Image-analysis-based assessment of the effects of the "Ca2+-jump" technique on sarcomere uniformity

A new image analysis-based technique was used to quantitatively examine the effects of the "Ca2+-jump" activation protocol on the maintenance of fiber quality in skinned rabbit psoas muscle fiber segments. Specifically, contractions in pCa 4.6 were preceded by short-duration "preactivation" soaks in a solution in which EGTA was replaced with the low-Ca2+ buffering capacity analog hexamethylenediamine-N, N, N', N'-tetraacetate, which facilitated rapid Ca2+ equilibration within the fiber segments. Fiber quality was assessed by examining the Fourier spectra of the muscle fiber images before, during, and after activation. Segment lengths were typically below 500 micrometer, thus allowing the majority of the sarcomeres to be visualized in the field of view (x200 and x400 magnification). The preactivation protocol resulted in less deterioration of fiber quality with repetitive activation. In addition, there was also a significant reduction in the time required to reach the 50% level of maximum tension, with no significant change in the maximum tension level.     

Persistently exaggerated startle responses in rats treated with pyridostigmine bromide

Troops in the Persian Gulf War have registered complaints consistent with CNS dysfunction that emerged after returning from the Gulf. A common experience among Persian Gulf War veterans was exposure to pyridostigmine bromide (PB) for prophylaxis against nerve gas exposure. To determine whether PB causes emergent CNS dysfunction, Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats were given PB for 7 consecutive days in their drinking water. The WKY, but not the SD, rats exhibited a delayed-onset, persistently exaggerated startle response. The WKY rats exhibited exaggerated startle responses that appeared 15 days after the end of PB treatment and were still evident 22 days after the end of treatment. Both the duration and the magnitude of the exaggerated startle responses were related to the dosage of PB. The PB-treated rats exhibited normal short-term and long-term habituation. However, exaggerated startle responses were related to the development of enhanced short-term sensitization. Treating the rats for a second time, 7 weeks after the end of the first PB treatment, induced an exaggerated startle response that appeared sooner and dissipated faster than was evident after the first PB treatment. Inasmuch as the WKY rat has inherently low butyrylcholinesterase activity, a scavenger for PB, these results suggest that prophylactic PB may influence CNS function in individuals with low butyrylcholinesterase activity. Elaboration of the factors that mediate enhanced sensitization in the WKY rat may provide insight into some of the complaints registered by veterans of the Persian Gulf War.     

Cystic prostatic disease associated with adrenocortical lesions in the ferret (Mustela putorius furo)

CONTEXT: In British Columbia, human immunodeficiency virus (HIV)-infected persons eligible for antiretroviral therapy may receive it free but the extent to which HIV-infected injection drug users access it is unknown. OBJECTIVE: To identify patient and physician characteristics associated with antiretroviral therapy utilization in HIV-infected injection drug users. DESIGN: Prospective cohort study with record linkage between survey data and data from a provincial HIV/AIDS (acquired immunodeficiency syndrome) drug treatment program. SETTING: British Columbia, where antiretroviral therapies are offered free to all persons with HIV infection with CD4 cell counts less than 0.50 x 10(9)/L (500/microL) and/or HIV-1 RNA levels higher than 5000 copies/mL. SUBJECTS: A total of 177 HIV-infected injection drug users eligible for antiretroviral therapy, recruited through the prospective cohort study since May 1996. MAIN OUTCOME MEASURES: Patient use of antiretroviral drugs through the provincial drug treatment program and physician experience treating HIV infection. RESULTS: After a median of 11 months after first eligibility, only 71 (40%) of 177 patients had received any antiretroviral drugs, primarily double combinations (47/71 [66%]). Both patient and physician characteristics were associated with use of antiretroviral drugs. After adjusting for CD4 cell count and HIV-1 RNA level at eligibility, odds of not receiving antiretrovirals were increased more than 2-fold for females (odds ratio [OR], 2.53; 95% confidence interval [CI], 1.08-5.93) and 3-fold for those not currently enrolled in drug or alcohol treatment programs (OR, 3.49; 95% CI, 1.45-8.40). Younger drug users were less likely to receive therapy (OR, 0.47/10-y increase; 95% CI, 0.28-0.80). Those with physicians having the least experience treating persons with HIV infection were more than 5 times less likely to receive therapy (OR, 5.55; 95% CI, 2.49-12.37). CONCLUSIONS: Despite free antiretroviral therapy, many HIV-infected injection drug users are not receiving it. Public health efforts should target younger and female drug users, and physicians with less experience treating HIV infection.     

