Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.     

Informed consent: how much information is enough?

BACKGROUND: Recent judicial decisions involving informed consent have led to some medical practitioners altering the way they obtain consent. The aim of this study was to determine the degree to which patients understood the risks associated with a surgical procedure after giving routine consent and whether providing additional detailed verbal and/or written information improved their understanding. It was further determined whether the provision of more extensive information altered patients' anxiety levels. METHODS: Patients undergoing femoral popliteal bypass or carotid surgery were randomized to obtain either routine consent only or routine consent with verbal or written or verbal and written consent. Patients undertook a pre-operative risk and complication questionnaire, a pre- and postoperative anxiety and depression evaluation and a follow-up questionnaire 6 weeks after discharge. RESULTS: Thirty-two patients were included in the trial. The comprehension questionnaire resulted in a correct percentage response of 48% for the routine information only, 59% with added verbal information, 59% with added written information and 55% with added written and verbal information. Twenty-five per cent of patients stated that they had a poor understanding of the risks and complications of the procedure. CONCLUSIONS: Additional written or verbal information did not improve a patient's understanding of risks and complications of the procedure. It also did not improve patients' perceived understanding of the operation or its complications. Patients' anxiety levels were unaltered by the increase in the information they were given. The information provided to patients should be simple, easy to understand and list any possible major complications to enable the patient to determine whether to undergo or decline a procedure.     

Prevalence and demographic determinants of metronidazole resistance by Helicobacter pylori in a large cosmopolitan cohort of Australian dyspeptic patients

1. Heterozygous naked neck birds were raised under natural spring (average 21.2 degrees C) and summer temperatures (average 27.1 degrees C) to investigate the influence of dietary energy on broiler performance, carcase yield and nutrient composition of breast meat. 2. Birds were fed on a low energy diet of 12.12 MJ ME/kg, a medium energy diet of 12.96 MJ ME/kg and a high energy diet of 13.79 MJ ME/kg with 2 protein concentrations per energy treatment, 230 and 200 g/kg, from 0 to 3 and 3 to 7 weeks of age, respectively. 3. Summer rearing resulted in a decrease in body weight, body weight gain, carcase weight and carcase part yields of birds. 4. Increasing dietary energy from 12.12 to 13.79 MJ ME/kg increased body weight at 3 and 7 weeks, body weight gains from 0 to 3 and 3 to 7 weeks, carcase weights and relative abdominal fat weights of birds in a linear manner. There was no effect of dietary energy on the nutrient composition of breast meat. 5. It was concluded that there was no differences in dietary energy requirements of heterozygous naked neck birds when grown under natural optimum (21.2 degrees C) and summer temperatures (27.1 degrees C).     

5-HT4 receptor antagonism does not affect motor and reward mechanisms in the rat

5-HT4 receptors are concentrated in areas of the brain which are rich in dopamine neuronal markers, which may suggest that they influence motor and reward processes. We tested this hypothesis by examining the effects of a 5-HT4 receptor antagonist, 8-amino-7-chloro-(N-butyl-4-piperidyl)methylbenzo-1,4-dioxan-5-car boxylate hydrochloride (SB-204070-A) on amphetamine- and nicotine-induced locomotor stimulation in intact rats. In rats with unilateral 6-hydroxydopamine-induced lesions of the ascending nigrostriatal dopaminergic projection, SB-204070-A was tested for its effects on amphetamine-induced rotation. SB-204070-A was also tested for its effects on rewarded behaviour maintained by intracranial self-stimulation. SB-204070-A did not alter behaviour under any of these conditions, suggesting a lack of involvement of the 5-HT4 receptor in motor and reward processes.     

