beta-adrenergic receptor population is up-regulated by increased cyclic adenosine monophosphate concentration in chicken skeletal muscle cells in culture.

Skeletal muscle hypertrophy is promoted in vivo by administration of beta-adrenergic receptor (betaAR) agonists. Chicken skeletal muscle cells were treated with 1 microM isoproterenol, a strong betaAR agonist, between days 7 and 10 in culture. betaAR population increased by approximately 40% during this treatment; however, the ability of the cells to synthesize cyclic adenosine monophosphate (cAMP) was diminished by twofold. Neither the basal concentration of cAMP nor the quantity of myosin heavy chain (MHC) was affected by the 3-d exposure to isoproterenol. To understand further the relationship between intracellular cAMP levels, betaAR population, and muscle protein accumulation, intracellular cAMP levels were artificially elevated by treatment with 0-10 betaM forskolin for 3 d. The basal concentration of cAMP in forskolin-treated cells increased up to sevenfold in a dose-dependent manner. Increasing concentrations of forskolin also led to an increase in betaAR population, with a maximum increase of approximately 40-60% at 10 microM forskolin. A maximum increase of 40-50% in the quantity of MHC was observed at 0.2 microM forskolin, but higher concentrations of forskolin reduced the quantity of MHC back to control levels. At 0.2 microM forskolin, intracellular levels of cAMP were higher by approximately 35%, and the betaAR population was higher by approximately 30%. Neither the number of muscle nuclei fused into myotubes nor the percentage of nuclei in myotubes was affected by forskolin at any of the concentrations studied.

Effect of electrical stimulation on beta-adrenergic receptor population and cyclic amp production in chicken and rat skeletal muscle cell cultures.

Expression of the beta-adrenergic receptor (betaAR) and its coupling to cyclic AMP (cAMP) synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this study was to determine if electrical stimulation in a pattern simulating slow muscle contraction would alter the betaAR response in primary cultures of avian and mammalian skeletal muscle cells. Specifically, chicken skeletal muscle cells and rat skeletal muscle cells that had been grown for 7 d in culture were subjected to electrical stimulation for an additional 2 d at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. In chicken skeletal muscle cells, the betaAR population was not significantly affected by electrical stimulation; however, the ability of these cells to synthesize cyclic AMP was reduced by approximately one-half. In contrast, the betaAR population in rat muscle cells was increased slightly but not significantly by electrical stimulation, and the ability of these cells to synthesize cyclic AMP was increased by almost twofold. The basal levels of intracellular cyclic AMP in neither rat muscle cells nor chicken muscle cells were affected by electrical stimulation.

Beta-adrenergic receptor gene expression in bovine skeletal muscle cells in culture.

Beta-adrenergic receptors (betaAR) are abundant in fetal, neonatal, and adult skeletal muscles of cattle; however, only minimal levels of functional betaAR were detected in multinucleated muscle cell cultures prepared from 90- to 150-d fetal bovine skeletal muscle. Two other lines of evidence were consistent with low levels of betaAR expression in bovine muscle cultures. First, treating the cells with 10(-6)M isoproterenol for up to 20 min did not increase intracellular cAMP concentration. Second, neither the quantity of myosin heavy chain (MHC) nor its apparent synthesis rate were changed by treating the cells for 4 d with 10(-7) or 10(-6) M isoproterenol. Despite these results, the mRNA for the beta2AR could be detected in muscle cultures by PCR and on slot blots. Thus, the beta2AR mRNA was expressed, but significant levels of functional receptors could not be detected. Glucocorticoids are known to activate expression of OAR genes in several tissues, and the effect of dexamethasone on OAR gene expression in bovine multinucleated muscle cell cultures was evaluated. The intracellular concentration of cAMP following treatment with isoproterenol was elevated 10-fold by dexamethasone, and the population of functional receptors was elevated by approximately 50%. The effect of dexamethasone on muscle protein synthesis and accumulation was analyzed after pretreating the cells with dexamethasone for 24 h, followed by treatment with dexamethasone and 10(-6)M isoproterenol for an additional 48 h. The quantity of MHC synthesized and the apparent synthesis rate of MHC were stimulated by 10 to 35%. These effects seem to be due to posttranscriptional events, because the quantity of beta2AR receptor mRNA on slot blots was not increased by treatment with dexamethasone. Results of this study emphasize the importance of verifying that muscle cells contain functional betaAR when they are used to study the effects of betaAR agonists on muscle protein metabolism.

