Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene.

Antimicrobial susceptibility of canine Clostridium perfringens strains from Switzerland

Fifty Clostridium perfringens strains were isolated from individual dogs with acute diarrhoea that were not given antibiotics. Toxin types and minimal inhibitory concentrations of 15 antibiotics were determined for each of them. All strains harboured the alpha-toxin gene, 12 of them had both the alpha- and entero-toxin gene and 5 had both the alpha- and beta2-toxin gene. Eighteen percent of the isolates showed resistance to tetracycline and 54 % showed decreased susceptibility to metronidazole which is one of the most frequently used antibiotics in the treatment of canine diarrhoea. Apart from that, all isolates were susceptible to the remaining antibiotics tested. These findings lead to the conclusion that despite a general susceptibility to antibiotics in C. perfringens, resistance is developing in isolates from dogs. Therefore, careful identification of the pathogenic agent and antibiotic susceptibility testing should be performed prior to therapy in order to minimise further selection of antibiotic resistance.

Rapid and accurate G M1-gangliosidosis diagnosis using a parentage testing microsatellite

The G M1-gangliosidosis is an autosomal recessive lysosomal storage disease caused by structural defects of the beta-galactosidase gene (GLB1) which lead to a severe phenotypical impairment in homozygous individuals, whereas heterozygous carriers remain clinically normal. Currently employed DNA parentage tests include the analysis of microsatellites, which also have a diagnostic predictive value. The aim of this study was to provide a reliable tool for genotyping the canine GLB1 which can be effectively integrated in parentage testing investigations. For this purpose the association between the GLB1 gene and the AHT K253 microsatellite was analyzed in 30 Alaskan huskies (11 GLB1+/+, 17 GLB1+/- and 2 GLB1-/- dogs). The 143 bp AHT K253 microsatellite allele was identified only in GLB1+/- and GLB1-/- animals and was in strong linkage disequilibrium with the causative mutation for G M1-gangliosidosis, a 19 bp duplication within exon 15 of the GLB1 gene. The results of the present study revealed a 100% concordance between the previous established genotypes and those obtained after the analysis of the AHT K253 microsatellite. Thus, the genotype of the AHT K253 microsatellite, which is routinely determined during dog parentage testing, has a high predictive value for the G M1-gangliosidosis carrier status.

A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), which contain large fragments of genomic DNA, have been successfully used as transgenes to create mouse models of dose-dependent diseases. They are also potentially valuable as transgenes for dominant diseases given that point mutations and/or small rearrangements can be accurately introduced. Here, we describe a new method to introduce small alterations in BACs, which results in the generation of point mutations with high frequency. The method involves homologous recombination between the original BAC and a shuttle vector providing the mutation. Each recombination step is monitored using positive and negative selection markers, which are the Kanamycin-resistance gene, the sacB gene and temperature-sensitive replication, all conferred by the shuttle plasmid. We have used this method to introduce four different point mutations and the insertion of the β-galactosidase gene in a BAC, which has subsequently been used for transgenic animal production.

Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.

We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery significantly enhanced the efficiency of gene transfer. After injection into the brain of adult rats, sustained long-term expression of the transgene was obtained in the absence of detectable pathology. A high proportion of the neurons in the areas surrounding the injection sites of the vector expressed the transduced beta-galactosidase gene. This pattern was invariant in animals sacrificed several months after a single administration of the vector. Transduction occurs by integration of the vector genome, as it was abolished by a single amino acid substitution in the catalytic site of the integrase protein incorporated in the vector. Development of clinically acceptable derivatives of the lentiviral vector may thus enable the sustained delivery of significant amounts of a therapeutic gene product in a wide variety of somatic tissues.

The herpes simplex virus major regulatory protein ICP4 blocks apoptosis induced by the virus or by hyperthermia.

