Demonstration of an inwardly rectifying K+ current component modulated by thyrotropin-releasing hormone and caffeine in GH3 rat anterior pituitary cells

 Reduction of an inwardly rectifying K⁺ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical activity increases in GH₃ rat anterior pituitary cells. However, the existence of an inwardly rectifying K⁺ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target in pituitary cells, including GH₃ cells. In this report, an inwardly rectifying component of K⁺ current is indeed demonstrated in perforated-patch voltage-clamped GH₃ cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active. Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current. Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K⁺ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is probably mediated by a HERG-like K⁺ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production. Thus, an inwardly-rectifying K⁺ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH₃ cells. Received: 15 May 1997 / Received after revision and accepted: 24 July 1997     

Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection.

Enveloped whole virions and nucleocapsids of human herpesvirus 6 (HHV-6) strain Z29 were purified from supernatant fluids of infected human cord blood lymphocytes by filtration through polyvinylpyrrolidone-treated filters, banding on a Nycondenz step gradient, and centrifugation through two successive continuous sucrose gradients. More than 20 proteins ranging in molecular weight from less than 30,000 to more than 200,000 were identified in preparations of purified whole virions labeled with [35S]methionine and [35S]cysteine. Immunogenic virion proteins of HHV-6 were identified in immunoblot assays with human immune sera, immune sera generated from mice immunized with purified whole virions or purified nucleocapsids, and a monoclonal antibody generated from a mouse immunized with purified nucleocapsids. The sera and the monoclonal antibody reacted strongly with a 101-kilodalton protein in the immunoblots, suggesting that the protein is a component of the nucleocapsid. Human sera lacking HHV-6-specific antibodies and seropositive for one or more of the other human herpesviruses failed to react with this protein, indicating that it is a specific serologic marker for HHV-6 infection.     

A project to reduce the burden of diabetes in the African-American Community: Project DIRECT.

Project DIRECT (Diabetes Interventions Reaching and Educating Communities Together) is the first comprehensive community diabetes demonstration project in the United States in an African-American community. This article describes its intervention components and evaluation design. The development and implementation of Project DIRECT has included the community since the project''s beginning. Interventions are targeted in three areas: health promotion (improving diet and physical activity levels), outreach (improving diabetes awareness, detection of undiagnosed diabetes, and ensuring that persons with diabetes who are not receiving continuing diabetes care are integrated into the health-care system), and diabetes care (improving self-care, increasing access, and improving the quality of diabetes preventive care received within the health-care system). Evaluation will be internal (conducted by Project DIRECT staff to assess process outcomes in persons directly exposed to each specific intervention) and external (review of outcomes to assess the impact of the multi-intervention program at the level of the entire community). Because diabetes exacts a disproportionate toll among African Americans, the findings from this project should aid in developing strategies to lessen the burden of this disorder, particularly among minority populations.     

Antimicrobial resistance of Shiga toxin (verotoxin)-producing Escherichia coli O157:H7 and non-O157 strains isolated from humans, cattle, sheep and food in Spain

A total of 722 Shiga toxin-producing Escherichia coli (STEC) isolates recovered from humans, cattle, ovines and food during the period from 1992 to 1999 in Spain were examined to determine antimicrobial resistance profiles and their association with serotypes, phage types and virulence genes. Fifty-eight (41%) out of 141 STEC O157:H7 strains and 240 (41%) out of 581 non-O157 STEC strains showed resistance to at least one of the 26 antimicrobial agents tested. STEC O157:H7 showed a higher percentage of resistant strains recovered from bovine (53%) and beef meat (57%) than from human (23%) and ovine (20%) sources, whereas the highest prevalence of antimicrobial resistance in non-O157 STEC was found among isolates recovered from beef meat (55%) and human patients (47%). Sulfisoxazole (36%) had the most common antimicrobial resistance, followed by tetracycline (32%), streptomycin (29%), ampicillin (10%), trimethoprim (8%), cotrimoxazole (8%), chloramphenicol (7%), kanamycin (7%), piperacillin (6%), and neomycin (5%). The multiple resistance pattern most often observed was that of streptomycin, sulfisoxazole, and tetracycline. Ten (7%) STEC O157:H7 and 71 (12%) non-O157 strains were resistant to five or more antimicrobial agents. Most strains showing resistance to five or more antimicrobial agents belonged to serotypes O4:H4 (4 strains), O8:H21 (3 strains), O20:H19 (6 strains), O26:H11 (8 strains eae-beta1), O111:H- (3 strains eae-gamma2), O118:H- (2 strains eae-beta1), O118:H16 (5 strains eae-beta1), O128:H- (2 strains), O145:H8 or O145:H- (2 strains eae-gamma1), O157:H7 (10 strains eae-gamma1), O171:H25 (3 strains), O177:H11 (5 strains eae-beta1), ONT:H- (3 strains/1 eae-beta1) and ONT:H21 (2 strains). Interestingly, most of these serotypes, i.e., those indicated in bold) were found among human STEC strains isolated from patients with hemolytic uremic-syndrome (HUS) reported in previous studies. We also detected, among non-O157 strains, an association between a higher level of multiple resistance to antibiotics and the presence of the virulence genes eae and stx(1). Moreover, STEC O157:H7, showed an association between certain phage types, PT21/28 (90%), PT23 (75%), PT34 (75%), and PT2 (54%), with a higher number of resistant strains. We conclude that the high prevalence of antimicrobial resistance detected in our study is a source of concern, and cautious use of antibiotics in animals is highly recommended.     

