Cytosolic and mitochondrial isoenzymes of branched-chain amino acid aminotransferase during development of the rat.

The isoenzymic forms of branched-chain amino acid aminotransferase in mitochondria of rat tissues were compared with the better-known cytosolic forms in order to find any regular pattern of expression of these isoenzymes during development. Mitochondria of all tissues examined except brain contained only a type-I isoenzyme differing from the cytosolic type-I isoenzyme in heat stability and activation by mercaptoethanol. Foetal and adult brain mitochondria contained isoenzymes type III as well as type I. The large excess of type-I isoenzyme in foetal liver was localized in mitochondria, apparently of haematopoietic cells. The activity of this isoenzyme declined precipitously (by 80%) from day 19 of gestation at the same period and rate as does the volume fraction of haematopoietic cells that are then leaving the liver. Cortisol treatment accelerated the loss of these cells, and proportionally accelerated loss of the mitochondrial isoenzyme I. A development succession of type-I isoenzyme by the unique type II of liver parenchymal cell cytosols could not be demonstrated, since small, about equal, amounts of types I and II were always present in cytosols of foetal and adult liver. Developmental succession of isoenzymes within tissues was limited to cytosols and was demonstrated by the presence of cytosolic isoenzyme III in foetal and newborn skeletal muscle and kidney, organs which contain only isoenzyme I in the adult.     

Contribution of serum albumin to the transport of orally administered L-tryptophan into liver of rats with L-tryptophan depletion

Summary The role of serum albumin in the transport of orally administered L-tryptophan (Trp) into rat tissues was examined using analbuminemic and Sprague-Dawley (SD) rats with and without a-methyl-DL-tryptophan (AMT)-induced Trp depletion. Trp was orally administered to rats 16h after AMT or 0.85% NaCl administration, when liver tryptophan 2,3-dioxygenase and protein synthetic activities in AMT-treated rats were similar to those of 0.85% NaCl-treated rats. After oral Trp administration, regardless of the presence or absence of Trp depletion, free serum Trp concentrations were similar in the analbuminemic and SD rats, while total serum Trp concentrations were lower in analbuminemic rats than in SD rats. Although liver, brain, and muscle Trp concentrations after oral Trp administration under Trp depletion were lower in analbuminemic rats than in SD rats, the ratio of the liver Trp concentration in analbuminemic rats to that in SD rats was smaller than that of the brain or muscle Trp concentration. These results suggest that variations in serum albumin levels could affect the transport of orally administered Trp into the liver of rats with Trp depletion.     

p38 MAPK regulates COPII recruitment

Here, we investigate regulation of coat protein complex II (COPII) recruitment onto ER export sites in permeabilized cells. In cytosols from nocodazole treated HeLa cells we find COPII loading is inhibited. The stress kinase p38 MAPK is activated in these cytosols and COPII loading can be rescued by depletion of p38 MAPK α or by the p38 MAPK inhibitor (SB203580) but not by inhibition/depletion of cdc2. These observations indicate regulation of the early secretory pathway by p38 MAPK.     

Incorporation of 14C-thymidine into liver and brain DNA of protein-deficient rats.

The authors studied the effect of a protein-free diet on 14C-thymidine incorporation into rat liver DNA in vivo and found, after 2-3 weeks, a marked decrease in uptake of the radioactive base into the liver DNA, followed by a decrease in the proportion of DNA in liver cell homogenates. The total nuclear count/mg liver tissue displayed an increase during protein depletion, except for the 5th week, when a decrease was recorded. The incorporation of 14C thymidine into the brain DNA likewise displayed no great differences, although a significant drop was observed during the 2nd to 4th week of depletion. In the 5th week we recorded an increase in uptake of the radioactive base by brain DNA, exceeding the incorporation values in the controls.     

Differential regulation of the ascorbic acid transporter SVCT2 during development and in response to ascorbic acid depletion

The sodium-dependent vitamin C transporter-2 (SVCT2) is the only ascorbic acid (ASC) transporter significantly expressed in brain. It is required for life and is critical during brain development to supply adequate levels of ASC. To assess SVCT2 function in the developing brain, we studied time-dependent SVCT2 mRNA and protein expression in mouse brain, using liver as a comparison tissue because it is the site of ASC synthesis. We found that SVCT2 expression followed an inverse relationship with ASC levels in the developing brain. In cortex and cerebellum, ASC levels were high throughout late embryonic stages and early post-natal stages and decreased with age, whereas SVCT2 mRNA and protein levels were low in embryos and increased with age. A different response was observed for liver, in which ASC levels and SVCT2 expression were both low throughout embryogenesis and increased post-natally. To determine whether low intracellular ASC might be capable of driving SVCT2 expression, we depleted ASC by diet in adult mice unable to synthesize ASC. We observed that SVCT2 mRNA and protein were not affected by ASC depletion in brain cortex, but SVCT2 protein expression was increased by ASC depletion in the cerebellum and liver. The results suggest that expression of the SVCT2 is differentially regulated during embryonic development and in adulthood.     

pI(Cln) and cytosolic F-actin constitute a heteromeric complex with a constant molecular mass in rat skeletal muscles.

