Hepatitis E Virus (HEV) Infection in Ireland.

Hepatitis E virus (HEV) is a single stranded RNA virus causing infection worldwide. In developing countries HEV genotypes 1 and 2 spread faeco-orally via water. Recently, infections with HEV have been detected in Europe and North America in patients with no travel history. These are food-borne HEV genotypes 3 and 4, a pig-associated zoonosis. Most infections are asymptomatic but morbidity and chronic infection may occur with prior liver disease or immunosuppression. International seroprevalence rates vary and with improved diagnostics have increased. To determine the current prevalence in this region we studied anonymised serum samples submitted in 2015 for routine testing. We detected anti-HEV IgG in 16/198 (8%) individuals, highest rate in 40-59 year olds (43.8%). This is higher than reported for the same region in 1995 (0.4%) using a previous generation assay. This study provides evidence of HEV circulation in Ireland and reinforces the need for ongoing surveillance.

HBV reactivation after fludarabine chemotherapy identified on investigation of suspected transfusion-transmitted Hepatitis B virus.

BACKGROUND & AIMS: Multi-transfused patients often receive treatments inducing various levels of immunodeficiency. Acute viral infections may then be attributed either to transfusion-transmitted infection (TTI) or reactivation of a past infection. METHODS: A patient with chronic lymphocytic leukemia (CLL) who had >250 blood donor exposures developed acute Hepatitis B virus (HBV) infection. Routine donor testing for HB core antibodies (anti-HBc) was in place in the relevant period and investigations undertaken on the blood donors were negative. RESULTS: Review of historical, molecular, and antigenic evidence demonstrated reactivation of a recovered HBV infection dating >30 years and the selection of a rare escape mutant that briefly replicated and caused acute liver disease. This mutant was unreactive with several HBsAg assays and poorly reactive with an HBV vaccine plasma. Correcting the C139Y substitution by site directed mutagenesis of recombinant surface proteins re-established assay reactivity. CONCLUSIONS: Fludarabine, but not Chlorambucil, appeared sufficiently immunosuppressive to trigger reactivation despite low levels of neutralizing antibodies. Differentiating between TTI and reactivation of HBV becomes more challenging with the increasing frequency of immunocompromised blood recipients. Chemotherapy with Fludarabine alone should be considered as carrying high risk of viral reactivation. Pre-treatment testing and peripheral blood sample archiving may be indicated in HBsAg negative patients.

Chinese hamster ovary cell mutants affecting cholesterol metabolism.

The approach of somatic cell and molecular genetics for the study of intracellular regulation of cholesterol metabolism has blossomed in recent years. This review lists all the Chinese hamster ovary cell mutants involved in cholesterol metabolism. In addition, it summarizes the characteristics of mutants involved in three different processes: (1) sequential cleavage of the membrane-bound sterol regulatory element-binding proteins; (2) cholesterol esterification; and (3) intracellular cholesterol transport from the lysosome/endosome.

Microwave sputtering of conductors and insulators for optical emission and mass spectrometry.

A microwave-powered slab-line cavity was used to excite a discharge in low pressure argon or neon and to demonstrate the sputtering of conducting and non-conducting samples by a microwave excited discharge. Both optical emission spectroscopy and mass spectrometry were used as detection systems. The dependence of the signals on gas pressure and net microwave power was investigated.

Long-term potentiation in mice lacking synapsins.

Synapsin I and synapsin II are widely expressed synaptic vesicle phosphoproteins that have been proposed to play an important role in synaptic transmission and synaptic plasticity. To gain further insight into the functional significance of the phosphorylation sites on the synapsins, we have examined a number of synaptic processes thought to be mediated by protein kinases in knockout mice lacking both forms of synapsin (Rosahl et al., 1995). Long-term potentiation (LTP) at both the mossy fiber (MF)-CA3 pyramidal cell synapse and the Schaffer collateral-CA1 pyramidal cell synapse appears normal in hippocampal slices prepared from mice lacking synapsins. Moreover, the effects on synaptic transmission of forskolin at MF synapses and H-7 at synapses on CA1 cells are also normal in the mutant mice. These results indicate that the synapsins are not necessary for: (1) the induction or expression of two different forms of LTP in the hippocampus, (2) the enhancement in transmitter release elicited by activation of the cAMP-dependent protein kinase (PKA) and (3) the depression of synaptic transmission caused by H-7. Although disappointing, these results are important in that they exclude the most abundant family of synaptic phosphoproteins as an essential component of long-term synaptic plasticity.

