Differential effects of acute changes in cell Ca2+ concentration on myofibrillar and non-myofibrillar protein breakdown in the rat extensor digitorum longus muscle in vitro. Assessment by production of tyrosine and N tau-methylhistidine.

The influence of Ca2+ on myofibrillar proteolysis was evaluated in the isolated extensor digitorum longus muscle incubated in vitro with agents previously shown to increase the intracellular concentration of Ca2+. Myofibrillar proteolysis was evaluated by measuring the release of N tau-methylhistidine, and total proteolysis was evaluated by measuring tyrosine release by incubated muscles after the inhibition of protein synthesis with cycloheximide. Incubated muscles released measurable quantities of N tau-methylhistidine, and muscle contents of the amino acids remained stable over 2 h of incubation. The release of N tau-methylhistidine by incubated muscles was similar to its release by perfused rat muscle in response to brief starvation, indicating the integrity of the incubated muscles. Ca2+ ionophore A23187, dibucaine, procaine, caffeine and elevated K+ concentration increased lactate release by incubated muscles and decreased tissue contents of ATP and phosphocreatine to varying degrees, indicating the metabolic effectiveness of the agents tested. Only A23187 and dibucaine increased total cell Ca2+, and they increased tyrosine release. Caffeine and elevated [K+] increased neither cell Ca2+ nor tyrosine release; however, only A23187 and dibucaine increased tyrosine release significantly. On the other hand, these agents were without effect on myofibrillar proteolysis as assessed by N tau-methylhistidine release by incubated muscles and changes in tissue contents of the amino acid. In fact, some of the agents tested tended to decrease myofibrillar proteolysis slightly. These results indicate that acute elevation of intracellular Ca2+ is associated with increased breakdown of non-myofibrillar but not myofibrillar proteins. Because of this, the role of elevated Ca2+ in muscle atrophy in certain pathological states is questioned. The data also indicate that the breakdown of myofibrillar and non-myofibrillar proteins in muscle is regulated independently and by different pathways, a conclusion reached in previous studies with perfused rat muscle.     

Acute alterations in sodium flux in vitro lead to decreased myofibrillar protein breakdown in rat skeletal muscle.

Myofibrillar protein breakdown was evaluated by measuring the release of N tau-methylhistidine by isolated rat skeletal muscles or perfused rat muscles in the presence of a variety of agents known to affect Na+ flux. Total cell proteolysis was evaluated simultaneously by measuring tyrosine release by muscles after the inhibition of protein synthesis with cycloheximide. Treatment of muscles with the Na+ ionophore monensin or inhibitors of Na+-K+ ATPase (ouabain, digoxin or vanadate) decreased N tau-methylhistidine release by muscles by 21-35%. A phorbol ester (phorbol 12-myristate 13-acetate) as well as a synthetic diacylglycerol known to activate protein kinase C and a Na+/H+ antiport also decreased N tau-methylhistidine release by muscles. Removal of extracellular Na+ blocked the ability of these agents to attenuate N tau-methylhistidine release by muscles, suggesting that their effectiveness required a change in Na+ flux. In contrast with N tau-methylhistidine release by muscles, these agents, except for monensin, did not effect the release of tyrosine, suggesting that they attenuate specifically the breakdown of myofibrillar proteins. Overall these results indicate a link between Na+ and the regulation of protein breakdown in rat skeletal muscle, whereby an influx of Na+ can result in a decrease in myofibrillar proteolysis. Left unresolved is whether phospholipid hydrolysis is involved in this scheme.     

Nucleotide sequences and properties of the sites involved in lysogenic insertion of the bacteriophage HP1c1 genome into the Haemophilus influenzae chromosome.

Bacteriophage HP1c1 lysogenizes its host Haemophilus influenzae Rd by inserting its genome into the bacterial chromosome. The DNA segments corresponding to the integration regions on the phage and host chromosomes and the two junctions formed between phage and host sequences on lysogenic insertion were isolated and propagated in Escherichia coli HB101 as hybrid plasmids by using pBR322 as the vector. The nucleotide sequences in the vicinity of the point of recombinational insertion were determined. Phage and host DNA shared an extensive, nearly identical, segment that was 183 base pairs long. This segment consisted of 93 identical residues and a 27-residue portion containing 6 mismatches, followed by 63 identical residues. Recombinational insertion occurred within the 63-residue identical segment and involved neither duplication nor deletion of any residues. Short inverted repeats consisting of clustered A-T base pairs were present within the two 27-residue segments. Two additional sites on the host chromosome showed significant hybridization to the phage-host homology region.     

Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR

An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.     

