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Biomedical subjects

C Block

Publications and source records attributed to C Block.

At least 37 records · Page 2Linked to original sources
PubMed1997

Discrimination of amino acids mediating Ras binding from noninteracting residues affecting raf activation by double mutant analysis.

The contribution of residues outside the Ras binding domain of Raf (RafRBD) to Ras-Raf interaction and Ras-dependent Raf activation has remained unresolved. Here, we utilize a double mutant approach to identify complementary interacting amino acids that are involved in Ras-Raf interaction and activation. Biochemical analysis demonstrates that Raf-Arg59 and Raf-Arg67 from RafRBD are interacting residues complementary to Ras-Glu37 located in the Ras effector region. Raf-Arg59 and Raf-Arg67 also mediate interaction with Ras-Glu37 in Ras-dependent Raf activation. The characteristics observed here can be used as criteria for a role of residues from other regions of Raf in Ras-Raf interaction and activation. We developed a quantitative two-hybrid system as a tool to investigate the effect of point mutations on protein-protein interactions that elude biochemical analysis of bacterially expressed proteins. This assay shows that Raf-Ser257 in the RafCR2 domain does not contribute to Ras-Raf interaction and that the Raf-S257L mutation does not restore Raf binding to Ras-E37G. Yet, Raf-S257L displays high constitutive kinase activity and further activation by Ras-G12V/E37G is still impaired as compared with activation by Ras-G12V. This strongly suggests that the RafCR2 domain is an independent domain involved in the control of Raf activity and a common mechanism for constitutively activating mutants may be the interference with the inactive ground state of the kinase.

Amino Acid Substitution↗
PubMed1997

Ras-binding domains: predicting function versus folding.

Ras interacts with a number of effector molecules to achieve its prolific signalling. Based on iterative sequence profile and motif searches of databases a novel family of Ras-binding domains was recently identified (Ponting and Benjamin, Trends Biochem. Sci. 21: 422-425, 1996). Among them the rat unconventional myosin and Rho-GTPase-activating protein myr 5 was predicted to contain a Ras-binding domain at its N-terminus. Here we report that direct binding experiments between the proposed Ras-binding domain of myr 5 and Ras failed to demonstrate any interaction. Molecular modelling suggests that this domain in myr 5 adopts a similar folding topology as the Ras-binding domain of Raf kinase. However, unlike the Ras-binding domain of Raf kinase, the myr 5 domain lacks the positive surface charges necessary for binding the negatively charged Ras contact site. This result exemplifies the functional diversity of similar structures and suggests that the identified Ras-binding motif does not reliably predict Ras-binding domains.

Animals↗
PubMed1997

Double-blind vaccine-controlled randomised efficacy trial of an investigational Shigella sonnei conjugate vaccine in young adults.

