Myosin synthesis in embryonic chicken fibroblasts.

The rate of constitutive myosin synthesis was measured in cultures of replicating embryonic chicken skin fibroblasts by pulse labeling with [3H]leucine. These cells synthesized the 200,000-dalton heavy chain of myosin (MHC) at a rate of 3.2 x 10(3) molecules/cell/min. Additionally, an independent estimate of the MHC synthesis rate needed to maintain a constant level of constitutive MHC/cell was calculated from total protein content, percentage MHC, fibroblast doubling time, and MHC half-life. This calculated rate of approximately 2.9 x 10(3) molecules/cell/min was in close agreement with the measured rate. By comparison, the synthesis rate of myofibrillar MHC in fully activated muscle cell cultures was approximately 2.9 x 10(4) molecules/nucleus/min.

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.

Stimulation of myosin heavy chain synthesis in steady-state muscle cultures by the ionophore, A23187, requires transcription of messenger RNA.

After approximately one week in culture, embryonic chick skeletal muscle cells are at a steady state with respect to myosin heavy chain (MHC) concentration and synthesis rate. Muscle cells normally synthesize MHC at a maximum rate of 2.3 X 10 4 MHC/min/nucleus and contain approximately 3 X 10 7 MHC/nucleus. These cells also contain approximately 3500 copies/nucleus of MHC mRNA associated with polysomes and 1600 copies/nucleus of MHC mRNA localized in the nonpolysomal fraction. To determine if nonpolysomal MHC mRNA in mature muscle cultures could be recruited into active translation complexes when MHC synthesis was stimulated, muscle cultures were treated with the Ca 2+ ionophore, A23187 (0-1 micro M). The MHC synthesis rate was stimulated by 25 to 50% relative to stimulation of the rate of total protein synthesis in the presence of A23187, but this stimulation was blocked when 10 microgram/ml actinomycin D was also present. These results suggest that even though 30% of MHC mRNA is not actively engaged in MHC synthesis in mature muscle cultures, stimulation of MHC synthesis by A23187 results from transcription of new MHC mRNA rather than from utilization of pre-existing mRNA.

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.

Cytogenetics of recurrent abortions.

Chromosome banding studies were carried out on both partners of 37 couples who had had two or more spontaneous abortions. Three patients had chromosome disorders; one was a triple-X female and the other two (one male and one female) were t(13;14) translocation carriers. Review of the literature indicates that the over-all frequency of major chromosome disorders in couples with repeated abortions is 2.6%. About three-fourths of these disorders are reciprocal and Robersonian translocations.

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 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.

Cell differentiation, protein synthesis rate and protein accumulation in muscle cell cultures isolated from embryos of layer and broiler chickens.

Muscle cell cultures were prepared from the leg muscle of 12-d layer and broiler chicken embryos. Cultures were then compared over a 10-d period from their capacity to differentiate into multinucleated myotubes and to synthesize and accumulate protein. Differentiation was qualitatively similar in the two cell types as evidenced by myoblast fusion that occurred rapidly during the first 2 d and remained essentially constant between d 3 and 10. However, several quantitative differences were observed. Even though the number of myotubes per culture was comparable between layers and broilers throughout development, layer muscle cultures usually exhibited a higher percentage fusion and more myonuclei per culture than broiler muscle cultures. Additionally, the nuclear density (i.e., the number of nuclei per myotube segment) was approximately 25% greater in layer cultures than in broilers between d 2 and 10 in culture. The rate of incorporation of 3H-leucine into total protein during pulse labeling experiments was comparable in muscle cultures of layers and broilers; however, broiler muscle cells accumulated approximately 40% more total protein per nucleus between d 6 and 10. Myosin heavy chain synthesis rate was higher in layer than in broiler muscle cultures, but broiler muscle cultures accumulated approximately 30% more myosin heavy chain than layers between d 6 and 10. The half-life of myosin heavy chain was 45 h in layer muscle cultures and 103 h in broiler muscle cultures. Thus, the capacity of broiler cells to accumulate more muscle protein was primarily due to a drastically slower protein breakdown rate.

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.

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.

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.

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.

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.

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.

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.