Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry

Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism. Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme. These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da. Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate. The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme. Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme. Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70. A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130. A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde. Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.     

Extrahepatic metabolism of 4-methylumbelliferone and lidocaine in the anhepatic rabbit

OBJECTIVE: The initially well-fixed implants of total hip replacement (THR) are in the long-term subject to aseptic loosening. Many cytokines can contribute to osteolysis due to osteoclast recruitment and/or activation. However, in this respect tumor necrosis factor-alpha (TNF-alpha) plays a pivotal role, because it upregulates interleukin-1 and 6 and granulocyte-macrophage colony stimulating factor. The aim of this study was to assess the eventual presence, cellular localization and extent of expression of TNF-alpha in the synovial-like membrane at the implant or at the cement to bone interface compared to control synovial membrane. METHODS: Twenty samples from the synovial-like membrane of the periprosthetic tissues were compared to control samples. TNF-alpha containing cells were visualized using an avidin-biotin-peroxidase complex (ABC) method and analyzed by light microscopy, double labelling and image analysis. RESULTS: TNF-alpha was found in the periprosthetic tissues in fibroblasts and vascular endothelial cells, but mainly in the macrophages was it found to coincide with areas containing implant-derived debris. TNF-alpha containing cells were more numerous in the synovial-like membrane in the interface tissue from the proximal stem area (2816 +/- 318 cells) than in the control synovial membrane (565 +/- 93 cells, p < 0.01). Interestingly, similarly high TNF-alpha expression (3452 +/- 582 cells) was also seen in the synovial-like membrane of the pseudocapsule. CONCLUSION: These findings suggest that the foreign body-type host reaction caused by THR is characterized by the high expression of TNF-alpha. Because such expression occurred in the interface tissue between the implant and surrounding bone, TNF-alpha, due to its pivotal direct and indirect role in the activation and recruitment of osteoclasts, may contribute to periprosthetic osteolysis and to the loosening of THR.     

Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34 h post human bloodmeal in midguts of P. papatasi and 34-48 h in P. langeroni; all endo- and exoprotease activities were completed by 50 h in P. papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was < 2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P. langeroni was the high level of aminopeptidase recorded within 10 h of the bloodmeal.     

The influence of breast size on late radiation effects and association with radiotherapy dose inhomogeneity

A prospective assessment of late changes in breast appearance in 559 patients after tumour excision and radiotherapy for early breast cancer noted a strong association with breast size. Only 3/48 (6%) patients with small breasts developed moderate or severe late changes compared with 94/423 (22%) with medium sized breasts and 34/88 (39%) patients with large breasts (p < 0.001). One possibility is that greater radiation changes are related to greater dose inhomogeneity in women with large breasts. To explore this hypothesis, radiation dose distributions were assessed in a separate group of 37 women in whom three-level transverse computer tomographic images of the breast in the treatment position were available. A significant correlation was found between breast size and dose inhomogeneity which may account for the marked changes in breast appearance reported in women with large breasts.     

Dental plaque flora of the dog with reference to fastidious and anaerobic bacteria associated with bites

Animal bite wounds are amongst the most common types of traumatic injuries in humans. The organisms isolated from these wounds generally reflect the oral flora of the biting animal, and may be fastidious in nature and difficult to identify. This study was undertaken to determine the prevalence of Eikenella corrodens, Actinobacillus actinomycetemcomitans, Porphyromonas and Prevotella spp. in supragingival dental plaque collected from the right maxillary canine and carnassial teeth and the right mandibular canine tooth of dogs. In part one of the study, 30 dogs were used. E. corrodens was found in 62% of these dogs and 44% of individual plaque samples. A. actinomycetemcomitans was not detected in any of the dogs sampled. In part two, 34 dogs were used to determine the prevalence of the black pigmented anaerobic bacilli (Porphyromonas and Prevotella spp.). Porphyromonas gingivalis was present in 68% of these dogs and 47% of individual plaque samples. Prevotella intermedia was present in 44% of the dogs and 23% of individual plaque samples. The recently described Porphyromonas canoris, Porphyromonas salivosa, Porphyromonas cangingivalis, Porphyromonas cansulci, Porphyromonas crevioricanis and Prevotella denticola species were isolated from only 9%, 6%, 3%, 3%, 3% and 3% of dogs respectively. Porphyromonas gingivicanis was not isolated from any of the animals sampled. In conclusion, black-pigmented anaerobic bacilli were isolated from 91% of the animals sampled and therefore constitute a significant risk with respect to bite wound infections. It is also suggested that the prevalence of E. corrodens in wound infections has been underestimated in previous reports because of use of inappropriate techniques for detecting this organism.     