Tumescent mini abdominoplasty.

The tumescent technique for liposuction has become a widely accepted procedure in the plastic surgical community. We have used this technique as primary anesthesia for a limited abdominoplasty (mini abdominoplasty) in a series of 35 patients over a 2-year period on an outpatient basis. Anesthesia for the procedure consists of tumescent lidocaine solution and minimal sedation with oral Valium or low-dose intravenous Versed. All patients had good hemodynamic stability and tolerated the procedure well. No complications were noted intra- or postoperatively. The tumescent technique provides adequate and safe anesthesia for mini abdominoplasty with supplemental liposuction. The main advantages of the procedure include avoidance of risks associated with general anesthesia, less bleeding, faster recovery, and probably reduced cost of the operation.

Myosin heavy chain gene expression in bovine fetuses and neonates representing genotypes with contrasting patterns of growth.

Fetal and neonatal myosin heavy chain (MHC) gene expression was examined in bovine genotypes that differed in their postnatal growth pattern and mature size. Pregnancies were established that would be expected to produce early-, intermediate-, or late-maturing postnatal growth. Fetal skeletal and cardiac muscles were collected at 100 and 200 days of gestation and at 30 days of age. Muscle tissue was analyzed for relative levels of MHC RNA and protein. Longissimus muscle MHC RNA/microgram RNA was greater at the 100-day time point for the intermediate maturity type (P < 0.05), which differed from the 200-day time point where the early maturity type had the greater RNA level (P < 0.05). Triceps muscle MHC RNA/microgram RNA weights differed due to genotype at 200 days gestation but did not differ at 100 days gestation or at 30 days postnatal. Ventricular muscle MHC RNA did not differ due to genotype at any of the three developmental stages. Differences due to maturity type in MHC protein/mg DNA were observed at 30 days of age, but no differences due to maturity type were observed at the prenatal time points. These results indicate that bovine fetal skeletal muscle MHC RNA production can be influenced by genotype and that genotype may be an important factor for future studies examining the role of external influences on fetal muscle growth.

Growth and development of bovine fetuses and neonates representing three genotypes.

Growth was examined in bovine fetuses and neonates that typically differ in mature size and postnatal developmental pattern. Pregnancies were established from matings expected to produce early (E), late (L), and intermediate (I) maturing postnatal growth patterns. Tissues were collected at 100 and 200 d of gestation and 30 d postnatal. Muscle:body weight ratios were lower at 100 and 200 d for the E maturity type than for the L maturity type (P < .05). This differs from observations of muscle:body weight ratios made at 30 d postnatal, at which time ratios for E were either greater than (triceps brachii, P < .05) or similar to those for L. Few differences due to maturity type were observed at 100 d for bone weight:body weight ratios; however, at 200 d of gestation E bone weight:body weight ratios were generally lower (P < .05) than those for L. The genotypic relationship for bone weight:body weight ratio at 30 d postnatal was similar to that observed at 200 d of gestation. Observations of organ weight:body weight ratios revealed no clear patterns due to maturity type. The genotypic relationship for total muscle DNA content was similar to that observed for muscle weight. These results indicate that fetal muscle development differs in cattle that have different postnatal growth patterns by as early as 100 d of gestation and that differences in fetal muscle growth are related to differences in muscle hyperplasia.

Expression pattern and partial sequence analysis of a fetal bovine myosin heavy-chain gene.