Sublingual immunotherapy with a standardised grass pollen extract; a double-blind placebo-controlled study

Congenital dislocation of the knee is a uncommon malformation. Frequently there is an association with other congenital deformities including congenital dislocation of the hip. The classification of Leveuf and Pais into three separate groups (Type A, Type B, Type C) is now widely accepted. Between October 1989 and April 1995 we evaluated ten children (five girls and five boys) with twelve dislocated knees. Clinical, radiographic and different ultrasonography examinations were carried out on both knees. The sonographic classification showed Type A in 3, Type B in 5 and Type C in 4 cases. The sonographic examination revealed the essential anatomic structures and their pathomorphology. The anterior and the posterior cruciate ligament could be demonstrated in the majority of the cases. Three dislocated knees showed an obliteration of the recessus suprapatellaris and fibrotic changes in the m. quadriceps. These cases required operative treatment. With conservative treatment we achieved a satisfactory result in eight children with nine disease knees. There was a good functional result with an average flexion of 106 degrees. Early conservative treatment recommend as the therapy of choice. Ultrasonography imaging offers the possibility of reliable differentiation into one of the three groups of the classification of Leveuf and Pais. Radiographic documentation during the therapy has become the exception. Ultrasonography imaging helped substantially in the decision making and the timing of operative treatment.     

Analysis of the association of a heat shock protein70-1 gene promoter polymorphism with myocardial infarction and coronary risk traits

In 12 depressed inpatients referred for bilateral electroconvulsive therapy (ECT), each patient was titrated at the first treatment session by using an ascending method-of-limits procedure with a step-wise increase in pulse frequency (frequency titration) or train duration (duration titration). At the second treatment session, seizure threshold was redetermined by using the method (frequency or duration titration) not used at the first treatment. Frequency or duration was maintained at the lowest level when the other parameter was titrated. Seizure threshold was significantly lower with duration titration (mean, 90 mC; SD, 27.3) than frequency titration (mean, 114 mC; SD, 35.6; p = 0.03). On average, patients in the duration-titration group required 1.2 (SD, 0.6) subconvulsive stimulations before a seizure was elicited, and patients in the frequency-titration group required 1.7 (SD, 0.9) subconvulsive stimulations before a seizure was elicited, a nonsignificant difference. These findings suggest that to elicit a seizure during ECT, increasing train duration may be slightly more efficient than increasing frequency. Basic and other clinical research findings indicate that increasing pulse width may be an inefficient way to elicit a seizure. Therefore the following sequence in the determination of seizure threshold is worth considering when using dose-titration or related techniques: the train duration should be increased first before increasing pulse frequency, and the decision to increase pulse width should be reserved for patients who do not seize at the maximal duration and frequency settings. Further empiric research is needed to establish the utility of this approach.     

The dual phases of the response to neonatal exposure to a VH family-restricted staphylococcal B cell superantigen

In vitro studies of several naturally occurring proteins have characterized VH family-specific B lymphocyte binding and stimulatory properties that appear analogous to those of T cell superantigens. To examine the in vivo consequences of exposure to a putative B cell superantigen, we treated neonatal BALB/c mice with a form of staphylococcal protein A (MS) devoid of Fcgamma binding activity, which retains the clan VHIII Fab binding specificity. In naive adults, about 5% of peripheral B cells and >13% of splenic IgM-secreting cells display MS binding activity, in association with high IgM and low IgG circulating anti-MS Ab titers. Neonatal exposure to MS elicited two distinct temporal phases of immune responsiveness. The early phase, representing the first approximately 5 wk of life, was associated with MS-specific B cell and T cell tolerance. Microfluorometric assays revealed that exposure caused a dramatic MS-specific B cell clonal loss in bone marrow and spleen, but levels normalized by about 3 wk of life. The late phase (>6 wk of age) was associated with spontaneous priming for MS-specific T cell responses and production of MS-specific IgG1 Abs despite long term persistently depressed in vivo and in vitro MS-specific IgM responses. In vivo challenge during the late phase induced high frequencies of MS-specific IgG-secreting cells, indicating recruitment of highly focused Ab responses that were predominantly encoded by rearrangements of the S107 family, a member of the VHIII clan. These studies document the immunodominance of the VH-restricted Fab binding site on staphylococcal protein A and demonstrate the diverse effects of a B cell superantigen on the emerging peripheral B cell compartment.