Cells infected with herpes simplex virus 1 (HSV-1) undergo productive or latent infection without exhibiting features characteristic of apoptosis. In this report, we show that HSV-1 induces apoptosis but has evolved a function that blocks apoptosis induced by infection as well as by other means. Specifically, (i) Vero cells infected with a HSV-1 mutant deleted in the regulatory gene alpha 4 (that encodes repressor and transactivating functions), but not those infected with wild-type HSV-1(F), exhibit cytoplasmic blebbing, chromatin condensation, and fragmented DNA detected as a ladder in agarose gels or by labeling free DNA ends with terminal transferase; (ii) Vero cells infected with wild-type HSV-1(F) or cells expressing the alpha 4 gene and infected with the alpha 4- virus did not exhibit apoptosis; (iii) fragmentation of cellular DNA was observed in Vero cells that were mock-infected or infected with the alpha 4- virus and maintained at 39.5 degrees C, but not in cells infected with wild-type virus and maintained at the same temperature. Wild-type strains of HSV-1 with limited extrahuman passages, such as HSV-1 (F), carry a temperature-sensitive lesion in the alpha 4 gene and at 39.5 degrees C only alpha genes are expressed. These results indicate that the product of the alpha 4 gene is able to suppress apoptosis induced by the virus as well by other means.

Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.

The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta-glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI alpha-amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.

Comparison of environmental and genetic factors for Parkinson's disease between Chinese and Caucasians

This review paper compares the differences in prevalence, and environmental and genetic risk factors for Parkinson's disease between Chinese and Caucasian subjects. Comparison of age-specific prevalence between Chinese people and Caucasians suggests that the prevalence is lower in the Chinese ( at least in the past), although the prevalence rate in China appears to be rising. Distinctions in environmental risk factors and genetic factors are discussed. The difference in prevalence may be due to distinctions in environmental and genetic risk factors as well as the complex interaction between these environmental and genetic factors, although discrepancies in methodology for prevalence surveys can also be an explanation. Copyright (C) 2004 S. Karger AG, Basel.

The seeds of lotus japonicus lines transformed with sense, antisense, and sense/antisense galactomannan galactosyltransferase constructs have structually altered galactomannan in their endosperm cell walls

Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1-->6)-alpha-galactose (Gal) substitution of the (1-->4)-beta-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense ("hairpin loop") constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T-1 generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T-1 generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T-2 generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase.

Glycine receptors: A new therapeutic target in pain pathways

Although glycine receptor Cl- channels (GlyRs) have long been known to mediate inhibitory neurotransmission onto spinal nociceptive neurons, their therapeutic potential for peripheral analgesia has received little attention. However, it has been shown that alpha 3-subunit-containing GlyRs are concentrated into regions of the spinal cord dorsal horn where nociceptive afferents terminate. Furthermore, inflammatory mediators specifically inhibit alpha 3-containing GlyRs, and deletion of the murine alpha 3 gene confers insensitivity to chronic inflammatory pain. This strongly implicates GlyRs in the inflammation-mediated disinhibition of centrally projecting nociceptive neurons. Future therapies aimed at specifically increasing current flux through alpha 3-containing GlyRs may prove effective in providing analgesia.

Nickel dependent carcinogenesis and infections: structural and biophysical characterization of NDRG1 and SgSrnR

In prokaryotic organisms, lower eukaryotes and plants, some important biological reactions are catalyzed by nickel-dependent enzymes, making this metal ion essential microelement for their life. On the other hand, excessive concentration of nickel into the cell, or prolonged exposure to nickel compounds, has toxic effects in living organisms. In addition, nickel has been classified by IARC as Group I human carcinogen, because of the correlation between its inhalation and increased incidence of nasal and lung cancers. The aim of this work was to investigate the nickel impact on human health, considering both its direct role on human cells and its indirect effect as essential element for human important bacteria. In humans, nickel induces N-myc downstream regulated gene 1 (NDRG1) expression, recently proposed as new target in cancer therapy. CD, light scattering and ITC were applied on the recombinant full-length protein and its C-terminal intrinsically disordered domain, for studying the NDRG1 structural and functional properties. In particular, the fold and dynamics of the C-terminal region were examined by NMR spectroscopy and site-directed spin labeling coupled to EPR, showing the features of an intrinsically disordered region. In nickel-dependent bacteria, nickel metabolism is strictly regulated, through the activity of different transcription factors. In Streptomyces griseus the expression of two superoxide dismutases (SODs) is antagonistically regulated by nickel thanks to the transcriptional complex SgSrnR/SgSrnQ. The SgSrnR protein was heterologously expressed and its activity as possible nickel sensor studied. DNaseI footprinting and β-galactosidase gene reporter assays revealed that SgSrnR functions as transcriptional activator, prompting the hypothesis of a new model to describe the activity of this complex. In addition, ITC, NMR and X-ray crystallography demonstrated that SgSrnR presents the fold typical of ArsR/SmtB transcription factors and low metal binding affinity, non compatible with a role as a nickel-sensor, function probably played by its partner SgSrnQ.

