Differential expression of pathogen-responsive genes encoding two types of glycine-rich proteins in barley

This trial assessed whether behavioral treatment improves outcome during a 26-week outpatient opioid detoxification. Thirty-nine opioid-dependent adults were assigned randomly to a buprenorphine dose-taper combined with either behavioral or standard treatment. Behavioral treatment included (a) a voucher incentive program for providing opioid-free urine samples and engaging in verifiable therapeutic activities and (b) the community reinforcement approach, a multicomponent behavioral treatment. Standard treatment included lifestyle counseling. Fifty-three percent of the patients receiving behavioral treatment completed treatment, versus 20% receiving standard treatment. The percentage of patients achieving 4, 8, 12, and 16 weeks of continuous opioid abstinence were 68, 47, 26, and 11 for the behavioral group and 55, 15, 5, and 0 for the standard group, respectively. Behavioral treatment improved outcomes during outpatient detoxification.     

Functional morphology of serially linked skeletal muscle fibers

In the skeletal muscle fiber organization of many vertebrate muscles, serial arrangements or linkages of muscle fibers along the muscle or fascicle are commonly found. These serially linked muscle fibers employ distinct junctional morphologies from muscle to muscle. Notable are the end-to-end linkages of muscle fibers through tendinous intersections (TIs), where many fibers end onto a continuous connective tissue plate with folded terminations similar to myotendinous junctions. Besides this end-to-end linkage, overlapping linkages or arrangements occur among nonspanning fibers terminating intrafascicularly. These nonspanning fibers bear tapering terminations with direct cell-cell (myomuscular) junctions or without any specialized junctions. Despite their overlapping linkages or tapering profiles, nonspanning fibers maintain a uniform sarcomere length along the linked fibers, suggesting that the overlapping-linked nonspanning fibers are equivalent to the end-to-end linked fibers in their mechanical capacity. However, the junctional compliance could differ in their extracellular elastic components and their organization at junctional sites, e.g., direct mechanical (myomuscular) junctions vs. indirect linkages through connective tissue. Increasing evidence suggests that the elastic components, including muscle fibers as well as connective tissues, are more critical than previously thought for the mode and/or the efficiency of tension transmission among serially arranged fibers and thus for the mechanical properties of the muscle.     

Origin of genes encoding multi-enzymatic proteins in eukaryotes

In several biosynthetic pathways of eukaryotes, multiple steps are catalyzed by enzymes physically linked as domains of multi-enzymatic proteins. The same steps in prokaryotes are frequently carried out by mono-enzymatic proteins. If genes encoding mono-enzymatic proteins are the precursors to those genes encoding multi-enzymatic proteins, how these genes fused remains an open question. However, the recent discovery of a cleavage-polyadenylation signal within an intron of the GART gene provides clues to this process and might also have more general implications for the origin of genes that contain alternative RNA processing reactions at their 5' or 3' ends.     

Capillary supply of skeletal muscle fibers in untrained and endurance-trained men

Muscle fiber diameters and numbers of capillaries per fiber, per square millimeter, and around each fiber were determined in needle biopsies from the lateral part of the quadriceps muscle of 23 young men. Twelve subjects were untrained (UT) and eleven were endurance-trained (ET) athletes. Average values for maximal oxygen uptake were 51.3 (UT) and 72.0 ml/kg-min (ET). Mean fiber diameters were not significantly different in the two groups (48.8 and 49.1 micron). The capillaries per fiber ratios were 1.77+/-0.10 and 2.49+/-0.08 (mean+/-SE) in the UT and ET groups, respectively. The numbers of capillaries around each fiber were 4.43+/-0.19 (UT) and 5.87+/-0.18 (ET). The numbers of capillaries per mm2 were 585+/-40 (UT) and 821+/-28 (ET). Fiber diameters were 28% smaller in ultrathin than in fresh-frozen sections from the same biopsies. After correction for this difference, the numbers of capillaries per mm2 were 305 and 425 in the UT and ET, respectively. The capillaries per fiber ratio increased with increasing fiber diameter, but not sufficiently to maintain the number of capillaries per mm2. Fibers containing many mitochondria are surrounded by more capillaries than fibers with few mitochondria.     

