Identification of yeast proteins from two-dimensional gels: working out spot cross-contamination

With the complete sequence of the yeast genome now available, efforts by many laboratories are underway to identify each of the spots on two-dimensional (2-D) gels corresponding to the most abundant yeast proteins. The high mass accuracy now attainable using matrix assisted laser desorption/ionization (MALDI)-mass spectrometry equipped with delayed extraction simplifies the process of identification, such that many spots can be unambiguously identified in a short period of time merely by using peptide mass fingerprinting and generally available database matching programs. Although it is not always possible to match spots between gels run by different laboratories, proteins generally yield the same abundant proteolytic fragments when tryptic digestions are performed. Databases containing these signature peptides not only simplify the task of reidentifying proteins from different gels, but also make it possible to identify small amounts of cross-contaminating proteins from different spots, as well as common extraneous contaminants such as human keratins. In this paper, we present data on the identification of > 20 previously unreported yeast proteins from 2-D gels. Some novel proteins were identified from randomly analyzed spots. Focusing on 14 spots in a narrow-pH-range gel, we demonstrate how organizing peak-table data and peptide match-list data into databases enables the identification of a larger percentage of the peaks.     

Neuromodulation of hypoglossal motoneurons: cellular and developmental mechanisms

Hypoglossal motoneurons (HMs) in the caudal brainstem have a respiratory-related activity pattern and contribute to control of upper airway resistance. In this review, we focus primarily on signalling mechanisms utilized by neurotransmitters to enhance HM excitability. In particular, we consider: (1) the membrane depolarization induced by a number of different putative transmitters [thyrotropin-releasing hormone (TRH), serotonin (5-HT), norepinephrine (NE)]; and (2) the inhibition of a calcium-dependent spike after hyperpolarization (AHP) by 5-HT and its effect on firing behavior. Potential functional consequences on HM behavior of these different neurotransmitter effects is discussed. In addition, we describe postnatal changes in transmitter effects and suggest potential cellular mechanisms to explain those developmental changes. Most of the data discussed are derived from in vitro electrophysiological recordings performed in preparations from neonatal and adult rats.     

Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte-macrophage colony-stimulating factor gene transfer

Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced, irradiated tumor vaccines induce potent, T-cell-mediated antitumor immune responses in preclinical models. We report the initial results of a Phase I trial evaluating this strategy for safety and the induction of immune responses in patients with metastatic renal cell carcinoma (RCC). Patients were treated in a randomized, double-blind dose-escalation study with equivalent doses of autologous, irradiated RCC vaccine cells with or without ex vivo human GM-CSF gene transfer. The replication-defective retroviral vector MFG was used for GM-CSF gene transfer. No dose-limiting toxicities were encountered in 16 fully evaluable patients. GM-CSF gene-transduced vaccines were equivalent in toxicity to nontransduced vaccines up to the feasible limits of autologous tumor vaccine yield. No evidence of autoimmune disease was observed. Biopsies of intradermal sites of injection with GM-CSF gene-transduced vaccines contained distinctive macrophage, dendritic cell, eosinophil, neutrophil, and T-cell infiltrates similar to those observed in preclinical models of efficacy. Histological analysis of delayed-type hypersensitivity responses in patients vaccinated with GM-CSF-transduced vaccines demonstrated an intense eosinophil infiltrate that was not observed in patients who received nontransduced vaccines. An objective partial response was observed in a patient treated with GM-CSF gene-transduced vaccine who displayed the largest delayed-type hypersensitivity conversion. No replication-competent retrovirus was detected in vaccinated patients. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous GM-CSF gene-transduced tumor vaccine for RCC patients.     

Influence of dietary selenium on the disposition of arsenate in the female B6C3F1 mouse

Ecdysteroid titres have been determined in adult female house crickets (Acheta domesticus) in relation to reproductive maturation. Ecdysteroid levels in newly emerged adult females are low except in the gut and carcass, which probably reflects the remnants of the preecdysial ecdysteroid peak. Ecdysteroid levels in all compartments increase markedly once ovarian weight surpasses 10 mg. Apolar ecdysteroid conjugates (ecdysone 22-fatty acyl esters) predominate in ovarian tissue throughout ovarian maturation, but low levels of free ecdysteroid and polar conjugated ecdysteroids are also present. During this period, two peaks of ecdysteroids (mainly free and apolar conjugated ecdysteroids) are observed in the haemolymph, gut, and carcass compartments. The peaks in the haemolymph occur when the ovarian mass reaches 30 and 100 mg. The gut and carcass may be acting as sinks or sites of metabolism for the hormone released from the ovaries. The rate of ecdysone acylation by ovaries was found to be developmentally regulated, increasing from low levels in the immature ovaries of newly emerged females as the ovaries increase in size. A semiquantitative assay has been developed to identify compounds which inhibit the conversion of [3H]ecdysone into 22-fatty acyl [3H]ecdysone by ovaries in vitro. A number of ecdysteroids possessing a free hydroxyl group as C-22 as well as the side-chain stereochemistry of ecdysone effectively inhibit this conversion, probably by acting as competitive substrates. In the cases of 20-hydroxyecdysone and ponasterone A, it was clearly demonstrated that these compounds are converted to a mixture of C-22 fatty acyl esters. Several other compounds which have been suggested to affect ecdysteroid metabolism/mode of action in other systems were also tested for their effects on the acyltransferase activity of ovaries in vitro.     

