Herpes simplex virus type 1 DNase: functional analysis of the enzyme expressed by recombinant baculovirus

Herpes simplex virus type 1 DNase (HSV-1 DNase) was expressed in insect cells by recombinant baculovirus (NPVUL12) and purified by a combination of anionic exchanger chromatography and gel filtration. Two polypeptides of 85 and 75 kD, whose ratio varied during purification, were induced 24 h after infection. The 75-kD protein was isolated and shown to possess catalytic activity. Gel filtration analysis indicated that the active form of the enzyme at an ionic strength of I = 0.3 is a dimeric protein with an apparent molecular weight of 130,000. The recombinant enzyme exhibited the overall characteristics of the native enzyme such as 5'-3' exonuclease and endonuclease activities with a preferred degradation of DNA. In the absence of extraneously added Mg2+, the enzyme was capable of removing mononucleotides from 5'-end-labeled DNA, but not from RNA and 3'-end-labeled DNA. The peculiar mechanism of double-strand DNA degradation suggests a specific role of HSV-1 DNase in DNA recombination processes during viral replication.     

Life cycle of Ixodes minor (Acari: Ixodidae) in the laboratory

OBJECTIVE: It is commonly believed that the full moon exerts an influence on violence and aggression in psychiatric settings. The literature to date is contentious. This study used a robust methodology to examine the hypothesis that there was an increased frequency of violent and aggressive behaviour among hospitalised psychiatric clients at the time of the full moon. METHOD: Prospective data were collected in five inpatient psychiatric settings across the Northern Sydney Area Health Service. Morrison's hierarchy of violence and aggression was used to rate behaviour. Lunar phases were clearly defined and Poisson regression used to examine relationships between lunar phase and violence. Extraneous temporal variation was considered. RESULTS: No significant relationship was found between total violence and aggression or level of violence and aggression and any phase of the moon. CONCLUSION: Future research could profitably examine the implications of a belief in the lunar effect among health workers in the face of evidence that no relationship exists between violence, aggression and the lunar cycle.     

Aortic perforation during lumbar laminectomy

A method to determine the optimal replacement time for dye affinity adsorbents used in protein purification processes that are subjected to severe regeneration conditions has been developed. To demonstrate the utility of the method, an experimental fixed-bed decay model was employed to determine the optimum number of cycles for the adsorbent replacement. This number is a function of the column regeneration frequency and of the capital and operation costs. The implications of the results on the design and operation of dye-ligand chromatographic processes are discussed.     

Antibiotic susceptibility profile of group B streptococcus acquired vertically

OBJECTIVE: To determine the contemporary antibiotic susceptibility profile of vertically acquired group B streptococcal isolates. METHODS: Susceptibility to ampicillin, penicillin G, erythromycin, clindamycin, cefazolin, and gentamicin was assessed by two methods, minimal inhibitory concentration and disc diffusion. RESULTS: The susceptibility profiles of 119 colonizing and eight invasive strains of group B streptococcus isolated from January 1996 to September 1997 at two hospitals in Birmingham, Alabama-University of Alabama at Birmingham and Cooper Green-were studied. Minimal inhibitory concentration determinations indicated that all colonizing strains were susceptible or moderately susceptible to ampicillin and penicillin G. Resistance was noted by at least one strain to each of the other antibiotics; all were resistant to gentamicin, whereas 27 (21%) were resistant to erythromycin, five (4%) to clindamycin, and one (1%) to cefazolin. All of the eight invasive strains were susceptible or moderately susceptible to ampicillin, penicillin G, clindamycin, and cefazolin; one (13%) was resistant to erythromycin, and all were resistant to gentamicin. Disc diffusion results generally were concordant with minimal inhibitory concentration results, although by disc diffusion fewer isolates were classified as susceptible, and more as moderately susceptible, to ampicillin and penicillin G than by minimal inhibitory concentration. CONCLUSION: Universal susceptibility of group B streptococcus to members of the penicillin family supports the continued use of penicillin G or ampicillin for early onset neonatal group B streptococcal disease prevention. For patients allergic to beta-lactam agents, clindamycin (4% resistance) may be a better alternative than erythromycin (21% resistance).     

Evidence for a salt bridge between transmembrane segments 5 and 6 of the yeast plasma-membrane H+-ATPase

The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell. This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase. In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase. Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum. Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998) J. Biol. Chem. 273, 17411-17417). To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated. Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent H+ pumping. These results demonstrate that neither Arg-695 nor Asp-730 is required for enzymatic activity or proton transport, but suggest that there is a salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide.     

