Replication terminator protein-based replication fork-arrest systems in various Bacillus species

The replication terminator protein (RTP) of Bacillus subtilis interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region. A similar situation exists in Escherichia coli, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another. In the present work, RTP homologs in four species closely related to B. subtilis (B. atrophaeus, B. amyloliquefaciens, B. mojavensis, and B. vallismortis) have been identified and characterized. An RTP homolog could not be identified in another closely related species, B. licheniformis. The nucleotide and amino acid changes from B. subtilis among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of the rtp genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from B. licheniformis. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning. By coincidence, the single change (E30K) found in the B. mojavensis RTP corresponds exactly to that purposefully introduced by others into B. subtilis RTP to implicate a crucial role for E30 in the fork-arrest mechanism. The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in B. subtilis in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a Bacillus terminator consensus and identification of two more terminators in the B. subtilis 168 genome sequence to bring the total to nine.     

Hemodynamic rounds series II: Coronary hemodynamics for angioplasty and stenting after myocardial infarction: use of absolute, relative coronary velocity and fractional flow reserve

To assess the effects and site of action of a sub-anaesthetic concentration of isoflurane, desflurane and sevoflurane (0.1 minimum alveolar concentration (MAC)) on respiratory control, we measured the ventilatory response to square wave changes in PE1CO2 against a background of normoxia. Using the computer steered "end-tidal forcing system", 2 min of steady state ventilation were followed by a step increase in PE1CO2 (1-1.5 kPa). This level was maintained for 8 min, followed by a step decrease to the original value for another 8 min. Each hypercapnic response was separated into a fast, peripheral component and a slow, central component, characterized by a time constant, carbon dioxide sensitivity, time delay and off-set. We studied 25 healthy volunteers; they performed 2-3 studies without and 2-3 studies during inhalation of the anaesthetic agent. Level of sedation was scored using a subjective seven-point scale from 0 (= alert and awake) to 6 (unrousable). In the isoflurane (16 subjects, 33 control, 37 drug studies) and sevoflurane (15 subjects, 40 control, 41 drug studies) studies, peripheral carbon dioxide sensitivity was reduced by approximately 45% and approximately 27% (ANOVA, P < 0.05 vs control), respectively, without affecting central carbon dioxide sensitivity or apnoeic threshold. In the desflurane study (16 subjects, 36 control, 37 drug studies), no significant effect was observed for any of the variables measured. A significant relation was observed between sedation score and change from control in central carbon dioxide sensitivities in the isoflurane and desflurane studies and in the change in the ratio peripheral carbon dioxide sensitivity over total carbon dioxide sensitivity in the sevoflurane studies. At the highest level of sedation observed (score 3-arousal state comparable with "light sleep"--in three subjects) these latter variables differed significantly from those in the other observed sedation levels (scores 1 and 2-a state of drowsiness). We conclude that 0.1 MAC of isoflurane and sevoflurane depressed the peripheral chemoreflex loop, without affecting the central chemoreflex loop. Desflurane at the same MAC showed no effect on peripheral and central carbon dioxide sensitivity. When the level of sedation was considered, our data suggested that at levels of sedation comparable with sleep, a depressive effect of all three anaesthetics was observed on the central chemoreflex loop.     

Comparative efficacy evaluation of dicationic carbazole compounds, nitazoxanide, and paromomycin against Cryptosporidium parvum infections in a neonatal mouse model

The efficacies of dicationic carbazole compounds, nitazoxanide (NTZ), and paromomycin were evaluated against the AUCp1 isolate of Cryptosporidium parvum by using a neonatal mouse model. Compounds were solubilized or suspended in deionized water and administered orally by gavage to neonatal mice at a constant dose rate on days 0 to 5 (treatment started on day 0). Dose rates varied for individual carbazole compounds but ranged from 0.65 to 20 mg/kg of body weight. NTZ was tested at 100 and 150 mg/kg, and paromomycin was tested at 50 mg/kg. Efficacies were determined by comparing numbers of oocysts present in treated versus control mice at necropsy examination on day 6. Demonstrable efficacy was observed for several carbazole compounds, based on significant reductions in the numbers of oocysts recovered from treated mice versus control mice. Compounds 1, 7, and 10 (19.0 mg/kg) reduced oocyst passage in treated mice to less than 5% of that in control mice. Treatment with compounds 6, 8, and 9 (17.0 mg/kg) resulted in reductions of oocyst output to less than 10% of that in controls. Although they were not comparable in efficacy to compounds 1, 6, 7, 8, 9, and 10, treatment with other carbazole compounds resulted in statistically significant reductions in oocyst output in treated versus control mice. Compound 1 retained efficacy resulted in reduction of oocyst output to approximately 6% of that in controls when the dose was reduced to 5 mg/kg. Further reductions in the dose rate resulted in considerable reductions in anticryposporidial activity. Likewise, the efficacies of compounds 9 and 10 were reduced substantially when the doses were lowered to one-half the screening dose. Paromomycin yielded excellent activity (reduction of oocyst output to <2% of that in controls) at a dose of 50 mg/kg. NTZ yielded moderate efficacy as powder and injectable formulations administered at 100 mg/kg orally (reduction of oocyst output to 42 and 26% of that in controls, respectively). Oral administration of the injectable formulation of NTZ at a dose of 150 mg/kg resulted in improved efficacy (oocyst output, <5% of that in controls).     

