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Biomedical subjects

R Novak

Publications and source records attributed to R Novak.

At least 19 recordsLinked to original sources
PubMed2001

Clinical isolates of Streptococcus pneumoniae that exhibit tolerance of vancomycin.

The ability of Streptococcus pneumoniae to escape lysis and killing by vancomycin, a property termed "tolerance," has recently been noted in a laboratory strain of the species. Vancomycin tolerance in clinical isolates represents a potential new health risk. We determined the prevalence of vancomycin and penicillin tolerance among 116 clinical isolates of pneumococci by monitoring lysis and viability after exposure to the respective antibiotic for 4 hours. Eight percent of the strains were tolerant to penicillin and 3% were tolerant to vancomycin. The 3 vancomycin-tolerant isolates also had a high ratio of minimum bactericidal concentration to minimum inhibitory concentration, in contrast to nontolerant strains. They were of serotype 9V and had reduced susceptibility to penicillin. Only 1 was also tolerant to penicillin. Growth rate and ability to divide were not affected in the 3 vancomycin-tolerant strains, and they all lysed with deoxycholate, which indicates autolysin production. Vancomycin tolerance among clinical isolates of pneumococci will necessitate tracking to determine the magnitude of the evolving health risk, since tolerance may contribute to treatment failure (in particular, cases of meningitis, in which bactericidal activity is critical for eradication) and since it may also be a favored background for acquisition of resistance of vancomycin.

Amino Acid Sequence↗
PubMed2001

Competitive DNA hybridization in microtitre plates for chicken anaemia virus.

Unlabelled chicken anaemia virus (CAV) DNA probe, produced by PCR, was immobilized onto nitrocellulose discs that then were fitted into microtitre plate wells in order to develop a competitive, non-radioactive hybridization test for detection of CAV. The discs were hybridized with either DNA extracts of buffy coats or dilutions of CAV DNA (for standard curves), followed by hybridization with biotin-labelled CAV DNA probe in excess of the immobilized, capture probe. Thus, CAV from sample DNA extracts and standard DNA preparations competed with the biotin-labelled CAV DNA probe for the immobilized, capture probe, decreasing subsequent colour development by an avidin-biotin-alkaline phosphatase detection system. Standard curves were log linear from 5-100 ng viral DNA with r(2)> or =0.91. Tests were considered positive at 2 SD less than mean absorbence of samples from uninfected chickens, and ranged from 52 to 108 microm viral DNA or 2 to 4.2x10(10)virions microg(-1)buffy coat DNA. Blood samples from chickens infected and not infected with CAV at one day of age were tested for evidence of infection until 28 days of age by viral isolation, competitive hybridization in microtitre plates, dot-blots, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization on blood smears. None of the tests was positive for uninfected chickens. Viral isolation from buffy coats, though expensive and lengthy, was the most sensitive method. It detected virus in buffy coat from each infected chicken, while competitive hybridization detected 72% of infected chickens, in situ hybridization 69%, dot-blots 67%, and ELISA 36%. Sensitivity of competitive hybridization was 0.78, and its specificity was 1.00. Three chickens must be sampled from an infected flock for a 90% chance of detecting a positive chicken at the 0.025 one-tailed level of significance, assuming 100% prevalence.

Age Factors↗
PubMed2001

The rationale and design of the CPCRA (Terry Beirn Community Programs for Clinical Research on AIDS) 058 FIRST (Flexible Initial Retrovirus Suppressive Therapies) trial.

The CPCRA (Terry Beirn Community Programs for Clinical Research on AIDS) 058 FIRST (Flexible Initial Retrovirus Suppressive Therapies) trial is a large, long-term, randomized, prospective comparison of three different antiretroviral strategies in highly active antiretroviral therapy-naïve, HIV-1-infected persons. The trial was designed as a flexible framework upon which other studies could be added to answer more limited, but still important, questions. This article presents the study design, discusses the challenges we have faced in implementing the trial, and describes our preliminary experiences. Control Clin Trials 2001;22:176-190

Acquired Immunodeficiency Syndrome↗
PubMed2001

Slovene smart card and IP based health-care information system infrastructure.