Phosphorylation of contractile proteins in heart and skeletal muscle

Contractile performance of cardiac and skeletal muscles may be regulated by cyclic AMP or Ca2+, two second messengers that stimulate the phosphorylation of specific myofibrillar proteins. Cyclic AMP-dependent protein kinase catalyzed the rapid phosphorylation of a single site in the inhibitory subunit of cardiac troponin in vitro and in perfused hearts. Skeletal muscle troponin was not phosphorylated by this enzyme in vivo. Although there was a correlation between cardiac troponin phosphorylation and the positive inotropic response to catecholamines, a biochemical mechanism that could account for a functional relationship between the two processes has not been discovered. Phosphorylation of skeletal muscle myosin was catalyzed by myosin light chain kinase in the presence of Ca2+ and the ubiguitous, multifunctional Ca2+-dependent regulator protein (CDR). The activation of kinase activity appeared to proceed via a trimolecular reaction process in which Ca2+ bound to CDR and the Ca2+.CDR complex then interacted with the enzyme. In rat extensor digitorum longus muscle, a 1 sec tetanic contraction resulted in phosphorylation of myosin light chain with the maximal phosphate incorporated 20 sec after the contraction. The light chain phosphate content declined slowly and correlated to post-tetanic potentiation of isometric twitch tension. Phosphorylation of skeletal muscle myosin may be important in modulating contraction.     

Excitation-calcium release uncoupling in aged single human skeletal muscle fibers

The principal objective of this communication has been to determine the manner in which two tissue culture substrata (plastic dishes and type I collagen gels) modulate the response of adult skin fibroblasts to TGF-beta 1 with respect to hyaluronan (HA) synthesis. Our results indicate that (a) fibroblasts cultured on collagen gels synthesised more HA compared to cells plated at the same density on plastic dishes, (b) this up-regulation in total HA synthesis by collagen-cultured cells was accompanied by an increase in the relative proportion of high molecular mass species of newly synthesised HA, and (c) the specific effect of TGF-beta 1 on HA synthesis was dependent upon the substratum: i.e. TGF-beta 1 inhibited HA synthesis by subconfluent fibroblasts cultured on both substrata, had no apparent effect on confluent cells cultured on collagen gels, and stimulated HA synthesis by confluent cells cultured on plastic dishes. The TGF beta-stimulated of HA synthesis by confluent fibroblasts cultured on plastic dishes persisted when these cells were transferred to collagen gels in the absence of further TGF-beta 1: interestingly, a second exposure of these plastic pre-incubated cells to TGF-beta 1 whilst growing on collagen resulted in a down-regulation in HA synthesis. Confluent fibroblasts pre-incubated with TGF-beta 1 for 24 h on plastic dishes (i.e. under conditions which stimulate HA synthesis) also displayed an HA-dependent stimulation in cell migration when subsequently plated onto collagen gels in the absence of further cytokine.     

Degeneration and regeneration of striated skeletal muscle fibers in Duchenne muscular dystrophy

Like all other muscular dystrophies, Duchenne muscular dystrophy is characterized by the coexistence of degenerative lesions of the muscle fibers and of regenerative changes. The present study has been carried out in order to precise the degree of regeneration at different stages of the disease, by analyzing the expression of several markers of cell proliferation and of muscular differentiation. In the two affected foetuses of our series, the m. quadriceps is histologically normal, except for the absent expression of immunoreactive dystrophin. The quadriceps from the eight children of our series (20 months-16 years) all present clear dystrophic changes. Muscle regeneration is characterized by activation of the satellite cells, by their multiplication followed by their fusion giving birth to regenerative fibers. By studying the expression of muscular markers (vimentin, desmin, isoforms of the myosin heavy chains), it has been possible to define more precisely the degree of maturation and of differentiation of these regenerative fibers. Our results suggest that an abortive regeneration of the muscle fibers in Duchenne muscular dystrophy can explain, at least partly, the progressive evolution of this disease.     

