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Biomedical subjects

D J Weber

Publications and source records attributed to D J Weber.

At least 73 records · Page 4Linked to original sources
PubMed1998

Stability and bactericidal activity of chlorine solutions.

OBJECTIVES: To determine the stability of sodium hypochlorite (diluted household bleach) when stored for 30 days in various types of containers and to determine the efficacy of low concentrations of free available chlorine to inactivate test bacteria. DESIGN: Laboratory-based study. Solutions of standard household bleach were prepared using tap water or sterile distilled water at dilutions of 1:100, 1:50, and 1:5. Chlorine concentrations were measured, and then the solutions were placed into five polyethylene containers and left at room temperature (20 degrees C) under various conditions (translucent containers with light exposure and with or without air; brown opaque container without light or air exposure). Samples for chlorine and pH determinations were taken at time 0 and on days 7, 14, 21, 30, and 40. Bactericidal activity of chlorine solutions was assessed using the Association of Official Analytical Chemists Use-Dilution Method. Test bacteria included Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella choleraesuis. RESULTS: Chlorine concentrations at 30 days varied from the 40% to 50% range for 1:50 or 1:100 dilutions stored in containers other than closed brown containers to 83% to 85% for the 1:5 dilution stored in closed but non-opaque containers to 97% to 100% for 1:50 or 1:5 solutions stored in closed brown containers. The lowest concentration of sodium hypochlorite solution that reliably inactivated all the test organisms was 100 ppm. CONCLUSIONS: These data suggest that chlorine solutions do not need to be prepared fresh daily, as is recommended currently, and the lowest concentration of chlorine that reliably inactivates S aureus, S choleraesuis, and P aeruginosa is 100 ppm.

Chlorine↗
PubMed1997

The role of cysteine residues in S100B dimerization and regulation of target protein activity.

Previous studies have demonstrated that the two cysteine residues in the calcium-binding protein S100B are required for its extracellular functions. In the present study, a recombinant S100B protein and mutant S100Bs containing one or no cysteine residue(s) have been used to determine the contribution of cysteine residues to S100B dimerization and interaction with the intracellular target proteins aldolase, phosphoglucomutase, and the microtubule associated tau protein. Mutation of C68 to a valine or C84 to a serine, C68 to valine and C84 to serine, or C68 to valine and C84 to alanine did not significantly alter S100B activation of aldolase. However, mutation of C84 to serine resulted in calcium-independent S100B activation of phosphoglucomutase and a loss of S100B inhibition of tau phosphorylation by Ca2+/calmodulin-dependent protein kinase II. The altered functionality of the C84S mutant with phosphoglucomutase and tau was not due to altered physical properties or dimerization state. All of the mutants exhibited heat stability and calcium dependent conformational changes which were identical to recombinant S100B. In addition, S100B proteins containing two, one or no cysteine residues behaved as dimers in size exclusion chromatography experiments in the presence or absence of calcium as well as in the presence or absence of reducing agent. Dynamic light scattering and analytical ultracentrifugation experiments confirmed that dimerization was not affected by calcium or reducing agent. Altogether these results demonstrate that S100B dimerization is not calcium- or sulfhydryl-dependent. In summary, cysteine residues are not necessary for the noncovalent dimerization of S100B, but are important in certain S100B target protein-interactions.

Calcium-Binding Proteins↗
PubMed1997

Characterization of lanthanide ion binding to the EF-hand protein S100 beta by luminescence spectroscopy.

S100 beta is a member of a group of low-molecular weight acidic calcium binding proteins widely distributed in the vertebrate nervous system containing two helix-loop-helix calcium binding motifs (sites I and II). In addition, S100 beta also has auxiliary Zn2+ binding sites that are distinct from the Ca2+ binding sites. Luminescence spectroscopy using Eu3+ and Tb3+ as spectroscopic probes for Ca2+ is used to characterize the Ca2+ binding sites of this protein. Eu3+-bound S100 beta shows two distinct Eu3+ binding environments from both the excitation spectrum and Eu3+ excited state lifetimes. Eu3+ bound to the classical EF hand site II has a Kd of 660 +/- 20 nM, whereas the dissociation constant for the pseudo-EF hand site I is significantly weaker. Lifetimes in H2O and D2O lead to the finding that there are four water molecules coordinated to the Eu3+ in the weakly binding site I and two water molecules to the tightly binding site II. Site II in S100 beta expectedly is very similar to high-affinity Ln3+ binding domains I and II in calmodulin. Eu3+ luminescence experiments with Zn2+-loaded S100 beta show that the lifetime for Eu3+ in site I in Zn2+-loaded S100 beta is significantly different than that in the absence of Zn2+. Tyrosine-17-sensitized Tb3+ luminescence experiments indicate that the Tb3+ occupying the proximal weaker binding site I is sensitized, whereas Tb3+ in site II is not. The distance between sites I and II (15.0 +/- 0.4 A) in S100 beta was determined from Forster-type energy transfer in D2O solutions containing bound Eu3+ donor and Nd3+ acceptor ions. For Zn2+-S100 beta, the intersite distance is reduced to 13 +/- 0.3 A. Location of histidine-15 close to pseudo-EF site I suggests that Zn2+ binding likely changes the conformation of this site, causing a reduction of the intersite distance by approximately 2 A.

