A rapid and simple one step method for isolation of poly(A)+ RNA from cells in monolayer.

A simple method was developed to isolate low abundance hormone receptor poly(A)+ RNA from cells in tissue culture. Adherent cells in tissue culture plates were directly released with proteinase K and solubilized in SDS. Oligo(dT) cellulose was directly added to the lysate to obtain poly(A)+ RNA. Yields and purity of the poly(A)+ RNA were comparable to other more lengthy methods. IGF-I receptor and insulin receptor mRNA could be detected on Northern blot without any degradation.

N-cadherin gene maps to human chromosome 18 and is not linked to the E-cadherin gene.

cDNA clones encoding the human N-cadherin cell adhesion molecule have been isolated from an embryonic muscle library by screening with an oligonucleotide probe complementary to the chick brain sequence and chick brain cDNA probe lambda N2. Comparison of the predicted protein sequences revealed greater than 91% homology between chick brain, mouse brain, and human muscle N-cadherin cDNAs over the 748 amino acids of the mature, processed protein. A single polyadenylation site in the chick clone was also present and duplicated in the human muscle sequence. Immediately 3' of the recognition site in chick a poly(A) tail ensued; however, in human an additional 800 bp of 3' untranslated sequence followed. Northern analysis identified a number of major N-cadherin mRNAs. These were of 5.2, 4.3, and 4.0 kb in C6 glioma, 4.3 and 4.0 kb in human foetal muscle cultures, and 4.3 kb in human embryonic brain and mouse brain with minor bands of 5.2 kb in human muscle and embryonic brain. Southern analysis of a panel of somatic cell hybrids allowed the human N-cadherin gene to be mapped to chromosome 18. This is distinct from the E-cadherin locus on chromosome 16. Therefore, it is likely that the cadherins have evolved from a common precursor gene that has undergone duplication and migration to other chromosomal locations.

Atomic structure of the actin:DNase I complex.

The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease I both in the ATP and ADP forms have been determined by X-ray analysis at an effective resolution of 2.8 A and 3A, respectively. The two structures are very similar. The actin molecule consists of two domains which can be further subdivided into two subdomains. ADP or ATP is located in the cleft between the domains with a calcium ion bound to the beta- or beta- and gamma-phosphates, respectively. The motif of a five-stranded beta sheet consisting of a beta meander and a right handed beta alpha beta unit appears in each domain suggesting that gene duplication might have occurred. These sheets have the same topology as that found in hexokinase.

Sequence similarity of calreticulin with a Ca2(+)-binding protein that co-purifies with an Ins(1,4,5)P3-sensitive Ca2+ store in HL-60 cells.

HL-60 cells possess a 60 kDa Ca2(+)-binding protein that is contained in a discrete subcellular compartment, referred to as calciosomes. Subcellular fractionation studies have suggested that, in HL-60 cells, this intracellular compartment is an Ins(1,4,5)P3-sensitive Ca2+ store. In order to investigate the structural relationship of the 60 kDa Ca2(+)-binding protein of HL-60 cells to other Ca2(+)-binding proteins, we have purified the protein by ammonium sulphate extraction, acid precipitation, and DEAE-cellulose and phenyl-Sepharose column chromatography. The N-terminal sequence of the protein shows 93% identity with rabbit muscle calreticulin, a recently cloned sarcoplasmic reticulum Ca2(+)-binding protein. No amino acid sequence similarity with calsequestrin was found, although the purified protein cross-reacted with anti-calsequestrin antibodies. The calreticulin-related protein of HL-60 cells might play a role as an intravesicular Ca2(+)-binding protein of an Ins(1,4,5)P3-sensitive Ca2+ store.

Plasma, tissue, and urine carnitine concentrations in healthy adult cats and kittens.

