Verocytotoxin Escherichia coli-Associated Haemolytic Uraemic Syndrome.

Aims To describe laboratory data on clinical human Verotoxigenic E. coli (VTEC) strains causing haemolytic uraemic syndrome (HUS) and to characterise the VTEC strains, thus contributing to risk mitigation to decrease HUS incidence in Ireland. Methods Laboratory characterisation was performed on isolates from 52 VTEC-associated HUS cases identified in the National clinical VTEC Reference Laboratory (NRL-VTEC) for the years 2012-2014. Data were analysed with respect to age, gender, serogroup and verotoxin type and subtype. Results 52/83 (62.6%) culture positive HUS cases were identified from laboratory data; 30 (57.7%) cases occurred in females. Seven HUS-associated serogroups and eleven patterns of verotoxin subtypes are described. Conclusion Ireland has the highest incidence of VTEC infection in Europe and a variety of VTEC serogroups causing clinical infection, suggesting any viable VTEC may potentially cause HUS. A broad diagnostic approach, to detect uncommon serotypes, should be considered when analysing clinical and food samples for VTEC.

Fine-mapping of genes determining extrafusal fiber properties in murine soleus muscle.

Muscle fiber cross-sectional area (CSA) and proportion of different fiber types are important determinants of muscle function and overall metabolism. Genetic variation plays a substantial role in phenotypic variation of these traits; however, the underlying genes remain poorly understood. This study aimed to map quantitative trait loci (QTL) affecting differences in soleus muscle fiber traits between the LG/J and SM/J mouse strains. Fiber number, CSA, and proportion of oxidative type I fibers were assessed in the soleus of 334 genotyped female and male mice of the F34 generation of advanced intercross lines (AIL) derived from the LG/J and SM/J strains. To increase the QTL detection power, these data were combined with 94 soleus samples from the F2 intercross of the same strains. Transcriptome of the soleus muscle of LG/J and SM/J females was analyzed by microarray. Genome-wide association analysis mapped four QTL (genome-wide P < 0.05) affecting the properties of muscle fibers to chromosome 2, 3, 4, and 11. A 1.5-LOD QTL support interval ranged between 2.36 and 4.67 Mb. On the basis of the genomic sequence information and functional and transcriptome data, we identified candidate genes for each of these QTL. The combination of analyses in F2 and F34 AIL populations with transcriptome and genomic sequence data in the parental strains is an effective strategy for refining QTL and nomination of the candidate genes.

Divergent physiological characteristics and responses to endurance training among inbred mouse strains.

Both baseline values and adaptive changes in mice can vary depending on the genetic background. We aimed to assess variation in a battery of variables and their adaptations to endurance training in six inbred mouse strains. Males, n = 184, from A/J, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, and PWD/PhJ strains were assigned to a control or an endurance group (5 weeks swimming exercise). Enzyme activity, histology of soleus (SOL) muscle, swimming endurance, cardiac ventricular and hind limb muscle weight, and femur length were examined. Endurance capacity, morphological and histological variables, and enzyme activity substantially differed among strains. For example, SOL weight was twofold higher and cross-sectional area (CSA) of fibers was ≈ 30% greater in C57BL/6J than in PWD/PhJ strain. The CSA of type 1 fibers were larger than type 2A in PWD/PhJ (P < 0.01); however, the reverse was true in DBA/2J and BALB/cByJ strains (P < 0.05). Swimming endurance in DBA/2J strain was ≈ 9 times better than in BALB/cByJ. Endurance training increased the activity of citrate synthase in gastrocnemius across strains (P < 0.01), however, changes in endurance were strain-specific; the C57BL/6J and DBA/2J strains improved substantially, whereas A/J and BALB/cByJ strains did not. In conclusion, genetic background is a potent determinant of the physiological characteristics and adaptations to training in mice.

The vasopressin Avpr1b receptor: molecular and pharmacological studies.

