Biochemical changes in progressive muscular dystrophy, XVI. Effect of glutamic acid, aspartic acid and glycine on the amino acid content of skeletal muscle of dystrophic mice.

The effect of exogenous administration of glutamic acid (GL), aspartic acid (A) and glycine (G) on individual amino acids in the free amino acid pool was studied in skeletal muscles of 60- to 70-day-old normal (N) and dystrophic (D) mice. Both N and D mice received either 0.25 ml of saline (S) or 250 mg/kg weight of GL, A or G in 0.25 ml S subcutaneously for 13 days. GL, A, G or S did not cause any significant changes in the body and skeletal muscle weights of either group. Most of the individual amino acids were increased in skeletal muscles of GL-treated mice and were decreased in A- or G-treated animals compared to S administration in the N group. The picture was more dramatic in the D group: GL-induced amino acid elevations were more pronounced than the values of N- or S-treated D controls. A and G elicited amino acid increases in D mice compared to their S-treated counterparts. Most of the individual amino acids in skeletal of the D group were decreased relative to N mice after S, GL or A administration. This was evident when the D/N ratio was calculated for S, GL and A. The situation was very different after G administration since of the individual amino acids were augmented in the skeletal muscle of D mice compared to N animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice.

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of epidermal proteins for DNA-binding activity.

A group of DNA-binding proteins from the soluble extract of newborn rat epidermis have been separated by chromatography using DNA-cellulose columns. The electrophoretogram of the DNA-binding proteins eluted from a single stranded DNA-cellulose column shows five major proteins of molecular weights ranging between 25K to 40K. Both the epidermal protein filaggrin and most keratins, except two high molecular weight keratins, do not show in vitro DNA-binding activity.

Changes in myosin isoenzymes, ATPase activity, and contraction duration in rat cardiac muscle with aging can be modulated by thyroxine.

To determine whether the relative decline in cardiac myosin isoenzyme V1 with maturation continues progressively into senescence and whether thyroxine could reverse age-associated changes in the myosin isoenzyme profile and contraction, rats 2, 8, and 24 months old were treated with thyroxine, 6.4 mg/kg, for 7 days. Myosin isoenzymes, Ca2+-myosin ATPase activities, and isometric contractile function were measured in cardiac preparations from thyroxine-treated animals and age-matched controls. Right ventricular hypertrophy did not occur with aging in controls. Thyroxine increased right ventricular weight in each age group compared to the control group. Body weight decreased by 10% in all thyroxine-treated rats. The relative right ventricular V1 isoenzyme content progressively decreased from 75 +/- 1% to 54 +/- 1% and 14 +/- 1% in controls at 2, 8, and 24 months, respectively, and was associated with a reciprocal increase in V3 myosin isoenzyme. Ca2+-myosin ATPase activity also progressively declined monotonically with age in the control rats from 854 +/- 28 nmol Pi/mg prot/min at 2 months to 529 +/- 28 nmol Pi/mg prot/min at 24 months. Thyroxine administration increased right ventricular V1 at each age to 97 +/- 2%, 73 +/- 2%, and 59 +/- 2% at 2, 8, and 24 months, respectively. A thyroxine induced increase in the Ca2+-myosin ATPase activity could be detected only in the 24-month-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphoid organ mRNA translatability in rats: effect of protein energy undernutrition in early life.

Synthesis of mRNA was studied in the spleen and thymus of rats that had been exposed to undernutrition early in life. To achieve this objective, lactating females were separated into two groups 1 wk after they gave birth to offspring. These control and experimental dams suckled 8-11 and 13-16 pups, respectively, for a period of 2 wk. The young of both groups were then killed, and their thymus and spleen were isolated. Polyadenylated RNA (poly A+ RNA) was fractionated by affinity chromatography on an oligo-dT-cellulose column. Poly A+ RNA content as well as the percentage of poly A+ RNA in relation to total RNA were both lower in the undernourished pups than in the controls. Analysis of the in vitro translation product primed by poly A+ RNA of the thymus and spleen revealed a rise in [35S]methionine incorporation in the undernourished offspring, the increase being greater in the thymus than in the spleen. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis, autoradiography and densitometric autoradiographic tracings confirmed these findings and demonstrated that proteins were synthesized at a higher level in the spleen and thymus of the undernourished rats than in the controls. These results show that undernutrition early in life could modulate the metabolism of mRNA and, consequently, protein synthesis in the lymphoid organs of rats. Furthermore, the data suggest that cell-mediated immunity as well as humoral immunity are both deranged in protein energy undernutrition.