Expression of aquaporin-4 in fast-twitch fibers of mammalian skeletal muscle

OBJECT: Ependymomas in children continue to generate controversy regarding their histological diagnosis and grading. optimal management, and possible prognostic factors. To increase our knowledge of these tumors the authors addressed these issues in a cohort of children with prospectively staged ependymomas treated with radiotherapy and chemotherapy. METHODS: Children between the ages of 2 and 17.3 years harboring an intracranial ependymoma confirmed by a central review of the tumor's pathological characteristics were treated according to Children's Cancer Group Protocol 921 from 1986 to 1992. Treatment following surgery and postoperative tumor staging (including brain computerized tomography or magnetic resonance [MR] imaging, spinal MR imaging or myelography, and cerebrospinal fluid cytological investigation) included craniospinal irradiation with a local boost to the primary tumor and patient randomization to receive adjuvant chemotherapy with either 1) CCNU, vincristine, and prednisone, or 2) the eight-drugs-in-1-day regimen. Centralized review of the tumor pathological characteristics revealed 20 ependymomas and 12 anaplastic ependymomas in the 32 children included in the study. Diagnoses made at the individual institutions included anaplastic (malignant) ependymoma (15 patients), ependymoma (four patients), ependymoblastoma (nine patients), ependymoastrocytoma (one patient), and primitive neuroectodermal tumor (three patients), which were discordant with the centralized review diagnosis in 22 of 32 cases. Only three of the 32 patients had metastatic disease (two with M and one with M3 stages). At surgery, 47% of tumors were estimated to be totally resected. Among the 14 of 17 patients who suffered a relapse and were evaluated for site of relapse, 10 (71%) had an isolated local relapse, three (21%) had concurrent local and metastatic relapse, and only one (7%) had an isolated metastatic relapse. Kaplan-Meier estimates of 5-year progression-free survival (PFS) and overall survival rates were 50 +/- 10% and 64 +/- 9%, respectively. CONCLUSIONS: Predictors of PFS duration included an estimate of the extent of resection made at surgery (total compared with less than total, p = 0.0001) and the amount of residual tumor on postoperative imaging as verified by centralized radiological review (< or = 1.5 cm2 compared with > 1.5 cm2, p < 0.0001). No other factors, including centrally reviewed tumor histopathological type, location, metastasis and tumor (M and T) stages, patient age, race, gender, or chemotherapy treatment regimen significantly correlated with PFS duration. The pattern of predominantly local relapse and the important influence of residual tumor or the extent of resection on PFS duration confirms a prevailing impression that local disease control is the major factor in the prediction of outcome of ependymoma. Survival rates were comparable with those reported by other investigators who have treated patients with similar doses of radiation and no chemotherapy.     

Regulation of synaptic position, size, and strength in anuran skeletal muscle

An analysis of the physiology, morphology, and position of endplates on identified fibers in the Xenopus laevis pectoralis muscle has revealed the following. 1. The percentage of fibers with one endplate is lower in large muscles, and within the same muscle, singly innervated fibers are smaller than dually innervated fibers. 2. Single junctions tend to be stronger than junctions on dually innervated fibers. 3. Single junctions typically are located near the middle of their fibers, while the endplates on dually innervated fibers are located toward either end and usually are separated by at least 20% of the total fiber length. A significant proportion of dually innervated fibers appears to be innervated by the same axon at both junctions. 4. Junctions on the same dually innervated fiber tend to be more similar in length than do junctions on different fibers of the same input resistance. This observation is the same whether both junctions on a given fiber are formed by the same or different axons. There is no corresponding tendency for greater similarity in physiological strength of paired junctions, which frequently show large differences in endplate potential amplitude. 5. The total terminal length on dually innervated fibers of equivalent input resistance is inversely correlated with the mean release per unit length and total release of both junctions. There is no apparent correlation between the distance separating endplates and their strength or length. The data support a model of synaptic regulation in which nerve terminals are attracted, grow, and are maintained in proportion to the amount of a substance supplied by muscle fibers. Our findings suggest that such a substance is produced or distributed uniformly throughout each fiber in amounts proportional to the fiber size and inversely proportional to the total transmitter output of all junctions innervating the fiber. A form of competitive interaction between the terminals which helps to determine synaptic spacing may involve local depletion or inactivation of this substance.     

Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway

Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. One signal-response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably beta-hydroxybutyl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal-response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density-sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two-component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal-response pathways which converge to regulate expression of luminescence in V. harveyi.     

Developmental expression of dystrophin, dystrophin-associated glycoproteins and other membrane cytoskeletal proteins in human skeletal and heart muscle

Dystrophin, utrophin and the dystrophin-associated glycoproteins, beta-dystroglycan and adhalin, were analyzed, together with the membrane cytoskeletal proteins beta-spectrin, vinculin and talin, and adult and fetal myosin heavy chains, in 25 normal human fetuses from 8 to 24 weeks of gestation. Dystrophin was present in heart and skeletal muscle from 8 weeks although in the latter was mainly in the cytoplasm at this stage. Utrophin expression increased until around gestational weeks 19/21, but by 24 weeks immunostaining and immunoblot band intensities had reduced. Beta-dystroglycan was scarce in skeletal muscle at 8 weeks, increased with maturation and was more abundant in heart of the same age. Adhalin appeared later than beta-dystroglycan on skeletal muscle fiber surfaces, positivity became more intense as the fibers matured. In heart adhalin was detectable only in groups of cells at 12-16 weeks. From 8 weeks all fetal myotubes expressed beta-spectrin on their surfaces, while vinculin and talin positivity was mainly at the periphery of the fascicles, increasing with age. Adult slow myosin was seen in most myotubes at 10 weeks. Secondary myotubes then formed which increasingly expressed adult fast myosin, while still retaining fetal myosin. By 24 weeks most fibers expressing adult slow myosin had lost fetal myosin and were more mature in the expression of most membrane proteins. Muscle membrane organization during human fetal development is a complex process and takes place earlier in heart than skeletal muscle.     

Differential expression of rat brain synaptic proteins in development and aging

We have previously reported the differential involvement of synaptic proteins in Alzheimer's disease (AD). As AD is an aging-associated disease, in the present study we examined the developmental and aging-related changes in synaptic proteins such as synaptophysin, synaptobrevin, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), syntaxin 1/HPC-1 and drebrin in the rat brain. Immunoblot analyses of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that the protein level of synaptophysin and synaptobrevin increased after birth, being highest at 24 weeks, and then decreased with aging. Synaptotagmin was detected at E19, with levels increasing after birth to 96 weeks. SNAP-25 levels were highest at 4 weeks, and then decreased with aging. Syntaxin 1/HPC-1 levels were high at E19 and 1 week, decreasing rapidly from 2 weeks onwards, and drebrin levels were highest at E19 and 1 week, and decreased during aging. The present results suggest that the expression of each synaptic protein is differentially regulated in development and aging.     

Bed rest increases the amount of mismatched fibers in human skeletal muscle

The effects of a 37-day period of bed rest on myosin heavy chain (MHC) expression on both mRNA and protein level in human skeletal muscle fibers were studied. Muscle biopsies from vastus lateralis muscle were obtained from seven healthy young male subjects before and after the bed-rest period. Combined in situ hybridization, immunocytochemistry, and ATPase histochemistry analysis of serial sections of the muscle biopsies demonstrated that fibers showing a mismatch between MHC isoforms at the mRNA and protein level increased significantly after the bed-rest period, suggesting an increase in the amount of muscle fibers in a transitional state. Accordingly, fibers showing a match in expression of MHC-1 and of MHC-2A at the mRNA and protein level decreased, whereas fibers showing a match between MHC-2X mRNA and protein increased after bed rest. Overall, there was an increase in fibers in a transitional state from phenotypic type 1 --> 2A and 2A --> 2X. Furthermore, a number of fibers with unusual MHC mRNA and isoprotein combinations were observed after bed rest (e.g., type 1 fibers with only mRNA for 2X and type 1 fibers negative for mRNA for MHC-beta/slow, 2A, and 2X). In contrast, no changes were revealed after an examination at the protein level alone. These data suggest that the reduced load-bearing activity imposed on the skeletal muscles through bed rest will alter MHC gene expression, resulting in combinations of mRNA and MHC isoforms normally not (or only rarely) observed in muscles subjected to load-bearing activity. On the other hand, the present data also show that 37 days of bed rest are not a sufficient stimulus to induce a similar change at the protein level, as was observed at the gene level.     