Essential functions of synapsins I and II in synaptic vesicle regulation.

Synaptic vesicles are coated by synapsins, phosphoproteins that account for 9% of the vesicle protein. To analyse the functions of these proteins, we have studied knockout mice lacking either synapsin I, synapsin II, or both. Mice lacking synapsins are viable and fertile with no gross anatomical abnormalities, but experience seizures with a frequency proportional to the number of mutant alleles. Synapsin-II and double knockouts, but not synapsin-I knockouts, exhibit decreased post-tetanic potentiation and severe synaptic depression upon repetitive stimulation. Intrinsic synaptic-vesicle membrane proteins, but not peripheral membrane proteins or other synaptic proteins, are slightly decreased in individual knockouts and more severely reduced in double knockouts, as is the number of synaptic vesicles. Thus synapsins are not required for neurite outgrowth, synaptogenesis or the basic mechanics of synaptic vesicle traffic, but are essential for accelerating this traffic during repetitive stimulation. The phenotype of the synapsin knockouts could be explained either by deficient recruitment of synaptic vesicles to the active zone, or by impaired maturation of vesicles at the active zone, both of which could lead to a secondary destabilization of synaptic vesicles.

Translocation of both lysosomal LDL-derived cholesterol and plasma membrane cholesterol to the endoplasmic reticulum for esterification may require common cellular factors involved in cholesterol egress from the acidic compartments (lysosomes/endosomes).

Using a stable cell line 25-RA derived from wild-type Chinese hamster ovary (CHO) cells as the parental cell, this laboratory previously reported the isolation and characterization of CHO cell mutants (cholesterol-trafficking or CT) defective in transporting LDL-derived cholesterol out of the acidic compartment(s) (lysosomes/endosomes) to the endoplasmic reticulum (ER) for esterification. In this report, we show that the CT mutation can be complemented by fusion with human cells; however, attempts to complement the CT defect through DNA transfection have resulted in a collection of stable cell lines designated as ST cells. Under cholesterol starvation condition, the ST cells exhibit an elevated rate of cholesterol ester biosynthesis (by 3- to 5-fold) compared to both the parental CHO cells and the CT cells. The phenotypes of the ST cells are stable. ST cells are thus new cell lines arisen from the CT cells. When the plasma membranes of the parental, CT, and ST cells are labelled with [3H]cholesterol, ST cells show rates of [3H]cholesterol esterification much higher than that observed in CT cells but lower than that observed in the parental CHO cells. This result shows that translocation of plasma membrane cholesterol to the ER for esterification is defective in the CT cells. This result also suggests that ST cells acquire increased cholesterol trafficking activity between the lysosome and the ER without mixing the plasma membrane cholesterol pool. The characteristics of CT cells and ST cells reported here suggest that translocation of both lysosomal LDL-derived cholesterol and plasma membrane cholesterol to the ER for esterification may require common cellular factors involved in cholesterol egress from the acidic compartment(s) (lysosomes/endosomes).

Short-term synaptic plasticity is altered in mice lacking synapsin I.

Synapsin I, the major phosphoprotein of synaptic vesicles, is thought to play a central role in neurotransmitter release. Here we introduce a null mutation into the murine synapsin I gene by homologous recombination. Mice with no detectable synapsin I manifest no apparent changes in well-being or gross nervous system function. Thus, synapsin I is not essential for neurotransmitter release. Electrophysiology reveals that mice lacking synapsin I exhibit a selective increase in paired pulse facilitation, with no major alterations in other synaptic parameters such as long-term potentiation. In addition to potential redundant functions shared with other proteins, synapsin I in normal mice may function to limit increases in neurotransmitter release elicited by residual Ca2+ after an initial stimulus.

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.