Recovery of negative-strand RNA viruses from plasmid DNAs: a positive approach revitalizes a negative field.
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Animals↗
Biomedical subjects
A Roberts
Publications and source records attributed to A Roberts.
Asymmetries in sensory pathways from skin to motoneurons on each side of the body determine the direction of an avoidance response in hatchling Xenopus tadpoles.
1. When swimming is initiated by tail stimulation in hatchling Xenopus tadpoles, the first trunk contraction is usually on the opposite side and directs the animal away from the stimulus. We have investigated how asymmetries in the skin sensory pathways mediate this response. 2. In alpha-bungarotoxin-immobilized tadpoles, intracellular recordings were made of responses to ipsilateral (ISS) and contralateral skin stimulation (CSS) in thirty-two presumed motoneurons. ISS evokes an inhibitory postsynaptic potential (IPSP) followed by an excitatory postsynaptic potential (EPSP) whereas CSS only evokes an EPSP. Blocking the short latency IPSP evoked by ISS with strychnine reduced the difference in spike latency on the two sides but spikes still occurred first to CSS. 3. Motoneuron EPSPs evoked by ISS and CSS were therefore recorded during microperfusion of strychnine to block the short latency IPSP. We found: (a) the CSS-EPSPs have lower threshold, larger amplitude at a given intensity of stimulus, faster rising phase, and shorter latencies than those of ISS-EPSPs; (b) the ISS-EPSP onset latencies were longer than CSS-EPSPs and became shorter as the stimulus intensity increased while those of CSS-EPSPs remained little changed. At high stimulus intensities, EPSPs caused by CSS and ISS became similar; and (c) onset latencies of ISS-EPSPs had higher variance than those of CSS-EPSPs. However, this difference was reduced as the stimulus intensity was increased. 4. Since motoneuron EPSP onset latencies varied with stimulus intensity, we proposed that the pathway from the opposite side had stronger synapses from afferents to sensory interneurons. To test this proposal we built a neuronal population model of the spinal pathway from skin afferents, via sensory interneurons to ipsilateral and contralateral motoneurons incorporating this asymmetry. Inhibition was omitted from the model. 5. Simulated motoneuron EPSPs in response to skin stimulation on each side of the body showed the major asymmetries found experimentally. If the distribution and axonal projections of the interneurons in the two sensory pathways were made the same these differences remained. However, if the synaptic strength from sensory afferents onto interneurons projecting to the two sides were made equal, the difference between the two sides were lost. 6. We propose that the sensory pathway to contralateral motoneurons has more effective excitation from afferents to sensory interneurons which leads to these motoneurons firing first. At higher stimulus strengths, when population recruitment can blur these subtle differences in excitation between the two sides, inhibition normally plays a significant role to ensure that most first responses are still contralateral.
Action Potentials↗
Assessing the roles of glutamatergic and cholinergic synaptic drive in the control of fictive swimming frequency in young Xenopus tadpoles.
This paper investigates the proposal that the frequency of the swimming central pattern generator in young Xenopus tadpoles is partly determined by the population of glutamatergic premotor interneurons active on each cycle. During fictive swimming spinal neurons also receive cholinergic and electrotonic excitation from motoneurons. As frequency changes during swimming we make two predictions: first, since most motoneurons fire very reliably at all frequencies, the electrotonic and nicotinic drive from motoneurons should remain constant, and second, when swimming frequency decreases, the glutamatergic drive should decrease as the number of active premotor excitatory interneurons decreases. We have tested these predictions by measuring the excitatory synaptic drive to motoneurons as frequency changes during fictive swimming. The components of synaptic drive were revealed by the local microperfusion of strychnine together with different excitatory antagonists. After blocking the nicotinic acetylcholine receptor, the mainly glutamatergic excitatory synaptic drive still changed with frequency. However, when glutamate receptors or all chemical transmission was blocked, excitation did not change with frequency. Our predictions are confirmed, suggesting that premotor excitatory interneurons are a major factor in frequency control in the tadpole central pattern generator and that motoneurons provide a stable background excitation.