A fragment of a bovine myosin heavy-chain (MHC) gene approximately 15 kbp in size (designated MHC 67) was isolated from a bovine genomic DNA library. The direction of transcription was determined, and preliminary experiments indicated that the gene was expressed in fetal skeletal muscle. The expression pattern of this gene was, therefore, evaluated in detail using northern blots containing RNA from eleven different bovine muscle and nonmuscle tissues at three developmental ages. A restriction fragment of clone MHC 67 containing the 3' untranslated sequence (which is specific for each MHC gene) was used as a probe. This gene fragment hybridized predominantly to RNA from fetal skeletal muscles and did not hybridize to RNA from either neonatal or adult skeletal muscles (red or white), smooth muscle tissue, or nonmuscle tissue. A 7-kb EcoRI fragment containing both translated and untranslated regions surrounding the 3' end of the gene was subcloned into pBluescript II KS+ and partially sequenced. When these bovine sequences were aligned to that of the human and rat skeletal and cardiac MHC genes, we found that these sequences corresponded to exons 31, 32, and 33, and that they had homology with human perinatal and fetal MHC as high as 90% at the nucleotide level and 97% at the amino acid level. Comparison of the nucleotide sequences of isoform-specific 3' nontranslated regions from bovine, human, and rat genes further verify that the MHC 67 clone encodes the bovine fetal or perinatal MHC isoform.

Altered blood pressure reactivity in adolescent diabetics.

OBJECTIVE: We examined hemodynamic responses to a variety of physiologic stimuli in 14 normotensive adolescents with type I diabetes and 45 healthy controls to determine whether structural vascular changes contribute to a reduced vasodilator capacity in adolescent diabetics. We asked, in adolescents with type I diabetes: (1) Are structural vascular changes present? (2) Are changes in the systemic vascular bed reflected in abnormal blood pressure regulation? and (3) Is abnormal vascular reactivity associated with either diabetes duration or control? METHODOLOGY: Diabetic subjects were outpatients treated at the Medical College of Virginia, ages 13 to 18 years. Diabetes duration averaged 7.5 years. Each subject underwent an echocardiogram, dynamic and isometric exercise testing, and forearm plethysmography. RESULTS: Compared to controls, diabetic subjects had (1) higher systolic and diastolic blood pressure during dynamic and handgrip exercise, (2) decreased forearm vasodilator capacity in response to ischemia, and (3) an increased aortic peak velocity. Group diastolic filling abnormalities were found, but these did not persist after adjustment for heart rate. The following variables were related to both diabetes duration and control (average glycosylated hemoglobin): (1) diastolic blood pressure during dynamic exercise, (2) resting forearm vascular resistance, and (3) forearm vascular reactivity. In addition, diabetes duration correlated with isometric exercise diastolic blood pressure, and diabetes control correlated with resting diastolic blood pressure. CONCLUSION: In young diabetics we found that (1) abnormalities of the resistance vessels of the forearm may be present, (2) the degree of vascular change is related to diabetes duration and control, and (3) aortic distensibility may be impaired.

Diseases and disorders of muscle.

