplantation utilizing poorly matched related donors should be seriously considered for diabetic patients even before the onset of renal failure, as long as the transplants are carried out in association with DSTs.

Adult↗
PubMed1983

Properties of a thioredoxin purified from rabbit bone marrow which fails to serve as a hydrogen donor for the homologous ribonucleotide reductase.

A thioredoxin has been highly purified from rabbit bone marrow. This thioredoxin is heat-stable, has a molecular weight of approximately 13,000, and contains 4 half-cystines. It is a substrate for the NADPH-dependent thioredoxin reductase of rabbit bone marrow, catalyzes the reduction of insulin disulfides by dithiothreitol, and is a hydrogen donor for methionine sulfoxide reductase of yeast. Although active as a hydrogen donor for ribonucleotide reductase of Lactobacillus leichmannii, this activity could not be demonstrated when the bone marrow thioredoxin was tested with the ribonucleotide reductases of rabbit bone marrow and Corynebacterium nephridii. A regulatory role for the bone marrow thioredoxin was investigated by determining its ability to activate two of the enzymes of spinach chloroplasts known to require thioredoxin for their activation. The bone marrow thioredoxin effectively activates spinach NADP-malate dehydrogenase but not spinach fructose 1,6-diphosphatase. These observations suggest that instead of serving as a hydrogen donor for ribonucleotide reduction in bone marrow, this thioredoxin may be involved in the regulation of the activity of bone marrow enzyme(s).

Amino Acids↗