The distribution, pharmacology and function of the arginine vasopressin (Avp) 1b receptor subtype (Avpr1b) has proved more challenging to investigate compared to other members of the Avp receptor family. Avp is increasingly recognised as an important modulator of the hypothalamic-pituitary-adrenal (HPA) axis, an action mediated by the Avpr1b present on anterior pituitary corticotrophs. The Avpr1b is also expressed in some peripheral tissues including pancreas and adrenal, and in the hippocampus (HIP), paraventricular nucleus and olfactory bulb of the rodent brain where its function is unknown. The central distribution of Avpr1bs is far more restricted than that of the Avpr1a, the main Avp receptor subtype found in the brain. Whether Avpr1b expression in rodent tissues is dependent on differences in the length of microsatellite dinucleotide repeats present in the 5' promoter region of the Avpr1b gene remains to be determined. One difficulty of functional studies on the Avpr1b, especially its involvement in the HPA axis response to stress, which prompted the generation of Avpr1b knockout (KO) mouse models, was the shortage of commercially available Avpr1b ligands, particularly antagonists. Research on mice lacking functional Avpr1bs has highlighted behavioural deficits in social memory and aggression. The Avpr1b KO also appears to be an excellent model to study the contribution of the Avpr1b in the HPA axis response to acute and perhaps some chronic (repeated) stressors where corticotrophin-releasing hormone and other genes involved in the HPA axis response to stress do not appear to compensate for the loss of the Avpr1b.

Time-resolved molecular dynamics of single and double hydrogen migration in ethanol.

Being the lightest, most mobile atom that exists, hydrogen plays an important role in the chemistry of hydrocarbons, proteins and peptides and most biomolecules. Hydrogen can undergo transfer, exchange and migration processes, having considerable impact on the chemical behavior of these molecules. Although much has been learned about reaction dynamics involving one hydrogen atom, less is known about those processes where two or more hydrogen atoms participate. Here we show that single and double hydrogen migrations occurring in ethanol cations and dications take place within a few hundred fs to ps, using a 3D imaging and laser pump-probe technique. For double hydrogen migration, the hydrogens are not correlated, with the second hydrogen migration promoting the breakup of the C-O bond. The probability of double hydrogen migration is quite significant, suggesting that double hydrogen migration plays a more important role than generally assumed. The conclusions are supported by state-of-the-art molecular dynamics calculations.

Attenuated stress response to acute lipopolysaccharide challenge and ethanol administration in vasopressin V1b receptor knockout mice.

The arginine vasopressin (Avp) 1b receptor (Avpr1b) present on anterior pituitary corticotrophs is involved in the stimulation of adrenocorticotrophic hormone (ACTH) secretion, especially during times of stress. Corticotrophin-releasing hormone (CRH) is considered the major ACTH secretagogue during acute stress whereas Avp appears to be the more dominant mediator of the hypothalamic-pituitary-adrenal (HPA) axis response during chronic stress situations. To investigate the role of the Avpr1b in the HPA axis response to acute stress, we measured ACTH and corticosterone (CORT) plasma levels in Avpr1b knockout (KO) mice and wild-type controls in response to bacterial lipopolysaccharide (LPS) challenge and ethanol (EtOH) administration. Mice deficient in Avpr1b had markedly compromised plasma ACTH and CORT responses to acute (30 min) LPS, but normal ACTH and CORT response to more extended exposure (4 h) to the immune system activator. The plasma ACTH and CORT levels stimulated by intoxicating, sedative doses of EtOH (3.2 and 4 g/kg) were significantly decreased in the Avpr1b KO mice compared to wild-type littermates. Significantly higher EtOH-induced plasma ACTH and CORT secretion was measured in female than in male Avpr1b wild-type mice. There were no differences in the blood alcohol levels following acute EtOH administration in Avpr1b KO or wild-type mice of either gender. Our results clearly suggest that Avpr1b plays a significant role in the HPA axis response to acute immune stress and EtOH intoxication.

Biased T-cell receptor delta element recombination in scid thymocytes.