Mean carnitine concentrations [( carnitine]) were higher (P less than 0.05) in adult cats than in kittens for skeletal muscle (total and free carnitine), myocardium (free carnitine), and urine (total and free carnitine). The free/total carnitine ratio was lower (P less than 0.05) in kittens than in adults for liver, myocardium, and urine. Carnitine concentrations were similar between genders in kittens, but in adult cats, [carnitine] in plasma (total, free, and esterified carnitine) and liver (total and free carnitine) were higher (P less than 0.05) in female than in male cats. Total and free plasma [carnitine] were correlated to total and free liver [carnitine], respectively. Skeletal muscle [carnitine] was not correlated to plasma [carnitine]. Correlations in [carnitine] between plasma and myocardium, kidney, or urine were inconsistent.

Comparison of plasma, liver, and skeletal muscle carnitine concentrations in cats with idiopathic hepatic lipidosis and in healthy cats.

Concentrations of total, free, and esterified carnitine were determined in plasma, liver, and skeletal muscle from cats with idiopathic hepatic lipidosis and compared with values from healthy cats. The mean concentrations of plasma, liver, and skeletal muscle total carnitine; plasma and skeletal muscle free carnitine; and plasma and liver esterified carnitine were greater (P less than 0.05) in cats with idiopathic hepatic lipidosis than in control cats. The mean for the ratio of free/total carnitine in plasma and liver was lower (P less than 0.05) in cats with idiopathic hepatic lipidosis than in control cats. These data suggest that carnitine deficiency does not contribute to the pathogenesis of feline idiopathic hepatic lipidosis.

Effect of ATP on actin filament stiffness.

Actin is an adenine nucleotide-binding protein and an ATPase. The bound adenine nucleotide stabilizes the protein against denaturation and the ATPase activity, although not required for actin polymerization, affects the kinetics of this assembly Here we provide evidence for another effect of adenine nucleotides. We find that actin filaments made from ATP-containing monomers, the ATPase activity of which hydrolyses ATP to ADP following polymerization, are stiff rods, whereas filaments prepared from ADP-monomers are flexible. ATP exchanges with ADP in such filaments and stiffens them. Because both kinds of actin filaments contain mainly ADP, we suggest the alignment of actin monomers in filaments that have bound and hydrolysed ATP traps them conformationally and stores elastic energy. This energy would be available for release by actin-binding proteins that transduce force or sever actin filaments. These data support earlier proposals that actin is not merely a passive cable, but has an active mechanochemical role in cell function.

Localization of the alpha 1 and alpha 2 subunits of the dihydropyridine receptor and ankyrin in skeletal muscle triads.

We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the dihydropyridine (DHP) receptor and ankyrin in rat skeletal muscle with immunofluorescence and immunogold labeling techniques. All three proteins were concentrated in the triad junction formed between the T-tubules and sarcoplasmic reticulum. The alpha 1 and alpha 2 subunits of the DHP receptor were colocalized in the junctional T-tubule membrane, supporting their proposed association in a functional complex and the possible participation of the alpha 2 subunit in excitation-contraction coupling. Ankyrin label in the triad showed a distribution different from that of the DHP receptor subunits. In addition, ankyrin was found in longitudinally oriented structures outside the triad. Thus, ankyrin might be involved in organizing the triad and in immobilizing integral membrane proteins in T-tubules and the sarcoplasmic reticulum.

Efeito da inatividade física sobre parâmetros bioquímicos e grau de ateromatose aórtica em coelhos / Effect of lack of physical activity of rabbits in biochemical data and aortic atheromatosis

Foi realizada uma experiência durante 80 dias, usando 32 coelhas em fase de crescimento. Os animais foram distribuídos em quatro grupos (A, B, C e D) de oito animais cada. Os animais dos grupos A e C permaneceram soltos, sendo que os do grupo A foram alimentados com raçäo basal e os do grupo C com raçäo aterogênica (raçäo basal + 0,5% de colesterol). Os animais dos grupos B e D permaneceram praticamente imobilizados em gaiolas estreitas e foram alimentados, respectivamente, com raçäo basal e raçäo aterogênica. Os resultados obtidos permitem inferir que a inatividade física tem efeito nocivo no processo ateromatoso. De fato, foram observados aumentos (relacionados à quantidade de colesterol ingerido), maiores nos animais engaiolados em relaçäo aos soltos, de lipídios totais no plasma sangüineo e no fígado (significativo); e de colesterol total no fígado, na aorta e no músculo (significativo). Além disso, o grau de ateromatose aórtica foi significativamente mais severo nos animais engaiolados

Effect of neural activity on skeletal muscle phosphoproteins.