Amino Acid Sequence↗
PubMed1997

Solution structure for Pandinus toxin K-alpha (PiTX-K alpha), a selective blocker of A-type potassium channels.

PiTX-K alpha, a 35-residue peptide recently isolated from the venom of Pandinus imperator, blocks the rapidly inactivating (A-type) K+ channel(s) in rat brain synaptosomes and the cloned Kv 1.2 potassium channel at very low toxin concentrations (6 nM and 32 pM, respectively) [Rogowski, R. S., Collins, J. H., O'Neil, T. J., Gustafson, T. A., Werkman, T. A., Rogawski, M. A., Tenenholz, T. C., Weber, D. J., & Blaustein, M. P. (1996) Mol. Pharmacol. 50, 1167-1177]. The three-dimensional structure of PiTX-K alpha was determined using NMR spectroscopy in order to understand its selectivity and affinity toward K+ channels. PiTX-K alpha was found to have an alpha-helix from residues 10 to 21 and two beta-strands (betaI, 26-28; betaII, 33-35) connected by a type II beta-turn to form a small antiparallel beta-sheet. Three disulfide bonds, which are conserved in all members of the charybdotoxin family (alpha-K toxins), anchor one face of the alpha-helix to the beta-sheet. The N-terminal portion of PiTX-K alpha has three fewer residues than other alpha-K toxins such as charybdotoxin. Rather than forming a third beta-strand as found for other alpha-K toxins, the N-terminal region of PiTX-K alpha adopts an extended conformation. This structural difference in PiTX-K alpha together with differences in sequence at Pro-10, Tyr-14, and Asn-25 (versus Ser-10, Trp-14, and Arg-25 in CTX) may explain why PiTX-K alpha does not block maxi-K+ channels. Differences in three-dimensional structure between PiTX-K alpha and charybdotoxin are also observed in both the tight turn and the loop that connects the first beta-strand to the alpha-helix. As a result, side chains of two residues (Tyr-23 and Arg-31) are in regions of PiTX-K alpha that probably interact with rapidly inactivating A-type K+ channels. The analogous residues in charybdotoxin are positioned differently on the toxin surface. Thus, the locations of Tyr-23 and Arg-31 side chains in PiTX-K alpha could explain why this toxin blocks A-type channels at much lower concentrations than does charybdotoxin.

Amino Acid Sequence↗
PubMed1997

Oligomerization state of S100B at nanomolar concentration determined by large-zone analytical gel filtration chromatography.

S100B is a Ca(2+)-binding protein known to be a non-covalently associated dimer, S100B(beta beta), at high concentrations (0.2-3.0 mM) under reducing conditions. The solution structure of apo-S100B (beta beta) shows that the subunits associate in an antiparallel manner to form a tightly packed hydrophobic core at the dimer interface involving six of eight helices and the C-terminal loop (Drohat AC, Amburgey JC, Abildgaard F, Starich MR, Baldisseri D, Weber DJ. 1996. Solution structure of rat apo-S100B (beta beta) as determined by NMR spectroscopy. Biochemistry 35:11577-11588). The C-terminal loop, however, is also known to participate in the binding of S100B to target proteins, so its participation in the dimer interface raises questions as to the physiological relevance of dimeric S100B (beta beta). Therefore, we investigated the oligomerization state of S100B at low concentrations (1-10,000 nM) using large-zone analytical gel filtration chromatography with 35S-labeled S100B. We found that S100B exists (> 99%) as a non-covalently associated dimer, S100B (beta beta), at 1 nM subunit concentration (500 pM dimer) in the presence or absence of saturating levels of Ca2+, which implies a dissociation constant in the picomolar range or lower. These results demonstrate for the first time that in reducing environments and at physiological concentrations, S100B exists as dimeric S100B (beta beta) in the presence or absence of Ca2+, and that the non-covalent dimer is most likely the form of S100B presented to target proteins.

Amino Acid Sequence↗
PubMed1997

Detection and characterization of a protein isoaspartyl methyltransferase which becomes trapped in the extracellular space during blood vessel injury.

