Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo

Cellular protein trafficking has been studied to date only in vitro or with techniques that are invasive and have a low time resolution. To establish a gentle method for analysis of glucose transporter-4 (GLUT4) trafficking in vivo in fully differentiated rat skeletal muscle fibres we combined the enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week-old rats and peaked around 1 week after transfection. The gene gun was used subsequently with a plasmid coding for EGFP linked to the C-terminus of GLUT4 (GLUT4-EGFP). Rats were anaesthetised 5 days after transfection and insulin given i.v. with or without accompanying electrical hindleg muscle stimulation. After stimulation, the hindlegs were fixed by perfusion. GLUT4-EGFP-positive FDB fibres were isolated and analysed by confocal microscopy. The intracellular distribution of GLUT4-EGFP under basal conditions as well as after translocation to the plasma membrane in response to insulin, contractions, or both, was in accordance with previous studies of endogenous GLUT4. Finally, GLUT4-EGFP trafficking in quadriceps muscle in vivo was studied using time-lapse microscopy analysis in anaesthetised mice and the first detailed time-lapse recordings of GLUT4-EGFP translocation in fully differentiated skeletal muscle in vivo were obtained.     

DNA microarray technique for detection and identification of seven flaviviruses pathogenic for man

A flavivirus microarray was developed for detection and identification of yellow fever (YF), West Nile, Japanese encephalitis (JE), and the dengue 1-4 viruses, which are causing severe human disease all over the world. The microarray was based on 500-nucleotide probe fragments from five different parts of the seven viral genomes. A low-stringent amplification method targeting the corresponding regions of the viral genomic RNA was developed and combined with hybridization to the microarray for detection and identification. For distinction of the generated virus-specific fluorescence-patterns a fitting analysis procedure was adapted. The method was verified as functional for all seven flaviviruses and the strategy for the amplification, combined with the long probes, provided a high tolerance for smaller genetic variability, most suitable for these rapidly changing RNA viruses. A potentially high detection and identification capacity was proven on diverged strains of West Nile and dengue viruses. The lower limit for detection was equivalent, or better, when compared to routinely used RT-PCR methods. The performance of the method was verified on human patient samples containing dengue viruses, or normal human serum spiked with YF or JE viruses. The results demonstrated the ability of the flavivirus microarray to screen simultaneously a sample for several viruses in parallel, in combination with a good lower limit of detection.     

Frequent polymorphism of the mitochondrial DNA polymerase gamma gene (POLG) in patients with normal spermiograms and unexplained subfertility

BACKGROUND: Male fertility largely depends on the quality of sperm production, which may be affected by environmental and genetic factors. In this study, we explored a possible role of the polymerase gamma (POLG) gene polymorphism, recently reported to be associated with male infertility in some populations. METHODS: The polymorphic CAG repeat (usually 10 codons long) in the POLG gene was studied in 1298 male subjects: 429 patients with infertility/subfertility, and 869 controls (495 men from the general population with unknown fertility and 374 recent fathers). In all subjects, the POLG polymorphism was assessed in relation to their semen quality, and--in the fertile controls--with biological fecundity measured as waiting time-to-pregnancy (TTP) for the couples. In the patients lacking the common POLG allele, the outcome of the assisted reproductive techniques (ART) for the couples was evaluated. RESULTS: The absence of one (10/ not equal to 10) or both common POLG alleles (not equal to 10/not equal to 10) was more frequent among the subfertile patients than among fertile controls (P=0.021 and P=0.04 respectively). The estimated predictive value for infertility in a man homozygous for the POLG polymorphism was 15.5% (95% CI: 4.8-51%). There was a positive association with sperm concentration: 14.3% of the normospermic subfertile patients were homozygous for the absence of the common POLG allele (not equal to 10/not equal to 10), in comparison with 2.3% of unselected controls (P=0.001) and 0.9% of the fertile men (P=0.0001). No association with sperm motility, morphology and TTP was found. Spermatozoa of the three not equal to 10/not equal to 10 patients treated with IVF retained the ability to penetrate the egg, but the fertilization rate was low. Nine homozygous not equal to 10/ not equal to 10 patients were treated with ICSI, resulting in pregnancy in seven couples. CONCLUSIONS: The POLG gene polymorphism should be considered as a possible contributing factor in patients with unexplained subfertility and normal spermiograms. The oocyte penetration ability of sperm may be partially impaired in the not equal to 10/not equal to 10 patients but most of them can be successfully treated with ICSI.     