A number of phosphoproteins were found in the soluble fraction of rat skeletal muscle by two-dimensional polyacrylamide gel electrophoresis (PAGE). The pattern of some of these phosphoproteins differed in fast (extensor digitorum longus, EDL) or slow (soleus) muscles, was dependent on normal innervation, and was altered with denervation. In order to determine if the pattern was maintained by electrical activity or trophic factors, we compared the effect of electrical block by local neural application of tetrodotoxin with the effect of complete nerve section. Both methods produced similar alterations in phosphoproteins, indicating that the pattern is dependent on nerve activity, not trophic factors. Such phosphoproteins are possible mediators of neural activity on gene expression.

Translocation of the brain-type glucose transporter largely accounts for insulin stimulation of glucose transport in BC3H-1 myocytes.

Insulin-stimulated glucose transport was examined in BC3H-1 myocytes. Insulin treatment lead to a 2.7 +/- 0.3-fold increase in the rate of deoxyglucose transport and, under the same conditions, a 2.1 +/- 0.1-fold increase in the amount of the brain-type glucose transporter (GLUT 1) at the cell surface. It has been shown that some insulin-responsive tissues express a second, immunologically distinct, transporter, namely GLUT 4. We report here that BC3H-1 myocytes and C2 and G8 myotubes express only GLUT 1; in contrast, rat soleus muscle and heart express 3-4 times higher levels of GLUT 4 than GLUT 1. Thus translocation of GLUT 1 can account for most, if not all, of the insulin stimulation of glucose transport in BC3H-1 myocytes. On the other, hand, neither BC3H-1 myocytes nor the other muscle-cell lines are adequate as models for the study of insulin regulation of glucose transport in muscle tissue.

Supplemental fat source affects feedlot performance and carcass traits of finishing yearling steers and estimated diet net energy value.

Two trials that utilized 356 yearling steers were conducted to evaluate the effects of fat sources (3.5% of diet dry matter) in steam-flaked milo finishing diets. Fats differed in fatty acid composition and level of free fatty acids. In Trial 1, soybean oil, tallow and yellow grease were compared to a nonfat control. Feeding fat increased (P less than .05) daily gain, feed efficiency, estimated diet NE concentration, carcass weight and dressing percentage of steers. In Trial 2, fat treatments were control, acidulated soybean soapstock (SBSS), tallow, a blend of 70% SBSS:30% tallow, and yellow grease. Feeding tallow or the SBSS:tallow blend improved (P less than .05) feed efficiency and estimated dietary NE compared to control. Proportions of palmitic, stearic, oleic, linoleic and linolenic acid in longissimus muscle of steers were altered (P less than .05) by source of supplemental fat. Potential variability in animal response to fat blends was demonstrated by differences in animal response to yellow grease in the two trials. It was concluded that fats vary in feeding value and may alter carcass composition, contrary to putative thought. Further, potential associative effects of fat blends and interactions of fat with other dietary components in high-grain finishing diets require further investigation.

Performance, carcass, cartilage calcium, sensory and collagen traits of longissimus muscles of open versus 30-month-old heifers that produced one calf.