Slovenia initiated a nation-wide project to introduce smart cards in the health sector in 1995 and its full-scale deployment started in September 2000. Although the basic aim of the project was to support insurance related procedures, the system was designed in a flexible and open manner to present an infrastructure for the whole health sector. The functionality of the current system is described in this paper along with lessons learned so far. The upgrade of the system is outlined, with emphasis on technical details, the objective being to provide a real-time EDI based environment for a general set of applications in the medical sector, supported by the flexibility and security of modern smart card technologies. Integration with similar systems in other EU countries is discussed.

Computer Communication Networks↗
PubMed2000

Signal transduction by a death signal peptide: uncovering the mechanism of bacterial killing by penicillin.

The binding of bactericidal antibiotics like penicillins, cephalosporins, and glycopeptides to their bacterial targets stops bacterial growth but does not directly cause cell death. A second process arising from the bacteria itself is necessary to trigger endogenous suicidal enzymes that dissolve the cell wall during autolysis. The signal and the trigger pathway for this event are completely unknown. Using S. pneumoniae as a model, we demonstrate that signal transduction via the two-component system VncR/S triggers multiple death pathways. We show that the signal sensed by VncR/S is a secreted peptide, Pep27, that initiates the cell death program. These data depict a novel model for the control of bacterial cell death.

Amino Acid Sequence↗
PubMed2000

Extracellular targeting of choline-binding proteins in Streptococcus pneumoniae by a zinc metalloprotease.

A genetic-based search for surface proteins of Streptococcus pneumoniae involved in adhesion identified a putative zinc metalloprotease (ZmpB). ZmpB shared high amino acid sequence similarities with IgA1 proteases of Gram-positive bacteria, but ZmpB had neither IgA1 nor IgA2 protease activity. Analysis of a family of surface-expressed proteins, the choline-binding proteins (Cbp's), in a zmpB-deficient mutant demonstrated a global loss of surface expression of CbpA, CbpE, CbpF and CbpJ. CbpA was detected within the cytoplasm. The zmpB-deficient mutant also failed to lyse with penicillin, a sign of lack of function of the Cbp LytA. Immunodetection studies revealed that the autolysin (LytA), normally located on the cell wall, was trapped in the cytoplasm colocalized with DNA and the transformation protein CinA. Trafficking of CinA and RecA to the cell membrane during genetic competence was also not observed in the zmpB-deficient mutant. These results suggest a protease dependent regulatory mechanism governing the translocation of CinA and the Cbp's LytA and CbpA of S. pneumoniae.

Alleles↗
PubMed2000

Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC.

The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones-regulators of proteolysis. By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC. The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis. Northern blot experiments showed that expression of clpC was induced in response to high temperature (40-42 degrees C) versus 37 degrees C, suggesting that ClpC is a heat shock protein. Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases. The clpC-deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin. The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells. Finally, the clpC disruption resulted in decreased genetic transformation. Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline-binding proteins LytA, CbpA, CbpE, CbpF, CbpJ. These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.

Bacterial Adhesion↗
PubMed2000

Isolation and characterization of vancomycin-tolerant Streptococcus pneumoniae from the cerebrospinal fluid of a patient who developed recrudescent meningitis.

The emergence of tolerance to vancomycin has recently been reported in Streptococcus pneumoniae, the most common cause of bacterial meningitis. A vancomycin- and cephalosporin-tolerant strain of S. pneumoniae, the Tupelo strain, was isolated from the cerebrospinal fluid of a patient who then developed recrudescence of meningitis despite treatment with vancomycin and a third-generation cephalosporin. The Tupelo strain evidenced no lysis in the exponential or stationary phase of growth when exposed to vancomycin and only minimal loss of viability. Further characterization revealed normal autolysin expression, localization, and triggering by detergents, indicating that the defect leading to tolerance in the Tupelo strain is in the control pathway for triggering of autolysis. Because tolerance is a precursor phenotype to resistance and may lead to clinical failure of antibiotic therapy, these observations may have important implications for vancomycin use in infections caused by S. pneumoniae.

