Impaired Growth and Force Production in Skeletal Muscles of Young Partially Pancreatectomized Rats: A Model of Adolescent Type 1 Diabetic Myopathy?

This present study investigated the temporal effects of type 1 diabetes mellitus (T1DM) on adolescent skeletal muscle growth, morphology and contractile properties using a 90% partial pancreatecomy (Px) model of the disease. Four week-old male Sprague-Dawley rats were randomly assigned to Px (n = 25) or Sham (n = 24) surgery groups and euthanized at 4 or 8 weeks following an in situ assessment of muscle force production. Compared to Shams, Px were hyperglycemic (>15 mM) and displayed attenuated body mass gains by days 2 and 4, respectively (both P<0.05). Absolute maximal force production of the gastrocnemius plantaris soleus complex (GPS) was 30% and 50% lower in Px vs. Shams at 4 and 8 weeks, respectively (P<0.01). GP mass was 35% lower in Px vs Shams at 4 weeks (1.24±0.06 g vs. 1.93±0.03 g, P<0.05) and 45% lower at 8 weeks (1.57±0.12 vs. 2.80±0.06, P<0.05). GP fiber area was 15–20% lower in Px vs. Shams at 4 weeks in all fiber types. At 8 weeks, GP type I and II fiber areas were ∼25% and 40% less, respectively, in Px vs. Shams (group by fiber type interactions, P<0.05). Phosphorylation states of 4E-BP1 and S6K1 following leucine gavage increased 2.0- and 3.5-fold, respectively, in Shams but not in Px. Px rats also had impaired rates of muscle protein synthesis in the basal state and in response to gavage. Taken together, these data indicate that exposure of growing skeletal muscle to uncontrolled T1DM significantly impairs muscle growth and function largely as a result of impaired protein synthesis in type II fibers.     

Influence of nutrients and currents on the genomic composition of microbes across an upwelling mosaic

Metagenomic data sets were generated from samples collected along a coastal to open ocean transect between Southern California Bight and California Current waters during a seasonal upwelling event, providing an opportunity to examine the impact of episodic pulses of cold nutrient-rich water into surface ocean microbial communities. The data set consists of ∼5.8 million predicted proteins across seven sites, from three different size classes: 0.1–0.8, 0.8–3.0 and 3.0–200.0 μm. Taxonomic and metabolic analyses suggest that sequences from the 0.1–0.8 μm size class correlated with their position along the upwelling mosaic. However, taxonomic profiles of bacteria from the larger size classes (0.8–200 μm) were less constrained by habitat and characterized by an increase in Cyanobacteria, Bacteroidetes, Flavobacteria and double-stranded DNA viral sequences. Functional annotation of transmembrane proteins indicate that sites comprised of organisms with small genomes have an enrichment of transporters with substrate specificities for amino acids, iron and cadmium, whereas organisms with larger genomes have a higher percentage of transporters for ammonium and potassium. Eukaryotic-type glutamine synthetase (GS) II proteins were identified and taxonomically classified as viral, most closely related to the GSII in Mimivirus, suggesting that marine Mimivirus-like particles may have played a role in the transfer of GSII gene functions. Additionally, a Planctomycete bloom was sampled from one upwelling site providing a rare opportunity to assess the genomic composition of a marine Planctomycete population. The significant correlations observed between genomic properties, community structure and nutrient availability provide insights into habitat-driven dynamics among oligotrophic versus upwelled marine waters adjoining each other spatially.     

Skeletal muscle regeneration is delayed by reduction in Xin expression: consequence of impaired satellite cell activation?

Xin is a striated muscle-specific actin-binding protein whose mRNA expression has been observed in damaged skeletal muscle. Here we demonstrate increased Xin protein expression early postinjury (≤ 12 h) and localization primarily to the periphery of damaged myofibers. At 1 day postinjury, Xin is colocalized with MyoD, confirming expression in activated satellite cells (SCs). By 5 days postinjury, Xin is evident in newly regenerated myofibers, with a return to preinjury levels by 14 days of regeneration. To determine whether the increased Xin expression is functionally relevant, tibialis anterior muscles of wild-type mice were infected with Xin-short hairpin RNA (shRNA) adenovirus, whereas the contralateral tibialis anterior received control adenovirus (Control). Four days postinfection, muscles were harvested or injured with cardiotoxin and collected at 3, 5, or 14 days thereafter. When compared with Control, Xin-shRNA infection attenuated muscle regeneration as demonstrated by Myh3 expression and fiber areas. Given the colocalization of Xin and MyoD, we isolated single myofibers from infected muscles to investigate the effect of silencing Xin on SC function. Relative to Control, SC activation, but not proliferation, was significantly impaired in Xin-shRNA-infected muscles. To determine whether Xin affects the G0-G1 transition, cell cycle reentry was assessed on infected C2C12 myoblasts using a methylcellulose assay. No difference in reentry was noted between groups, suggesting that Xin contributes to SC activation by means other than affecting G0-G1 transition. Together these data demonstrate a critical role for Xin in SC activation and reduction in Xin expression results in attenuated skeletal muscle repair.     