One hundred eleven Simmental x Hereford (3/8 to 5/8 Simmental) heifers were used to determine the effects of age, parturition and implantation on performance, carcass and meat-sensory traits, muscle-collagen characteristics and thoracic-button calcification. Eighty-five heifers that calved at about 2 yr of age, designated as single-calf heifers (SCH), were either implanted (I-SCH) with Synovex-H or not implanted (NI-SCH). The remaining 26, 2-yr-old non-pregnant heifers (2-OH) served as controls. Additionally, 24, 1-yr-old open heifers (1-OH) from the same genetic source were utilized as the standard heifer-production system. The 1-OH and 2-OH were slaughtered after being fed a high-grain diet for 137 and 112 d, respectively. The SCH were fed the same high-grain diet beginning about 1 mo after calving and were fed 137 d before slaughter. The 33 I-SCH were implanted when started on the high-grain diet. Calves were weaned about 5 wk before the SCH were slaughtered. The 2-OH had the highest (P less than .05) feedlot ADG, whereas no differences (P greater than .05) occurred among other treatments. Dressing percentages were higher (P less than .01) for I-SCH than for NI-SCH. Carcass weights were lowest (P less than .05) and percentage kidney, pelvic and heart fat was highest (P less than .01) for 1-OH. Fat thickness, yield grades, marbling scores and quality grades were similar (P greater than .05) and desirable for all treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat myogenic c-mos cDNA: cloning sequence analysis and regulation during muscle development.

We have isolated and sequenced a cDNA clone, homologous to the rat c-mos gene, from a cDNA library of rat skeletal muscles. The 3220 nucleotide cDNA clone codes for a protein of 339 amino acids (37.4 kDa). Both the nucleotide sequence and the deduced amino acid sequence show 60-90% overall homology to Xenopus, chicken, mouse and human mos. By Northern blot analysis, we detected two c-mos transcripts, one major of about 3.6 Kb long, and one minor of about 1.7 Kb long. These are differently regulated during the development of cardiac and skeletal muscles. By Western blot with two antibodies directed against two different portions of the mos protein, we observed in rat muscle two polypeptides of 43 kDa, and 75 kDa respectively.

Two-dimensional spectra of intact tissue: homonuclear Hartmann-Hahn spectroscopy provides increased sensitivity and information content as compared to COSY.

Hartmann-Hahn (HOHAHA) spectroscopy led to good definition of proton spin systems of metabolites such as lactate, (phospho) creatine, and carnosine from a 3-g sample of excised frog gastrocnemius muscle in a 17-min experiment. The sign of the HOHAHA cross-peak intensity appears to provide additional information about interactions between metabolites and their protein binding sites.

Metabolic response to exercise in malignant hyperthermia-sensitive patients measured by 31P magnetic resonance spectroscopy.

The currently favored theory of pathogenesis of malignant hyperthermia (MH) implicates an abnormality in skeletal muscle calcium ion transport. During a MH crisis a profound lactic acidosis occurs and in MH-sensitive individuals a delayed recovery of venous lactate has been previously noted postexercise. We have used 31P magnetic resonance spectroscopy to follow noninvasively in vivo changes in muscle of intracellular pH and high-energy phosphate metabolites during rest, exercise, and recovery of MH-sensitive subjects. Eleven biopsy-positive MH-sensitive patients have been studied and compared to 26 normal subjects. The MH-sensitive subjects as a group prematurely dropped their intracellular pH during mild aerobic exercise and they demonstrated a marked delay before the recovery of pH after maximal exercise. PCr/(PCr + Pi) ratios also dropped early during exercise but recovered normally. The observed changes in pH and PCr/(PCr + Pi) are consistent with a myopathy in MH-susceptible individuals.

Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle.

One slow and three fast myosin heavy chains have been described in typical skeletal muscles of the adult rat using immunocytochemical analysis. Electrophoretic isolation and immunochemical identification of these four isoforms has not been achieved. An electrophoretic procedure is described which, by altering the cross-linkage and polymerization kinetics of 5% polyacrylamide gels, allows resolution of these four distinct myosin heavy chains. Using specific monoclonal antibodies and double immunoblotting analysis, the identity and electrophoretic migration order of the myosin heavy chains was established to be: 2A less than 2X less than 2B less than beta/slow.