Bacteriolysis↗
PubMed2000

Uncontrolled oxygen administration and respiratory failure in acute asthma.

STUDY OBJECTIVES: To determine if 100% oxygen administration adversely influences gas exchange in acutely ill asthmatic subjects. DESIGN: Prospective preinterventional and postinterventional comparison. SETTING: University hospital emergency department. PATIENTS: Thirty-seven asthmatic subjects seeking care for symptomatic exacerbations. INTERVENTIONS: Twenty minutes of 100% oxygen administration by face mask. MEASUREMENTS AND RESULTS: Arterial blood gases and FEV(1) were measured before and during the last minute of oxygen administration. On presentation, the subjects had moderately severe airway obstruction (FEV(1), 49.1 +/- 3.6% of predicted); hypocarbia (PaCO(2), 36.8 +/- 1.1 mm Hg); hypoxemia (PaO(2), 70.2 +/- 2.5 mm Hg); and respiratory alkalosis (pH, 7.43 +/- 0.01). During oxygen breathing, 25 patients (67.6%) experienced elevations in PaCO(2) ranging from 1 to 10 mm Hg (mean, 4.1 +/- 0.6 mm Hg; p = 0.0003). The increase was considered to be a physiologic manifestation of the Haldane effect (ie, < or = 2 mm Hg) in 10 subjects, but in the remaining 15 subjects (40.5% of the total studied), the elevation represented worsening gas exchange. In seven of these patients (46.7%), hypercapnic respiratory failure developed (PaCO(2) before oxygen, 39.6 +/- 0.6; during oxygen, 44.7 +/- 0.7 mm Hg; p = 0.005), and in six patients (40%), it worsened (PaCO(2) before oxygen, 46.8 +/- 1.9; during oxygen, 52.0 +/- 3.1 mm Hg; p = 0.03). In general, the tendency toward hypercarbia was the greatest in the participants with the most severe airway obstructions. CONCLUSIONS: Our data demonstrate that the administration of 100% oxygen to acutely ill asthmatics may adversely influence carbon dioxide elimination.

Acute Disease↗
PubMed2000

Laboratory evaluations of methylated soy oil and monoterpenes as mosquito larvicides.

The larvicidal toxicities of methylated soy oil (MSO) and surfactant combinations were compared to 2 commercially available oil larvicides (Golden Bear Oil 1111 and Bonide) in standard laboratory bioassays of 4th-stage larvae of Culex pipiens Linn. The dose lethal to 50% of the test organisms (LD50) and the dose lethal to 95% of the test organisms (LD95) values are presented as microliters (microl) per beaker (treatment surface area of 54 cm2). The 2 surfactant-MSO mixtures differed significantly in their toxicity to Cx. pipiens larvae; 2% Pyroter CPI-40 in MSO was more toxic than 2% Pluronic L121 in MSO (LD50 = 3.8 microl per 54 cm2 and 11.3 microl per 54 cm2, respectively). The 2 most active larvicides were Golden Bear Oil (LD50 = 3.6 microl per 54 cm2) and the 2% Pyroter-MSO mixture. These 2 were not significantly different from each other. Bonide (LD50 = 6.2 microl per 54 cm2) and the Pluronic L121-MSO mixture (LD50 = 11.3 microl per 54 cm2) were less toxic than Golden Bear Oil and the MSO-Pryroter mixture and they were significantly different from each other. Bioassays with 4th-stage larvae of Anopheles stephensi Liston showed that toxicity of the Pyroter-MSO formulations increased about 2-fold from 18 degrees C to 24 degrees C (LD50 = 20.5 microl per 54 cm2 and 11.8 microl per 54 cm2, respectively). The laboratory bioassays suggest that MSO mixed with surfactants are potential mosquito larvicides. We also evaluated the influence of the 2 surfactants on the toxicity of 3 monoterpenes. The larvicidal activity of citral and limonene increased with the addition of surfactants, but neither surfactant enhanced the toxicity of cineole. All 3 monoterpenes, with and without surfactants, were considered poor candidates as surface larvicides because of their high volatilities.

