Immunoprecipitation of a cytoplasmic precursor of rat-liver cytochrome oxidase.

A simple method for the isolation of rat liver cells is described. The cells are shown, by an isotope dilution method, to maintain a constant rate of protein synthesis for 8 h of incubation. Antibodies to purified rat liver cytochrome oxidase were raised in rabbits and used to investigate the labeling of cytochrome oxidase in isolated rat liver cells and in vivo. The data demonstrate the occurrence of a precursor of the subunits of cytochrome oxidase that are synthesized in the cytoplasm. 1. Dodecylsulfate gel electrophoresis of the immunoprecipitates from isolated rat liver cells that had been labeled with [35S]methionine for 1 h showed a single radioactive peak with a molecular weight of 50000. 2. Judged by the effects of cycloheximide and chloramphenicol the labeled protein is synthesized on cytoplasmic ribosomes. 3. After labeling for 1 h in vivo with [3H]leucine the labeled protein appears to be exclusively associated with the hepatic microsomal fraction. 4. Ouchterlony double-diffusion analysis demonstrated immunological relationship between the precipitates from microsomes and cytochrome oxidase. In addition to the precipitates derived from mitochondria and microsomes immunoprecipitates were also obtained from the cytosol in comparable amounts; these again were immunologically related. The occurrence of large amounts of precursor(s) (or degradation products) of cytochrome oxidase in rat liver fractions is interpreted in terms of a regulatory pool for amino acid homeostasis in the organism.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Characterization of four members of a multigene family encoding outer membrane proteins of Helicobacter pylori and their potential for vaccination

In search of protective antigens which can be used in a vaccine to prevent Helicobacter pylori infection, we report on the identification of four genes, hopV, hopW, hopX and hopY, and the characterization of the corresponding proteins which belong to the H. pylori outer membrane protein (Hop) family containing 32 homologous members, some of which were shown to function as porins. Sequence analysis of 16 different H. pylori strains revealed that the proteins HopV, HopW, HopX and HopY are highly conserved. Localization of HopV, HopW, HopX and HopY at the surface of the bacteria was investigated by immunofluorescence. Using a planar lipid bilayer system the proteins HopV and HopX were shown to form pores with single-channel conductances of 1.4 and 3.0 nS, respectively.     

Sensitive liquid chromatography-tandem mass spectrometry method for the determination of clarithromycin in human plasma

A sensitive method for the determination of clarithromycin in plasma is described, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Samples were prepared using liquid-liquid extraction and separated on a Supelco Discovery C18 column with a mobile phase consisting of acetonitrile, methanol and acetic acid. Detection was performed by a PE SCIEX API 2000 mass spectrometer in the multiple reaction monitoring (MRM) mode (LC-MS-MS) using TurbolonSpray ionization and monitoring the transition of the protonated molecular ion for clarithromycin at m/z 748.5 (M+1) to the predominant product ion of m/z 158.2. The mean recovery of clarithromycin was 87.3%, with a lower limit of quantification of 2.95 ng/ml when using 0.3-ml plasma. This high-throughput method was used to quantify 230 samples per day, and is sufficiently sensitive to be employed in pharmacokinetic studies.     

Cultivation of Mycobacterium bovis BCG in bioreactors

The Mycobacterium bovis BCG vaccine for commercial use is classically produced as surface pellicles by culture on synthetic medium. Under these conditions, reproducibility of the cultures and quality assessment are hampered by slow growth of the bacilli, the formation of bacterial aggregates and a high proportion of dead bacilli after processing and final formulation of the vaccine. Here, we established dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors. These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of live bacteria. In the murine model, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than the vaccine produced under the classical conditions. The protective efficacy against challenge with M. tuberculosis was identical for both vaccine preparations.     

The Inhibitory Effects of Anacardic Acid on Hepatitis C Virus Life Cycle

Hepatitis C virus (HCV) is a small positive-sense single-stranded RNA virus that causes severe liver diseases. Current anti-HCV therapies involving direct-acting antivirals have significantly enhanced efficacy in comparison to traditional interferon and ribavirin combination. However, further improvement is needed to eradicate HCV. Anacardic acid (AnA) is a phytochemical compound that can inhibit the activity of various cellular enzymes including histone acetyltransferases (HATs). In this study, we investigated the effects of AnA on different phases of HCV life cycle. Our data showed that AnA can inhibit HCV entry, replication, translation, and virion secretion in a dose-dependent manner with no measurable effects on cell viability. In addition, we showed that two HAT inhibitors and knocking down HAT (PCAF) by RNAi can reduce HCV replication, suggesting a mechanism of AnA’s inhibitory effects on HCV. Elucidation of the AnA-mediated inhibitory mechanism should facilitate the development of new drug candidates for HCV infection.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Immunoprophylaxis of respiratory syncytial virus: global experience

In sarcoidosis, host genetic factors are discussed as contributing to disease susceptibility and course. Since tumor necrosis factor (TNF)-α is a central mediator of granuloma formation and since elevated TNF-α levels are found during active phases of sarcoidosis, genetic polymorphisms correlating with influences on TNF-α levels are of special interest. The complete sequencing of the MHC region and the increase in the number of identified gene polymorphisms in this locus associated with TNF-α production offer the opportunity of detecting new genes associated with sarcoidosis and perhaps of defining disease-associated haplotypes that bear the potential of serving as predictive markers for this disease.