A high performance liquid chromatography method to obtain rat epidermal filaggrin.

Sodium pyrophosphate-glycerol buffer, a non-denaturing buffer has been used to solubilize epidermal proteins. The extracted proteins are different in their electrophoretic profile in various mammalian species, 47 K in rat and guinea pig, 31 K and 60 K in mouse and 43 K and 60 K proteins in human epidermis were most predominant. Electrophoretic analyses show synthesis of new proteins as a function of age in rat tissue. Purification of the major rat protein fraction was achieved using a reverse phase high-performance liquid chromatography using a gradient of 5-50% acetonitrile. Based upon the molecular size, amino acid data and immunodiffusion analysis, we conclude the purified rat protein to be filaggrin.

ATPase activity and force production in myofibrils and twitch characteristics in intact muscle from neonatal, adult, and senescent rat myocardium.

To determine whether age-associated alterations in the cardiac muscle twitch could be related to altered myofibrillar ATPase activity or to an altered force-pCa relationship, these variables were measured in rat cardiac preparations across a broad age range. Between 2 and 6 months, maximum ATPase activity in detergent treated myofibrils decreased approximately two fold (0.166 +/- 0.01 v. 0.078 +/- 0.02 microM Pi/min X mg protein, P less than 0.001), but did not change with further aging (12 or 24 months). The Ca2+-dependent force in thin 'Triton skinned' papillary muscles was not age-related. ATPase activity and force exhibited identical Ca2+ sensitivity from the submicromolar to micromolar range: for ATPase activity pCa for 50% activity averaged 6.1 and Hill coefficients averaged 4.5; pCa for 50% force development was 6.1 and Hill coefficients of the force-pCa relation averaged 4.5; no age differences in these parameters were observed. In the intact muscles prior to skinning, neither twitch force nor the maximum rate of force production were age-related; however, indices of the time course of contraction, time to peak force, half relaxation time, and their sum, increased progressively, changing by approximately 30% from 2 to 24 months (P less than 0.001). Since the decline in ATPase activity occurred over the maturational period only, and did not change with further ageing, while the twitch duration changed progressively with age, it is concluded that the twitch prolongation of the senescent myocardium cannot be directly related to the age-related decline in myofibrillar ATPase activity.

Scattered-light intensity fluctuations in diastolic rat cardiac muscle caused by spontaneous Ca++-dependent cellular mechanical oscillations.

Laser light scattered by nonstimulated rat cardiac muscle bathed in physiological saline containing a [Ca++] of 0.4-2.5 mM displays scattered-light intensity fluctuations (SLIF); the frequencies of both SLIF and resting force are Ca++ dependent. Direct inspection of these muscles by phase-contrast microscopy under incoherent illumination revealed the presence of spontaneous asynchronous cellular motions that are also Ca++ dependent. The physical properties of the scattered light are compatible with the hypothesis that SLIF are due to the diastolic motion, except for the dependence on scattering angle, which may be perturbed because the muscles are optically thick. To determine whether diastolic SLIF and motion are an intrinsic property of activated myofilaments, photon-counting auto-correlation of the scattered light was performed both in rat right-ventricular papillary muscles skinned with the detergent Triton X-100 (1%) and in muscles with intact membranes under conditions that alter cellular Ca++ fluxes. In skinned muscles activated over a range of Ca++ from threshold to maximum force production, neither SLIF nor asynchronous motion was observed when Ca++ was buffered to constant values. In intact muscles the frequency of SLIF and the amplitude of diastolic motion were (a) markedly increased by substituting K+ or Li+ for Na+ in the bath; (b) not altered by verapamil (1 microM); and (c) reversibly abolished by caffeine (greater than or equal to 10 mM). These properties are exactly those of mechanical oscillations that have been observed in isolated cardiac cell fragments, which are the result Ca++ oscillations caused by Ca++ release from the sarcoplasmic reticulum (SR). We infer that mechanical oscillations caused by spontaneous Ca++-induced Ca++ release from the SR occur in intact nonstimulated cardiac muscle even in the absence of Ca++ overload and are the principle cause of SLIF, and that myoplasmic [Ca++] in "resting" muscle is not in a microscopic steady state.