Animals↗
Autocrine secretion of TGF-beta 1 and TGF-beta 2 by pre-adipocytes and adipocytes: a potent negative regulator of adipocyte differentiation and proliferation of mammary carcinoma cells.
We have developed an in vitro system to examine the influence of adipocytes, a major mammary stromal cell type, on the growth of a murine mammary carcinoma, SP1. Previously, we have shown that 3T3-L1 adipocytes release a mitogenic factor, hepatocyte growth factor, which strongly stimulates proliferation of SP1 cells. We now show that 3T3-L1 pre-adipocytes secrete active inhibitory molecules which inhibit DNA synthesis in SP1 cells. In addition, latent inhibitory activity is present in conditioned media (CM) from both pre-adipocytes and adipocytes, and is activated following acid treatment. CM also inhibited DNA synthesis in Mv1Lu wild type epithelial cells, but not DR27 mutant epithelial cells which lack TGF-beta type II receptor. Inhibitory activity of CMs was partially abrogated by neutralizing anti-TGF-beta1 and anti-TGF-beta2 antibodies, and was removed following ultrafiltration through membranes of 10,000 Mr but not 30,000 Mr pore size. These results show that the inhibitory effect on DNA synthesis is mediated by TGF-beta1-like and TGF-beta2-like molecules. In addition, acid-treated CM as well as purified TGF-beta inhibited differentiation of pre-adipocytes. Untreated pre-adipocyte CM, but not mature adipocyte CM, spontaneously inhibited adipocyte differentiation. Together, these findings indicate that pre-adipocytes spontaneously activate their own secreted TGF-beta, whereas mature adipocytes do not, and suggest that activation of TGF-beta has a potent negative regulatory effect on adipocyte differentiation and tumor growth. Thus, TGF-beta may be an important modulator of tumor growth and adipocyte differentiation via both paracrine and autocrine mechanisms. These findings emphasize the importance of adipocyte-tumor interactions in the regulation of tumor microenvironment.
3T3 Cells↗
Fetal blood sampling immediately before and within 24 hours of death in monochorionic twin pregnancies complicated by single intrauterine death.
OBJECTIVE: Our goal was to investigate the mechanisms that play a role in intrauterine death in monochorionic twins and that contribute to the high perinatal mortality and morbidity in the survivors. STUDY DESIGN: In 8 monochorionic twin pregnancies complicated by the intrauterine death of a single twin, we took samples from 5 twin fetuses immediately before death and from 4 of their cotwins and also from 4 surviving fetuses within 24 hours after death of the cotwin. RESULTS: Four of the 5 fetuses sampled who subsequently died were acidemic and 3 were hypoxemic. None of these fetuses or their cotwins were anemic at that time. All 4 survivors sampled within 24 hours of the death of each cotwin had low hematocrits. CONCLUSION: Fetal anemia, probably the consequence of acute blood loss just before the time of death of the cotwin, may play a role in the high mortality and morbidity found in the surviving twin. It is unlikely that immediate delivery of the surviving twin after death could affect the outcome.
Acid-Base Equilibrium↗
Structural and experiential neighborhood contexts, developmental stage, and antisocial behavior among urban adolescents in poverty.
This study explored the effects of structural and experiential neighborhood factors and developmental stage on antisocial behavior, among a sample of poor urban adolescents in New York City. Conceptually and empirically distinct profiles of neighborhood experience were derived from the data, based on measures of perceived neighborhood cohesion, poverty-related hassles, and involvement in neighborhood organizations and activities. Both the profiles of neighborhood experience and a measure of census-tract-level neighborhood hazard (poverty and violence) showed relationships to antisocial behavior. Contrary to expectation, higher levels of antisocial behavior were reported among adolescents residing in moderate-structural-risk neighborhoods than those in high-structural-risk neighborhoods. This effect held only for teens in middle (not early) adolescence and was stronger for teens perceiving their neighborhoods as hassling than for those who did not. Implications for future research and preventive intervention are discussed.