Thymocytes in mutant mice with severe combined immunodeficiency (scid thymocytes) show ongoing recombination of some T-cell receptor delta gene elements, generating signal joints quantitatively and qualitatively indistinguishable from those in wild-type fetal thymocytes. Excised D delta 2-J delta 1 and D delta 1-D delta 2 rearrangements are detectable at levels equivalent to or greater than those in thymocytes from wild-type mice on fetal day 15. Signal junctional modification, shown here to occur frequently in wild-type adult but not newborn excised D delta 2-J delta 1 junctions, can occur normally in adult scid thymocytes. Excised D delta 1-D delta 2 scid junctions, similar to wild-type thymocytes, include pseudonormal coding junctions as well as signal junctions. Inversional D delta 1-D delta 2 rearrangements, generating conventional hybrid junctions, are also reproducibly detectable in scid thymus DNA. These hybrids, unlike those reported for artificial recombination constructs, do not show extensive nucleotide loss. In contrast to the normal or high incidences of D delta 1-, D delta 2-, and J delta 1-associated signal junctions in scid thymocytes, V delta 1, V gamma 3, and V gamma 1.2 signal products are undetectable in scid thymocytes or are detectable at levels at least 10-fold lower than the levels in wild-type fetal thymocytes. These findings confirm biased T-cell receptor element recombination by V(D)J recombinase activity of nontransformed scid thymocytes and indicate that analysis of in vivo-mediated gene rearrangements is important for full understanding of how the scid mutation arrests lymphocyte development.

Starvation-sensitive UCP 3 protein expression in thymus and spleen mitochondria.

To date, UCP 3 has only been associated with skeletal muscle and brown adipose tissue (BAT). Using RT-PCR/PCR methodology, we show that human spleen and human thymus contain UCP 3. In addition, using peptide antibodies, previously demonstrated to be selective for UCP 3, we show that UCP 3 protein is present in mitochondria isolated from rat thymus and mitochondria isolated from reticulocytes, monocytes and lymphocytes of rat spleen. UCP 3 protein expression is also starvation-sensitive. UCP 3 abundance is augmented in mitochondria isolated from thymus and mitochondria isolated from lymphocytes of the spleen from fasted rats when compared to fed controls. The results are consistent with a role for UCP 3 in developing lymphocytes, thymus atrophy and fatty acid utilisation in spleen and thymus.

Distribution of mRNA encoding B78/apj, the rat homologue of the human APJ receptor, and its endogenous ligand apelin in brain and peripheral tissues.

The human APJ receptor is a G protein-coupled receptor which functions as an efficient alternative co-receptor for a number of human immunodeficiency virus type 1 and simian immunodeficiency virus strains. We have cloned the rat APJ receptor, which we term B78/apj, and have mapped the mRNA distribution of both the receptor and its natural ligand apelin in rat tissues. Northern blot analysis showed a similar pattern of expression for B78/apj and apelin mRNAs with hybridising transcripts seen in the lung, heart, skeletal muscle, kidney, brain and liver. In situ hybridisation histochemistry studies revealed intense B78/apj gene expression in the parenchyma of the lung, a sub-population of glomeruli in the kidney, the corpora lutea of the ovary and isolated cells of the anterior lobe of the pituitary. B78/apj mRNA had a striking and unique distribution within the central nervous system (CNS) where receptor expression was found in cells within the meninges around the brain, in the posterior magnocellular and medial parvocellular areas of the hypothalamic paraventricular nucleus and in the supraoptic nucleus. This hypothalamic distribution offers a possible specific role of this receptor in mediating neuroendocrine responses in the CNS.

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation.

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.

Classification and nomenclature of somatostatin receptors.

There is considerable controversy about the classification and nomenclature of somatostatin receptors. To date, five distinct receptor genes have been cloned and named chronologically according to their respective publication dates, but two were unfortunately given the same appellation (SSTR4). Consensually, a nomenclature for the recombinant receptors has been agreed according to IUPHAR guidelines (sst1, sst2, sst3, sst4, and sst5). However, a more informative classification is to be preferred for the future, employing all classification criteria in an integrated scheme. It is already apparent that the five recombinant receptors fall into two classes or groups, on the basis of not only structure but also pharmacological characteristics. One class (already referred to by some as SRIF1) appears to comprise sst2, sst3 and sst5 receptor subtypes. The other class (SRIF2) appears to comprise the other two recombinant receptor subtypes (sst1 and sst4). This promising approach is discussed but it is acknowledged that much more data from endogenous receptors in whole tissues are needed before further recommendations on somatostatin receptor nomenclature can be made.

T cell leakiness in scid mice.

