Biases associated with genotype and sex in prediction of fat-free lean mass and carcass value in hogs

Carcass and live measurements of 165 market hogs that represented seven genotypes were used to investigate genotype and sex biases associated with the prediction of fat-free lean mass (FFLM) and carcass value. Carcass value was determined as the sum of the product of weight of individual cuts and their average unit prices adjusted for slaughter and processing costs. Independent variables used in the prediction equations included carcass measurements, such as optical probe, midline ruler, ribbed carcass measurements, and electromagnetic scanning (EMSCAN), and live animal ultrasonic scanning. The effect of including subpopulation mean values of independent variables in the prediction equations for FFLM and carcass value was also investigated. Genotype and sex biases were found in equations in which midline backfat, ribbed carcass, EMSCAN, and live ultrasonic scanning were used as single technology sets of measurements. The prediction equations generally undervalued genotypes with above-average carcass value. Biases were reduced when measurements of combined technologies and mean adjusted variables were used. The FFLM and carcass value of gilts were underestimated, and they were overestimated of barrows. Equations that combined OP and EMSCAN technologies were the most accurate and least biased for both FFLM and carcass value. Equations that included carcass weight and midline last-rib backfat thickness measurements were the least accurate and most biased. Genotype and sex biases must be considered when predicting FFLM and carcass value.     

Isolation of marine polycyclic aromatic hydrocarbon (PAH)-degrading Cycloclasticus strains from the Gulf of Mexico and comparison of their PAH degradation ability with that of puget sound Cycloclasticus strains

Phenanthrene- and naphthalene-degrading bacteria were isolated from four offshore and nearshore locations in the Gulf of Mexico by using a modified most-probable-number technique. The concentrations of these bacteria ranged from 10(2) to 10(6) cells per ml of wet surficial sediment in mildly contaminated and noncontaminated sediments. A total of 23 strains of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were obtained. Based on partial 16S ribosomal DNA sequences and phenotypic characteristics, these 23 strains are members of the genus Cycloclasticus. Three representatives were chosen for a complete phylogenetic analysis, which confirmed the close relationship of these isolates to type strain Cycloclasticus pugetii PS-1, which was isolated from Puget Sound. PAH substrate utilization tests which included high-molecular-weight PAHs revealed that these isolates had similar, broad substrate ranges which included naphthalene, substituted naphthalenes, phenanthrene, biphenyl, anthracene, acenaphthene, and fluorene. Degradation of pyrene and fluoranthene occurred only when the strains were incubated with phenanthrene. Two distinct partial PAH dioxygenase iron sulfur protein (ISP) gene sequences were PCR amplified from Puget Sound and Gulf of Mexico Cycloclasticus strains. Phylogenetic analyses of these sequences revealed that one ISP type is related to the bph type of ISP sequences, while the other ISP type is related to the nah type of ISP sequences. The predicted ISP amino acid sequences for the Gulf of Mexico and Puget Sound strains are identical, which supports the hypothesis that these geographically separated isolates are closely related phylogentically. Cycloclasticus species appear to be numerically important and widespread PAH-degrading bacteria in both Puget Sound and the Gulf of Mexico.     

Calmodulin binding to myosin light chain kinase begins at substoichiometric Ca2+ concentrations: a small-angle scattering study of binding and conformational transitions

We have used small-angle scattering to study the calcium dependence of the interactions between calmodulin (CaM) and skeletal muscle myosin light chain kinase (MLCK), as well as the conformations of the complexes that form. Scattering data were measured from equimolar mixtures of a functional MLCK and CaM or a mutated CaM (B12QCaM) incompetent to bind Ca2+ in its N-terminal domain, with increasing Ca2+ concentrations. To evaluate differences between CaM-enzyme versus CaM-peptide interactions, similar Ca2+ titration experiments were performed using synthetic peptides based on the CaM-binding sequence from MLCK (MLCK-I). Our data show there are different determinants for CaM binding the isolated peptide sequence compared to CaM binding to the same sequences within the enzyme. For example, binding of either CaM or B12QCaM to the MLCK-I peptide is observed even in the presence of EGTA, whereas binding of CaM to the enzyme requires Ca2+. The peptide studies also show that the conformational collapse of CaM requires both the N and C domains of CaM to be competent for Ca2+ binding as well as interactions with each end of MLCK-I, and it occurs at approximately 2 mol of Ca2+/mol of CaM. We show that CaM binding to the MLCK enzyme begins at substoichiometric concentrations of Ca2+ (< or = 2 mol of Ca2+/mol of CaM), but that the final compact structure of CaM with the enzyme requires saturating Ca2+. In addition, MLCK enzyme does bind to 2Ca2+ x B12QCaM, although this complex is more extended than the complex with native CaM. Our results support the hypothesis that CaM regulation of MLCK involves an initial binding step at less than saturating Ca2+ concentrations and a subsequent activation step at higher Ca2+ concentrations.     