Adolescent↗
TGF-beta1 is essential for the homeostasis of the dentin-pulp complex.
Among the complex network of cytokines that influence odontoblast function during development and repair, TGF-beta1 is unique in its dual abilities to function as a potent immunosuppressant and as an inducer of extracellular matrix production. These properties underscore the importance of this molecule in maintaining the homeostasis of the dentin-pulp complex after injury. The purpose of this paper is to describe new findings of our phenotypic analysis of dentition in mice in which the TGF-beta1 gene has been disrupted. The major phenotype of TGF-beta1(-/-) offspring is one of diffuse immune system activation with progressive inflammation, wasting and death. Our studies of adult TGF-beta1(-/-) dentition show widespread pulpal and periapical inflammation and necroses. In addition, the coronal surfaces of occluding molars show marked attrition. To determine whether the phenotypic changes in TGF-beta1(-/-) dentition are directly linked to the loss of TGF-beta1 rather than the inflammatory process itself, we studied adult dentition in TGF-beta1(-/-) mice backcrossed into immunodeficient backgrounds. Results of our histopathologic and radiographic analyses show that teeth of TGF-beta1(-/-) immunodeficient mice retain vitality in pulpal and periapical regions but show excessive wear of occlusal surfaces.
Animals↗
Vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge.
Since the development of a system for generating vesicular stomatitis virus (VSV) from plasmid DNAs, our laboratory has reported the expression of several different glycoproteins from recombinant VSVs. In one of these studies, high-level expression of an influenza virus hemagglutinin (HA) from a recombinant VSV-HA and efficient incorporation of the HA protein into the virions was reported (E. Kretzschmar, L. Buonocore, M. J. Schnell, and J. K. Rose, J. Virol. 71:5982-5989, 1997). We report here that VSV-HA is an effective intranasal vaccine vector that raises high levels of neutralizing antibody to influenza virus and completely protects mice from bronchial pneumonia caused by challenge with a lethal dose of influenza A virus. Additionally, these recombinant VSVs are less pathogenic than wild-type VSV (serotype Indiana). This vector-associated pathogenicity was subsequently eliminated through introduction of specific attenuating deletions. These live attenuated recombinant VSVs have great potential as vaccine vectors.
Administration, Inhalation↗
Carotid stenting and angioplasty: a statement for healthcare professionals from the Councils on Cardiovascular Radiology, Stroke, Cardio-Thoracic and Vascular Surgery, Epidemiology and Prevention, and Clinical Cardiology, American Heart Association.
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Angioplasty, Balloon↗
Nurse-patient communication in palliative care: an evaluation of a communication skills programme.
Good communication between nurses and patients is a central aspect of palliative care. However, evaluation of courses designed to improve nurses' communication skills has been inconclusive. Most courses have concentrated on skills training, although communication training programmes which have been integrated into clinical practice over time and have also focused on attitudes and used a range of teaching methods, have been shown to be effective. A study was set up to evaluate whether a communication skills course which would focus on knowledge, attitudes and skills would improve nurses' communication skills. One-hundred-and-ten nurses completed a 26 h training programme over six months and completed precourse and postcourse audiotape recordings of a patient assessment. An overall statistically significant improvement in assessment skills between pretest and post-test mean total scores (P < 0.001) was found, with statistically significant improvements in six of the nine key areas assessed. The nurses reported that although some elements of the programme, such as role play, had been stressful they felt more confident in handling difficult situations. The longer integrated communication skills programme which allows nurses to explore attitudes, raise self-awareness and develop knowledge and skills appears to be effective.
Adult↗
What is SGML (Standard Generalised Markup Language)?
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Database Management Systems↗
Effect of lymphatic and splenic pump techniques on the antibody response to hepatitis B vaccine: a pilot study.
