Use of electrospray mass spectrometry for mass determination of grape (Vitis vinifera) juice pathogenesis-related proteins: a potential tool for varietal differentiation.

Methods based on liquid chromatography-mass spectrometry (LC-MS) and protein trap mass spectrometry (trap-MS) were developed to determine the complement of pathogenesis-related (PR) proteins in grape juice. Trap-MS was superior to LC-MS in terms of simplicity, rapidity, and sensitivity. Proteins with a wide range of masses (13--33 kDa) were found in the juices of 19 different varieties of grape (Vitis vinifera) and were identified as mostly PR-5 type (thaumatin-like) and PR-3 type (chitinases) proteins. Although the PR proteins in juices of grapes are highly conserved, small consistent differences in molecular masses were noted when otherwise identical proteins were compared from different varieties. These differences persisted through different harvest years and in fruits grown in different Australian locations. With the definition of four different masses for PR-5 proteins (range = 21,239--21,272 Da) and nine different masses of PR-3 proteins (range = 25,330--25,631 Da) and using statistical analysis, the methods developed could be used for varietal differentiation of grapes grown in several South Australian locations on the basis of the PR protein composition of the juice. It remains to be seen whether this technology can be extended to grapes grown worldwide and to wine and other fruit-derived products to assist with label integrity to the benefit of consumers.

Clonal relationships among bloodstream isolates of Escherichia coli.

The clonal relationships among 187 bloodstream isolates of Escherichia coli from 179 patients at Boston, Mass., Long Beach, Calif., and Nairobi, Kenya, were determined by multilocus enzyme electrophoresis (MLEE), analysis of polymorphisms associated with the ribosomal operon (ribotyping), and serotyping. MLEE based on 20 enzymes resolved 101 electrophoretic types (ETs), forming five clusters; ribotyping resolved 56 distinct patterns concordant with the analysis by MLEE. The isolates at each study site formed a genetically diverse group and demonstrated similar clonal structures, with the same small subset of lineages accounting for the majority of isolates at each site. Moreover, two ribotypes accounted for approximately 30% of the isolates at each study site. One cluster contained the majority (65%) of isolates and, by direct comparison of the ETs and ribotypes of individual isolates, was genetically indistinguishable from the largest cluster for each of two other collections of E. coli causing pyelonephritis and neonatal meningitis (R. K. Selander, T. K. Korhonen, V. Väisänen-Rhen, P. H. Williams, P. E. Pattison, and D. A. Caugent, Infect. Immun. 52:213-222, 1986; M. Arthur, C. E. Johnson, R. H. Rubin, R. D. Arbeit, C. Campanelli, C. Kim, S. Steinbach, M. Agarwal, R. Wilkinson, and R. Goldstein, Infect. Immun. 57:303-313, 1989), thus defining a virulent set of lineages. The isolates within these virulent lineages typically carried DNA homologous to the adhesin operon pap or sfa and the hemolysin operon hly and expressed O1, O2, O4, O6, O18, O25, or O75 antigens. DNA homologous to pap was distributed among isolates of each major cluster, whereas hly was restricted to isolates of two clusters, typically detected in pap-positive strains, and sfa was restricted to isolates of one cluster, typically detected in pap- and hly-positive strains. The occurrence of pap-positive isolates in the same geographically and genetically divergent lineages suggests that this operon was acquired early in the radiation of E. coli, while hly and sfa were acquired subsequently, most likely by pap-positive and pap- and hly-positive precursors, respectively.

Insulin sensitivity in cystic fibrosis.

Cystic fibrosis (CF) patients demonstrate a spectrum of pancreatic beta-cell abnormalities. Those with no exocrine insufficiency (NEXO) have normal insulin secretion. Exocrine-insufficient CF patients with overt diabetes (EXO-IT) have impaired insulin secretion and fasting hyperglycemia. Exocrine-insufficient patients without diabetes (EXO) have impaired insulin secretion but maintain normoglycemia. We postulated that EXO individuals compensate for insulin deficiency by increasing insulin sensitivity and investigated glucose utilization in CF. To examine hepatic and peripheral insulin sensitivity, euglycemic-hyperinsulinemic clamp studies were performed by using the hot GINF isotope dilution technique. Insulin was sequentially infused at 0.25, 1.0, and 10.0 mU.kg-1.min-1. Glucose-mediated glucose uptake (GMGU) was assessed on another day with hyperglycemic clamp studies, during which insulin and somatostatin were infused to hold insulin-mediated glucose uptake constant between the two clamp studies. Skeletal muscle GLUT4 levels were assessed in EXO and control patients with Western blotting. Three patterns of peripheral and hepatic insulin sensitivity were seen that were related to the degree of pancreatic beta-cell dysfunction. NEXO individuals had normal peripheral and hepatic insulin sensitivity. EXO individuals had enhanced peripheral insulin sensitivity that was not associated with a change in skeletal muscle glucose transporter abundance compared with control patients; paradoxically, EXO subjects demonstrated hepatic insulin resistance. EXO-IT had peripheral and hepatic insulin resistance. GMGU was diminished in both EXO and EXO-IT subjects. The unique combination of increased hepatic glucose production and increased peripheral glucose utilization seen in EXO may be a metabolic adaptation to increased peripheral energy needs.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of a needleless system with conventional heparin locks.