Isolation and characterization of a calpain activator in chicken skeletal muscle

The calpains (E.C. 3.4.22.17) and calpastatin constitute an ubiquitous, intracellular, Ca2+-dependent protease/inhibitor system. This system has been implicated as a principal regulator of myofibrillar protein degradation in both ante-mortem and postmortem muscle. Although proteolytic activity of the calpains is primarily controlled through interaction of calpain and calpastatin, evidence for an activator(s) has been limited and the reported characteristics varied. The function of the activator has not been elucidated. A putative calpain activator has been isolated from the Pectoralis muscle of broiler breeders (Cobb x Cobb). The activator elutes from an ion-exchange column at approximately 200 mM NaCl. Addition of activator increased apparent m-calpain activity to a level demonstrating a fourfold increase in proteolysis. The activator/calpain complex maintains a requirement for Ca2+ for proteolytic activity. Under physiological conditions, presence of the activator negates the ability of calpastatin to inhibit m-calpain. Additionally, the activator alone does not demonstrate proteolytic activity. Effect of the activator is pH-dependent; in a physiological pH range, the activator enhances m-calpain proteolytic activity but at pH less than 6.75 the effect is to inhibit m-calpain. The activator's ability to modulate m-calpain activity and eliminate calpastatin's effect provides a further means of regulating this important enzyme system.     

A High Molecular-Mass Anoxybacillus sp. SK3-4 Amylopullulanase: Characterization and Its Relationship in Carbohydrate Utilization

An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.     

Immunocytochemistry of mucosal changes in patients infected with the intestinal nematode Strongyloides stercoralis

AIM: To investigate the immunopathological changes in duodenal tissues induced by strongyloidiasis and to relate these to degrees of clinical severity. METHODS: Tissues taken from 21 patients showing mild, moderate or severe symptoms of strongyloidiasis, and from non-infected controls, were sectioned and stained immunocytochemically for IgA, secretory component (SC) and HLA-DR. Immunopathology was assessed by changes in numbers, intensity and distribution of stained cells. RESULTS: Parasitised individuals showed villous atrophy and crypt hyperplasia. There was notable infiltration of the lamina propria by IgA positive plasma cells and of the epithelium by intraepithelial lymphocytes. Infection was also associated with increased expression of SC and decreased expression of HLA-DR in epithelial cells. Changes in all parameters correlated with degree of clinical severity. CONCLUSIONS: Profound mucosal changes are induced by strongyloidiasis. Some are analogous to those seen in coeliac disease, but others seem quite unusual. It is likely that these changes are functionally related to the immunopathophysiological consequences of infection seen in patients with severe disease.     

Dominant-negative effect of a mutant cardiac troponin T on cardiac structure and function in transgenic mice

Hypertrophic cardiomyopathy (HCM) is a disease of sarcomeric proteins. The mechanism by which mutant sarcomeric proteins cause HCM is unknown. The leading hypothesis proposes that mutant sarcomeric proteins exert a dominant-negative effect on myocyte structure and function. To test this, we produced transgenic mice expressing low levels of normal or mutant human cardiac troponin T (cTnT). We constructed normal (cTnT-Arg92) and mutant (cTnT-Gln92) transgenes, driven by a murine cTnT promoter, and produced three normal and five mutant transgenic lines, which were identified by PCR and Southern blotting. Expression levels of the transgene proteins, detected using a specific antibody, ranged from 1 to 10% of the total cTnT pool. M-mode and Doppler echocardiography showed normal left ventricular dimensions and systolic function, but diastolic dysfunction in the mutant mice evidenced by a 50% reduction in the E/A ratio of mitral inflow velocities. Histological examination showed cardiac myocyte disarray in the mutant mice, which amounted to 1-15% of the total myocardium, and a twofold increase in the myocardial interstitial collagen content. Thus, the mutant cTnT-Gln92, responsible for human HCM, exerted a dominant-negative effect on cardiac structure and function leading to disarray, increased collagen synthesis, and diastolic dysfunction in transgenic mice.     

Short-term exercise enhances insulin-stimulated GLUT-4 translocation and glucose transport in adipose cells

This investigation examined the effects of short-term exercise training on insulin-stimulated GLUT-4 glucose transporter translocation and glucose transport activity in rat adipose cells. Male Wistar rats were randomly assigned to a sedentary (Sed) or swim training group (Sw, 4 days; final 3 days: 2 x 3 h/day). Adipose cell size decreased significantly but minimally (approximately 20%), whereas total GLUT-4 increased by 30% in Sw vs. Sed rats. Basal 3-O-methyl-D-[14C]glucose transport was reduced by 62%, whereas maximally insulin-stimulated (MIS) glucose transport was increased by 36% in Sw vs. Sed rats. MIS cell surface GLUT-4 photolabeling was 44% higher in the Sw vs. Sed animals, similar to the increases observed in MIS glucose transport activity and total GLUT-4. These results suggest that increases in total GLUT-4 and GLUT-4 translocation to the cell surface contribute to the increase in MIS glucose transport with short-term exercise training. In addition, the results suggest that the exercise training-induced adaptations in glucose transport occur more rapidly than previously thought and with minimal changes in adipose cell size.     

Expression patterns of transmembrane and released forms of neuregulin during spinal cord and neuromuscular synapse development

We mapped the distribution of neuregulin and its transmembrane precursor in developing, embryonic chick and mouse spinal cord. Neuregulin mRNA and protein were expressed in motor and sensory neurons shortly after their birth and levels steadily increased during development. Expression of the neuregulin precursor was highest in motor and sensory neuron cell bodies and axons, while soluble, released neuregulin accumulated along early motor and sensory axons, radial glia, spinal axonal tracts and neuroepithelial cells through associations with heparan sulfate proteoglycans. Neuregulin accumulation in the synaptic basal lamina of neuromuscular junctions occurred significantly later, coincident with a reorganization of muscle extracellular matrix resulting in a relative concentration of heparan sulfate proteoglycans at endplates. These results demonstrate an early axonal presence of neuregulin and its transmembrane precursor at developing synapses and a role for heparan sulfate proteoglycans in regulating the temporal and spatial sites of soluble neuregulin accumulation during development.