Molecular epidemiology of an outbreak caused by methicillin-resistant Staphylococcus aureus in a health care ward and associated nursing home

Our point-prevalence survey followed an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in a long-term care facility and identified five MRSA strains, of which two possessed an outbreak genotype not encountered previously and three had another profile. All of them possessed SCCmec type V. Six methicillin-sensitive S. aureus strains were genotypically related to the epidemic strains.     

Treatments of pelvic girdle pain in pregnant women: adverse effects of standard treatment,acupuncture and stabilising exercises on the pregnancy,mother, delivery and the fetus/neonate

Background  

Previous publications indicate that acupuncture is efficient for the treatment of pelvic girdle pain, PGP, in pregnant women. However, the use of acupuncture for PGP is rare due to insufficient documentation of adverse effects of this treatment in this specific condition. The aim of the present work was to assess adverse effects of acupuncture on the pregnancy, mother, delivery and the fetus/neonate in comparison with women that received stabilising exercises as adjunct to standard treatment or standard treatment alone.     

An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.     

Linkage analysis between manic-depressive illness and markers on the long arm of chromosome 11

The long arm of chromosome 11 is one of the most interesting regions in the search for major genes involved in the etiology of manic-depressive illness. Several candidate genes have been identified, including the gene encoding the dopamine D2 receptor, the M1 muscarinic receptor, and porfobillinogen deaminase. Furthermore, different families with co-segregation of psychiatric illness and structural chromosome abnormalities involving regions 11q21, 11q22.3, and 11q25 have been reported. Using narrow as well as broad phenotypic models, conservative genetic parameters, models with dominant or recessive modes of inheritance, and various methods to reduce misclassification, the present study did not find evidence for a major gene causing manic-depressive illness on the long arm of chromosome 11. In the broader phenotypic models multi-point analyses excluded at least 11q14 to 11q23.3, approximately 60 cM, even in one large family. Assuming homogeneity close linkage to DRD2 was excluded for all dominant models, and also in the affecteds-only analyses in the large family alone. © 1995 Wiley-Liss, Inc.     

Web-based virtual microscopy in teaching and standardizing Gleason grading

Gleason grading forms the basis of prognostic and therapeutic assessment in prostatic carcinoma despite its subjective nature and substantial interobserver variation. The accuracy of Gleason grading can be improved by the use of educational tools such as reference images. However, conventional microscopy images are of limited educational value because it is neither possible to view the sample at different magnifications nor to navigate into different areas of the specimen. This limitation can be overcome by the use of virtual microscopy, which allows viewing entire digitized microscope slides. We created an interactive Web site ( www.webmicroscope.net/gleason ) featuring a comprehensive set of prostatic needle biopsies as virtual slides, which can be viewed with a standard Web browser (Internet Explorer or Netscape). To evaluate the validity of Web-based virtual microscopy for Gleason grading, an experienced uropathologist (TK) scored a series of 62 biopsies from the original glass slides and 6 weeks later from virtual slides on the Web site using an ordinary desktop computer. The intraobserver agreement was excellent, with identical Gleason scores found in 48 of the 62 cases ( kappa = 0.73). The 14 remaining scores differed only by 1 point on the Gleason scale (2-10). The virtual slides were viewed by 2 other uropathologists (PM and HH), with interobserver kappa coefficients ranging from 0.55 to 0.62, which is within the range of previously reported studies using glass slides. The 3 uropathologists' Gleason scores were included as reference scores on the Web site, which now serves as a publicly open platform for self-testing and learning of Gleason grading. We conclude that Web-based virtual microscopy is a promising new tool for teaching and standardizing Gleason grading.