Immunocytochemical localization of aldolase in normal, denervated, and dystrophic chicken muscles

To investigate whether immunocytochemical localization of muscle-specific aldolase can be used for fiber phenotype determination, we produced specific antibodies against the enzyme and studied its distribution in adult chicken skeletal muscles by indirect immunofluorescence microscopy. Monoclonal antibodies against the myosin heavy chains of fast-twitch (MF-14) and slow-tonic (ALD-58) muscle fibers were also used to correlate aldolase levels with the fiber phenotype. The goat anti-aldolase antibody was found to be specific for the A form of aldolase, as evidenced by sodium dodecyl sulfate gel electrophoresis, immunotitration experiments, and immunoblot analysis. The antibody reacted strongly with the fast-twitch myofibers of normal pectoralis and posterior latissimus dorsi muscles; the phenotype of these muscle fibers was confirmed by a positive immunofluorescent reaction after incubation with MF-14 antibody. By contrast, the slow-tonic myofibers of normal anterior latissimus dorsi, which react positively with ALD-58 antibody, reacted weakly with anti-aldolase antibodies. In denervated chicken muscles, reaction to anti-aldolase antibodies was markedly reduced in fast-twitch fibers, although reaction to MF-14 was not diminished. By contrast, in dystrophic muscle, fast-twitch fibers showed reduced reactivity to anti-aldolase and marked to moderate reduction in MF-14 reactivity. Our results show that: (a) in normal muscles, reactivity to anti-aldolase matches the phenotype obtained by using anti-fast or anti-slow myosin heavy chain antibodies, and therefore can serve to identify mature fibers as fast or slow; and (b) in denervated or dystrophic muscles, the intracellular expressions of aldolase and fast-twitch myosin heavy chains are regulated independently.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Myosin heavy chain expression during development and following denervation of fast fibers in the red strip of the chicken pectoralis

The myosin heavy chain composition of muscle fibers that comprise the red strip of the pectoralis major was determined at different stages of development and following adult denervation. Using a library of characterized monoclonal antibodies we found that slow fibers of the red strip do not react with antibodies to any of the fast myosin heavy chains of the superficial pectoralis. Immunocytochemical analysis of the fast fibers of the adult red strip revealed that they contain the embryonic fast myosin heavy chain rather than the adult pectoral isoform found throughout the adult white pectoralis. This was confirmed using immunoblot analysis of myosin heavy chain peptide maps. We show that during development of the red strip both neonatal and adult myosin heavy chains appear transiently, but then disappear during maturation. Furthermore, while the fibers of the superficial pectoralis reexpress the neonatal isoform as a result of denervation, the fibers of the red strip reexpress the adult isoform. Our data demonstrate a new developmental program of fast myosin heavy chain expression in the chicken and suggest that the heterogeneity of myosin heavy chain expression in adult fast fibers results from repression of specific isoforms by innervation.     

Vinculin in subsarcolemmal densities in chicken skeletal muscle: localization and relationship to intracellular and extracellular structures

Using immunocytochemical methods we have studied the distribution of vinculin in the anterior and posterior latissimus dorsi skeletal (ALD and PLD, respectively) muscles of the adult chicken. The ALD muscle is made up of both tonic (85%) and twitch (15%) myofibers, and the PLD muscle is made up entirely of twitch myofibers. In indirect immunofluorescence, antivinculin antibodies stained specific regions adjacent to the sarcolemma of the ALD and PLD muscles. In the central and myotendinous regions of the ALD, staining of the tonic fibers was intense all around the fiber periphery. Staining of the twitch fibers of both ALD and PLD muscles was intense only at neuromuscular junctions and myotendinous regions. Electron microscopy revealed subsarcolemmal, electron-dense plaques associated with the membrane only in those regions where vinculin was localized by immunofluorescence. Using antivinculin antibody and protein A conjugated to colloidal gold, we found that the electron-dense subsarcolemmal densities in the tonic fibers of the ALD contain vinculin; no other structures were labeled. The basal lamina overlying the densities appeared to be connected to the sarcolemma by fine, filamentous structures, more enriched at these sites than elsewhere along the muscle fiber. Increased amounts of endomysial connective tissue were often found just outside the basal lamina near the densities. In tonic ALD muscle fibers, the subsarcolemmal densities were present preferentially over the I-bands. In partially contracted ALD muscle, subsarcolemmal densities adjacent to the Z-disk appeared to be connected to that structure by short filaments. We propose that in the ALD muscle, through their association with the extracellular matrix, the densities stabilize the muscle membrane and perhaps assist in force transmission.     

KINETICS OF EXTRACELLULAR RELEASE IN AXENIC ALGAE AND IN MIXED ALGAL-BACTERIAL CULTURES: SIGNIFICANCE IN ESTIMATION OF TOTAL (GROSS) PHYTOPLANKTON EXCRETION RATES1

In axenic Chlorella pyrenoidosa Chick cultures, extracellular release was linear with time, but plateau-type curves were obtained in cultures with added bacteria. Initial rates of excretion were identical in both, systems. Kinetics of extracellular release in axenic Anabaena flos-aquae (Lyng.) Bréb. cultures were more complex than in Chlorella but the initial excretion rates were identical in axenic and mixed algal-bacterial cultures. In lakewater, extracellular release kinetics resemble the pattern in mixed Chlorella-bacteria cultures. An explanation is an initial lag in bacterial utilization of algal extracellular products. As a result, both in situ and in the laboratory, consecutive short, experiments give higher excretion rates than single long incubations. It is suggested that the former are close to total or gross extracellular release rates whereas the latter give net values, detecting only substances not, removed by heterotrophs.     

Patterns of population genetic variation in sympatric chiltoniid amphipods within a calcrete aquifer reveal a dynamic subterranean environment

Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km² region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.     

An approach to optimal wetland mitigation using mathematical programming and geographic information system based wetland function estimation

We developed and tested a quantitative geographic information system (GIS)-based approach for selecting wetland restoration sites. Our approach uses a combination of an existing wetland function evaluation program, a GIS and integer programming methodology with an objective to minimize cost of restoration subject to meet environmental requirements. Investigations were conducted on the formulation to examine the effects of problem size, site ordering for input, and restoration targets. The formulation could be solved for the largest problem size tested of 996 integer variables. The larger the problem, the more time it took to solve. Larger restoration targets usually took more sites and more time to solve. Sorting sites by size was found to lead to inefficient and often unfeasible solutions. Random sorting of sites was found to be the more efficient method of inputting restoration sites into analysis.     

Beverage specific alcohol intake in a population-based study: Evidence for a positive association between pulmonary function and wine intake

Background  

Lung function is a strong predictor of cardiovascular and all-cause mortality. Previous studies suggest that alcohol exposure may be linked to impaired pulmonary function through oxidant-antioxidant mechanisms. Alcohol may be an important source of oxidants; however, wine contains several antioxidants. In this study we analyzed the relation of beverage specific alcohol intake with forced expiratory volume in one second (FEV₁) and forced vital capacity (FVC) in a random sample of 1555 residents of Western New York, USA.     

Comparison of ribotyping and repetitive extragenic palindromic-PCR for identification of fecal Escherichia coli from humans and animals

This report compares the performances of two popular genotypic methods used for tracking the sources of fecal pollution in water, ribotyping and repetitive extragenic palindromic-PCR (rep-PCR). The rep-PCR was more accurate, reproducible, and efficient in associating DNA fingerprints of fecal Escherichia coli with human and animal hosts of origin.