Characterization of a calmodulin kinase II inhibitor protein in brain

Ca2+/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII alpha and beta and potently inhibited kinase activity with an IC50 of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i. e., in the presence of Ca2+/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.     

Insulin activates the hepatitis B virus X gene through the activating protein-1 binding site in HepG2 cells

OBJECTIVES: To study the turnaround time (TAT) for rendering diagnoses on routine biopsy specimens, to examine pathology practice variables that influence TAT, and to assess the level of surgeons' satisfaction with biopsy TAT. DESIGN: Over a 3-month period, voluntary participants in the College of American Pathologists Q-Probes laboratory quality improvement program prospectively collected TAT data on up to 20 biopsy specimens performed on elective surgical cases, completed questionnaires profiling their institution's practice characteristics, and had surgeons complete questionnaires indicating their satisfaction with biopsy report TAT. SETTING AND PARTICIPANTS: One hundred fifty-seven private and public small hospitals located in 43 American states (n = 153), Canada (n = 1), and Australia (n = 3). MAIN OUTCOME MEASURES: The routine surgical biopsy report TATs for 2 testing intervals, each commencing when surgeons acquired the biopsy specimens. One interval concluded when pathologists signed off the biopsy diagnoses, and the other concluded when surgeons received the hard-copy reports. RESULTS: Pathologists signed off 85.9% of 5384 biopsy diagnoses by the second working day, and surgeons received 88.3% of the hard-copy reports by the fourth working day. In 90% of hospitals participating in this study, pathologists signed off half their biopsy diagnoses between the second and third postcollection days, and 90% of surgeons received half their final hard-copy reports by the fourth postcollection day. Institutional practice variables associated with fewer sign-off and/or hard-copy receipt TATs exceeding the institutional 90th percentile performance benchmarks included yearly surgical caseloads greater than 2000 cases per full-time equivalent pathologist, provision of pathology support services on site, and accreditation of the hospital by the Joint Commission on Accreditation of Healthcare Organizations and of the laboratory by the College of American Pathologists. Most (96.4%) surgeons indicated that they were satisfied with hard-copy TATs and that they believed most (98.1%) of the hard-copy TATs had no effect on the lengths of their patients' hospital stays. CONCLUSIONS: Pathologists are capable of signing off most routine biopsy diagnoses within 2 working days and delivering the final hard-copy reports to surgeons within 4 working days (both intervals measured from the time that surgeons collect biopsy specimens). Most surgeons report they are satisfied with this level of performance.     

Angioplasty and primary stenting of the subclavian, innominate, and common carotid arteries in 83 patients

A new macrocyclic of the bis(benzylisoquinoline) alkaloid family, d-(+)-tubocurarine chloride (DTC), has been evaluated as a chiral selector for the separation of optical isomers of organic carboxylates using capillary electrophoresis (CE). The pertinent physicochemical properties, such as absorption spectrum, isoionic point, and solution conformation, of DTC were determined. The effects of varying such experimental parameters as DTC concentration, pH, and methanol content in the running buffer were assessed. CE separation of the enantiomers of 18 different compounds was achieved using DTC as the chiral selector under optimized background electrolytic conditions.     

