Plasma S-nitrosothiol status in neonatal calves: ontogenetic associations with tissue-specific S-nitrosylation and nitric oxide synthase

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (<1 sec), its functionality can be prolonged, and in many regards more discretely modulated, when it reacts with low-molecular-weight and protein-bound thiols to form S-nitrosothiols (RSNO), from which NO subsequently can be rereleased. We used the calf as a model to test the hypothesis that plasma concentrations of RSNO are elevated at birth in mammals, correlate with ascorbate and urate levels, are selectively generated in critical tissue beds, and are generated in a manner temporally coincident with changes in tissue levels of active NO synthases (NOS). Plasma concentrations of RSNO, ascorbate, and urate were highest immediately after birth (Day 0), dropped >50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.     

Acute regulation of IGF-I by alterations in post-exercise macronutrients

This investigation sought to examine the contributions of exercise and nutrient replenishment on in vivo regulation of the insulin-like growth factor-I (IGF-I) axis components. Eight college-aged males completed three high-intensity interval training (HIIT) protocols followed by three post-exercise nutritional protocols: (1) placebo (EX); (2) carbohydrate only (CHO); and (3) essential amino acid/carbohydrate (EAA/CHO). Samples were analyzed for growth hormone (GH), free IGF-I, IGFBP-1, IGFBP-2, insulin, hematocrit, hemoglobin, serum leucine, matrix metalloproteinase-9 (MMP-9) proteolytic activity, and presence of IGFBP-3 protease activity. No evidence for IGFBP-3 proteolysis was observed. Significant increases in [free IGF-I] and [leucine] were observed in the EAA/CHO group only. Significant differences were noted in [IGFBP-1] and [IGFBP-2] across conditions. Significant increases in [GH] and MMP-9 activity were observed in all groups. These results indicate that post-exercise macronutrient ratio is a determinant of [free IGF-I], [IGFBP-1 and -2] and may play a role in modulating the IGF-I axis in vivo.     

Isolation and characterization of polymorphic microsatellite loci in muskrat, Ondatra zibethicus

We describe the isolation and characterization of 12 highly polymorphic microsatellite loci for the muskrat, Ondatra zibethicus. Microsatellite markers from three other rodent species were cross-amplified in muskrat and one of them was polymorphic. We observed moderate to high levels of genetic variability in these 13 polymorphic loci (five to 22 alleles per locus) with observed heterozygosity ranging from 0.48 to 0.96. These markers will be useful for further studies on population genetic structure in muskrat and potentially in other rodent species.     

Enhanced mitochondrial complex gene function and reduced liver size may mediate improved feed efficiency of beef cattle during compensatory growth

Growing ruminants under extended dietary restriction exhibit compensatory growth upon ad libitum feeding, which is associated with increased feed efficiency, lower basal energy requirements, and changes in circulating concentrations of metabolic hormones. To identify mechanisms contributing to these physiological changes, 8-month-old steers were fed either ad libitum (control; n = 6) or 60–70% of intake of control animals (feed-restricted; n = 6) for a period of 12 weeks. All steers were fed ad libitum for the remaining 8 weeks of experimentation (realimentation). Liver was biopsied at days −14, +1, and +14 relative to realimentation for gene expression analysis by microarray hybridization. During early realimentation, feed-restricted steers exhibited greater rates of gain and feed efficiency than controls and an increase in expression of genes functioning in cellular metabolism, cholesterol biosynthesis, oxidative phosphorylation, glycolysis, and gluconeogenesis. Gene expression changes during feed restriction were similar to those reported in mice, indicating similar effects of caloric restriction across species. Based on expression of genes involved in cell division and growth and upregulation of genes encoding mitochondrial complex proteins in early realimentation, it was concluded that reduced hepatic size and increased mitochondrial function may contribute to improved feed efficiency observed during compensatory growth.     

Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome

The selective recognition of ubiquitin conjugates by proteasomes is a key step in protein degradation. The receptors that mediate this step have yet to be clearly defined although specific candidates exist. Here we show that the proteasome directly recognizes ubiquitin chains through a specific subunit, Rpn10, and also recognizes chains indirectly through Rad23, a reversibly bound proteasome cofactor. Both binding events can be observed in purified biochemical systems. A block substitution in the chain-binding ubiquitin interacting motif of RPN10 when combined with a null mutation in RAD23 results in a synthetic defect in protein degradation consistent with the view that the direct and indirect recognition modes function to some extent redundantly in vivo. Rad23 and the deubiquitinating enzyme Ubp6 both bind proteasome subunit Rpn1 through N-terminal ubiquitin-like domains. Surprisingly, Rad23 and Ubp6 do not compete with each other for proteasome binding. Thus, Rpn1 may act as a scaffold to assemble on the proteasome multiple proteins that act to either bind or hydrolyze multiubiquitin chains.     

Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail

The medullary bone serves as a source of labile calcium mobilized during calcification of the egg shell in birds. Quantitative histological methods demonstrate that the numbers of medullary bone osteoclasts and nuclei per osteoclast remain unchanged during the egg cycle in the Japanese quail (Coturnix). Therefore, cyclic changes in bone resorption cannot be explained by modulations of osteoclasts from and into other bone cells, a mechanism previously suggested for certain species of birds. Rather, dramatic changes in osteoclast cell-surface features occur during the egg cycle, which might account for cyclic variations in resorptive activity. During egg shell calcification, osteoclasts with ruffled borders are closely apposed to bone surfaces; the cytoplasm is rich in vacuoles that contain mineral crystals and seem to derive from the ruffled border. At the completion of egg shell calcification, the ruffled borders and vacuoles move away from the bone surface, although the osteoclast remains attached to the bone along the filamentous or "clear" zone. Associated with the disappearance of the ruffled borders is the appearance of extensive interdigitated cell processes along the peripheral surface of the osteoclast away from the bone. These unusual structures, which may serve as a reservoir of membrane, largely disappear when ruffled borders and associated structures reappear. Therefore, in these hens, the osteoclasts modulate their cell surface rather than their population during the egg cycle.     

Characterization of calves exhibiting a novel inheritable TNF-alpha hyperresponsiveness to endotoxin: associations with increased pathophysiological complications.

A subpopulation of calves, herein termed "hyperresponders" (HPR), was identified and defined by the patterns of plasma TNF-alpha concentrations that developed following two challenges with endotoxin (LPS, 0.8 mug Escherichia coli 055:B5 LPS/kg(0.75) live body wt) separated by 5 days. The principle characteristic of HPR calves was a failure to develop tolerance to repeated LPS challenge that was evident in the magnitude of the TNF-alpha concentrations and prolonged severity of pathological sequellae. Whereas calves failing to develop LPS tolerance were identified on the basis of their excessive in vivo plasma TNF-alpha concentration responses, in vitro TNF-alpha responses of peripheral blood mononuclear cells isolated from each calf and challenged with LPS or PMA did not correlate or predict the magnitude of in vivo plasma TNF response of the calf. Intentional breeding to obtain calves from bulls and/or cows documented as HPR resulted in offspring displaying the HPR character when similar progeny calves were tested with LPS in vivo, with extensive controls in place to account for sources of variability in the general TNF-alpha response to LPS that might compromise interpretation of the data. Feed intake, clinical serology and hematology profiles, and acute-phase protein responses of HPR calves following LPS were significantly different from those of calves displaying tolerance. These results suggest that the pattern of plasma TNF-alpha changes that evolve from a low-level double LPS challenge effectively reveal the presence of a genetic potential for animals to display excessive or prolonged pathological response to LPS-related stress and compromised prognosis for recovery.     

Effects of sex chromosome dosage on corpus callosum morphology in supernumerary sex chromosome aneuploidies

Background

Supernumerary sex chromosome aneuploidies (sSCA) are characterized by the presence of one or more additional sex chromosomes in an individual’s karyotype; they affect around 1 in 400 individuals. Although there is high variability, each sSCA subtype has a characteristic set of cognitive and physical phenotypes. Here, we investigated the differences in the morphometry of the human corpus callosum (CC) between sex-matched controls 46,XY (N =99), 46,XX (N =93), and six unique sSCA karyotypes: 47,XYY (N =29), 47,XXY (N =58), 48,XXYY (N =20), 47,XXX (N =30), 48,XXXY (N =5), and 49,XXXXY (N =6).

Methods

We investigated CC morphometry using local and global area, local curvature of the CC boundary, and between-landmark distance analysis (BLDA). We hypothesized that CC morphometry would vary differentially along a proposed spectrum of Y:X chromosome ratio with supernumerary Y karyotypes having the largest CC areas and supernumerary X karyotypes having significantly smaller CC areas. To investigate this, we defined an sSCA spectrum based on a descending Y:X karyotype ratio: 47,XYY, 46,XY, 48,XXYY, 47,XXY, 48,XXXY, 49,XXXXY, 46,XX, 47,XXX. We similarly explored the effects of both X and Y chromosome numbers within sex. Results of shape-based metrics were analyzed using permutation tests consisting of 5,000 iterations.

Results

Several subregional areas, local curvature, and BLDs differed between groups.Moderate associations were found between area and curvature in relation to the spectrum and X and Y chromosome counts. BLD was strongly associated with X chromosome count in both male and female groups.

Conclusions

Our results suggest that X- and Y-linked genes have differential effects on CC morphometry. To our knowledge, this is the first study to compare CC morphometry across these extremely rare groups.