Injury to rat blood vessels in vivo was found to release intracellular pools of protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) into the extracellular milieu, where it becomes trapped. This trapped cohort of PIMT is able to utilize radiolabeled S-adenosyl-L-methionine (AdoMet) introduced into the circulation to methylate blood vessel proteins containing altered aspartyl residues. As further shown in this study, methylated substrates are detected only at the specific site of injury. In vitro studies more fully characterized this endogenous PIMT activity in thoracic aorta and inferior vena cava. Methylation kinetics, immunoblotting, and the lability of methylated substrates at mild alkaline pH were used to demonstrate that both types of blood vessel contain an endogenous protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT). At least 50% of the PIMT activity is resistant to nonionic detergent extraction, suggesting that the enzyme activity becomes trapped within or behind the extracellular matrix (ECM). Quantities of lactate dehydrogenase (LDH), another soluble enzyme of presumed intracellular origin, were found to be similarly trapped in the extracellular space of blood vessels.

Animals↗
PubMed1997

Injury-induced enzymatic methylation of aging collagen in the extracellular matrix of blood vessels.

As a result of blood vessel injury, protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT), a normally intracellular enzyme, becomes trapped within the meshwork of the vascular extracellular matrix where it can methylate substrate proteins. In this investigation we examined the distribution of such altered aspartyl-containing substrate proteins in the vascular wall. Nearly 90% of all the altered aspartyl residues were inaccessible to intracellular PIMT. Proteins of the extracellular matrix were found to be the major repository of altered aspartyl-containing polypeptides in the blood vessel wall, accounting for approximately 70% of the total amount. Proteolytic cleavage of extracellular matrix proteins with cyanogen bromide (CNBr) revealed that collagens account for most of the altered aspartyl-containing proteins of the ECM. As a consequence of blood vessel injury, both type I and type III collagen along with other proteins were found to become methylated by injury-released PIMT. It is estimated that 1 cm of vein contains on the order of 5 x 10(14) altered aspartyl residues involving between 1% and 5% of the total extracellular protein.

Animals↗
PubMed1997

Susceptibility of antibiotic-susceptible and antibiotic-resistant hospital bacteria to disinfectants.

OBJECTIVE: To evaluate whether hospital strains of antibiotic-resistant bacteria exhibited altered susceptibility to disinfectants. DESIGN: Antibiotic-susceptible bacteria were obtained from American Type Culture Collection: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Salmonella choleraesuis, and Pseudomonas aeruginosa. Hospital strains of antibiotic-resistant bacteria were obtained from clinical isolates, including: S aureus, S epidermidis, E coli, Enterococcus species, K pneumoniae, and P aeruginosa. The Association of Official Analytical Chemist's use-dilution method was used to test these 12 strains of 7 bacterial pathogens for their susceptibility to a phenol and a quaternary ammonium compound. For five pathogens, we tested a susceptible and a more resistant strain in 20 comparative trials (5 pathogens, 2 disinfectants, 2 dilutions per disinfectant). RESULTS: In our 20 comparative trials, the antibiotic-resistant strains exhibited an increased resistance to the disinfectant in only a single instance. CONCLUSIONS: Our data demonstrate that the development of antibiotic resistance does not appear to be correlated to increased resistance to disinfectants.

Bacteria↗
PubMed1997

Uses of inorganic hypochlorite (bleach) in health-care facilities.

Hypochlorite has been used as a disinfectant for more than 100 years. It has many of the properties of an ideal disinfectant, including a broad antimicrobial activity, rapid bactericidal action, reasonable persistence in treated potable water, ease of use, solubility in water, relative stability, relative nontoxicity at use concentrations, no poisonous residuals, no color, no staining, and low cost. The active species is undissociated hypochlorous acid (HOCl). Hypochlorites are lethal to most microbes, although viruses and vegetative bacteria are more susceptible than endospore-forming bacteria, fungi, and protozoa. Activity is reduced by the presence of heavy metal ions, a biofilm, organic material, low temperature, low pH, or UV radiation. Clinical uses in health-care facilities include hyperchlorination of potable water to prevent Legionella colonization, chlorination of water distribution systems used in hemodialysis centers, cleaning of environmental surfaces, disinfection of laundry, local use to decontaminate blood spills, disinfection of equipment, decontamination of medical waste prior to disposal, and dental therapy. Despite the increasing availability of other disinfectants, hypochlorites continue to find wide use in hospitals.

Chlorine↗
PubMed1996

Solution structure of rat apo-S100B(beta beta) as determined by NMR spectroscopy.