BACKGROUND: The aim of this double-blind randomised vaccine-controlled trial was to assess the efficacy of a conjugate vaccine composed of Shigella sonnei O-specific polysaccharide bound to Pseudomonas aeruginosa recombinant exoprotein A (S sonnei-rEPA) and of an oral, live-attenuated Escherichia coli/S flexneri 2a (EcSf2a-2) hybrid vaccine among military recruits in Israel at high risk of exposure to Shigella spp. We report here our preliminary findings on the efficacy of S sonnei-rEPA; we have not documented sufficient cases to assess the efficacy of EcSf2a-2. METHODS: Between April, 1993, and August, 1994, male Israeli Military recruits aged 18-22 years were asked to take part in our study. We enrolled 1446 soldiers from seven separate field sites (groups A-G). Soldiers were randomly allocated one injection of S sonnei-rEPA and four doses of oral placebo (n = 576), four oral doses of EcSf2a-2 and one injection of saline placebo (n = 580), or one injection of meningococcal tetravalent control vaccine and four doses of oral placebo (n = 290). Because there were no cases of S flexneri 2a, the EcSf2a-2 and meningococcal vaccines were the control group. We defined S sonnei shigellosis as diarrhoea with a positive faecal culture for S sonnei. Each group of soldiers was followed up for 2.5-7.0 months. The primary endpoint was protective efficacy of S sonnei-rEPA against S sonnei shigellosis. FINDINGS: Cases of culture-proven S sonnei shigellosis occurred in four groups of soldiers (groups A-D), which comprised 787 volunteers (312 received S sonnei-rEPA, 316 received EcSf2a-2, and 159 received meningococcal control vaccine). In groups A-C, cases of shigellosis occurred 70-155 days after vaccination, whereas in group D cases occurred after 1-17 days. In groups A-C, the attack rate of shigellosis was 2.2% in recipients of S sonnei-rEPA compared with 8.6% in controls (protective efficacy 74% [95% CI 28-100], p = 0.006). S sonnei-rEPA also showed significant protection against shigellosis in group D (43% [4-82], p = 0.039). Prevaccination and postvaccination ELISA measurements of antibody to S sonnei lipopolysaccharide among recipients of S sonnei-rEPA showed that the vaccinees who developed S sonnei shigellosis had significantly lower serum IgG and IgA responses to the homologous lipopolysaccharide than those who did not (p = < 0.05). INTERPRETATION: One injection of S sonnei-rEPA confers type-specific protection against S sonnei shigellosis. The high antibody concentration induced by the conjugate vaccine in volunteers who did not develop shigellosis suggests that there is an association between serum antibody titre and protection.

Adolescent↗
PubMed1997

Advanced glycation end products in human penis: elevation in diabetic tissue, site of deposition, and possible effect through iNOS or eNOS.

OBJECTIVES: We hypothesized that advanced glycation end product (AGE) formation contributes to erectile dysfunction (ED) by quenching nitric oxide. Our first goal was to identify the specific AGE pentosidine in the diabetic human penis. Because AGE-mediated effects may involve inducible nitric oxide synthase (iNOS), we performed immunohistochemical and Western blot analysis of diabetic and nondiabetic human penile tissue for iNOS. Finally, because AGEs may act intracellularly to affect proteins, we set out to identify endothelial NOS (eNOS) in the human penis as an initial step in examining a possible intracellular interaction between eNOS and AGEs. METHODS: We performed high-performance liquid chromatographic analysis of diabetic human penile corpus cavernosum and serum for pentosidine and performed immunohistochemical, electron microscopic (EM), and Western blot analysis of the diabetic and nondiabetic penile corpus cavernosum and tunica for pyrraline, iNOS, and eNOS (and neural NOS [nNOS] for comparative purposes) via standard methods. RESULTS: We found a significant elevation of pentosidine in the penile tissue but not the serum of diabetic patients (average age 55.6 +/- 2.3 years) compared with that of nondiabetic patients (average age 61.8 +/- 3.6 years). Pentosidine was 117.06 +/- 9.19 pmol/mg collagen in the diabetic tunica versus 77.58 +/- 5.5 pmol/mg collagen in the nondiabetic tunica (P < 0.01) and 74.58 +/- 8.49 pmol/mg collagen in the diabetic corpus cavernosum versus 46.59 +/- 2.53 pmol/mg collagen in the nondiabetic corpus cavernosum (P < 0.01), suggesting a tissue-specific effect of the AGEs. We localized the site of deposition of the specific AGE pyrraline to the human penile tunica and the penile corpus cavernosum collagen. Immunohistochemical and EM analysis localized eNOS and iNOS to the cavernosal endothelium and smooth muscle. Western blot analysis in 6 patients revealed the following: iNOS, but no eNOS, in penile tissue from 1 insulin-dependent diabetic man; eNOS only in 1 man after radical prostatectomy; both eNOS and iNOS in 2 men with Peyronie's disease, as well as in 2 other men with impotence and hypertension. Finally, the specific iNOS inhibitor PNU-19451A significantly augmented relaxation of precontracted human cavernosal tissue, from 64.7% +/- 5.58 to 80.03% +/- 4.55 at 10 microM acetylcholine and 65.06% +/- 2.84 to 86.16% +/- 3.96 at 0.1 mM acetylcholine (n = 4, P < 0.002 and P < 0.02, respectively). CONCLUSIONS: AGEs are elevated in diabetic human penile tissue, but not in serum, and are localized to the collagen of the penile tunica and corpus cavernosum. We identified eNOS and iNOS in the human penile cavernosal smooth muscle and endothelium. The augmentation of cavernosal relaxation with a specific iNOS inhibitor, combined with the identification of iNOS protein, but not eNOS, in a patient with severe diabetes and ED, allows for speculation of a pathophysiologic mechanism for AGE-mediated ED via upregulation of iNOS and downregulation of eNOS. These data provide further insight into the mechanisms of advanced glycation end product-mediated ED and provide a foundation for further study.