FACS analysis showed that the incidence of leaky T cells increases with age, such that virtually all old scid mice (greater than 1 year) contain detectable CD3+ cells. The number of detectable T cells remained very low; individual old scid mice generally contained less than 10(5) CD3+ cells. When CD3+ populations in individual leaky mice were analyzed for expression of the T cell subset markers, CD4 and CD8, the ratios of CD4/CD8 were found to be markedly skewed relative to normal mice. This suggested the presence of very few T cell clones. Indeed, the analysis of TCR gene rearrangements in polyclonally stimulated T cell cultures revealed only 1-5 clones in the pooled spleen and lymph nodes of individual old scid mice. These studies also indicated that TCR gene rearrangements in the majority of the stimulated T cell cultures did not contain abnormal J-associated deletions that are characteristic of antigen receptor genes of scid lymphomas. Four of five alloreactive T cell clones from leaky scid mice also apparently lacked abnormal J-associated deletions in their rearranged TCR alleles. Therefore, most leaky lymphocytes appear to derive from progenitors with normal or near-normal scid recombinase activity. However, one of five leaky T cell clones (S1233) and one Con A stimulated monoclonal culture (8706) contained both normally and abnormally rearranged TCR genes. The configuration of TCR loci in such clones may reflect the ability of the defective scid recombinase to mediate normal rearrangements at a low frequency.

Magnaporthe grisea pathogenicity genes obtained through insertional mutagenesis.

We have initiated a mutational analysis of pathogenicity in the rice blast fungus, Magnaporthe grisea, in which hygromycin-resistant transformants, most generated by restriction enzyme-mediated integration (REMI), were screened for the ability to infect plants. A rapid primary infection assay facilitated screening of 5,538 transformants. Twenty-seven mutants were obtained that showed a reproducible pathogenicity defect, and 18 of these contained mutations that cosegregated with the hygromycin resistance marker. Analysis of eight mutants has resulted in the cloning of seven PTH genes that play a role in pathogenicity on barley, weeping lovegrass, and rice. Two independent mutants identified the same gene, PTH2, suggesting nonrandom insertion of the transforming DNA. These first 7 cloned PTH genes are described.

Widespread distribution of somatostatin receptor messenger ribonucleic acids in rat pituitary.

The expression of five somatostatin receptor subtypes, rsstr1-5, was examined in rat pituitary by in situ hybridization histochemistry. The anterior lobe of the pituitary expressed mRNA encoding all five rsstr subtypes. Relatively high levels of rsstr3 mRNA expression were also observed in the intermediate lobe of the pituitary. If all five rsstr proteins are expressed in the pituitary, the effects of somatostatin and somatostatin-28 on pituitary function may therefore represent the composite activation of more than one sstr. Co-localization studies on the same pituitary sections revealed a widespread distribution of rsstr mRNA in the major endocrine cell groups. Somatotrophs showed a relatively high level of rsstr4 and -5 mRNA expression while thyrotrophs predominantly expressed rsstr2 mRNA. These data may point to the potential roles for sstr subtypes in mediating SRIF physiology in the pituitary.

The localization of messenger ribonucleic acids for somatostatin receptors 1, 2, and 3 in rat testis.

Somatostatin (SRIF) exerts multiple inhibitory actions throughout the body by binding to specific SRIF receptors (sst). In recent years, five subtypes of SRIF receptors (sst1-5) have been cloned. In this study, 35S-labeled complementary RNA probes were used for in situ hybridization to localize the sst1-5 messenger RNAs (mRNAs) in the rat testis and examine the changes in their distribution during the cycle of the seminiferous epithelium. We found that sst 1-3 mRNAs were visualized in rat testes and were mainly localized within the seminiferous tubules. The signal for sst3 mRNA was also found in interstitial cells. sst4 and 5 mRNAs were not detected in rat testes with the method used in this study. In Sertoli cells, the most intense labeling for sst1 and 3 mRNAs was in stages IV-VII of the cycle of the seminiferous epithelium, which coincided with the lowest labeling intensity for sst2. In germ cells, sst1-3 mRNAs showed similar patterns of distribution. In these cells, sst1-3 mRNA was not observed at the early steps of spermatogenesis. Positive signals for sst1-3 mRNAs were first apparent in the pachytene spermatocytes at stage VII and last until stage XII and in the diplotene spermatocyte at stage XIII. Positive signals for sst1-3 were also detected in round spermatids at stages I-VIII. Labeling of spermatids dramatically decreased at stage IX, when these cells began their elongating changes. The presence of three sst in testis suggests that SRIF may play an essential role in testicular function.

Distribution of V1a and V2 vasopressin receptor messenger ribonucleic acids in rat liver, kidney, pituitary and brain.