Respiratory arrest following peribulbar anesthesia for cataract surgery: case report and review of the literature

Hypnic headache syndrome is a rare benign disorder which occurs in the elderly. It presents as a holocephalic or, less frequently, hemicranial headache during night sleep, lasting between 20 and 180 min; it is not associated with autonomic signs and responds well to lithium carbonate. We report a new case which responded well to indomethacin, and suggest that indomethacin may be used as an alternative treatment in this rare headache syndrome of the elderly.     

Subacute and reproductive effects in mink from exposure to Fusarium fujikuroi culture material (M-1214) containing known concentrations of moniliformin

Okadaic acid (OA) is a potent inhibitor of PP1 and PP2A serine/threonine phosphatases and an inhibitor of phosphatidylinositol 3'-kinase (PI 3-kinase) recruitment/ activation. Here we report that PI 3-kinase associates with a serine kinase activated by OA. Whole cell phosphorylation studies showed that PI 3-kinase associates with a wortmannin insensitive 76 kDa serine phosphoprotein (pp76) distinct from the p85 subunit of PI 3-kinase. Serine kinase assays demonstrated that pp76 phosphorylation was dependent upon a wortmannin insensitive serine kinase contained within PI 3-kinase/pp76 complexes and that this kinase had different cation requirements than PI 3-kinase serine kinase. Treatment of whole cells with OA lead to a wortmannin-independent 7.6-fold increase in pp76 serine phosphorylation and to a 7-fold rise in pp76 kinase activity. Together, these findings indicate that pp76 is a PI 3-kinase associated phosphoprotein and suggest that pp76 may be a novel PI 3-kinase associated serine kinase that is activated by OA.     

Cancer early-detection services in community health centers for the underserved. A randomized controlled trial

BACKGROUND: Achieving cancer early-detection goals remains a challenge, especially among low-income and minority populations. DESIGN/SETTING: A randomized trial based in 62 community health centers for the underserved in New York, New Jersey, and western Connecticut. Family physicians were on staff at most of the centers. INTERVENTION: Workshops, materials, and ongoing advice for center leaders promoted implementation of a preventive services office system to identify patients in need of services at each visit through use of medical record flow sheets, other tools, and staff involvement. EVALUATION END POINTS: The proportion of randomly selected patients by center who were up to date for indicated services at baseline (n = 2645) and follow-up (n = 2864) record review. RESULTS: Only 1 service (breast self-examination advice) increased more in intervention centers. Seven of 8 target services increased significantly for the 62 centers overall. During the study, the medical director changed in 26 centers (42%). Keeping the same medical director at intervention centers was associated with improvements in services. CONCLUSIONS: Cancer early-detection services are improving in community health centers, but the intervention had only a small impact, as determined by record review. To have an impact, the intervention required that there be no change in medical director. The relationship of changes in the practice environment to services delivered is complex and deserves more study.     

Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation

Recent data suggest that aberrant function of the wild type p53 protein (WTp53) may alter cellular survival following DNA damage through cellular pathways involving apoptosis and cell-cycle checkpoints, but little is known concerning it's possible role in DNA repair. In the present study, the ionizing radiation sensitivity was determined for a series of rat embryo fibroblast (REF) cell lines transfected with an activated form of the H-ras oncogene alone, or in combination with a variety of missense-mutant p53 (MTp53) alleles. Transformed REF clones which expressed exogenous MTp53 and p21ras proteins (CLASS II clones) were generally radioresistant in culture as determined by higher values for the surviving fraction after 2 Gy (SF2 value) and the radiation dose required to reduce survival to a fraction of 0.1 (D10 value), compared either to transformed REF clones expressing p21ras protein alone (CLASS I clones), or to non-transfected REF control cell lines expressing baseline endogenous levels of p21ras and WTp53 protein. The increased radioresistance observed in the CLASS II clones (following both HDR- and LDR-irradiation), was significantly correlated with increased expression of MTp53 protein, and a decreased radiation-induced G1 arrest response. The variability observed in clonogenic radiosensitivity among REF clones was not explained by differential radiation-induced apoptosis. Using the Comet assay performed after continuous low dose-rate (LDR)-irradiation, MTp53-expressing REF clones were also found to be more proficient at the rejoining of DNA double-strand breaks (DNA-dsb), compared to WTp53-expressing REF clones. These results suggest that an enhanced DNA and cellular repair capacity may, in part, explain the increased radiation survival observed in some MTp53-expressing transformed fibroblasts and tumours.     