Acyclic Monoterpenes↗
PubMed1999

Identification of two sources of carbon monoxide in comet Hale-Bopp.

The composition of ices in comets may reflect that of the molecular cloud in which the Sun formed, or it may show evidence of chemical processing in the pre-planetary accretion disk around the proto-Sun. As carbon monoxide (CO) is ubiquitous in molecular clouds, its abundance with respect to water could help to determine the degree to which pre-cometary material was processed, although variations in CO abundance may also be influenced by the distance from the Sun at which comets formed. Observations have not hitherto provided an unambiguous measure of CO in the cometary ice (native CO). Evidence for an extended source of CO associated with comet Halley was provided by the Giotto spacecraft, but alternative interpretations exist. Here we report observations of comet Hale-Bopp which show that about half of the CO in the comet comes directly from ice stored in the nucleus. The abundance of this CO with respect to water (12 per cent) is smaller than in quiescent regions of molecular clouds, but is consistent with that measured in proto-stellar envelopes, suggesting that the ices underwent some processing before their inclusion into Hale-Bopp. The remaining CO arises in the coma, probably through thermal destruction of more complex molecules.

Carbon Monoxide↗
PubMed1999

Emergence of vancomycin tolerance in Streptococcus pneumoniae.

Streptococcus pneumoniae, the pneumococcus, is the most common cause of sepsis and meningitis. Multiple-antibiotic-resistant strains are widespread, and vancomycin is the antibiotic of last resort. Emergence of vancomycin resistance in this community-acquired bacterium would be catastrophic. Antibiotic tolerance, the ability of bacteria to survive but not grow in the presence of antibiotics, is a precursor phenotype to resistance. Here we show that loss of function of the VncS histidine kinase of a two-component sensor-regulator system in S. pneumoniae produced tolerance to vancomycin and other classes of antibiotic. Bacterial two-component systems monitor environmental parameters through a sensor histidine-kinase/phosphatase, which phosphorylates/dephosphorylates a response regulator that in turn mediates changes in gene expression. These results indicate that signal transduction is critical for the bactericidal activity of antibiotics. Experimental meningitis caused by the vncS mutant failed to respond to vancomycin. Clinical isolates tolerant to vancomycin were identified and DNA sequencing revealed nucleotide alterations in vncS. We conclude that broad antibiotic tolerance of S. pneumoniae has emerged in the community by a molecular mechanism that eliminates sensitivity to the current cornerstone of therapy, vancomycin.

Animals↗
PubMed1999

Neuroprotection by a caspase inhibitor in acute bacterial meningitis.

Half of the survivors of bacterial meningitis experience motor deficits, seizures, hearing loss or cognitive impairment, despite adequate bacterial killing by antibiotics. We demonstrate that the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (z-VAD-fmk) prevented hippocampal neuronal cell death and white blood cell influx into the cerebrospinal fluid compartment in experimental pneumococcal meningitis. Hippocampal neuronal death was due to apoptosis derived from the inflammatory response in the cerebrospinal fluid. Apoptosis was induced in vitro in human neurons by inflamed cerebrospinal fluid and was blocked by z-VAD-fmk. As apoptosis drives neuronal loss in pneumococcal meningitis, caspase inhibitors might provide a new therapeutic option directed specifically at reducing brain damage.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Pneumolysin, a protein toxin of Streptococcus pneumoniae, induces nitric oxide production from macrophages.

