Assignment of the porcine calcium release channel gene, a candidate for the malignant hyperthermia locus, to the 6p11----q21 segment of chromosome 6

Several studies point to the possibility that malignant hyperthermia (MH) in pigs is caused by a defect in the calcium release channel (CRC) of skeletal muscle sarcoplasmic reticulum. The locus for MH is closely linked to the glucosephosphate isomerase (GPI) locus, near the centromere of chromosome 6. We demonstrate synteny of the genes for CRC and GPI using somatic cell hybrid lines, and assign the CRC gene to chromosome 6p11----q21 by in situ hybridization.     

Influence of temperature and ambient oxygen on the swimming energetics of cyprinid larvae and juveniles

Synopsis The relationship between respiration and swimming speed of larvae and juveniles (2–100 mg fresh mass) of Danube bleak, Chalcalburnus chalcoides (Cyprinidae), was measured at 15° and 20° C under hypoxic (50% air saturation), normoxic, and hyperoxic (140% air saturation) conditions. In a flow-tunnel equipped with a flow-through respirometer the animals swam at speeds of up to 8 lengths · s^-1; speeds were sustained for at least two minutes. The mass specific standard, routine, and active respiration rates declined with increasing body mass at both temperatures. Metabolic intensity increased with temperature, but also the critical swimming speed (at which oxygen uptake reached its maximum) was higher at 20° than at 15° C by about 30%. Nevertheless, the oxygen debt incurred by the fish at the highest speeds was about 40%, and the net cost of swimming about 32%, lower at 20° than at 15°C. The standard metabolic rate was more strongly dependent on temperature (Q₁₀ around 2.5) than the maximum active rate (Q₁₀ below 2). Whereas standard and routine respiration rates were well regulated over the pO₂-range investigated (8.5–25.8 kPa), the active rates showed a conformer-like pattern, resulting in factorial scopes for activity between 2 and 4. Under hypoxia, the critical swimming speed was lower than under normoxia by about 1.51 · s^-1, but the net cost of swimming was also lower by about 30%. On the other hand, hyperoxia neither increased the swimming performance nor did it lead to a further increase of the metabolic cost of swimming. The hypoxia experiments suggest that in response to lowered tensions of ambient oxygen maintenance functions of metabolism not directly related to swimming may be temporarily reduced, leading to increased apparent swimming efficiency under these conditions. The responses of the larvae of Danube bleak to low temperature and low ambient oxygen are discussed in terms of the metabolic strategies by which energy-limited animals meet the challenge of environmental deterioration.     

Two novel mutations in exons 19a and 20 and a BsaI polymorphism in a newly characterized intron of the neurofibromatosis type 1 gene

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. It is caused by mutations in the NF1 gene, which comprises 60 exons and is located on chromosome 17q11.2. A total of 170 unrelated NF1 patients were screened for mutations in four exons by temperature-gradient gel electrophoresis. Preparatory work revealed the presence of a previously uncharacterized intron (19a) in what was previously designated exon 19; this allowed us to develop assays for genomic mutation screening in the newly defined exons 19a and 19b. Two novel NF1 mutations were detected: a single-base insertion in exon 19a creating a frameshift, and a second mutation affecting the splice donor site of intron 20 and leading to skipping of exon 20. A novel BsaBI polymorphism was identified in intron 19a. Received: 11 August 1997 / Accepted: 13 November 1997     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Hyperdiploidy and apparent aneusomy in mesothelial cells from non-malignant effusions as detected by fluorescence in situ hybridization (FISH)

Interphase cytogenetics by fluorescence in situ hybridization (FISH) can be used to detect malignant cells characterized by chromosomal aneuploidy. However, apparent aneusomy in normal "control" tissues has to be considered when using FISH as diagnostic tool. In effusions as model tissue exposed to metastasis, the definition of cut-off levels for background aneusomy by FISH was aimed in this study. Using centromeric probes representing chromosomes 7, 8, 11, 12, 17 and 18, extensive chromosome copy number enumeration by single-color FISH analysis was performed in pleural and ascitic effusions derived from 15 patients with various, non-malignant diseases. In all effusions, cells with gain of hybridization signals for several or all chromosomes tested were found (in up to 1.94% of cells). A consistent finding was high grade hyperdiploidy (>4 centromeric signals). Mesothelial elements mainly contributed to hyperdiploidy in effusions, as demonstrated by a combined analysis of FISH and immunocytochemistry with staining for cytokeratin. Dual-color FISH analysis showed that hyperdiploidy was predominantly corresponding to polyploidization; however, there were always minor cell populations classified as aneuploid by dual-color FISH. In conclusion, stringent criteria have to be applied to distinguish malignancy-related aneuploidy from background aneusomy by FISH.     

Bacterial virulence, proinflammatory cytokines and host immunity: how to choose the appropriate Salmonella vaccine strain?

Salmonella infection in its mammalian host can be dissected into two main components. The co-ordinate expression of bacterial virulence genes which are designed to evade, subvert or circumvent the host response on the one hand, and the host defence mechanisms which are designed to restrict bacterial survival and replication on the other hand. The outcome of infection is determined by the one which succeeds in disturbing this equilibrium more efficiently. This delicate balance between Salmonella virulence and host immunity/inflammation has important implications for vaccine development or therapeutic intervention. Novel Salmonella vaccine candidates and live carriers for heterologous antigens are attenuated strains with defined genetic modifications of metabolic or virulence functions. Although genetic defects of different gene loci can lead to similar degrees of attenuation, effects on the course of infection may vary, thereby altering the quality of the elicited immune response. Studies with gene-deficient animals indicate that Salmonella typhimurium strains with mutations in aroA, phoP/phoQ or ssrA/ssrB invoke different immune responses and that a differential repertoire of pro-inflammatory cytokines is required for clearance. Consequently, Salmonella mutants defective in distinct virulence functions offer the potential to specifically modulate the immune response for defined medical applications.     