Microbiological and chemical properties of soil associated with macropores at different depths in a red-duplex soil in NSW Australia

Some agricultural soils in South Eastern Australia with duplex profiles have subsoils with high bulk density, which may limit root penetration, water uptake and crop yield. In these soils, a large proportion (up to 80%) of plant roots maybe preferentially located within the macropores or in the soil within 1–10 mm of the macropores, a zone defined as the macropore sheath (MPS). The chemical and microbiological properties of MPS soil manually dissected from a 1–3 mm wide region surrounding the macropores was compared with that of adjacent bulk soil (>10 mm from macropores) at 4 soil depths (0–20 cm, 20–40 cm, 40–60 cm and 60–80 cm). Compared to the bulk soil, the MPS soil had higher organic C, total N, bicarbonate-extractable P, Ca⁺, Cu, Fe and Mn and supported higher populations of bacteria, fungi, actinomycetes, Pseudomonas spp., Bacillus spp., cellulolytic bacteria, cellulolytic fungi, nitrifying bacteria and the root pathogen Pythium.In addition, analysis of carbon substrate utilization patterns showed the microbial community associated with the MPS soil to have higher metabolic activity and greater functional diversity than the microbial community associated with the bulk soil at all soil depths. Phospholipid fatty acids associated with bacteria and fungi were also shown to be present in higher relative amounts in the MPS soil compared to the bulk soil. Whilst populations of microbial functional groups in the MPS and the bulk soil declined with increasing soil depth, the differentiation between the two soils in microbiological properties occurred at all soil depths. Soil aggregates (< 0.5 mm diameter) associated with plant roots located within macropores were found to support a microbial community that was quantitatively and functionally different to that in the MPS soil and the bulk soil at all soil depths. The microbial community associated with these soil aggregates thus represented a third recognizable environment for plant roots and microorganisms in the subsoil.     

Dynamics of sediment carbon stocks across intertidal wetland habitats of Moreton Bay,Australia

Coastal wetlands are known for high carbon storage within their sediments, but our understanding of the variation in carbon storage among intertidal habitats, particularly over geomorphological settings and along elevation gradients, is limited. Here, we collected 352 cores from 18 sites across Moreton Bay, Australia. We assessed variation in sediment organic carbon (OC) stocks among different geomorphological settings (wetlands within riverine settings along with those with reduced riverine influence located on tide‐dominated sand islands), across elevation gradients, with distance from shore and among habitat and vegetation types. We used mid‐infrared (MIR) spectroscopy combined with analytical data and partial least squares regression to quantify the carbon content of ~2500 sediment samples and provide fine‐scale spatial coverage of sediment OC stocks to 150 cm depth. We found sites in river deltas had larger OC stocks (175–504 Mg/ha) than those in nonriverine settings (44–271 Mg/ha). Variation in OC stocks among nonriverine sites was high in comparison with riverine and mixed geomorphic settings, with sites closer to riverine outflow from the east and south of Moreton Bay having higher stocks than those located on the sand islands in the northwest of the bay. Sediment OC stocks increased with elevation within nonriverine settings, but not in riverine geomorphic settings. Sediment OC stocks did not differ between mangrove and saltmarsh habitats. OC stocks did, however, differ between dominant species across the research area and within geomorphic settings. At the landscape scale, the coastal wetlands of the South East Queensland catchments (17,792 ha) are comprised of approximately 4,100,000–5,200,000 Mg of sediment OC. Comparatively high variation in OC storage between riverine and nonriverine geomorphic settings indicates that the availability of mineral sediments and terrestrial derived OC may exert a strong influence over OC storage potential across intertidal wetland systems.     

Expression and purification of the angiogenesis inhibitor 16-kDa prolactin fragment from insect cells

The 16-kDa fragment of prolactin (16-kDa PRL), derived from proteolytic cleavage of 23-kDa PRL, was shown to have antiangiogenic activity. Previous studies have shown that recombinant 16-kDa PRL produced from bacteria often contained endotoxins, which are cytotoxic to endothelial cells, and varied in its biological activity due to changes in its refolding from inclusion bodies. These problems limited the use of recombinant 16-kDa PRL. To improve the generation of recombinant 16-kDa PRL, we expressed 16-kDa PRL in Sf9 insect cells using a baculoviral expression system. The signal sequence of the human PRL gene and codons for seven histidines were added to the N- and C-termini, respectively, of the 16-kDa PRL cDNA construct. Recombinant 16-kDa PRL was detected in both the cell pellet and the medium. About 0.28 mg purified protein was isolated from the cell pellet of 4 x 10(7) infected cells using nickel affinity chromatography. Sixteen kilodalton PRL was posttranslationally modified with apparent molecular weights of 16 and 18 kDa on SDS-PAGE. The level of 18-kDa protein was significantly reduced after digestion with peptidyl-N-glycosidase, suggesting that the heterogeneity was due to glycosylation of 16-kDa PRL. N-terminal sequence analysis confirmed the fact that both proteins were human 16-kDa PRL and the signal sequences were cleaved at the same position as that of human PRL. Consistent with its role as an angiogenesis inhibitor, purified recombinant 16-kDa PRL inhibits the proliferation of endothelial cells with a potency similar to that previously reported for the protein generated in Escherichia coli. This 16-kDa PRL expressed in Sf9 cells is a useful reagent for functional studies and for the purification and identification of its receptor.     

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