Adult↗
PubMed1997

Convenience store robberies in selected metropolitan areas. Risk factors for employee injury.

Circumstances of injury were abstracted from police reports for 1835 convenience store robberies that occurred during 1992 or 1993 in selected metropolitan areas of seven eastern states. Subset analyses were performed using the data (758 robberies) from four states with relatively complete risk factor information. The purpose of this study was to estimate the risk of injury in a robbery situation for various risk factors. The overall risk of employee robbery-related injury could not be estimated because the probability of robbery is unknown. Of the 1835 robberies, 59% of the total robberies occurred at nighttime (9 p.m. to 3 a.m.), 47% occurred in stores previously robbed in the study period, 63% involved the use of a firearm, and 12% were associated with an injury to at least one employee. In the subset analysis of 758 robberies in four states, the employee probability of injury in a robbery was lower with firearm use compared with no weapon or use of a blunt instrument, and the probability of severe injury (defined as death, or an injury necessitating a trip to a hospital) was lower with a firearm compared with the use of a blunt instrument. However, all five fatalities were firearm-related. Other factors that were associated with a lower probability of employee injury included robbery occurrence in stores that had been robbed multiple times, compared with stores robbed only once; having 1 to 999 dollars stolen, compared with having no money stolen; and the presence of a customer(s) in the store at the time of the robbery. The employee risk of injury was not significantly different between one- (0.106) and multiple-employee (0.111) stores. Similarly, the employee risk of severe injury was not significantly different between one- (0.029) and multiple-employee stores (0.022). We conclude that there are several potential risk factors for employee injury in convenience store robberies, some of which are amenable to interventions. Further research on these factors and their relationship to employee injury is indicated.

Chi-Square Distribution↗
PubMed1997

CD11b mRNA expression in neutrophils isolated from peripheral blood and gingival crevicular fluid.

Adhesion molecule CD11b/CD18 expressed by neutrophils (PMNs) participates in cell migration and phagocytosis of C3bi derivatized bacteria. It is this phagocytic function that eliminates some of the known periodontal pathogens in periodontal pockets. In patients with advanced periodontitis, homotypic aggregation of crevicular fluid PMNs (CF-PMNs) may occur due to overexpression of CD11b/CD18 and this may lead to ineffective elimination of periodontal pathogens. We have previously shown that CF-PMNs isolated from the periodontal pockets overexpress CD11b compared to PB-PMNs. This study tested the hypotheses that (1) overexpression of surface CD11b correlates with expression of CD11b mRNA in CF-PMNs isolated from advanced periodontitis subjects, and (2) the intrinsic capacity of CD11b mRNA upregulation by PB-PMNs from periodontitis patients differs from that of control subjects. CF-PMNs and peripheral blood PMNs (PB-PMNs) were isolated from 13 subjects with healthy gingiva (control group) and 13 subjects with advanced periodontitis (patient group). The surface expression of CD11b was determined by flow cytometry and CD11b mRNA was determined by extraction of mRNA and reverse transcription to cDNA followed by DNA amplification using primers to detect a segment of the cDNA which encodes CD11b. The results of this study confirm that the surface expression of CD11b on CF-PMNs is significantly higher in periodontitis subjects vs control subjects (p = 0.03), whereas surface CD11b expression on PB-PMNs does not differ significantly between groups (p = 0.06). The level of surface CD11b expression on CF-PMNs did not correlate with the amount of mRNA present in CF-PMNs in either group (p = 0.056, 0.07 for control and periodontitis patients, respectively). Most (9 of 13) individuals in the patient group expressed CD11b mRNA whereas very few control subjects (2 of 11) had CD11b mRNA in their CF-PMNs. This difference between groups was statistically significant (p = 0.004). The capacity to upregulate CD11b mRNA upon stimulation with fMLP and/or GM-CSF was highly variable and there was no statistical difference between the 2 groups.