BACKGROUND: Despite the improvements in needle disposal systems, needlesticks to health care workers continue to occur at unacceptably high rates. Needleless systems have been shown to reduce the risk of needlesticks. METHODS: This pilot study examined the safety of such a system for patients by comparing the rates of intravenous infection-related indicators between a conventional heparin lock and a needleless system. Patients (n = 97) were categorized on the basis of the duration of intravenous placement into 24-, 48-, and 72-hour groups. Within each group, half of the patients received conventional heparin locks and half received the needleless system. Intravenous infection-related indicators included catheter tip culture, adaptor fluid culture, intravenous site erythema, induration and tenderness, and elevated oral temperature. RESULTS: Prevalence of one or more indicators was 48% for the conventional and 40% for the needleless system, a difference that was not statistically significant. CONCLUSIONS: The needleless system appeared to pose no greater risk of infection to patients and nurses preferred it for its reduced risk of potential needlesticks.

The role of bacterial adhesins in the outcome of childhood urinary tract infections.

OBJECTIVES: To compare the efficacy of 3-day vs 10-day treatment with a combination of amoxicillin and clavulanate potassium for children with uncomplicated urinary tract infections and to determine the role of host factors, including vesicoureteral reflux, and of bacterial virulence factors, including adhesins, in treatment outcome. DESIGN: Randomized, double-blind, controlled trial. SETTING: A pediatric infectious diseases clinic at an urban medical center. PATIENTS: Thirty-seven children with uncomplicated urinary tract infections. INTERVENTIONS: Treatment with 3 days or 10 days of antibiotics at a dosage of 20 mg/kg per day of amoxicillin and 5 mg/kg per day of clavulanate potassium in three divided doses. MEASUREMENTS AND MAIN RESULTS: The success rate for 10-day treatment was 82% (14/17) compared with 55% (11/20) for 3-day treatment (P = .09). Among the 35 patients infected with Escherichia coli, all 10 patients infected with adhesin-negative isolates were treated successfully regardless of the duration of treatment, whereas only 14 (56%) of the 25 infections involving adhesin-positive isolates were clinically cured (P = .015). Two of the three failures in the 10-day treatment group were in patients with reflux. CONCLUSIONS: We conclude that 3-day treatment with amoxicillin and clavulanate is insufficient for afebrile childhood urinary tract infections and that both bacterial and host factors affect treatment outcome.

Relationship between indole production and differentiation of Klebsiella species: indole-positive and -negative isolates of Klebsiella determined to be clonal.

Klebsiellae are an important cause of nosocomial infections. The two clinically relevant species, Klebsiella pneumoniae and Klebsiella oxytoca, are differentiated by the ability to produce indole from tryptophan, K. oxytoca being indole positive. We report here the detailed biochemical and molecular analysis of two isolates of Klebsiella, cultured from the same urine specimen, that differed only in their ability to produce indole. The two isolates were identical as determined by ribotyping and pulsed-field gel electrophoresis, and they differed from 10 epidemiologically unrelated strains. Probing with the Escherichia coli tryptophanase operon, tna, revealed seven restriction fragment length polymorphisms (RFLP) among the 12 strains. The two index strains had identical RFLP; no single RFLP could account for all of the indole-positive or -negative strains. Thus, the identification of epidemiologically related strains of Klebsiella differing only in indole production may warrant further examination to determine whether the strains are clonal.

Bacterial adhesins and host factors: role in the development and outcome of Escherichia coli bacteremia.

To assess the contributions of bacterial virulence factors and defects in host defense to Escherichia coli bacteremia, we examined isolates and available medical records from 169 episodes at two hospitals. Adhesins and hemolysin virulence factors were documented in 84% of bacteremia-associated isolates originating from the urinary and respiratory tracts and in 50% of such isolates originating from other extraurinary foci. Of the evaluable episodes of bacteremia involving 35 adhesin-negative isolates, 21 (60%) were in patients who had mucocutaneous defects at the primary site of infection, and another 13 (37%) were in patients who had comorbid systemic diseases (i.e., chronic renal failure or alcoholism) associated with impaired leukocyte function. In contrast, of 93 evaluable episodes with adhesin-positive isolates, 22 (24%) were in patients who had none of these host conditions (P = .008). Overall, of evaluable episodes of bacteremia involving 128 isolates, 71 (55%) had both bacterial virulence-associated and host-related risk factors. Mortality was related to the severity of comorbid illnesses and was not affected by the presence of the bacterial virulence factors assessed.