Drosophila coracle, a member of the protein 4.1 superfamily, has essential structural functions in the septate junctions and developmental functions in embryonic and adult epithelial cells

Although extensively studied biochemically, members of the Protein 4. 1 superfamily have not been as well characterized genetically. Studies of coracle, a Drosophila Protein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor of EgfrElp, a hypermorphic form of the Drosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis of coracle, one of the first for a member of the Protein 4. 1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical-basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults.     

Effect of topical anesthetic use on initial patient satisfaction and overall success with rigid gas permeable contact lenses

BACKGROUND: Rigid gas permeable (RGP) contact lenses have numerous benefits; however, RGP lens use is not increasing in the United States. An important factor for this trend has been initial comfort. Studies have demonstrated that how RGPs are presented to patients, in addition to lens design, can play an important role in the initial comfort process. Another important factor could be the use of a topical anesthetic during the fitting and dispensing visits. The purpose of this study was to use a multicenter format to determine if topical anesthetic use increased the likelihood of patient satisfaction and success. METHODS: A total of 80 subjects, with no previous rigid lens wear experience, was entered into this 1-month study, including 20 subjects from each of 4 institutions. Subjects were randomly divided into the following two groups: (A) anesthetic or (B) placebo, with the former group receiving one drop of a topical anesthetic before lens insertion at both the diagnostic fitting and dispensing visits, whereas the latter group received a placebo. Subjects completed a questionnaire on their perception of rigid lens wear both immediately before fitting and at the 1-month visit. After diagnostic fitting with rigid lenses, subjects completed an adaptation questionnaire after 15 min, 1 week, 2 weeks, and 1 month of lens wear. RESULTS: Seventy of the 80 subjects completed the study and, of the 10 subjects who discontinued, 8 were in the placebo group. In all categories evaluated, the anesthetic group experienced a more optimum adaptation experience at each visit vs. the placebo group. Specifically, overall comfort was rated significantly higher at both dispensing and 2 weeks. In addition, the anesthetic group exhibited significantly greater overall satisfaction with rigid lens wear at 2 and 4 weeks. Also, the anesthetic group perceived their adaptation, sensitivity, and adaptation time to be significantly better at the 1-month visit. There was no significant difference in corneal staining between these two groups at each visit, with the exception of a greater amount of staining in the central quadrant for the placebo group at the 1-month visit. CONCLUSIONS: The use of a topical anesthetic at the fitting and dispensing visits for first-time wearers of RGP lenses resulted in significantly fewer dropouts, improved initial comfort, an enhanced perception of the adaptation process, and greater overall satisfaction after 1 month of lens wear as compared to the use of a nonanesthetizing placebo at those visits. This result, in combination with both presenting RGP lenses in a nonthreatening manner and optimizing the lens design and fitting relationship, should result in a positive adaptation process and successful wear of RGP contact lenses.     

Chromatin structural changes in sperm after scrotal insulation of Holstein bulls

The reported effects on semen quality ascribed to testicular heat stress generally relate to traits impacting sperm transport and fertilizing ability but not to the genetic material contained by the sperm. To characterize the effects of testicular heat stress on sperm chromatin, susceptibility of DNA in sperm nuclear chromatin to in situ acid denaturation was measured by flow cytometry after staining with acridine orange using the sperm chromatin structure assay (SCSA). Semen was collected from Holstein bulls at 3-day intervals, before and after 48-hour scrotal insulation, until the morphologically abnormal sperm content in raw semen exceeded 50%. After cryopreservation in egg yolk-citrate extender, semen was thawed and sampled during incubation in vitro at 38.5 degrees C. Overall, SCSA results showed that chromatin susceptibility to denaturation was increased for sperm collected post- vs. preinsulation and was more pronounced for sperm presumably in the testes during insulation than for those sperm presumably in the epididymides. Increased susceptibility was detected as early as the first collection postinsulation; however, chromatin of sperm presumably in the proximal epididymis during insulation did not appear to have been detrimentally affected. Chromatin susceptibility to denaturation increased with increased incubation time in vitro, but the rate of change in susceptibility during incubation did not differ among pre- vs. postinsulation specimens. We conclude that elevated scrotal temperatures adversely affect both epididymal and testicular sperm by reducing sperm chromatin stability. The effects of heat stress on the chromatin of epididymal sperm were more subtle than those exhibited by testicular sperm but detectable within close proximity to the heat stress event.