Adult↗
PubMed1996

Quantitative structure-activity analysis correlating Ras/Raf interaction in vitro to Raf activation in vivo.

Binding of Ras to c-Raf-1 is a pivotal step of many mitogenic signalling pathways. Based on the recent crystal structure of the complex of Rap1A with the Ras-binding domain of Raf, mutations were introduced in c-Raf-1 and their effects on Ras/Raf binding affinity in vitro and Ras/Raf regulated gene expression in vivo were analysed. Our data reveal an empirical semilogarithmic correlation between dissociation constants and Raf-induced gene activity. The functional epitope that primarily determines binding affinity consists of residues Gln 66, Lys 84 and Arg 89 in Raf. This quantitative structure-activity investigation may provide a general approach to correlate structure-guided biochemical analysis with biological function of protein-protein interactions.

Amino Acid Sequence↗
PubMed1996

Ras/Rap effector specificity determined by charge reversal.

Members of the Ras subfamily of small GTP-binding proteins have been shown to be promiscuous towards a variety of putative effector molecules such as the protein kinase c-Raf and the Ral-specific guanine nucleotide exchange factor (Ral-GEF). To address the question of specificity of interactions we have introduced the mutations E30D and K31E into Rap and show biochemically, by X-ray structure analysis and by transfection in vivo that the identical core effector region of Ras and Rap (residues 32-40) is responsible for molecular recognition, but that residues outside this region are responsible for the specificity of the interaction. The major determinant for the switch in specificity is the opposite charge of residue 31--Lys in Rap, Glu in Ras--which creates a favourable complementary interface for the Ras-Raf interaction.

Amino Acid Sequence↗
PubMed1996

Employee injuries and convenience store robberies in selected metropolitan areas.

The number of robberies and robbery-related injuries to employees in convenience stores (C-stores) during 1992 or 1993 were estimated for selected metropolitan areas around Miami and Tampa, Florida; Atlanta, Georgia; Chicago, Illinois; Baltimore, Maryland; Boston, Massachusetts; Detroit, Michigan; Pittsburgh and Philadelphia, Pennsylvania; Charleston, Columbia, Greenville, and Spartanburg, South Carolina; and Arlington, Chesterfield, and Henrico counties, Virginia. Of the 1835 C-store robberies that occurred during 1992 or 1993 in all selected areas (excluding Atlanta and Chicago), there were 12 homicides of C-store employees; 219 nonfatal injuries of C-store employees; 1071 robberies in which there were no injuries but a weapon was used, displayed, or implied toward a C-store employee; and 132 robberies in which there was no injury and no weapon used, but an employee was struck, pushed, or shoved. Corresponding figures for the 238 robberies that occurred in Chicago during January to June 1993, and for which victim employment status was unknown (customer or employee) were three homicides, 53 nonfatal injuries, 120 attacks in which a weapon was used but there was no injury, and 57 attacks in which a person was struck, pushed, or shoved but there was no injury. The proportion of robberies that resulted in a homicide or injury to an employee varied among selected areas from .03 to .25. The proportion of homicides and injuries to an employee was. 14 or higher for target areas in Baltimore (.24), Detroit (.25), and Virginia (.14); the proportion to an employee or customer was .24 in Chicago. The conclusions from these data are that the risk of employee injury in C-store robberies was high in selected metropolitan areas. This underscores the need for effective robbery prevention programs to reduce injury. In addition, further research is needed to determine the effectiveness of prevention programs in the C-store industry and the application of these programs to other retail industries.

Commerce↗
PubMed1996

Limitations of paperless on-line reporting of diagnostic bacteriology culture results.