Molecular mechanisms of chondrocyte-specific expression on the mouse pro$\alpha$1(II) collagen gene

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro$\alpha1$(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a $\beta$-galactosidase reporter gene. A construction containing a 3000-bp promoter and a 3020-bp intron 1 fragment directed high levels of $\beta$-galactosidase expression specifically to chondrocytes. Successive deletions of intron 1 delineated a 48-bp fragment which targeted $\beta$-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. When the Col2a1 promoter was replaced with a minimal $\beta$-globin promoter, the 48-bp intron 1 sequence was still able to target expression of the transgene to chondrocytes, specifically. Therefore a 48-bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression to a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression. Nuclear proteins present selectively in mouse primary chondrocytes and rat chondrosarcoma cells bind to the three putative HMG (High-Mobility-Group) domain protein binding sites in this 48-bp sequence and the chondrocyte-specific proteins likely bind the DNA through minor groove. Together, my results indicate that a 48-bp sequence in Col2a1 intron 1 controls chondrocyte-specific expression in vivo and suggest that chondrocytes contain specific nuclear proteins involved in enhancer activity. ^

Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors

Lima GA, Anhe GF, Giannocco G, Nunes MT, Correa-Giannella ML, Machado UF. Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors. Am J Physiol Endocrinol Metab 296: E132-E138, 2009. First published October 28, 2008; doi: 10.1152/ajpendo.90548.2008.-Skeletal muscle is a target tissue for approaches that can improve insulin sensitivity in insulin-resistant states. In muscles, glucose uptake is performed by the GLUT-4 protein, which is encoded by the SLC2A4 gene. SLC2A4 gene expression increases in response to conditions that improve insulin sensitivity, including chronic exercise. However, since chronic exercise improves insulin sensitivity, the increased SLC2A4 gene expression could not be clearly attributed to the muscle contractile activity per se and/or to the improved insulin sensitivity. The present study was designed to investigate the role of contractile activity per se in the regulation of SLC2A4 gene expression as well as in the participation of the transcriptional factors myocyte enhancer factor 2D (MEF2D), hypoxia inducible factor 1a (HIF-1a), and thyroid hormone receptor-alpha (TR alpha). The performed in vitro protocol excluded the interference of metabolic, hormonal, and neural effects. The results showed that, in response to 10 min of electrically induced contraction of soleus muscle, an early 40% increase in GLUT-4 mRNA (30 min) occurred, with a subsequent 65% increase (120 min) in GLUT-4 protein content. EMSA and supershift assays revealed that the stimulus rapidly increased the binding activity of MEF2D, HIF-1a, and TR alpha into the SLC2A4 gene promoter. Furthermore, chromatin immunoprecipitation assay confirmed, in native nucleosome, that contraction induced an approximate fourfold (P < 0.01) increase in MEF2D and HIF-1a-binding activity. In conclusion, muscle contraction per se enhances SLC2A4 gene expression and that involves MEF2D, HIF-1a, and TR alpha transcription factor activation. This finding reinforces the importance of physical activity to improve glycemic homeostasis independently of other additional insulin sensitizer approaches.

Tamoxifen inhibits transforming growth factor-alpha gene expression in human breast carcinoma samples treated with triiodothyronine

Objectives: To examine the effects of triiodothyronine (T(3)), 17 beta-estradiol (E(2)), and tamoxifen (TAM) on transforming growth factor (TGF)-alpha gene expression in primary breast cancer cell cultures and interactions between the different treatments. Methods and results: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3-mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T(3); dish 3: T(3)+TAM; dish 4: TAM; dish 5: E(2); dish 6: E(2)+TAM. TGF-alpha mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T(3) for 48 h significantly increased TGF-alpha mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-alpha mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. Conclusion: We demonstrate that TGF-alpha mRNA expression is more efficiently upregulated by T(3) than E(2). Concomitant treatment with TAM had a mitigating effect on the T(3) effect, while E(2) induced TGF-alpha upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-alpha, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER alpha and beta; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E(2).. Endocrinol. Invest. 31: 1047-1051, 2008) (c) 2008, Editrice Kurtis