Nitric oxide (NO) production by inducible NO synthase (iNOS) during inflammation is an essential element of antimicrobial immunity but can also contribute to host-induced tissue damage. Under conditions of bacterial sepsis, large amounts of NO are produced, causing hypotension, a critical pathological feature of septic shock. In sepsis caused by gram-positive organisms, the bacterial factors contributing to host NO production are poorly characterized. We show that a soluble toxin of Streptococcus pneumoniae, pneumolysin (Pln), is a key component initiating NO production from macrophages. In contrast to wild-type bacteria, a mutant of S. pneumoniae lacking Pln failed to elicit NO production from murine macrophages. Purified recombinant Pln induced NO production at low concentrations and independently of exogenous gamma interferon (IFN-gamma) priming of RAW 264.7 macrophages. However, IFN-gamma was essential for Pln-induced NO production, since primary macrophages from mice lacking the IFN-gamma receptor or interferon regulatory factor 1, a transcription factor essential for iNOS expression, failed to produce NO when stimulated with Pln. In addition, Pln acts as an agonist of tumor necrosis factor alpha and interleukin 6 production in macrophages. The properties of Pln, previously identified as a pore-forming hemolysin, also include a role as a general inflammatory agonist.

Animals↗
PubMed1999

Identification of a Streptococcus pneumoniae gene locus encoding proteins of an ABC phosphate transporter and a two-component regulatory system.

The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.

ATP-Binding Cassette Transporters↗
PubMed1999

Pathogenesis of pneumococcal pneumonia.

Pneumococcal pneumonia is the most severe of the common community-acquired pulmonary infections. The recent release of the complete DNA sequence reveals the entire capability of the bacteria and the current challenge is to map gene products to the mechanics of disease. This process will reveal antibiotic targets and protein vaccine candidates crossing serotype boundaries. Choline is a major constituent of surfactant and it is also a required nutrient for the pneumococcus. It appears that choline incorporated into the cell wall can bind the bacteria to the receptor for platelet activating factor, the gateway to invasion. The choline also serves as an antenna to which multifunctional proteins dock, thereby decorating the bacterial surface. This set of 12 choline binding proteins is subject to phase variation of expression resulting in display of different combinations of proteins that adapt the bacteria to survival on the mucosa versus the blood stream. These changes affect protective antigens, adhesions, and lytic proteins tying together the major elements of pneumococcal physiology: natural DNA transformation, adherence and invasion of host cells, and autolysis. Taking these components and building an understanding of disease is challenging. Clearly, the toxin pneumolysin is a major mediator of cell damage in the lung. Inflammation is also incited by cell wall components. The signal transduction pathways that explain pneumococcal inflammation are more complex than those for gram-negative endotoxin.

Animals↗
PubMed1999

Modification of the Kock's pouch for bladder replacement.

AIM: To describe a modification of the Kock's pouch and present our clinical experience in its application. METHOD: After isolating an adequate ileal segment, the bowel is split by a longitudinal incision, with both ends remaining unopened for about 3-4 cm in length. Previously anastomozed ureters are inserted into the proximal end so that a nipple valve is created. The distal unsplit end serves for a direct anastomosis with the urethra. During the past 9 years, this surgery was performed in 41 patients who underwent radical cystectomy due to invasive carcinoma. Seventeen patients died for unknown reasons or were lost to follow-up. The remaining 24 were included in the follow-up for a period of 7 months to 6 years post surgery. RESULTS: Half of the patients included in the follow-up urinated spontaneously, with post void residuals less than 120 mL and no need for catheterization. Four patients did not urinate spontaneously and needed continuos catheterization, whereas 8 patients had post void residuals of 150-200 mL and needed catheterization once a day or once a week. None of the patients had a stricture at the site of ureteroileal anastomosis. CONCLUSION: We believe that our modification of the Kock's pouch makes the uretero-intestinal anastomosis much easier to perform; both ureters are inserted into the unsplit bowel end, thus preventing reflux; anastomosis with the urethra is performed under visual control; and direct anastomosis of widely opened distal end of the bowel reduces the possibility of scar stenosis.

Aged↗