AIMS: To estimate the extent to which microbiology laboratory results made available on a computerised reporting system do not reach their intended destination. METHODS: Prospective observational study of 180 urine cultures submitted from patients seen at the accident and emergency department of a 250 bed university affiliated general hospital. Observations were made of: telephone requests for results; whether results were noted in patients' charts; and antibiotic administration to patients sent home. RESULTS: Results were requested/recorded for 73% of 37 patients admitted to hospital and for only 23% of 143 patients sent home (p < 1 x 10(-7)). Overall, results were more frequently recorded for patients with positive cultures (p = 0.04). When determined separately for admitted and discharged groups, this association was not shown. Three of 14 culture positive patients sent home and for whom results were not recorded received inappropriate therapy; 19 culture negative patients were given antibiotics. CONCLUSIONS: In view of the results, measures were instituted to ensure delivery of printed reports to the health care providers of patients not admitted from the accident and emergency department. Organisations operating computerised reporting systems in evolving health care settings must ensure that system design guarantees delivery of reports to all end-users. This will minimise therapeutic problems, reduce wastage of laboratory resources, and limit risks of litigation.

Bacteriology↗
PubMed1995

Switching to Rac and Rho.

Could the recent elucidation of the structure of the Rap-Raf complex have been the first glimpse of a universal arrangement between GTPase switches and kinase cascades, as a number of recent reports show that Ras is not unique in its ability to start a signalling 'chain reaction'?

Animals↗
PubMed1995

Shigella lipopolysaccharide antibodies in pediatric populations.

Shigellosis is the most common cause of bacterial dysentery. To study the specific immunity to the two major groups causing shigellosis, we assayed antibodies to lipopolysaccharide (LPS) by enzyme-linked immunoadsorbent assay to both Shigella sonnei and Shigella flexneri 2a serotypes in the following populations: (1) women immediately after delivery and their infants to assess the transfer of passive immunity by placenta and the presence of secretory antibodies in breast milk; (2) children of different ages; and (3) the kinetics of antibody production in pediatric patients, who had culture-proved shigellosis. The sera of these women showed variable concentrations of antibodies of all three isotypes to LPS of S. sonnei and S. flexneri 2a. These serotype-specific antibodies were not cross-reactive. Transfer of IgG anti-LPS across the placenta was significantly correlated with concentration of the specific antibody in the mother (S. sonnei, r = 0.96; S. flexneri, r = 0.84). Varying concentrations of anti-LPS IgA were present in colostrum, which was correlated with serum anti-LPS IgA titers in the case of S. sonnei (r = 0.44; P < 0.05) but not S. flexneri (r = 0.17). Healthy children between the ages of 6 months and 4 years in our population had undetectable or relatively low titers of anti-S. sonnei IgG. More children had detectable antibody titers to S. flexneri 2a than to S. sonnei. The relatively high concentrations of these natural antibodies are particularly noteworthy because there is a far lower incidence (< 10% of patients) with S. flexneri than with S. sonnei disease in this population.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Meningococcal disease in the Israel Defense Force: epidemiologic trends and new challenges.

To determine recent trends in its epidemiology and the need to reconsider prophylactic interventions, meningococcal disease in the Israel Defense Force (IDF) from 1975 through 1993 was studied. All cases of meningitis or meningococcemia were included. A considerable increase in the number of cases has been observed since 1991, with serogroup C becoming predominant (76% of cases) since then. Serogroup Y was the second most frequent serogroup during this period, while serogroup B, predominant in the civilian population of Israel, was rare. Most cases occurred during the first 6 months of military service. Seasonality was important, with most of the cases occurring between December and March, although a small summer peak was also noted. Since 1992, three small clusters of meningococcal disease were encountered in the IDF, for the first time, with all cases caused by group C meningococci. In one cluster, the emergence of rifampicin resistance resulted in failure of chemoprophylaxis. The rise in group C and Y cases since 1991, and the occurrence of rifampicin resistance, necessitate considering meningococcal vaccines and new antimicrobial agents for prophylaxis.

