Direct syncytial assay for the quantitation of bovine leukemia virus.

A direct in vitro tissue culture method is described for the quantitation of bovine leukemia virus, utlizing a feline S+L-- cell line.     

IMPT1, an imprinted gene similar to polyspecific transporter and multi- drug resistance genes

Human chromosome 11p15.5 and distal mouse chromosome 7 include a megabase-scale chromosomal domain with multiple genes subject to parental imprinting. Here we describe mouse and human versions of a novel imprinted gene, IMPT1 , which lies between IPL and p57 KIP2 and which encodes a predicted multi-membrane-spanning protein similar to bacterial and eukaryotic polyspecific metabolite transporter and multi- drug resistance pumps. Mouse Impt1 and human IMPT1 mRNAs are highly expressed in tissues with metabolite transport functions, including liver, kidney, intestine, extra-embryonic membranes and placenta, and there is strongly preferential expression of the maternal allele in various mouse tissues at fetal stages. In post-natal tissues there is persistent expression, but the allelic bias attenuates. An allelic expression bias is also observed in human fetal and post-natal tissues, but there is significant interindividual variation and rare somatic allele switching. The fact that Impt1 is relatively repressed on the paternal allele, together with data from other imprinted genes, allows a statistical conclusion that the primary effect of human chromosome 11p15.5/mouse distal chromosome 7 imprinting is domain-wide relative repression of genes on the paternal homolog. Dosage regulation of the metabolite transporter gene(s) by imprinting might regulate placental and fetal growth.      

Small Conductance Ca2+-Activated K+ Channels Formed by the Expression of Rat SK1 and SK2 Genes in HEK 293 Cells

The rat SK1 gene ( rSK1 ) does not form functional Ca²⁺-activated potassium channels when expressed alone in mammalian cell lines. Using a selective antibody to the rSK1 subunit and a yellow fluorescent protein (YFP) tag we have discovered that rSK1 expression produces protein that remains largely at intracellular locations. We tested the idea that rSK1 may need an expression partner, rSK2, in order to form functional channels. When rSK1 was co-expressed with rSK2 in HEK 293 cells it increased the current magnitude by 77 ± 34 % (as compared with cells expressing rSK2 alone). Co-expression of rSK1 with rSK2 also changed the channel pharmacology. The sensitivity of SK current to block by apamin was reduced ~16-fold from an IC₅₀ of 94 p m (for SK2 alone) to 1.4 n m (for SK2 and SK1 together). The sensitivity to block by UCL 1848 (a potent small molecule blocker of SK channels) was similarly reduced, ~26-fold, from an IC₅₀ of 110 p m to 2.9 n m . These data clearly demonstrate that rSK1 and rSK2 subunits interact. The most likely explanation for this is that the subunits are able to form heteromeric assemblies.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.      

Characterization of a gene encoding survival motor neuron (SMN)-related protein, a constituent of the spliceosome complex

Mutations in the gene encoding the Survival Motor Neuron (SMN) protein are responsible for autosomal recessive proximal spinal muscular atrophy (SMA). SMN orthologues have been identified in the nematode worm Caenorhabditis elegans and the yeast Schizosaccharomyces pombe but, to date, no human paralogues have been described. Here we describe identification and characterization of an SMN-related protein (SMNrp) gene that encodes a novel protein of 239 amino acids, which has recently been identified as a constituent of the spliceosome complex and designated SPF30. Significant similarity to the SMN protein is apparent only within a central region of SMNrp that represents a tudor domain. The SMNrp/SPF30 gene has been mapped to chromosome 10q23. It is differentially expressed, with abundant levels in skeletal muscle. An exclusively nuclear localization for SMNrp in cultured cells and muscle sections was revealed using GFP fusion constructs and thereafter confirmed with a polyclonal antibody raised against SMNrp. Overexpression of SMNrp as a fusion protein in HeLa cells in culture induced dose-dependent apoptosis with positive TUNEL staining. In addition to a possible role for this protein as a pro-apoptotic factor, SMN and its related protein share significant similarities in sequence and cellular function.      

1141610241Altered expression of HuR, an mRNA stabilizing protein, mediates aberrant Placenta Growth Factor (PlGF) expression in preeclampsia

Background:  Control of mRNA stability is an essential regulatory process in eukaryotic gene expression. HuR, a 3'UTR mRNA binding protein, can protect AU-rich mRNA from degradation in response to stresses. PlGF, an angiogenic growth factor, contains two consensus AU-rich sites suggesting that under normal conditions HuR may protect PlGF mRNA from degradation. Trophoblast expression of PlGF is significantly decreased in preeclampsia and by hypoxia in vitro . We hypothesize that decreased levels of cytoplasmic HuR may contribute to decreased PlGF expression in hypoxic and preeclamptic trophoblast.
Methods:  Western blots were used to determine relative effects of in vitro hypoxia on HuR protein expression and subcellular localization in trophoblast. Immunohistochemistry was used to compare HuR expression patterns in trophoblast of preeclamptic and normal placentae.
Results:  Cytoplasmic expression of HuR was decreased 1.4 fold in the cytoplasm and 1.2 fold in the nucleus of JEG3 cells. A shift in HuR was more apparent in primary trophoblast with a greater than 2-fold decrease in the cytoplasm and a 1.4 fold decrease in the nucleus following 24 hr of hypoxia. Immunohistochemical analyses detected HuR expression in near term trophoblast in situ . However, this technical approach did not detect a significant change in HuR expression between normal and preeclamptic trophoblast.
Conclusions:  HuR expression is decreased in hypoxic trophoblast, at least in vitro , which may provide a causal link to decreased PlGF mRNA expression. Down regulation of trophoblast PlGF expression is thought to contribute to the pathophysiology associated with preeclampsia including the relative lack of perfusion of the placenta and systemic renal effects.     