Osteopathic manipulative treatment (OMT) facilitates the movement of lymphatic fluid and may enhance the immunologic response to infection or injected antigen. In this investigation, two groups of volunteers were vaccinated with recombinant hepatitis B vaccine, given at 0, 5, and 25 weeks. The experimental group (n = 20) received OMT (lymphatic and splenic pump) three times per week for 2 weeks after each vaccination. Control subjects (n = 19) received vaccine but no OMT. Resultant serum antibody levels were measured by enzyme immunoassay. Fifty percent of subjects in the treatment group achieved protective antibody titers (> or = 10 mIU/mL) on the 13th week with an average titer of 374 mIU/mL. Only 16% of the control subjects had positive antibody responses, with average titers of 96 mIU/mL. At all time points from week 6 on, the average anti-hepatitis B titer was higher in the treatment group than in the control group. These data suggest an enhanced immunologic response in subjects who received OMT.
Enzyme-Linked Immunosorbent Assay↗
Efficacy of anticoagulation post-inferior vena caval filter placement.
Anticoagulation with heparin or Coumadin is usually effective in the management of deep vein thrombosis; however, if anticoagulation treatment is contraindicated, is ineffective, or leads to adverse effects, inferior vena caval (IVC) placement of a filter is indicated. The purpose of this study is to determine whether increased early adverse effects are present in patients not given anticoagulation medication after placement of an IVC filter. The records of 240 consecutive patients who received IVC filter placement were reviewed for indications for placement, as well as for whether patients received anticoagulation medication on discharge. There were 41 in-patient mortalities. Of the remaining 199 patients, 100 were discharged and prescribed anticoagulation medication, and 99 were not. Patients were followed for 3 to 60 months. These results suggest that no early adverse effects are seen in patients who are not given anticoagulants after IVC filter placement.
Adult↗
Intensive cardiovascular risk factor intervention in a rural practice: a glimmer of hope?
BACKGROUND: Large trials of primary care-based health promotion to modify coronary heart disease risks have shown only modest benefits. Could more intensive intervention, with doctors sharing with practice nurses in health promotion, produce better health outcomes in the context of the small family practice? How cost-effective might these interventions be? AIM: To assess the cost-effectiveness of an intensive programme of coronary heart disease (CHD) risk factor modification in a rural general practice in which doctors had a major input. METHOD: A longitudinal study of changes in risk factors in a group of adult patients identified as having one or more major CHD risk factor and monitored for one to seven years. Patients were recruited from and followed up in health promotion clinics, routine practice nurse appointments, or routine doctors' surgeries. All received the practice's routine interventions to modify risk, and changes in risk factors were recorded. Time spent by members of the primary health care team on CHD health promotion was recorded over a two-year period. RESULTS: From a practice list of 2040, 760 patients with one or more CHD risk factors were identified and followed up over a mean of 3.61 years (range six months to seven years). Significant improvements in each of the risk factors occurred, except in body mass index (BMI). Mean Dundee risk scores fell from 7.4 to 5.7 (by 23.3%). The annual cost to the practice (including doctor/nurse/secretarial time plus sundry practice expenses and laboratory costs, but excluding drug costs) was 6000 pounds. Cost per coronary death prevented was calculated as approximately 10,000 pounds. CONCLUSION: The results show an effect on risk factors broadly similar but slightly greater in magnitude than that achieved in the OXCHECK and British Family Heart Studies of nurse-delivered risk factor intervention in primary care. The results suggest that more intensive effort in lifestyle modification and health promotion, with more active involvement of doctors, could produce significant additional benefit. The cost-effectiveness of this approach compares favourably with many other accepted measures in coronary heart disease prevention.
Aged↗
Heterogeneity of air pollution effects is related to average temperature.
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Air Pollution↗
Evidence for decreased splanchnic glucose uptake after oral glucose administration in non-insulin-dependent diabetes mellitus.