Surfactant protein A enhances Mycobacterium avium ingestion but not killing by rat macrophages

Mycobacterium avium complex (MAC) is a significant cause of opportunistic infection in patients with acquired immunodeficiency syndrome. Although the major route of entry of MAC is via the gastrointestinal tract, MAC can infect humans through the respiratory tract and eventually encounter alveolar macrophages within the lung. Once in the lung, MAC can potentially interact with surfactant protein A (SP-A), an important component of the pulmonary innate-immune response. Previous work on other pulmonary pathogens including Mycobacterium bovis Bacillus Calmette-Guerin (BCG) suggests that SP-A participates in promoting efficient clearance of these organisms by alveolar macrophages. In the present study, we investigated the role of SP-A in clearance of MAC by cultured rat macrophages. SP-A bound to MAC organisms and enhanced the ingestion of the mycobacteria by macrophages. Infection of macrophages with SP-A-MAC complexes induced the production of nitric oxide (NO) and tumor necrosis factor-alpha. However, intracellular survival of MAC was not altered by preopsonization with SP-A. In addition, inhibitors of inducible NO synthase did not alter MAC clearance. These results suggest that SP-A can bind to and enhance the uptake of MAC by alveolar macrophages, similar to previous findings with BCG and Mycobacterium tuberculosis.However, unlike BCG and other pulmonary pathogens that are cleared effectively in the presence of SP-A via a NO-dependent pathway, macrophage-mediated clearance of MAC is not enhanced by SP-A.     

Assessment of the COBAS Amplicor HBV Monitor Test for quantitation of serum hepatitis B virus DNA levels

Treatment of patients with chronic hepatitis B virus (HBV) infections with potent antiviral therapy often results in dramatic reductions in the levels of viremia to very low levels. Monitoring of serum HBV DNA levels is a consistent method for the assessment of antiviral potency; however, widely used hybridization assays for the monitoring of HBV DNA levels have limited sensitivities and are not effective for the monitoring of patients whose serum HBV DNA levels have decreased to below approximately 700,000 HBV genomes/ml. The objective of the present study was to assess a PCR-based assay (the COBAS-AM assay) for quantitation of serum HBV DNA levels and to compare the results of the COBAS-AM assay with those of a solution hybridization assay with a radiolabeled probe. The precision and accuracy of the assay were determined with low-positive and high-positive controls consisting of a plasmid DNA molecule containing HBV-specific primer binding regions, and the sensitivity of the assay was determined by using serial dilutions of sera from subjects with chronic HBV infection. HBV DNA levels were quantitated in 1,695 serum samples from subjects with chronic HBV infection who were enrolled in clinical trials of lamivudine in North America or Asia. The COBAS-AM assay demonstrated high levels of inter- and intra-assay precision and accuracy, and the linear range of the COBAS-AM assay was greater than that of the solution hybridization assay. The assay is linear over a 3-log(10) range and is able to quantitate serum HBV DNA at levels 3 log(10) lower than those that can be detected by the solution hybridization assay. We found that the COBAS-AM assay is an accurate PCR-based assay for quantitation of serum HBV DNA levels in subjects with chronic HBV infection.     

Pregnancy with frozen-thawed and fresh testicular biopsy after motile and immotile sperm microinjection, using the mechanical touch technique to assess viability

BACKGROUND: There are few reports of pregnancy using immotile sperm, and none using a purely mechanical assessment of viability. METHODS: In this pilot study, we retrospectively analysed 66 cycles in 61 patients with determinant male factor, recording rates of fertilization, implantation, normal pregnancy and take-home babies achieved with ICSI. Sperm selection was based on morphologically normal appearance under the inverted microscope. Viability of immotile spermatozoa was assessed by the mechanical touch technique to observe tail flexibility and tail shape recovery. RESULTS: Of 17 ICSI cycles using frozen-thawed testicular sperm, six microinjected with immotile and 11 with motile sperm, we achieved fertilization rates of 65.7 and 74.3%, respectively, and five pregnancies (two and three, respectively). Of 49 ICSI cycles using fresh testicular sperm, 10 microinjected with immotile and 39 with motile sperm, we achieved fertilization rates of 73.4 and 64.4%, respectively, and 12 pregnancies (three and nine, respectively). CONCLUSIONS: Immotile (fresh and frozen-thawed) testicular sperm of normal morphological appearance can be used to achieve clinical pregnancy with ICSI. Our results strongly suggest that immotile sperm viability can be assessed by the mechanical touch technique.     

Adaptive Wiener Filtering for Improved Acquisition of Distortion Product Otoacoustic Emissions

An innovative acoustic noise canceling method using adaptive Wiener filtering (AWF) was developed for improved acquisition of distortion product otoacoustic emissions (DPOAEs). The system used one microphone placed in the test ear for the primary signal. Noise reference signals were obtained from three different sources: (a) pre-stimulus response from the test ear microphone, (b) post-stimulus response from a microphone placed near the head of the subject and (c) post-stimulus response obtained from a microphone placed in the subjects nontest ear. In order to improve spectral estimation, block averaging of a different number of single sweep responses was used. DPOAE data were obtained from 11 ears of healthy newborns in a well-baby nursery of a hospital under typical noise conditions. Simultaneously obtained recordings from all three microphones were digitized, stored and processed off-line to evaluate the effects of AWF with respect to DPOAE detection and signal-to-noise ratio (SNR) improvement. Results show that compared to standard DPOAE processing, AWF improved signal detection and improved SNR. © 1998 Biomedical Engineering Society. PAC98: 4364Jb, 4360-c, 8790+y