Divergent and reticulate processes in evolution of Ethiopian Lophuromys flavopunctatus species complex: evidence from mitochondrial and nuclear DNA differentiation patterns

Molecular study of mitochondrial and nuclear genes and cytogenetic analysis were performed to examine possible patterns of speciation in the diverse Lophuromys flavopunctatus species complex of Ethiopia. Phylogenetic analysis of mtDNA data resulted in an unresolved bush of ten deeply diverged haplotype groups corresponding to potential species either well supported by various types of character or 'cryptic'. The cytogenetic analysis showed representatives of five of these mtDNA lineages to share an identical karyotype (2 n  = 70, NFa = 84), that has not been found previously in Ethiopia. One of them, L.  cf.  sikapusi , being a member of the L. flavopunctatus species complex, demonstrates remarkable morphological similarity to representatives of another species complex, L. sikapusi s.l ., which might be considered as a result of convergent evolution in analogous environments. Analysis of RAPD data suggests that at least two mtDNA types might have been subject to interspecific transfer due to hybridization. In the case of two sympatric haplotypes of L. brunneus we may assume that the contemporary pattern of variation between them can be explained by relatively recent hybridization with another distinct species, L. flavopunctatus . The formation of two groups belonging to distinct mitochondrial lineages within northern populations could be associated with more complex processes including ancient hybridization.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 301–316.     

Physiological dilation of uteroplacental arteries in the guinea pig depends on nitric oxide synthase activity of extravillous trophoblast

The trophoblast invasion of uteroplacental arteries in the guinea pig has been studied by means of electron microscopy and immunohistochemisty. To identify trophoblast cells, smooth muscle cells, and endothelial cells, antibodies against cytokeratins, smooth muscle myosin, desmin, and vimentin were employed. Furthermore, the immunohistochemical expression patterns of nitric oxide synthase isoforms (eNOS, mNOS and bNOS) were studied and were compared with the enzyme histochemical staining for NADPH-diaphorase. Dilation of uteroplacental arteries begins prior to day 30, when trophoblast cells that coexpress endothelial and macrophage nitric oxide synthase can be found in the vicinity of the vessels and replace the surrounding peritoneal mesothelium. Trophoblast invasion of the arterial walls and the subsequent wall destruction are only secondary effects. Starting around day 50, the final steps of pregnancy-dependent vessel modifications involve intraarterial trophoblast adhesion to the endothelium and subsequent replacement of the endothelium by the trophoblast cells. These may centrifugally invade the vessel media eventually forming intraluminal plugs. These findings led us to the conclusion that in the guinea pig pregnancy-induced physiological dilation of the uteroplacental arteries is due to the effect of nitric oxide rather than being caused by trophoblast-induced media destruction.Parts of this study were supported by Grant Ka 36017-2 from the Deutsche Forschungsgemeinschaft.     

The eighth component of human complement: molecular basis of C8A (C81) polymorphism

Using an exon-specific polymerase chain reaction (PCR) followed by direct DNA sequence analysis we have analyzed the polymorphism of the -chain of the eighth component of human complement (C8) at the DNA level. We found that two common alleles, C8A^*A and C8A^*B, are characterized by the substitution of a single amino acid (Gln to Lys), which is caused by a point mutation of a single nucleotide (C to A) in exon 3 at position 187 of the mature C8 cDNA sequence. Based on this mutation, an allele-specific PCR was designed detecting the two alleles of C8A. We applied this method to type the C8A polymorphism using DNA samples from a Chinese Han population. The comparison with the data of protein typing of the same samples proved that the described method is efficient and reliable for the identification of C8A genotypes and may be valuable for further application in population studies and forensic science.     

Voluntary anonymous linked study of the prevalence of HIV infection and hepatitis C among inmates in a Canadian federal penitentiary for women.

OBJECTIVE: To determine the seroprevalence of HIV infection and hepatitis C among inmates of a federal penitentiary for women. DESIGN: Voluntary, anonymous, linked, point-prevalence study involving testing of blood samples for antibodies to HIV and hepatitis C virus. PARTICIPANTS: All inmates of the multilevel security federal Prison for Women, Kingston, Ont., who volunteered to participate in the study. Inmates at this long-stay facility are from across Canada. OUTCOME MEASURE: Seroprevalence rate among participants of antibodies to HIV and hepatitis C virus. RESULTS: Of the 130 inmates available for study 113 (86.9%) agreed to donate a blood sample. One woman (0.9%) was HIV positive; 45 (39.8%) were positive for hepatitis C antibody. CONCLUSIONS: It is possible to obtain a high participation rate in a voluntary, anonymous, linked point-prevalence study in a long-stay penitentiary. The HIV seroprevalence rate of 0.9% is lower than that found in studies in provincial (short-stay) prisons. However, the high rate of antibodies to hepatitis C suggests a significant level of risk behaviour, most likely injection drug use, and suggests the potential for a rapid increase in the rate of HIV infection should the number of newly admitted HIV-positive inmates who use injection drugs rise.