S100B(beta beta), a member of the S100 protein family, is a Ca(2+)-binding protein with noncovalent interactions at its dimer interface. Each apo-S100 beta subunit (91 residues) has four alpha-helices and a small antiparallel beta-sheet, consistent with two predicted helix-loop-helix Ca(2+)-binding domains known as EF-hands [Amburgey et al. (1995) J. Biomol. NMR 6, 171-179]. The three-dimensional solution structure of apo-S100B(beta beta) from rat has been determined using 2672 distance (14.7 per residue) and 88 dihedral angle restraints derived from multidimensional nuclear magnetic resonance spectroscopy. Apo-S100B (beta beta) is found to be globular and compact with an extensive hydrophobic core and a highly charged surface, consistent with its high solubility. At the symmetric dimer interface, 172 intermolecular nuclear Overhauser effect correlations (NOEs) define the antiparallel alignment of helix I with I' and of helix IV with IV'. The perpendicular association of these pairs of antiparallel helices forms an X-type four-helical bundle at the dimer interface. Whereas, the four helices within each apo-S100 beta subunit adopt a unicornate-type four-helix bundle, with helix I protruding from the parallel bundle of helices II, III, and IV. Accordingly, the orientation of helix III relative to helices I, II, and IV in each subunit differs significantly from that known for other Ca(2+)-binding proteins. Indeed, the interhelical angle (omega) observed in the C-terminal EF-hand of apo-S100 beta is -142 degrees, whereas omega ranges from 118 degrees to 145 degrees in the apo state and from 84 degrees to 128 degrees in the Ca(2+)-bound state for the EF-hands of calbindin D9k, calcyclin, and calmodulin. Thus, a significant conformational change in the C-terminal EF-hand would be required for it to adopt a structure typical of the Ca(2+)-bound state, which could readily explain the dramatic spectral effects observed upon the addition of Ca2+ to apo-S100B(beta beta).

Amino Acid Sequence↗
PubMed1996

Seroepidemiological survey of hepatitis B and C virus infections in Ghanaian children.

The seroprevalences of hepatitis B virus (HBV) and hepatitis C virus (HCV) markers were evaluated in a random sample of 803 children attending school in Ashanti-Akim North district in Ghana in order to gain a better understanding of transmission patterns of these viruses, particularly horizontal transmission of HBV. This rural district is typical of 70% of the Ghanaian population. The overall seroprevalence of at least one marker of HBV infection was 61.2%, with rates increasing from 48% to 80% between the ages of 6-18 years (P < 0.001). The overall HBsAg seroprevalence was 15.8%, with the proportion of HBsAg positives amongst those with anti-HBc increasing from 39.3% in 6-7-year-olds to 51.8% in 12-13-year-olds. It appears that horizontal transmission during this age period was accompanied by a high rate of HBsAg carriage. Among those infected but not carriers, i.e., those HBsAg negative and anti-HBc positive, > 50% lacked detectable levels of anti-HBs, an unusual pattern of convalescent immune response to HBV. The overall seroprevalence of anti-HCV was 5.4% and did not differ significantly by age or gender. Anti-HCV seroprevalence was not associated with the presence of any HBV marker. A better understanding of the unusually high prevalences of HBV and HCV infections demonstrated in this population is likely to influence vaccination and blood transfusion policies and to stimulate further evaluations of these infections and their vehicles of spread in highly endemic regions such as sub-Saharan Africa.

Adolescent↗
PubMed1996

Prevention and control of varicella-zoster infections in healthcare facilities.

Varicella-zoster virus (VZV) is the causative agent of two diseases: varicella (chickenpox) and zoster (shingles). Although varicella generally is a mild disease in children, serious morbidity and mortality are common if infection occurs in neonates, pregnant women, adults, or immunocompromised patients. For this reason, the Centers for Disease Control and Prevention recommends that all the hospitals institute control measures. Healthcare workers should be screened for VZV immunity and, if susceptible, should receive the recently licensed Oka/Merck vaccine (unless contraindicated). This article reviews nosocomial outbreaks associated with VZV and provides detailed algorithms for preexposure immunization and postexposure management of healthcare workers exposed to VZV.

Adolescent↗
PubMed1996

Management of healthcare workers with pharyngitis or suspected streptococcal infections.

The group A streptococcus may cause pharyngitis, rheumatic fever, streptococcal toxic shock syndrome, and serious skin and soft-tissue infections. More than 50 nosocomial outbreaks have been reported since 1966. For this reason, healthcare facilities should develop policies for the diagnosis and treatment of symptomatic hospital employees, and for the recognition and management of potential outbreaks. The clinical diagnosis of streptococcal pharyngitis is unreliable. Rapid streptococcal tests may be used for initial screening, but a negative rapid test should be confirmed with a properly obtained culture. Penicillin remains the treatment of choice, but new alternatives now include a 5-day course of either azithromycin or cefpodoxime.

Anti-Bacterial Agents↗