In vitro studies of activity of voriconazole (UK-109,496), a new triazole antifungal agent, against emerging and less-common mold pathogens

The in vitro activity of voriconazole was compared with that of itraconazole. Eighty-six isolates of pathogenic molds belonging to 23 species were tested by an agar dilution method in High Resolution medium. Voriconazole was more active than itraconazole against a number of hyaline molds, including several Fusarium spp. and Scedosporium prolificans. Voriconazole and itraconazole showed comparable good activity against several hyaline molds, including Penicillium marneffei and Scedosporium apiospermum, and a number of dematiaceous molds, including Bipolaris australiensis, Cladophialophora bantiana, several Exophiala spp., and several Fonsecaea spp. Our results suggest that voriconazole could be effective against a wide range of mold infections in humans.     

Enhancing motivation to reduce the risk of HIV infection for economically disadvantaged urban women

This research evaluated a motivation-based HIV risk reduction intervention for economically disadvantaged urban women. Participants completed a survey that assessed HIV-related knowledge, risk perceptions, behavioral intentions, sexual communication, substance use, and risk behavior. A total of 102 at-risk women (76% African American) were randomly assigned to either the risk reduction intervention or to a waiting list. Women were reassessed at 3 and 12 weeks. Results indicated that treated women increased their knowledge and risk awareness, strengthened their intentions to adopt safer sexual practices, communicated their intentions with partners, reduced substance use proximal to sexual activities, and engaged in fewer acts of unprotected vaginal intercourse. These effects were observed immediately, and most were maintained at follow-up.     

Computer intuition: guiding scientific research in imaging and oral implantology

As the costs associated with clinical research and new drug development increase, it is incumbent upon us to develop alternative research methodologies. A new Computer Intuition (CI) program identifies literature-based evidence with the potential to generate a hypothesis that is most likely to be clinically confirmed regarding the cause of a disease or the questions being posed. The objective of this study was to demonstrate whether computer intuition can be used to guide scientific research in solving important biomedical questions. The establishment of CI as an alternative research methodology has the potential to accelerate the translation of basic science findings into clinical practice. The specific aim of this study was to develop a CI-driven hypothesis related to the controversial issue debating which radiographic imaging technology is most suitable for diagnostic purposes prior to the placement of oral implants. This hypothesis was then compared, in retrospect, with a known opinion established following a recent literature review on this issue. In his review of the literature, Frederiksen (1995) suggests that multiplanar reformatted computed tomography (CT) is the diagnostic imaging modality of choice prior to implant placement. To compare the CI hypothesis with Frederiksen's (1995) opinion, we subjected 34 relevant papers to CI analysis. The output consisted of clusters of important statements, phrases, and thought processes from the given dataset rated with the greatest potential to generate testable hypotheses. Both CI's output and Frederiksen (1995) indicated that CT scanning is the diagnostic imaging modality of choice prior to implant placement. Although the ultimate utility of CI is dependent on the successful testing of its derived hypothesis, this preliminary retrospective study suggests that CI might be useful in guiding scientific research.     

The characteristics of bone injuries in childhood

All aboard for an imaginary journey to the village of Health Care Chaos where the different actors and issues involved in the very real state of health care come alive. The author incorporates an interview with Virginia Trotter Betts, President of the American Nurses Association, to highlight the controversy surrounding the involvement and role of advanced practice nurses.     

Role of lipids in the permeabilization of membranes by class L amphipathic helical peptides

We studied the mechanism of membrane permeabilization by the 18L model peptide (GIKKFLGSIWKFIKAFVG), which features the consensus class L sequence averaged from the number of naturally occurring lytic peptides. Two aspects of membrane lipid composition significantly affected peptide-membrane interactions: the presence of acidic lipids and, in zwitterionic membranes, and the presence of nonbilayer forming lipids. In zwitterionic membranes, 18L peptide destabilizes the membrane, leading to a transient formation of large defects in the membrane which result generally in contents leakage, but in the presence of bilayer-bilayer contact can alternatively lead to vesicle fusion. In membranes containing acidic lipids (DOPC:DOPG, DOPG), 18L caused leakage but not fusion, probably due to mutual repulsion of acidic vesicles. While the extent of contents leakage was approximately the same as for zwitterionic membranes, the kinetics of leakage could be resolved only by using stopped-flow, leakage being essentially complete within the first minute. Previously, we reported that apolipoprotein (class A) and lytic (class L) peptide analogs have opposing effects on some properties of biological membranes. This reciprocal effect of 18L and Ac-18A-NH2, class A model peptide, is restricted to membranes with a high propensity for nonbilayer phase formation (DOPE, Me-DOPE, DOPC:DOPE, DOPC:Me-DOPE). The decrease in the content of nonbilayer phase forming lipid or the addition of acidic lipids reduces or eliminates the reciprocal effects. This suggests the importance of nonbilayer phase propensity for certain functions of biological membranes.