Veterinary practice and occupational health. An epidemiological study of several professional groups of Dutch veterinarians. I. General physical examination and prevalence of allergy, lung function disorders, and bronchial hyperreactivity

The prevalence of allergy, lung function disorders, and bronchial hyperreactivity was studied in 102 Dutch veterinarians, subdivided into five professional groups (predominantly working with either swine, cattle, poultry, companion animals, or as a non-practitioner). The mean age of the participants was 43 years; 6 participants were females. Twenty-two per cent of the participants were overweight, and relatively more non-practitioners than practitioners were overweight. Approximately 23% of the vets reported complaints of prolonged fatigue. The data suggest a relationship between complaints of prolonged fatigue and a more than average number of daily working hours. Only a small proportion of vets were sensitized against several allergens. There were no significant differences in prevalence of distinct lung function disorders or bronchial hyperreactivity between professional groups. It is hypothesized that the respiratory complaints (chronic coughing, chronic phlegm production, stuffed nose, sneezing) reported by the vets predominantly working in swine and/or poultry practice could be caused by irritation and/or inflammation of the first part of the trachea-bronchial tree that has no measurable and permanent consequences for changes in lung function or increased bronchial hyperreactivity. The results of a skin test against allergens and determination of allergen-specific IgE in blood indicated that the respiratory complaints were probably not related to allergy against the panel of allergens tested.     

Quality of care problems among Medicare and Medicaid patients

The purpose of this study was to characterize quality of care problems among Medicare and Medicaid inpatients in New York State. The patients selected for this study comprised 1991 and 1992 Medicare and all 1992 Medicaid inpatients in whom quality of care problems with actual or potential adverse effects were found. The patients in this study were drawn from public, proprietary, voluntary and teaching hospitals. A total of 1000 quality of care problems with either actual or potential adverse effects were found in 706 Medicare patients. Two hundred and seventy-five (275) quality of care problems with actual or potential adverse effects were found in 154 Medicaid patients. Premature death occurred in 53 (7.4%) of the 706 Medicare and in 42 (27.2%) of the 154 Medicaid patients. Treatment problems and monitoring failures accounted for the majority of quality of care problems with actual or potential adverse effects for both Medicare (63.0%) and Medicaid (75.7%) patients. Among Medicare patients, the treatment of infections and antibiotic use, fluid and electrolyte management, and inappropriate drug use were among the leading causes of quality of care problems. Attending physicians were associated with the majority of Medicare quality of care problems while house staff and attending physicians were associated with the majority of those among Medicaid patients. The results of this study indicate that there are several leading causes of quality of care problems among Medicare and Medicaid patients. Treatment problems and monitoring failures together comprise the majority of such problems. Among Medicare patients, it was found that most quality of care problems were associated with the treatment of infections and antibiotic use, fluid and electrolyte management, and inappropriate drug use. Most quality of care problems among Medicaid patients were associated with these categories as well as with labor and delivery problems, and poor discharge planning. The results of this study reflect the peer-review process in which providers are given an opportunity to respond to physician-reviewer decisions about the presence of actual or potential adverse effects. Such a process, which permits the presentation of additional data and information by providers, produces fewer final adverse outcome determinations than a process uniquely based on chart review. The quality of care problems observed in this study are amenable to focused educational interventions. Such remedial interventions could yield significant improvements in the quality of care for all patients.     

Meeting a person with AIDS in the classroom: an evaluation

Meeting a person with AIDS in the classroom was evaluated to determine if it had an impact on students' perceived susceptibility to HIV infection and attitudes toward persons with AIDS. The meeting was incorporated into the Grade 9 AIDS education program of a school district in Nova Scotia. Four schools participated in this study. Two schools were randomly assigned to the treatment group, which met the person with AIDS, and the remaining two schools formed the comparison group. Measures of the two attitudinal variables were collected using a self-report questionnaire that was administered both prior to and two weeks after the educational intervention. Meeting a person with AIDS in the classroom had no measurable impact on students perceived susceptibility to HIV infection nor on their attitudes toward persons with AIDS. Suggestions for using the educational intervention more effectively and for further research are made.     

Midbrain raphe cell firing and hippocampal theta rhythm in urethane-anaesthetized rats

This study aimed to examine the functional coupling between midbrain raphe and the septohippocampal system at the level of neuronal firing. Raphe unit activity and hippocampal EEG were simultaneously recorded in urethane-anaesthetized rats and their relationship was examined in the frequency domain. Subsets of presumably non-serotonergic neurones in both the dorsal and median raphe nuclei fired rhythmically in synchrony with hippocampal theta activity. Theta cells in the median raphe showed higher coherence than those in dorsal raphe and formed a more homogeneous group of cells, according to their firing rates. Since the raphe-septal serotonergic system is known to desynchronize the hippocampal EEG, activation of a subset of nonserotonergic cells during theta in this nucleus indicates a feedback from the limbic circuitry on the ascending raphe control of forebrain activity.