Identification of a mutation in synapsin I, a synaptic vesicle protein, in a family with epilepsy

A four generation family is described in which some men of normal intelligence have epilepsy and others have various combinations of epilepsy, learning difficulties, macrocephaly, and aggressive behaviour. As the phenotype in this family is distinct from other X linked recessive disorders linkage studies were carried out. Linkage analysis was done using X chromosome microsatellite polymorphisms to define the interval containing the causative gene. Genes from within the region were considered possible candidates and one of these, SYN1, was screened for mutations by direct DNA sequencing of amplified products. Microsatellite analysis showed that the region between MAOB (Xp11.3) and DXS1275 (Xq12) segregated with the disease. Two point linkage analysis demonstrated linkage with DXS1039, lod score 4.06 at theta = 0, and DXS991, 3.63 at theta = 0. Candidate gene analysis led to identification of a nonsense mutation in the gene encoding synapsin I that was present in all affected family members and female carriers and was not present in 287 control chromosomes. Synapsin I is a synaptic vesicle associated protein involved in the regulation of synaptogenesis and neurotransmitter release. The SYN1 nonsense mutation that was identified is the likely cause of the phenotype in this family.     

Susceptibility testing of multidrug-resistant Staphylococcus aureus with the Sceptor microdilution system.

The antimicrobial susceptibilities of Staphylococcus aureus isolates were concurrently determined by the Sceptor system (BBL Microbiology Systems, Cockeysville, Md.) and by the standard disk diffusion method. For the methicillin-resistant isolates, there was greater than 98% agreement between the two test results with penicillin G, erythromycin, clindamycin, tetracycline, gentamicin, and tobramycin. Major disagreements (susceptible by one method and resistant by the other) were 7% for methicillin, 13.5% for cephalothin, 3.5% for cefamandole, and 27% for amikacin. The major discrepancies for methicillin were eliminated by supplementing the inoculum broth with salt. For methicillin-susceptible isolates, agreement between the two methods was 96 to 100% for all antibiotics except amikacin.     

Comparison of three isolation systems for the culture of mycobacteria from respiratory and non-respiratory samples

AIMS: To compare the recovery of mycobacteria from clinical samples using the MB/BacT rapid culture system with that obtained using egg medium or the Bactec radiometric method. METHODS: The three methods were compared using 681 clinical samples (462 respiratory and 219 non-respiratory samples) and eight external quality control strains. Culture media were incubated at 35-37 degrees C for six weeks in the MB/BacT system and for 12 weeks in the Bactec system and on egg medium. Solid media were examined macroscopically once a week and the Bactec vials were read six times in the first two weeks, and then weekly for the next 10 weeks (a growth index > 50 indicated a positive vial). The MB/BacT system positive vials were unloaded from the machine as soon as possible after detection. Confirmation of growth for all systems was by Ziehl-Neelson stained smears. Isolates were identified by a combination of phenotypic and molecular methods. RESULTS: Of the 681 clinical samples, 59 (8.7%) were positive on culture, including 23 strains of Mycobacterium tuberculosis. None of the three systems recovered all of the isolates, but each recovered mycobacteria not detected by either of the other two systems. After six weeks incubation, isolation rates were 87%, 78%, and 90%, and mean times to detection were 13, 19, and nine days for the MB/BacT, egg medium, and Bactec systems, respectively. Although the MB/BacT system was slightly slower than the Bactec system, the biomass was greater, allowing earlier use of molecular probes and earlier inoculation of susceptibility tests. CONCLUSIONS: The MB/BacT system provides comparable performance to the Bactec radiometric system, without the problems of disposal of radioactive waste. Optimal recovery is obtained when culture on egg medium is used in conjunction with a rapid culture system.     

Macropotrema, pertinax gen. et sp. nov. (Digenea: Paramphistomidae) from a wallaby, Macropus agilis, in northern Australia, and associated pathology

Macropotrema pertinax gen. et sp. nov. (Digenea: Paramphistomidae) is described from the caecum of the macropodid marsupial Macropus agilis (Gould, 1842) from northern Australia. The new genus is characterised as follows: ventro-terminal acetabulum bearing many papillae; oral sucker with constriction and paired diverticula; oesophagus with muscular bulb; cirrus sac and genital sucker absent; testes symmetrical, unlobed, preovarian; Laurer's canal opening anterior to excretory pore. The genus is placed in the subfamily Pseudodiscinae N?smark, 1937. At the point where the worm attaches to the caecal wall of the host, the entire mucosa is destroyed and there is an inflammatory cell infiltration in the intact mucosa surrounding the attachment site.     

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.     

High-Throughput Generation of P. falciparum Functional Molecules by Recombinational Cloning

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.