Muscle may suffer from a number of diseases or disorders, some being fatal to humans and animals. Their management or treatment depends on correct diagnosis. Although no single method may be used to identify all diseases, recognition depends on the following diagnostic procedures: (1) history and clinical examination, (2) blood biochemistry, (3) electromyography, (4) muscle biopsy, (5) nuclear magnetic resonance, (6) measurement of muscle cross-sectional area, (7) tests of muscle function, (8) provocation tests, and (9) studies on protein turnover. One or all of these procedures may prove helpful in diagnosis, but even then identification of the disorder may not be possible. Nevertheless, each of these procedures can provide useful information. Among the most common diseases in muscle are the muscular dystrophies, in which the newly identified muscle protein dystrophin is either absent or present at less than normal amounts in both Duchenne and Becker's muscular dystrophy. Although the identification of dystrophin represents a major breakthrough, treatment has not progressed to the experimental stage. Other major diseases of muscle include the inflammatory myopathies and neuropathies. Atrophy and hypertrophy of muscle and the relationship of aging, exercise, and fatigue all add to our understanding of the behavior of normal and abnormal muscle. Some other interesting related diseases and disorders of muscle include myasthenia gravis, muscular dysgenesis, and myclonus. Disorders of energy metabolism include those caused by abnormal glycolysis (Von Gierke's, Pompe's, Cori-Forbes, Andersen's, McArdle's, Hers', and Tauri's diseases) and by the acquired diseases of glycolysis (disorders of mitochondrial oxidation). Still other diseases associated with abnormal energy metabolism include lipid-related disorders (carnitine and carnitine palmitoyl-transferase deficiencies) and myotonic syndromes (myotonia congenita, paramyotonia congenita, hypokalemic and hyperkalemic periodic paralysis, and malignant hyperexia). Diseases of the connective tissues discussed include those of nutritional origin (scurvy, lathyrism, starvation, and protein deficiency), the genetic diseases (dermatosparaxis, Ehlers-Danlos syndrome, osteogenesis imperfecta, Marfan syndrome, homocystinuria, alcaptonuria, epidermolysis bullosa, rheumatoid arthritis in humans, polyarthritis in swine, Aleutian disease of mink, and the several types of systemic lupus erythematosus) and the acquired diseases of connective tissues (abnormal calcification, systemic sclerosis, interstitial lung disease, hepatic fibrosis, and carcinomas of the connective tissues). Several of the diseases of connective tissues may prove to be useful models for determining the relationship of collagen to meat tenderness and its other physical properties. Several other promising models for studying the nutrition-related disorders and the quality-related characteristics of meat are also reviewed.

Protein metabolism in chicken muscle cell cultures treated with cimaterol.

Primary muscle cell cultures were prepared from the leg muscle of 12-d broiler chicken embryos. The partitioning agent cimaterol (10(-6) to 10(-10) M) was added on d 1 and each day thereafter, and cells were studied after 7 d in culture. Cimaterol had no effect at any level either on the percentage of nuclei within multinucleated myotubes or on the total number of nuclei within myotubes. At 10(-7) M cimaterol, the quantity of the myofibrillar protein fraction was increased by 25.1 +/- 8.0% (P less than .05) and the quantity of myosin heavy chain was increased by 30.9 +/- 4.5% (P less than .05). To understand the basis for the increase in myofibrillar protein, the incorporation rate of [3H]Leu was measured in pulse labeling experiments. The apparent synthesis rate of the soluble protein fraction and the crude myofibrillar fraction was not significantly increased by cimaterol; however, cimaterol levels greater than 10(-8) M caused a 10 to 12% increase (P less than .05) in the incorporation rate of [3H]Leu into myosin heavy chain. The effect of cimaterol on release of [3H]Leu from prelabeled protein also was assessed in pulse-chase experiments; the apparent rate of protein degradation was inhibited by 10 to 15% (P less than .05) at the higher levels of cimaterol. Dot blot analysis indicated that the quantity of myosin heavy chain mRNA was elevated in cimaterol-treated cultures. Thus, the increased quantity of myofibrillar proteins in embryonic broiler muscle cell cultures is the combined result of a stimulation in the rate of protein synthesis and an inhibition in the rate of protein degradation.

Effect of insulin on phosphorus metabolism in red blood cells from patients with schizophrenia and psychotic depressions.

In an exploratory study of the metabolism of selected intermediates of glycolysis in rbc of patients with schizophrenia and with major depressive disorders, statistically significant decreases in the RSA of 2,3-DPG were found in the rbc of patients with psychotic disorders compared to normal subjects. No statistically significant differences were observed in the RSA of any of the other glycolytic intermediates studied comparing patients to normal subjects.

Genomic clones encoding chicken myosin heavy-chain genes.

