Insertion signal sequence fused to minimal peptides elicits specific CD8+ T-cell responses and prolongs survival of thymoma-bearing mice

CD8+ T-lymphocytes (TCD8+) recognize minimal peptides of 8-10 residues which are the products of intracellularly processed proteins and are presented at the cell surface by major histocompatibility complex class I molecules. An important step in this process is the translocation of processed proteins from the cytosol across the endoplasmic reticulum membrane, mediated by transporter associated with antigen-processing proteins or alternatively by endoplasmic reticulum-insertion signal sequences located at the NH2-terminus of the precursor molecules. We report here that the addition of an endoplasmic reticulum-insertion signal sequence at the NH2-terminus of TCD8+ epitopes from chicken ovalbumin (amino acids 257-264) or a naturally occurring tumor antigen expressed by the murine mastocytoma P815 (P1A amino acids 35-43) significantly enhanced the priming of specific TCD8+ in vivo. The signal sequence did not enhance peptide immunogenicity by merely increasing the hydrophobicity of the peptide, since ovalbumin amino acids 257-264 peptide with the signal sequence at its COOH-terminus did not demonstrate enhanced efficacy. The signal sequence did not act as a helper epitope, since TCD8+ responses were not diminished in class II-deficient transgenic mice or in mice depleted of CD4+ T-cells in vivo. Importantly, a single immunization with the fusion peptide significantly prolonged survival of mice challenged with E.G7OVA, a thymoma transfected with the complementary DNA of chicken ovalbumin.     

Differentiating recruitment strategies for direct patient care, clerical, and fundraising hospice volunteers

A survey of direct patient care and nondirect patient care volunteers in two hospice programs generated information about various aspects of their volunteer experience. This paper focuses on the effectiveness of identified recruitment strategies for direct patient care, clerical, and fundraising volunteers. People learned about the opportunity to volunteer at hospice most frequently from personal experiences, friends, and newspapers. Significantly more direct patient care volunteers learned about the volunteer opportunity through newspaper feature stories than did nondirect volunteers. Friends recruited significantly more fundraising volunteers than direct patient care or clerical volunteers. The events triggering direct care and clerical volunteers to serve were most often personal experiences, whereas friends appear to have triggered direct care and fundraising volunteers to serve.     

Pharmacist influence on economic and morbidity outcomes in a tertiary care teaching hospital

The influence of pharmacist participation on economic and morbidity outcomes at a tertiary care teaching hospital was studied. Patients admitted to internal medicine wards during a nine-month period were assigned to either a treatment team or a control team. Each team consisted of an attending physician, senior and junior medical residents, and medical students; the treatment team included a pharmacist who reviewed all patient charts, made rounds with the team, and recommended modifications of drug therapy. Pharmacy interaction with the control team was limited to contacting physicians about potentially dangerous orders, answering questions from the medical team, and handling orders for items not on the formulary or otherwise unavailable. After discharge, data from patient records were analyzed for pharmacy costs and total hospital costs and length of stay (as markers of the pharmacist's effect on economics and morbidity, respectively). Analysis of baseline characteristics showed that the two groups of patients were statistically comparable. Treatment team patients who were included in the data analysis (414) had significantly shorter stays (by a mean of 1.3 days) and lower pharmacy and total hospital costs (by a mean of $301 and $1654, respectively) than those included in the control team analysis (453). The direct participation of a pharmacist on a patient care team significantly decreased pharmacy and hospital costs, as well as length of stay, compared with minimal participation of a pharmacist.     