The role of splanchnic glucose uptake (SGU) after oral glucose administration as a potential factor contributing to postprandial hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM) has not been established conclusively. Therefore, we investigated SGU in six patients with NIDDM and six weight-matched control subjects by means of the hepatic vein catheterization (HVC) technique. In a second part, we examined the applicability of the recently developed OG-CLAMP technique in NIDDM by comparing SGU and first-pass SGU during HVC with SGU during the OG-CLAMP experiment. The OG-CLAMP method combines a euglycemic, hyperinsulinemic clamp and an oral glucose tolerance test (75 g) during steady state glucose infusion (GINF). During HVC, SGU equals the splanchnic fractional extraction times the total (oral and arterial) glucose load presented to the liver. For OG-CLAMP, SGU was calculated as first-pass SGU by subtracting the integrated decrease in GINF over 180 min from 75 g. Cumulative splanchnic glucose output after oral glucose correlated significantly between both methods and was increased significantly in NIDDM patients (73.1+/-5.1 g for HVC, 76.5+/-5.5 for OG-CLAMP) compared with nondiabetic patients (46.7+/-4.4 g for HVC, 57.5+/-1.9 for OG-CLAMP). Thus, in NIDDM patients, SGU (7.4+/-2.1 vs. 37.8+/-5.9% in nondiabetic patients, P < 0.001) and first-pass SGU (4.7+/-1.7 vs. 26.5+/-5.1% in nondiabetic patients, P < 0.01) were decreased significantly during HVC, as was SGU during OG-CLAMP (3.9+/-1.7 vs. 23.4+/-2.5% in nondiabetic patients, P < 0.0001). SGU measured during OG-CLAMP correlated significantly with SGU (r = 0.87, P < 0.05 for NIDDM patients; r = 0.94, P < 0.01 for nondiabetic patients) and first-pass SGU (r = 0.87, P < 0.05 for NIDDM patients; r = 0.84, P < 0.05 for nondiabetic patients) during HVC. In conclusion, (a) SGU after oral glucose administration is decreased in NIDDM as measured by both methods, and (b) SGU during the OG-CLAMP is well-correlated to SGU and first-pass SGU during HVC in NIDDM. The decrease in SGU in NIDDM might contribute to postprandial hyperglycemia in diabetic subjects.
Administration, Oral↗
Targeting vectors for intracellular immunisation.
We define intracellular immunization as the inhibition or inactivation of the function of a molecule by the ectopic intracellular expression of antibody binding domains which recognise the molecule. Such recombinant antibodies can be directed to different compartments of eukaryotic cells by means of previously defined targeting signals, thus permiting the study of any molecule in any cellular compartment for which an antibody is available. For this purpose, we have created a set of vectors based on the VHExpress vector described [Persic, L., Roberts, A., Wilton, J., Cattaneo, A., Bradbury, A. and Hoogenboom, H.R. (1997) An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187, 000-000], which has been modified to express scFvs (single chain fragments) linked to specific targeting signals. These permit the localisation of scFvs to different intracellular compartments: the endoplasmic reticulum (scFvE-er), the nucleus (scFvE-nuclear), the mitochondria (scFvE-mit), the cytoplasm (scFvE-cyto), and as secreted proteins (scFvE-sec). The function of these vectors has been assessed by the immunofluorescence of COS cells transiently transfected with constructs containing the alphaD11 scFv.
Amino Acid Sequence↗
An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries.
Phage display is now an established method to select antibody fragments specific for a wide range of diverse antigens. In particular, isolation of human monoclonal antibodies has become a reality and for most purposes bacterial expression of the selected recombinant antibody fragments is sufficient. However, there are some cases where the expression of complete human immunoglobulin in mammalian cells is, if not essential, at least desirable. For this reason we have designed and constructed a set of mammalian expression vectors which permit facile and rapid cloning of antibody genes for both transient and stable expression in mammalian cells. Immunoglobulin genes may be cloned into these expression vectors as V regions or as Fabs for expression as either complete antibodies or as Fab fragments, using restriction sites which are rare in human V genes. All the important elements in the vectors--promoter, leader sequence, constant domains and selectable markers--are flanked by unique restriction sites, allowing simple substitution of elements. The vectors have been evaluated using the variable regions from the neutralizing anti-nerve growth factor (NGF) antibody, alphaD11, and the V regions from 2E10, a scFv selected from a scFv phagemid library.
Animals↗