Hybrid bilayer membranes in air and water: infrared spectroscopy and neutron reflectivity studies

In this report we describe the fabrication and characterization of a phospholipid/alkanethiol hybrid bilayer membrane in air. The bilayer is formed by the interaction of phospholipid with the hydrophobic surface of a self-assembled alkanethiol monolayer on gold. We have characterized the resulting hybrid bilayer membrane in air using atomic force microscopy, spectroscopic ellipsometry, and reflection-absorption infrared spectroscopy. These analyses indicate that the phospholipid added is one monolayer thick, is continuous, and exhibits molecular order which is similar to that observed for phospholipid/phospholipid model membranes. The hybrid bilayer prepared in air has also been re-introduced to water and characterized using neutron reflectivity and impedance spectroscopy. Impedance data indicate that when moved from air to water, hybrid bilayers exhibit a dielectric constant and thickness that is essentially equivalent to hybrid bilayers prepared in situ by adding phospholipid vesicles to alkanethiol monolayers in water. Neutron scattering from these samples was collected out to a wave vector transfer of 0.25 A(-1), and provided a sensitivity to changes in total layer thickness on the order of 1-2 A. The data confirm that the acyl chain region of the phospholipid layer is consistent with that observed for phospholipid-phospholipid bilayers, but suggest greater hydration of the phospholipid headgroups of HBMs than has been reported in studies of lipid multilayers.     

Entropy-driven binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles

This study employed large unilamillar vesicles composed of purchased stratum corneum lipids to investigate the binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles. The partition coefficients of amino acids/dipeptides between the stratum corneum lipid vesicles and the acetate buffer were determined by HPLC. In addition, the binding/partition enthalpy of amino acids/dipeptides with the stratum corneum lipid vesicles was derived by directly measuring the binding/partition heat with isothermal titration calorimetry. According to the binding/petition Gibbs free energy and the binding/partition enthalpy, all the binding/partition of amino acids/dipeptides with the stratum corneum lipid vesicles is endothermic, implying an entropy-driven binding/partition. Also, the equilibrium binding/partition results demonstrate that the partition coefficients of amino acids/dipeptides do not correlate with the transdermal permeability. This finding suggests that either the interaction between the penetrants and the lipid bilayer between corneocytes may not be a determining step or that the paracellular path is not a dominant route of transdermal penetration.     

Evolution of the trappin multigene family in the Suidae

The purpose of the study was to examine the effects of manipulating lung volume (LV) on phonatory and articulatory kinematic behavior during sentence production in healthy adults. Five men and five women repeated the sentence "I sell a sapapple again" under five LV conditions. These included (1) speaking normally, (2) speaking after exhaling most of the air from the lungs, (3) speaking at end expiratory level (EEL), (4) speaking after a maximal inhalation, and (5) speaking after a maximal inhalation while attempting to maintain as normal a mode of speech as possible. From a multichannel recording, measures were made of LV, sound pressure level (SPL), fundamental frequency (F0) and semitone standard deviation (STSD), and upper and lower lip displacements and peak velocities. When compared with the reference condition, the sentence was spoken significantly more quickly at the lowest LV. SPL increased significantly for the high LV condition, as did the women's F0 and STSD. Upper lip displacements and peak velocities generally decreased for LVs other than the reference condition. Lower lip movements showed inconsistent changes as a function of LV. Adjustments to the LV for speech led to SPL and F0 changes consistent with a coordinated control of the respiratory system and the larynx. However, less consistent effects were observed in the articulatory kinematic measures, possibly because of a less direct biomechanical and neural control linkage between respiratory and articulatory structures.     

Observations on the risk of resistance with the extended use of vancomycin

OBJECTIVE: To document the risk of the development of vancomycin-resistant bacteria in a population of seriously burned patients during a 10-year period of common vancomycin hydrochloride use. DESIGN: Retrospective study. SETTING: The US Army Institute of Surgical Research, Burn Center, Fort Sam Houston, Tex. POPULATION AND METHODS: Microbiology, infection, and antibiotic use records collected during the hospitalization of 2266 consecutively admitted seriously burned patients were reviewed. Vancomycin was the primary therapeutic agent used for gram-positive infections and was also used as a perioperative prophylactic antibiotic during burn wound excision. This policy was established prior to this review because of a high incidence of methicillin-resistant Staphylococcus aureus colonization and an anecdotal association of increased beta-lactam resistance in endemic gram-negative pathogens associated with the use of penicillinase-resistant penicillins and cephalosporins. MAIN OUTCOME MEASURES: Isolation of vancomycin-resistant enterococci (VRE) or other gram-positive organisms resistant to vancomycin. RESULTS: Examinations of 15 125 gram-positive isolates, including 957 enterococci, for in vitro sensitivity to vancomycin yielded 3 VRE isolates in 3 patients. Vancomycin was used prior to VRE isolation in one of these patients. Resistance was found in 3 other organisms (2 Corynebacterium species, 1 Lactobacillus species). Vancomycin was used prior to these isolations in 2 of 3 patients. None of the vancomycin-resistant organisms was associated with infection and all 6 patients survived. Vancomycin-resistant enterococci or other vancomycin-resistant gram-positive organisms were not found in 663 patients treated with vancomycin for documented gram-positive infections or in 1027 patients where perioperative vancomycin was used. CONCLUSION: Use of vancomycin as the primary therapeutic agent in seriously burned patients was not associated with increased risk of VRE isolation or VRE infection.