Does practice make perfect? Examining the relationship between hospital surgical volume and outcomes for hip fracture patients in Quebec

OBJECTIVES: Most tests of the practice-makes-perfect hypothesis have used cross-sectional data, which reveal that patients receiving surgery in high-volume hospitals tend to experience better postsurgery outcomes. This study uses longitudinal data to explicitly examine whether any given hospital's patient outcomes change as its surgery volume varies with time. METHODS: Longitudinal data from all hospitals conducting hip fracture surgery in Quebec between 1990 and 1993 were used to examine the relationship between surgery volume and outcomes. The longitudinal data allowed volume to be measured using the actual number of surgeries performed by the admitting hospital in the 12 months before a patient's surgery. Determinants of postsurgery length of stay were assessed using ordinary least squares regression, and the explanators of inpatient mortality were identified using logistic regression. The regressions included fixed effects (hospital-specific dummy variables) to control for systematic differences in outcomes across hospitals that persist with time. Therefore, the coefficient on hip fracture surgery volume in the regression models captured differences in outcomes that were attributable to changes in surgery volume within hospitals with time. RESULTS: The fixed effects were significant explanators of both postsurgery length of stay and inpatient mortality, indicating that there were significant differences in outcomes across hospitals that persisted with time. In regressions that excluded the fixed effects, the coefficient on surgery volume was significant. In contrast, the coefficient on surgery volume was insignificant when the fixed effects were included. CONCLUSIONS: Longitudinal data revealed that after controlling for differences in hospital outcomes that were fixed with time, hospitals performing more surgeries in one period than in another experienced no significant improvement in outcomes. These results do not support the "practice makes perfect" hypothesis. The volume-outcome relationship for hip fracture patients thus appears to reflect fixed differences in quality between high-volume and low-volume hospitals.     

Vpx is required for dissemination and pathogenesis of SIV(SM) PBj: evidence of macrophage-dependent viral amplification

The viral accessory protein Vpx is required for productive in vitro infection of macrophages by simian immunodeficiency virus from sooty mangabey monkeys (SIV(SM)). To evaluate the roles of Vpx and macrophage infection in vivo, we inoculated pigtailed macaques intravenously or intrarectally with the molecularly cloned, macrophage tropic, acutely pathogenic virus SIV(SM) PBj 6.6, or accessory gene deletion mutants (deltaVpr or deltaVpx) of this virus. Both wild-type and SIV(SM) PBj deltaVpx viruses were readily transmitted across the rectal mucosa. A subsequent 'stepwise' process of local amplification of infection and dissemination was observed for wild-type virus, but not for SIV(SM) PBj deltaVpx, which also showed considerable impairment of the overall kinetics and extent of its replication. In animals co-inoculated with equivalent amounts of wild-type and SIV(SM) Pbj deltaVpx intravenously or intrarectally, the deltaVpx mutant was at a strong competitive disadvantage. Vpx-dependent viral amplification at local sites of initial infection, perhaps through a macrophage-dependent mechanism, may be a prerequisite for efficient dissemination of infection and pathogenic consequences after exposure through either mucosal or intravenous routes.     

Vitellin processing and protein synthesis during cricket embryogenesis

At the start of insect embryogenesis most of the protein mass of the egg cytoplasm exists as vitellin (Vt) obtained endocytically during vitellogenesis. Of the new embryo polypeptides (EP) appearing in the egg during embryogenesis, many are synthesized de novo, while, in some species, others derive from developmentally programmed partial proteolysis of Vt. Earlier we showed that by the end of vitellogenesis the two native Vts in Acheta domesticus exist in opposing gradients along the longitudinal axis of the egg. Here we hypothesize that this ooplasmic Vt distribution presents a milieu for Vt processing out of which region-specific regulatory molecules could arise. The metabolic origin and stage-specific patterns of seven predominant EPs (EP 1-7) identified by SDS-PAGE were examined and the results correlated with developmental morphology during the 14 days of embryogenesis. Based on antibody reactivity, peptide mapping and in vitro radiolabeling, we determined that EPs 1-3, 6 and 7 are Vt-derived, while EPs 4 and 5 are produced de novo by the embryo. The five Vt-derived EPs appear during the first 24 h of embryogenesis when migrating cleavage nuclei and associated cytoplasm form the cellular blastoderm, and levels of EPs 4 and 5 increase during days 4-6 of embryogenesis when katatrepsis and yolk mass contraction occur. Positive periodic acid-Schiff staining indicated that EPs 1-3 and their Vt-precursor polypeptides are glycoproteins. This work shows that developmental stage-specific Vt processing occurs during A. domesticus embryogenesis and points next to investigation of the functional significance of Vt cleavage products during development.