A chicken genomic library was screened with a cDNA probe containing the 3' coding and noncoding portions of quail fast-twitch skeletal muscle myosin heavy chain (MHC). This probe hybridized to seven to nine bands on Southern blots of chicken genomic DNA, and 17 clones that hybridized to this probe were obtained from the genomic library. Partial restriction maps were constructed and probable orientation of transcription was determined for each of the 17 clones. These maps indicate the presence of at least 14 unique MHC genes or pseudogenes. Dot-blot hybridization analysis using DNA complementary to RNA from a variety of chicken tissues demonstrated that these genes are all related to the gene for sarcomeric MHC, and permitted tentative assignment of the tissue of expression for several of the MHC isoforms. To substantiate further the dot-blot data, a subclone of one of the genes (4b1), which showed significant homology with adult breast muscle RNA but which also showed weaker hybridization to RNA from other tissues, was sequenced. The sequence data verified that the clone contains a portion of a MHC gene, that it contains both 3' coding and noncoding regions, and that its predicted amino acid sequence is identical (with 96% nucleotide homology) to that of the 75-bp quail fast MHC cDNA clone published by Hastings and Emerson (1982). Thus, clone 4b1 contains a portion of one of the genes that is expressed in adult chicken breast skeletal muscle tissue.

Screening of a bovine genomic library for myosin heavy-chain genes.

Restriction enzyme digests of bovine genomic DNA were hybridized against a .37-kilobase (kb) quail embryonic myosin heavy-chain (MHC) copy deoxyribonucleic acid (cDNA) probe; containing both translated and nontranslated regions surrounding the 3' end of the gene. These experiments revealed seven to eight different bands of hybridization, indicative of multiple genes of MHC in the bovine genome. Additionally, a bovine genomic recombinant DNA library was screened with the .37-kb probe. Of the 10(6) phage screened, 11 clones containing portions of the MHC genome were identified, and four were selected for further analyses. Characterization of these four clones was carried out by constructing partial restriction enzyme maps of the inserts using six restriction enzymes singly or in combination. Orientation of the inserts with respect to the arms of the vector and with respect to direction of transcription was determined by hybridizing the DNA fragments against either the .37-kb Pst 1 fragment of pcC128 or to the .23-kb Pst I fragment of pcC128. The .23-kb fragment is located upstream from the .37-kg fragment and contains only coding sequence. Therefore, the differential hybridization pattern of these two probes provided a means for determining the probable direction of transcription. These data provide evidence for a myosin multigene family in cattle, as well as illustrating that the organization of these genes around the 3' end is unique for each of the genes analyzed.

Structural analysis of myosin genes using recombinant DNA techniques.

Myosin, the major protein of the myofibril, consists of two heavy chains with a molecular weight (MW) of 200,000, complexed with four light chains of MW 17,000 to 21,000. Both the heavy and light chains exhibit polymorphisms that are tissue-specific and developmental stage-specific. Myosin heavy chains and light chains appear to be represented in the genome as multigene families in various species, including chickens, cattle, humans, rats, rabbits and nematodes. Myosin heavy-chain proteins have a high amino acid sequence homology among isoforms, and the genes for each isoform likewise exhibit a high degree of nucleotide sequence conservation. The myosin heavy-chain genes have a complex structure and contain up to 65% intervening sequence composed of up to 20 or more individual introns. Partial sequence data, transcriptional orientation and tissue of expression have been determined for several myosin heavy-chain genes. The use of recombinant DNA and associated techniques will eventually yield definitive information on the control of expression of each individual gene, as well as factors that regulate expression of closely related isoforms.

Premature adrenarche. Clinical and diagnostic features.

Premature adrenarche (PA) or isolated growth of sexual hair in young children, is a benign condition that may initially be confused with true precocious puberty or pathologic virilizing disorders. The clinical findings, family history, and serum concentrations of dehydroepiandrosterone sulfate (DHEA-S) were compared in 24 children with PA (79% black females) seen in a 2-year period and in an age-matched control group of 17 black females. Twenty three of 24 patients, but none of the controls, had an adult-type axillary odor. There was a positive family history of PA in only three of 24 children with PA, and in one of 17 controls. The mean serum DHEA-S was significantly higher in the PA children than in the controls, but there was a broad range of concentrations (10-143 micrograms/dl), with values in 10 of 24 cases falling within the control range for age. We conclude that: (1) PA is a relatively common finding in black females between ages 3 and 8, (2) an axillary odor is almost always present in children with PA, and (3) determination of serum DHEA-S may be of some help in confirming the clinical impression of a modest increase in adrenal androgen secretion and in ruling out a more serious disorder. In most cases, however, the diagnosis of PA can be made on the basis of the history, physical examination, and lack of rapid progression over time; the use of laboratory tests to rule out a pathologic virilizing process may best be reserved for those children with very early onset, increased linear growth, or other signs of virilization.