Immunofluorescent visualization of myosin in human epidermal cells.

An antiserum against guinea pig epidermal myosin was used for the localization of myosin in frozen sections of human epidermis and cultured human epidermal cells. The antiserum gave a single precipitin band with epidermal myosin but did not cross react with muscle myosin or epidermal keratin in immunodiffusion plates. Strong staining, in the indirect immunofluorescence technique, was observed in cells of the lower layers of human and guinea pig epidermis. The antibody also reacted with myofibrils, intracellular filaments in cultured human epidermal cells and fibers in 3T3, HeLa and PtK2 cells. Double immunofluorescence staining using antisera against myosin and keratin revealed no obvious differences in staining patterns in filaments of cultured human epidermal cells.

Contractile proteins in epidermis. Isolation and properties of guinea-pig epidermal myosin.

Proteins of apparent molecular weights between 10 000 and 250 000 could be solubilized from guinea pig epidermis using a Tris/sucrose/ATP buffer. When the ionic concentration of the solubilized extract was made 75 mM with respect to KCl and 2 mM with respect to MgCl2, a protein complex precipitated which on SDS-polyacrylamide gel electrophoresis resolved into bands corresponding in migration to myosin, actin and a number of low molecular weight proteins. Myosin was dissociated from the complex with 0.6 M KI and purified by gel filtration chromatography on an agarose column. The purified epidermal myosin fraction contained a polypeptide of 200 000 molecular weight andtwo low molecular weight polypeptides of 16 500 and 13 000. The amino acid composition of the epidermal myosin heavy chain was similar to that of muscle myosin. At high ionic strength epidermal myosin had high specific (K+ + Ca2+)- and (K+ + EDTA)-ATPase activities and low specific (K+ + Mg2+)-ATPase activity. The pH activity curves of the (K+ + Ca2+)- and (K+ + EDTA)-ATPase were different. ATP was hydrolyzed faster than other nucleoside triphosphates. At low ionic strength, the (K+ + Mg2+)-ATPase activity of epidermal myosin was stimulated two fold by skeletal muscle actin. The myosin formed bipolar filaments in 50 mM KCl in the presence of 5 mM Mg2+.

Biochemical and immunological analysis of a basic protein from newborn rat epidermis.

A basic protein, solubilized in buffered salt solutions from keratohyalin granules of newborn rat epidermis, has been purified by ion-exchange chromatography. The relative molecular weight of the protein was determined as 12 800 +/- 200 from its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is relatively rich in lysine, glycine, alanine, and valine which together comprise about 60% of the total amino acid residues. Using an antibody to this protein, which we have designated fraction 4, we have found that it is specific to rat epidermis and is not present in any other rat tissues or in epidermal extracts from other species. The cells of the four epidermal layers were separated and the amount of fraction 4 in each cell layer was measured by radioimmunoassay. The protein is localized mainly in the upper layers of epidermis. The protein, which binds to DNA, appears in the epidermis just prior to birth, increases during the first week of post-natal life and declines sharply thereafter. Fraction 4 represents about 7% of the total solubilized protein in 7-day-old rat epidermis.

Fractionation and characterization of low molecular weight solubilized proteins of newborn rat keratohyalin granules.

Newborn rat epidermis was extracted using methods reported to extract keratohyalin granules. All extraction techniques yielded preparations of solubilized proteins with similar sodium dodecyl sulfate-polyacrylamide electrophoretograms. The solubilized proteins were fractionated on a Sephadex G-200 column and six low molecular weight protein fractions (apparent molecular weights between 10000 and 18000) have been identified. Four of these have been isolated and partially characterized. Two of the fractions are characterized by high histidine, arginine, serine and glutamic acid concentrations and have an amino acid composition similar to that of the histidine-rich protein characteristic of keratohyalin granules. One of these histidine-rich fractions (molecular weight 13700) has ribonuclease activity. The other two isolated fractions are basic proteins, one of which (molecular weight 12800) is a basic lysine-rich protein. This protein is not found in any other tissues of the new born or adult rat.