Sulfite enhancement of diolepoxide mutagenicity: the role of altered glutathione metabolism.

Sulfur dioxide is a cocarcinogen for benzo[a]pyrene in the respiratory tract of rats and hamsters. Sulfur dioxide exists under physiological conditions as the sulfite ion. Sulfite enhances the mutagenic potency of (+-)-7r,8t-dihydroxy-9t,-10t- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) and 7r,8t-dihydroxy-9c10c-epoxy-7,8,9,10-tetrahydrobenzo[a]py ren e (syn-BPDE) in Salmonella typhimurium strains TA98 and TA100. This enhancement of diolepoxide mutagenicity is observed with sulfite concentrations between 1 and 20 mM, and the concentration dependence is identical for the two diolepoxides. Half-maximal enhancement of mutagenicity occurs at approximately 5 mM sulfite. Sulfite is neither toxic nor mutagenic to the bacteria under these conditions. The enhancement of diolepoxide mutagenicity requires that the bacteria be exposed to sulfite prior to the addition of the diolepoxide. Simultaneous addition of sulfite and diolepoxide significantly decreases the enhancing effect, and addition 15 min after the diolepoxide virtually abolishes the effect. This is consistent with sulfite serving to increase the efficiency of processes leading to DNA modification by the diolepoxides, rather than some effect subsequent to DNA adduct formation. Direct evidence for this hypothesis was provided by determining the effect of sulfite on mutagenicity and DNA binding in TA98 using [3H]anti-BPDE. Exposure of the bacteria to 10 mM sulfite for 5 min prior to the addition of the labeled mutagen led to as much as 170% increase in DNA binding levels relative to parallel incubations without sulfite. Corresponding increases in mutagenicity were seen as well. As sulfite can affect the glutathione/glutathione-S-transferase systems, the primary cellular defense against BPDE, the effect of sulfite on these pathways in Salmonella was determined. When strain TA98 was treated with N-acetoxy-2-acetamidofluorene, a direct-acting mutagen not scavenged by glutathione, prior addition of 10 mM sulfite to the bacteria had no effect on resultant viability or mutagenicity. Assessment of the bacterial glutathione levels revealed that 10 mM sulfite treatment results in an 82% decrease in the concentration of the cosubstrate. We were, however, unable to detect diolepoxide-glutathione conjugates in any of our incubations. Moreover, the presence of sulfite leads to significant trapping of the diolepoxide in the form of sulfonate derivatives. Based on these data, we conclude that the depletion of glutathione does indeed play a role in the enhancement of diolepoxide mutagenicity in S. typhimurium.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of conventional mechanical ventilation and high-frequency ventilation. A prospective, randomized trial in patients with respiratory failure.

Acute respiratory failure (ARF) following trauma or sepsis has a mortality rate of 50% to 85%. The mainstays of treatment are mechanical ventilation and positive end-expiratory pressure (PEEP). In the past decade, many reports have claimed superiority of high frequency ventilation (HFV) in the treatment of ARF. We structured a prospective randomized trial of HFV versus conventional mechanical ventilation (CMV) in the treatment of acute respiratory failure. All patients admitted to the Surgical Intensive Care Unit (SICU) were eligible for the study. On admission patients identified for being at risk of developing acute respiratory failure were randomized to receive either HFV or CMV. Patients were treated to the same therapeutic endpoint (pH greater than 7.35, PaCO2 35 to 45 torr, PaO2/FIO2 greater than 225). Daily ventilatory support, fluid and drug requirements, and cardiopulmonary variables were recorded. One hundred thirteen patients were entered into the study. Of these, 100 completed the study (HFV n = 52, CMV n = 48) and 60 developed acute respiratory failure (HFV n = 32, CMV n = 28). Patients on HFV reached the therapeutic endpoint at a lower level of continuous positive airway pressure and mean airway pressure; however there were no differences in mortality, SICU days, hospital days, incidence of barotrauma, number of blood gases, or cardiovascular interventions. This report suggests that HFV offers no concrete advantages over CMV when applied in a prospective fashion for the treatment of acute respiratory failure.

Cobalt plaque versus enucleation for uveal melanoma: comparison of survival rates.

Two hundred and twenty-three patients treated by cobalt plaque for uveal melanoma were compared with 416 patients treated by enucleation for uveal melanoma in terms of patient survival. The median follow-up time for the patients treated by cobalt plaque was 4.3 years. Kaplan-Meier survival curves were calculated up to five years following treatment based on time to tumour-related deaths. Cox's proportional hazards multivariate analysis was performed to determine which variables were related to melanoma-related deaths while controlling for age, size, and location of the tumours. Statistically significant predictive factors were location of tumour and largest tumour dimension. There was not a statistically significant difference in survival between patients treated by cobalt plaque and those treated by enucleation.