Calcium and phosphate metabolism in children with idiopathic hypoparathyroidism or pseudohypoparathyroidism: effects of 1,25-dihydroxyvitamin D3.

Two children with congenital hypoparathyroidism and two children with pseudohypoparathyroidism were given maintenance doses of 15 to 45 ng/kg/day 1,25-dihydroxyvitamin D3 for a total of 255 months. The urinary calcium excretion showed an upward elevation after the first 2 years of treatment but was not significantly higher than that in 10 normal control subjects. The renal threshold for phosphate excretion stayed within the normal ranges compared with control values. Two hypercalcemic and two hypocalcemic episodes occurred during this period of treatment. Hypercalcemia was reversed within 1 week after withdrawal of 1,25-dihydroxyvitamin D3. Hypocalcemia was countered by increasing the dose of 1,25-dihydroxyvitamin D3. Renal functions were not adversely affected, as estimated by creatinine clearance and reciprocals of serum creatinine concentrations. The mean serum calcium concentration during 1,25-dihydroxyvitamin D3 treatment was significantly higher (P = 0.001) compared with that obtained during vitamin D2 treatment at a dose of 500 to 3000 IU/kg/day. These data provide additional support for the long-term use of 1,25-dihydroxyvitamin D3 in idiopathic hypoparathyroidism and pseudohypoparathyroidism.

Elevated ammonia release from dystrophic chicken muscle cell and fibroblast cultures.

The activity of adenosine monophosphate (AMP)-aminohydrolase, the major NH3-producing enzyme in skeletal muscle, was approximately 35% lower in 7-day dystrophic muscle cell cultures than in normal muscle cell cultures. However, the release rate of NH3 from dystrophic muscle cells was 45% higher than that from normal muscle cells. The reasons for this apparent discrepancy are not clear. To determine indirectly if deamination of amino acids from protein degradation contributed to NH3 release, cells were incubated with 100 micrograms/ml of the protease inhibitor, leupeptin. Leupeptin reduced the rate of NH3 release by only 18.8% in normal muscle cells and 16% in dystrophic muscle cells. The release of NH3 was also higher from dystrophic chicken fibroblast cultures.

Myosin heavy chain concentration, synthesis rate and degradation rate in normal and dystrophic chicken muscle cells in culture.

Myosin heavy chain concentrations, synthesis rates and degradation rates were studied in muscle cell cultures prepared from the breast muscle of 13-day normal (white leghorn) and dystrophic (line 307) chicken embryos. Muscle cells were studied after 7 days in culture, at which time they had reached a steady state with respect to myofibrillar protein synthesis and degradation. The quantity of myosin heavy chain was 10.5 +/- 0.9 micrograms/culture (n = 32) in normal cells and 8.10 +/- 1.2 micrograms/culture (n = 31) in dystrophic cells. However, the myosin heavy chain synthesis rate was 23 500 molecules/min/nucleus in normal cells and 39 900 molecules/min/nucleus in dystrophic cells, as determined by pulse labeling with [3H]leucine and measurement of the specific radioactivity of tRNA precursor pools. Myosin heavy chain half-lives rates were calculated to be 30.6 h in normal cells and 15.6 h in dystrophic cells when corrections were made for reutilization of [3H]leucine. Thus, dystrophic muscle cultures accumulate less myosin heavy chain, despite their faster synthesis rate, because of faster degradation of myosin heavy chain.