Functional role of vacuolization of the T-system of skeletal muscle fibers

T-tubules of skeletal muscle fibres easily transform into large vacuoles under the influence of various factors. These include osmotic shock produced by the efflux of small molecular weight molecules (e.g. glycerol), hypertonic shock, muscle fatigue and muscle damage. In most cases, vacuolation is reversible but the molecular mechanisms involved are not clear. Also, the functional role of reversible vacuolation has not been established. However, three possibilities may be considered. (1) Redistribution of ions and water between the cytoplasm and the extracellular space comprised by the T-system. Thus, the formation of large vacuoles may be a mechanisms for rapid osmoregulation that corresponds to regulated volume decrease in other types of cell. However, in our hands, inhibitors of various pathways that participate in volume regulation had no effect on reversible vacuolation. (2) Resealing of mechanical damage of the plasma membrane. This is usually accompanied by the development near the damaged membrane of numerous vacuoles which we have observed by confocal microscopy and use of a hydrophobic dye (RH414), to arise in part from T-tubules. (3) By confocal microscopy, it has also been shown that extracellular fluorescein dextran (Mr = 10,000), and both plasmid DNA (pUC18) and sonicated high molecular weight DNA stained with YOYO, enter vacuoles derived from T-tubules. This finding may indicate that reversible vacuolation, in the absence of membrane damage, could provide a pathway from the extracellular environment to the cytoplasm that is additional or complimentary to endocytosis; it may also be particularly relevant to the ability of muscle to be transfected by the direct injection of DNA. These several observations strongly indicate that the function of the T-system in skeletal muscle fibers is not restricted to excitation-contraction coupling.     

The effect of autografting on the myosin composition in skeletal muscle fibers

The purpose of the study was to investigate the changes in myosin heavy chain (MHC) and myosin light chain (MLC) isoforms following autotransplantation of extensor digitorum longus muscles. Muscles were grafted in "standard" and "nerve-intact" conditions. MHC and MLC isoforms were analyzed by sodium dodecyl sulphate gel electrophoresis. Changes in MHC isoforms 10, 30, and 60 days after grafting were similar in the "standard" and the "nerve-intact" grafts. In contrast to MHC, changes in MLC were different in the 10th day groups, but the same in the 30th day groups. Sixty days after grafting the content of MLC isoforms was the same as the control muscles. These data indicate that transient loss of functional innervation, even for a short time, has permanent effect on the composition of MHC but not MLC isoforms in regenerating skeletal muscle fibers.     

Differential titin isoform expression in human skeletal muscle

Mammalian skeletal muscle expresses at least two isoforms of the cytoskeletal protein titin (connectin; MW approximately 3000 kDa). These isoforms are associated with different passive force curves, and thus may affect physical performance. To study the distribution of titin and its possible influence on performance in humans, muscle biopsies were obtained from 15 males (mean +/- SE; age = 25.4 +/- 2.9 years, height = 177.7 +/- 1.8 cm, weight = 76.5 +/- 2.2 kg). Two biopsies were obtained on separate occasions from both the right and left vastus lateralis, and one biopsy each from the lateral head of the right gastrocnemius and the right soleus, with all biopsies handled identically. Fibre type analyses were performed via mATPase histochemistry. Expression of titin and myosin heavy chain isoforms were determined by SDS-PAGE. Titin bands in the resulting gels were highly repeatable and were verified by migration patterns, as well as Western blot analysis. Two groups of subjects were identified: group 1 (n = 10) expressed only one titin isoform (titin-1) in all biopsies, and group 2 (n = 5) expressed two titin isoforms (titin-1 and titin-2) in all biopsies. No significant differences (P > 0.05) between groups were observed for percentage fibre types, percentage fibre type areas, fibre type cross-sectional areas, and percentage myosin heavy chain expression when comparing individual muscles, sampling times or bilateral comparisons. This is the first report of differential titin isoform expression in healthy, mature human skeletal muscle, but it is not clear why this occurs or what influence this may have on performance.     

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.