Construction of a safe, stable, efficacious vaccine against Photobacterium damselae ssp. piscicida

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.     

Streptozotocin induces G2 arrest in skeletal muscle myoblasts and impairs muscle growth in vivo

Streptozotocin (STZ) is used extensively to induce pancreatic -cell death and ultimately diabetes mellitus in animal models. However, the direct effects of STZ on muscle are largely unknown. To delineate the effects of STZ from the effects of hypoinsulinemia/hyperglycemia, we injected young rats with 1) saline (control), 2) STZ (120 mg/kg) or 3) STZ and insulin (STZ-INS; to maintain euglycemia). STZ rats demonstrated significantly elevated blood glucose throughout the 48-h protocol, while control and STZ-INS rats were euglycemic. Body mass increased in control (13 ± 4 g), decreased by 19 ± 2 g in STZ and remained unchanged in STZ-INS rats (–0.3 ± 2 g). Cross-sectional areas of gastrocnemius muscle fibers were smaller in STZ vs. control (1,480 ± 149 vs. 1,870 ± 40 µm², respectively; P < 0.05) and insulin treatment did not rescue this defect (STZ-INS: 1,476 ± 143 µm²). Western blot analysis revealed a detectable increase in ubiquitinated proteins in the STZ skeletal muscles compared with control and STZ-INS. To further define the effects of STZ on skeletal muscle, independent of hyperglycemia, myoblasts were exposed to varying doses of STZ (0.25–3.0mg/ml) in vitro. Both acute and chronic exposures of STZ significantly impaired proliferative capacity in a dose-dependent manner. Within STZ-treated myoblasts, increased reactive oxygen species was associated with significant G₂/M phase cell-cycle arrest. Taken together, our findings show that the effects of STZ are not -cell specific and reveal that STZ should not be used for studies examining diabetic myopathy. satellite cell; diabetes; diabetic model; type 1 diabetes mellitus; cell cycle; proliferation; hypertrophy     

Urinary Sex Steroids and Anthropometric Markers of Puberty - A Novel Approach to Characterising Within-Person Changes of Puberty Hormones

Background/Aims

The longitudinal relationships of within-individual hormone and anthropometric changes during puberty have not ever been fully described. The objectives of this study were to demonstrate that 3 monthly urine collection was feasible in young adolescents and to utilise liquid chromatography-tandem mass spectrometry assay methods for serum and urine testosterone (T), estradiol (E₂) and luteinizing hormone (LH) in adolescents by relating temporal changes in urine and serum hormones over 12 months to standard measures of pubertal development.

Methods

A community sample of 104 adolescents (57 female) was studied over 12 months with annual anthropometric assessment, blood sampling and self-rated Tanner staging and urine collected every 3 months. Serum and urine sex steroids (T, E₂) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LH by immunoassay.

Results

A high proportion (92%) of scheduled samples were obtained with low attrition rate of 6.7% over the 12 months. Urine hormone measurements correlated cross-sectionally and longitudinally with age, anthropometry and Tanner stage.

Conclusion

We have developed a feasible and valid sampling methodology and measurements for puberty hormones in urine, which allows a sampling frequency by which individual pubertal progression in adolescents can be described in depth.     

Beta-PrP form of human prion protein stimulates production of monoclonal antibodies to epitope 91-110 that recognise native PrPSc

Prion diseases are associated with accumulation of strain-dependent biochemically distinct, disease-related isoforms (PrP(Sc)) of host-encoded prion protein (PrP(C)). PrP(Sc) is characterised by increased beta-sheet content, detergent insolubility and protease resistance. Recombinant alpha-PrP adopts a PrP(C)-like conformation, while beta-PrP conformationally resembles PrP(Sc), to these we raised 81 monoclonal antibodies in Prnp(0/0) mice. The N-terminal residues 91-110 are highly immunogenic in beta-PrP-immunised mice and of (17/41) anti-beta-PrP antibodies that could be epitope-mapped, approximately 70%, recognised this segment. In contrast, only 3/40 anti-alpha-PrP antibodies could be mapped and none interacted with this region, instead recognising residues 131-150, 141-160 and 171-190. Native PrP(C) was recognised by both antibody groups, but only anti-beta-PrP antibodies directed to 91-110 residues recognised native PrP(Sc) with high affinity, where in addition, species heterogeneity was also evident. Within the six anti-beta-PrP antibodies studied, they all recognised PK-treated native human and mouse PrP(Sc), four failed to recognise PK-treated native bovine PrP(Sc), one of which also did not recognise native PK-treated ovine PrP(Sc), showing the epitope becomes exposed on unfolding and disaggregation. These results demonstrate strain-dependent variations in chain conformation and packing within the 91-110 region of PrP(Sc).     

Homer modulates NFAT-dependent signaling during muscle differentiation

While changes in intracellular calcium are well known to influence muscle contraction through excitation contraction coupling, little is understood of the calcium signaling events regulating gene expression through the calcineurin/NFAT pathway in muscle. Here, we demonstrate that Ca(+2) released via the inositol trisphosphate receptor (IP3R) increases nuclear entry of NFAT in undifferentiated skeletal myoblasts, but the IP3R Ca(+2) pool in differentiated myotubes promotes nuclear exit of NFAT despite a comparable quantitative change in [Ca(+2)]i. In contrast, Ca(+2) released via ryanodine receptors (RYR) increases NFAT nuclear entry in myotubes. The scaffolding protein Homer, known to interact with both IP3R and RYR, is expressed as part of the myogenic differentiation program and enhances NFAT-dependent signaling by increasing RYR Ca(+2) release. These results demonstrate that differentiated skeletal myotubes employ discrete pools of intracellular calcium to restrain (IP3R pool) or activate (RYR pool) NFAT-dependent signaling, in a manner distinct from undifferentiated myoblasts. The selective expression of Homer proteins contributes to these differentiation-dependent features of calcium signaling.     

Molecular phylogeny of the springhare, Pedetes capensis, based on mitochondrial DNA sequences

The phylogenetic position of the Pedetidae, represented by a single species Pedetes capensis, is controversial, reflecting in part the retention of both Hystricomorphous and Sciurognathous characteristics in this rodent. In an attempt to clarify the species evolutionary relationships, mtDNA gene sequences from 10 rodent species (representing seven families) were analyzed using phenetic, parsimony, and maximum-likelihood methods of phylogenetic inference; the rabbit, Oryctolagus cuniculus (Order Lagomorpha), and cow, Bos taurus (Order Artiodactyla), were used as outgroups. Investigation of 714 base pairs of the protein-coding cytochrome b gene indicate strong base bias at the third codon position with significant rate heterogeneity evident between the three structural domains of this gene. Similar analyses conducted on 816 base pairs of the 12S rRNA gene revealed a transversion bias in the loop sections of all taxa. The cytochrome b gene sequences proved useful in resolving associations between closely related species but failed to produce consistent tree topologies at the family level. In contrast, phylogenetic analysis of the 12S rRNA gene resulted in strong support for the clustering of Pedetidae/Heteromyidae/Geomyidae and Muridae in one clade to the exclusion of the Hystricidae/Thryonomyidae and Sciuridae, a finding which is concordant with studies of rodent fetal membranes as well as reproductive and other anatomical features.