Studies on the metabolism of retinol-binding protein by primary hepatocytes from retinol-deficient rats

Studies were conducted to explore the regulation of retinol-binding protein (RBP) metabolism in cultured primary hepatocytes from retinol-deficient rats. Newly isolated hepatocytes from retinol-deficient rats contained elevated levels (3.4-fold) of RBP, compared to hepatocytes from normal (retinol-adequate) rats. Addition of retinol to retinol-depleted hepatocytes stimulated RBP secretion by the cells in a concentration-dependent manner. Maximal stimulation of RBP secretion was seen with a retinol level of 0.3 micrograms/ml. The effect of retinol was quite rapid, and was evident by 20 minutes after addition of retinol to the medium. Stimulation of RBP secretion was only seen during the first few hours after retinol addition. The effect of retinol was specific for RBP; thus, retinol had no effect on the secretion rates of transthyretin or albumin. Addition of retinoic acid also stimulated RBP secretion by retinol-deficient hepatocytes. Addition of dexamethasone to retinol-deficient cells did not maintain the initial rate of RBP secretion. Dexamethasone also had no effect on the secretion of transthyretin or albumin by these cells. The effects of retinol and of dexamethasone seen here with retinol-depleted cells differed dramatically from effects seen in other studies with normal (retinol-adequate) hepatocytes. Thus, with normal cells, dexamethasone maintains RBP, TTR, and albumin production and secretion rates close to initial rates. Also in normal hepatocytes, with ample retinol available within the cell, addition of exogenous retinol does not appear to influence RBP secretion. In contrast, and as shown previously in intact rats, in retinol deficiency the availability of retinol specifically regulates the secretion of RBP by hepatocytes.     

Arsonate-specific murine T cell clones. IV. Properties of I-E- and I-A-restricted clones

The T cell antigen L-tyrosine-p-azobenzenearsonate is unique in being a simple determinant that can be presented in the context of both I-A and I-E. I-E-restricted T cell clones derived from B10.A(5R) mice were found to fall into three groups: Type I clones recognized antigen only in the context of syngeneic apcs, Type II clones recognized antigen with the same highly specific major histocompatibility complex restriction but in addition proliferated in response to allogeneic stimuli; Type III clones were "degenerate" in their major histocompatibility complex-restricted recognition of antigen and proliferated when antigen-presenting cells bearing Eb beta Ek alpha (syngeneic), Ek beta Ek alpha, or Ed beta Ed alpha were used. These observations allow some conclusions to be drawn about sites on the I-E molecule that may be functionally significant in the presentation of this antigen. By using the B cell hybridoma LK35.2 as target cells, some of these T cell clones act as cytotoxic cells in the Class II-restricted manner predicted from the results of proliferative assays. Class II-restricted cytotoxicity can therefore be controlled by both I-A and I-E mouse Ir gene loci.     

In situ detection of early replication phases of a gemini virus in legume protoplasts

Protoplasts of Phaseolus vulgaris L. (Top Crop), infected with bean golden mosaic virus, were isolated and fixed by various methods for in situ hybridization. An iodine-125 labeled probe was made from the replicative form of the virus. The localization and quantitation was done by autoradiography. Cell wall removal lowered the background and allowed a more accurate analysis. RNase was used to eliminate the possibility of hybrids to RNA. The evidence suggests a sequence of virus movements starting from rough endoplasm reticulum, moving to the nuclear membrane, and finally with the highest concentration inside the nucleus.Abbreviations BGMV bean golden mosaic virus - rfBGMV or rfDNA replicative double-stranded DNA virus - ssDNA single-stranded virus     

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.     

Size of the directing moiety at carbon 5 of cytosine and the activity of human DNA(cytosine-5) methyltransferase

M13 DNAs in which carbon 5 of each deoxycytidine residue in one strand is replaced with a bulky group are very good substrates for human DNA (cytosine-5) methyltransferase. Rate enhancements of up to 35 fold are obtained depending on the size of the moiety at C-5. The enzyme appears optimally suited to sense a methyl group in one strand at this position. Alkaline density gradient analyses of the distribution of methyl groups applied to 5-BrdCyd or 5-IdCyd substituted DNA reveal that these groups serve to direct the enzyme to methylate the unsubstituted strand.     

Distribution and levels of cellular retinol- and cellular retinoic acid-binding protein in various types of rat testis cells

The distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) were measured in rat testicular peritubular and Sertoli cells and in isolated rat pachytene spermatocytes and spermatids. Two Sertoli cell preparations, one containing some germ cells and another that had been osmotically shocked to destroy germ cells, were examined. CRBP and CRABP levels were measured by specific and sensitive radioimmunoassays. Testicular peritubular cell cytosol preparations were found to contain high levels of CRBP (1.48 +/- 0.87 microgram CRBP/mg protein) but CRABP could not be detected. The mean CRBP level in Sertoli cell preparations that contained some germ cells was 0.93 +/- 0.24 microgram CRBP/mg protein; this value was similar to the level of 1.11 +/- 0.20 microgram CRBP/mg protein measured for Sertoli cells free of germ cells. The level of CRABP found in Sertoli cell preparations containing germ cells (0.81 +/- 0.32 microgram CRABP/mg protein) was approximately five times greater than was observed in Sertoli cells free of germ cells (0.16 +/- 0.03 microgram CRABP/mg protein). CRBP and CRABP levels in cultured Sertoli cells were not affected by time in culture for up to five days of culture. Pachytene spermatocytes and spermatids were very enriched in CRABP (0.72 +/- 0.26 microgram CRABP/mg protein for spermatocytes and 0.65 +/- 0.21 microgram CRABP/ml protein for spermatids). A search for a high molecular weight retinol-binding protein did not demonstrate the existence of such a protein in Sertoli cell-conditioned medium. In summary, these studies provide quantitative information about the distribution of the cellular retinoid-binding proteins in the cell types that compose the rat testis.(ABSTRACT TRUNCATED AT 250 WORDS)