The hepatic, vascular-type (V1aR) and the renal, antidiuretic-type (V2R) vasopressin receptor cDNAs were recently cloned from rat liver and kidney libraries, respectively. DNA fragments containing the region encoding the putative 5/6 transmembrane loops of these receptors were subcloned, separately, into RNA polymerase promoter-containing vectors from which 35S-labeled sense and antisense riboprobes were synthesized. In situ hybridization histochemistry showed high levels of V1aR transcripts in the liver and the renal medulla among the vascular bundles. Sparser labeling was found in the renal cortex, but there were no grains over the glomeruli. V1aR mRNA was detected in many brain areas, including the hippocampal formation, central amygdala, dorsolateral septum, lateral hypothalamus, suprachiasmatic, ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus, nucleus of the solitary tract, cerebellum, spinal nucleus of the trigeminal tract, reticular formation, inferior olivary nucleus, and choroid plexus. Rare labeled cells were seen along the periphery of the posterior pituitary. V2R transcripts were not detected in the liver or brain, but were present in high amounts in the inner and outer renal medulla, primarily associated with collecting ducts. Sparser labeling was found in the renal cortex, and no grains were seen over the glomeruli. These data confirm the expression of the V1a vasopressin receptor in liver and brain and demonstrate that kidney expresses mRNAs encoding V1a and V2 vasopressin receptors.

Pharmacological characterization and region-specific expression in brain of the beta 2- and beta 3-subunits of the rat GABAA receptor.

The cDNA for a third beta-subunit of the rat GABAA receptor has been cloned using another beta-subunit, which we had previously cloned [(1989) FEBS Lett. 246, 145-148], as a probe. The approximately 8-kb cDNA for this beta-subunit (termed beta 2) encodes a protein of 474 amino acid residues that shares approximately 80% sequence identity with the rat and bovine beta 1- and beta 3-subunits. Coexpression of the cloned beta-subunit cDNA with the alpha 1-subunit cDNA of the rat GABAA receptor in Xenopus oocytes produced a functional receptor and Cl- channel with pharmacological characteristics of a GABAA receptor. In contrast to interchanging alpha-subunits [(1988) Nature 335, 76-79], exchange of beta 2- or beta 3-subunits in an alpha 1/beta receptor complex did not markedly alter the pharmacological properties of expressed receptors. In situ hybridization histochemistry with synthetic subunit-specific oligo-deoxynucleotide probes revealed a region-specific expression of alpha 1-, beta 2- and beta 3-subunit mRNAs in the rat central nervous system. These observations provide an additional molecular basis for the functional heterogeneity in the GABAA receptor complex.

Multiple GABAA receptor alpha subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain.

GABAA receptor alpha subunit transcripts were detected by Northern analysis of rat, chicken and human brain mRNA using a series of 32P-labelled antisense RNA probes derived from human alpha 1 subunit cDNAs. These alpha subunit mRNAs differ in their distribution among various brain regions in the rat and at least one species is detected primarily in fetal brain. GABAA receptor alpha 1 subunit probes encoding the putative extracellular domain detect at least five alpha subunit transcripts in rat brain, whereas probes encoding the putative intracellular domain detect only two mRNAs. These data suggest the presence in brain of multiple GABAA receptor alpha subunits having homologous extracellular domains and whose expression is regionally and developmentally regulated. These alpha subunit transcripts may encode proteins that comprise GABAA isoreceptors differing in their pharmacological and physiological properties.

Cloning and expression of a novel rat GABAA receptor.

Two full-length cDNA clones encoding alpha- and beta-subunits of a GABAA receptor have been isolated from a rat cerebral cortex cDNA library. The mature alpha-subunit protein consists of 428 amino acids with a calculated Mr of 48,680. This protein is highly homologous (approximately 99% amino acid identity) with the bovine brain alpha 1-subunit receptor [(1988) Nature 335, 76-79]. The mature rat beta-subunit receptor is a 448 amino acid polypeptide and shares approximately 80% amino acid identity with the previously characterized bovine GABAA receptor beta-subunit [(1987) Nature 328, 221-227]. Co-expression of the cloned DNA in Xenopus oocytes produces a functional receptor and ion channel with pharmacological characteristics of a GABAA receptor. GABAA alpha- and beta-subunit mRNA is detectable in the cortex, cerebellum and hippocampus.