Moderate alcohol consumption and loss of cerebellar Purkinje cells

OBJECTIVE: To examine the dose-response effect of alcohol consumption on the number of cerebellar Purkinje cells. DESIGN: A prospective necropsy study combined with detailed reports on use of alcohol from a relative or friend. The number of Purkinje cells was counted in the anterior midsagittal section of the cerebellar vermis, the area of which was measured by computer assisted morphometry. SETTING: Department of forensic medicine, University of Helsinki. SUBJECTS: 66 men, aged 35 to 69 years, subjected to medicolegal necropsy because of sudden or violent death. The average all year daily alcohol consumption over the year was 0 to 10 g in 17 men, 11 to 80 g in 24 men, and more than 80 g in 25 men. MAIN OUTCOME MEASURES: Number of Purkinje cells, alcohol consumption. RESULTS: The numbers and density of Purkinje cells in the cross section of vermis showed a consistent but weak decrease with increasing daily alcohol intake but not with age. A wide variation in the cell counts was observed, especially in men drinking more than 80 g, suggesting differences in the susceptibility to effects of alcohol. Compared with men drinking 40 g or less, a long term moderate consumption of an average of 41 to 80 g daily was associated with a significant average loss of 242 (95% confidence interval 45 to 439) Purkinje cells (15.2%) from a mean of 1583 to 1341 cells. In those drinking 81 to 180 g the average loss was 535 (259 to 811) cells (33.4%) to a mean of 1048 cells. The density of cells in the cross section of vermis also fell significantly by 0.9 cell/mm (0.1 to 1.7) when the daily consumption exceeded 40 g and by 1.4 cell/mm (0.3 to 2.5) when the intake was 81 to 180 g. Only three cases (4.5%) in the series showed macroscopical cerebellar atrophy. CONCLUSION: Long term intake of moderate doses of alcohol daily for 20-30 years may damage the cerebellum before the onset of macroscopical atrophy. Despite distinct individual differences an all year average daily alcohol intake of 41-80 g results in a risk of significant loss of Purkinje cells.     

Increase of 5-HT7 (serotonin-7) and 5-HT1A (serotonin-1A) receptor mRNA expression in rat hippocampus after adrenalectomy

Kinematic and electromyographic (EMG) analysis of a target-directed, maximal velocity movement was used to investigate the effects of high-force eccentric exercise on the neuromuscular control of elbow flexion. Ten non-weight-trained females [19.6 (1.6) years old] performed 50 maximal velocity elbow flexion movements from 0 to 1.58 rad (90 degrees), as rapidly as possible in response to a light stimulus, while kinematic and triphasic EMG parameters were measured. This was done three times pre-exercise, immediately and 1, 2, 3, 4, and 5 days following the 50 maximal eccentric elbow flexion actions. The eccentric exercise caused lengthening of kinematic parameters including total movement time and time to peak velocity. The EMG elements of the biceps brachii (b.) motor time, time to peak EMG, biceps b. burst duration, and the latency period between biceps b. and triceps b. bursts were lengthened post-exercise. These changes persisted for up to 5 days post-exercise. The exercise also caused a large increase in serum creatine kinase (CK) activity. It was concluded that high-force eccentric exercise in this population caused prolonged changes in neuromuscular control that were a function of exercise-induced disruption of the skeletal muscle. Compensation in the central motor program was such that the components of the triphasic EMG pattern were systematically lengthened.     

Automated docking of glucosyl disaccharides in the glucoamylase active site

To better understand the molecular basis of glucomylase selectivity, low-energy conformers of glucosyl disaccharides obtained from relaxed-residue conformational mapping were flexibly docked into the glucoamylase active site using AutoDock 2.2. This procedure ensures that significant conformational space is searched and can produce bound structures comparable to those obtained by protein crystallography. alpha-Linked glucosyl disaccharides except alpha,alpha-trehalose dock easily into the active site while exclusively beta-linked disaccharides do not, explaining why only the former are glucoamylase substrates. The optimized docking modes are similar at the nonreducing end of the different substrates. Individual atomic energies of intermolecular interaction allow the definite identification of key hydroxyl groups for each substrate. This approach confirmed the versatility of the second subsite of the glucoamylase active site in binding different substrates.     

The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid

The gamma-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (alpha1-6, beta1-3, gamma1-3, delta1, and epsilon1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the beta subunit subtype present within the receptor. Receptors containing beta2- or beta3-, but not beta1 subunits, are highly sensitive to the agent. Here, chimeric beta1/beta2 subunits coexpressed in Xenopus laevis oocytes with human alpha6 and gamma2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the beta3 subunit to Ser (the homologous residue in beta1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the beta1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk-) cells stably expressing the alpha6beta3gamma2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.