House dust mite allergen avoidance and self-management in allergic patients with asthma: randomised controlled trial

BACKGROUND: The efficacy of bed covers that are impermeable to house dust mites has been disputed. AIM: The aim of the present study was to investigate whether the combination of 'house dust mite impermeable' covers and a self-management plan, based on peak flow values and symptoms, leads to reduced use of inhaled corticosteroids (ICS) than self-management alone. DESIGN OF STUDY: Prospective, randomised, double blind, placebo-controlled trial. SETTING: Primary care in a south-eastern region of the Netherlands. METHOD: Asthma patients aged between 16 and 60 years with a house dust mite allergy requiring ICS were randomised to intervention and placebo groups. They were trained to use a self-management plan based on peak flow and symptoms. After a 3-month training period, the intervention commenced using house dust mite impermeable and placebo bed covers. The follow-up period was 2 years. Primary outcome was the use of ICS; secondary outcomes were peak expiratory flow parameters, asthma control, and symptoms. RESULTS: One hundred and twenty-six patients started the intervention with house dust mite impermeable or placebo bed covers. After 1 and 2 years, significant differences in allergen exposure were found between the intervention and control groups (P<0.001). No significant difference between the intervention and control groups was found in the dose of ICS (P = 0.08), morning peak flow (P = 0.52), peak flow variability (P = 0.36), dyspnoea (P = 0.46), wheezing (P = 0.77), or coughing (P = 0.41). There was no difference in asthma control between the intervention and control groups. CONCLUSION: House dust mite impermeable bed covers combined with self-management do not lead to reduced use of ICS compared with self-management alone.     

The effectiveness of a treatment protocol for male lower urinary tract symptoms in general practice: a practical randomised controlled trial

BACKGROUND: Randomised controlled trials have shown the efficacy of several treatment modalities for lower urinary tract symptoms (LUTS) in selected populations. The effectiveness in daily practice has hardly been investigated, especially in primary care and is dependent on choices between all possible treatment options and best investigated in a comprehensive study, including all treatment modalities (watchful waiting, alpha-blockers, 5-alpha-reductase inhibitors, and surgery). AIM: Assessment of the effectiveness of a comprehensive treatment protocol for LUTS in primary care. DESIGN OF STUDY: Randomised controlled trial. SETTING: Fourteen general practices in the Netherlands. METHOD: Intervention: treatment protocol based on a formalised expert opinion. Control condition: usual care. Study population: 208 subjects with moderate to severe LUTS (IPSS > or =8, median = 13). OUTCOME MEASURES: symptom severity (IPSS [International Prostate Symptom Score]), bother score (Dan-PSS [Danish Prostate Symptom Score]), and maximum urinary flow (Q(max)); incidence of acute urinary retention and urinary tract infections. RESULTS: In the intervention group markedly more subjects used an alpha-blocker at end of follow-up than in the usual care group (24% versus 6%). No significant differences were found between intervention and control group in IPSS, Q(max) or Dan-PSS. CONCLUSION: alpha-blockers and watchful waiting are the most frequent treatment modalities for LUTS in primary care. Our study showed no evidence that a protocol using well-defined indications for all possible treatment modalities based on a formalised expert opinion procedure has added value. Based on our results, we cannot recommend a broadening of the indication for alpha-blockers, which, however, seems to be the current trend.     

UK food and nutrition security during and after the COVID‐19 pandemic

The COVID‐19 pandemic is a major shock to society in terms of health and economy that is affecting both UK and global food and nutrition security. It is adding to the ‘perfect storm’ of threats to society from climate change, biodiversity loss and ecosystem degradation, at a time of considerable change, rising nationalism and breakdown in international collaboration. In the UK, the situation is further complicated due to Brexit. The UK COVID‐19 Food and Nutrition Security project, lasting one year, is funded by the Economic and Social Research Council and is assessing the ongoing impact of COVID‐19 on the four pillars of food and nutrition security: access, availability, utilisation and stability. It examines the food system, how it is responding, and potential knock on effects on the UK’s food and nutrition security, both in terms of the cascading risks from the pandemic and other threats. The study provides an opportunity to place the initial lessons being learnt from the on‐going responses to the pandemic in respect of food and nutrition security in the context of other long‐term challenges such as climate change and biodiversity loss.