Bacterial Vaccines↗
PubMed1994

Safety and immunogenicity of the oral E. coli K12-S. flexneri 2a vaccine (EcSf2a-2) among Israeli soldiers.

A double-blind placebo-controlled study was carried out on the safety and immunogenicity of the oral Shigella flexneri (EcSf2a-2) vaccine among Israeli soldiers. Sixty volunteers received the vaccine and 59 received placebo. Fifty-three were given the full vaccine regimen (four doses). Doses ranged between 4.1 x 10(8) and 1.1 x 10(9) c.f.u. Visits to the unit clinic for mild gastrointestinal symptoms were common after the first dose in vaccinees (13%) as compared with placebo recipients (5%), but the difference was not significant, p = 0.12. Similarly, there was no difference between the groups for either gastrointestinal or non-gastrointestinal complaints reported by questionnaire. The vaccine strain was excreted by 69% and 67% of the vaccinees one day after receiving the second and the fourth doses, respectively. As judged by antibiotic susceptibility, phage typing and restriction fragment length polymorphism (RFLP), the vaccine strain emerged as genetically stable after replication in human gut and shedding. There was neither bacteriological nor serological evidence of transmission of the vaccine from vaccinees to placebo recipients. Eighteen of 26 (69.2%) and 11 of 30 (36.7%) vaccinees had significant IgA secreting cell responses 7 and 21 days after the first dose, respectively. Significant IgA or IgG serum antibody response to S. flexneri 2a LPS was detected in 30% of the vaccinees. These results support further evaluation of EcSf2a-2 vaccine protective efficacy in field studies.

Administration, Oral↗
PubMed1994

Effect of corticosteroids on the maturation of neutrophil motility in very low birthweight neonates.

Neutrophil (PMN) chemotaxis and chemokinesis were longitudinally studied in a group of 17 neonates with birthweights between 750 and 1250 g. Five of the 17 neonates were treated with prenatal betamethasone to attempt to prevent hyaline membrane disease, six received postnatal dexamethasone in an effort to reduce bronchopulmonary dysplasia, three received both, and three were not treated with corticosteroids. The group of 17 neonates were tested on four separate occasions: (1-2, 3-4, 7-8, and 10-14 postnatal days). PMN chemotaxis and chemokinesis were determined using a standard micropore filter assay. A group of 36 adults was used as additional controls. There were no significant differences noted in PMN chemotaxis or chemokinesis for the corticosteroid vs the noncorticosteroid-treated groups. In the total group of 17 neonates, there was depression in PMN chemotaxis compared with adult values, which lasted at least through postnatal day 8. By day 13 to 14, PMN chemotactic values were similar to those of adults. In contrast, chemokinesis, was depressed during the initial 14 days (except for the first 2 postnatal days). These data suggest that perinatal corticosteroid administration does not affect PMN motility in newborn infants.

Betamethasone↗
PubMed1994

Epidemiology of pediatric meningitis caused by Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis in Israel: a 3-year nationwide prospective study. Israeli Pediatric Bacteremia and Meningitis Group.

In a 3-year nationwide prospective study on pediatric meningitis caused by Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis in Israel, 1258 invasive infections with a known focus were observed. Meningitis was found in 482 (38%): 56%, 16%, and 76% of all infections by H. influenzae type b, S. pneumoniae, and N. meningitidis, respectively. The incidence of meningitis during the first year of life was 67.1, 17.5, and 9.5/100,000 for H. influenzae type b, S. pneumoniae, and N. meningitidis, respectively, and in children < 5 years old it was 18.5, 5.3, and 5.2. Extrapolated for a population in which 100,000 live births occur yearly, 2097 hospital days were required. The case fatality rate was 2.2%, 5.9%, and 6.3% for H. influenzae type b, S. pneumoniae, and N. meningitidis, respectively. Boys were affected significantly more often than girls, but mortality was higher among girls. On the basis of the observed serotypes and age distribution, even with optimal vaccine development in the next 5 years, it is not likely that > 50% of all cases will be prevented.

Age Distribution↗