To elucidate the function of pI(Cln), its localization in subcellular organellae was investigated. A specific polyclonal anti-pI(Cln) antibody detected the soluble 38-kDa pI(Cln) exclusively in the cytosols of rat heart, lung, liver, spleen, skeletal muscle, testis, and brain, but not rat kidney. pI(Cln)-associated proteins in skeletal muscle were also analyzed. Native-gradient PAGE showed a single 340-kDa protein band reactive to anti-pI(Cln) antibody. This band also stained with anti-actin antibody. Two-dimensional PAGE and immunoprecipitation analysis indicated that all of the pI(Cln) was present in association with actin of a constant length: the molecular ratio of pI(Cln) to actin was roughly 1:7. In addition, all actin in the cytosol fractions was found in association with pI(Cln). These results suggest the possibility that skeletal muscle pI(Cln) controls the length of cytosolic F-actin.     

Metabolism of Collagen and Muscle Protein of Young Rats in Protein Depletion

The effect of protein depletion on the metabolism of body collagen and muscle protein has been investigated in young male rats fed with a protein-free diet for 14 and 28 days.

During the protein depletion, the protein content of the liver, intestine and skin decreased significantly, but the decrease of proteins was very little in the carcass, tail and bone (ossa cruris). An increase of tissue collagen in protein depletion was found in the carcass, bone, tail, skin and liver, while muscle protein in the carcass was evidently lost at a later stage of protein depletion. The increase of calcium in the bone was parallel to the increase of collagen, indicating continuous growth of the bones in spite of protein depletion. These results may indicate that the young animals continuously synthesize collagens of their special tissues from other tissue proteins even with severe protein deficiency. The metabolic responses of body collagens to dietary protein depletion in young rats have been discussed and compared with those in adult rats reported previously.     

Widespread, specific binding of 25-hydroxycholecalciferol in rat tissues.

In the vitamin D-depleted rat, all nucleated tissues examined (brain, lung, heart, pancreas, liver, cartilage, muscle, bone, kidney, and intestine) contained a soluble substance which bound 25-hydroxy[3H]cholecalciferol in vitro specifically and sedimented at 6.3 S in linear sucrose gradients. The serum-steroid complex sedimented a 4.1 S, and erythrocyte lysates were apparently devoid of specific binding activity. The ability of these cytosols to specifically bind the steroid was destroyed by treatment with trypsin, but not by RNase, DNase, or 1 mM p-hydroxymercuribenzoate. The sedimentation pattern was not altered in sucrose gradients containing 0.5 M KCl or following cytosol preparation and ultracentrifugation in gradients containing 0.012 M dithiothreitol. The apparent avidity for 25-hydroxycholecalciferol (KA similar to 2 times 10- M) was slightly higher in muscle and kidney cytosols than in serum, but serum contained a large number of specific binding sites. The presence of widespread, high affinity binding proteins for 25-hydroxycholecalciferol raises the possibility that tissues other than the intestine, bone, and kidney may respond directly to vitamin D metabolites.     

Liberation and analysis of protein-bound arsenicals

Protein-bound arsenicals were liberated from binding sites on liver cytosolic proteins by exposure to 0.1M CuCl at pH 1. This method released greater than 90% of the arsenicals associated with biological matrices. Ultrafiltrates of CuCl-treated cytosols were subjected to thin-layer chromatography to speciate and quantify inorganic and methylated arsenicals. For rat liver cytosol in an in vitro methylation assay and for liver and kidney cytosols from arsenite-treated mice, most inorganic arsenic was protein bound. Appreciable fractions of the organoarsenical metabolites present in these cytosols were also protein bound. Therefore, CuCl treatment of cytosols releases protein-bound arsenicals, permitting more accurate estimates of the pattern and extent of arsenic methylation in vitro and in vivo.     

Covalent linkage of 125I-insulin to a cytosolic insulin-degrading enzyme

Cytosol extracts high in insulin-degrading activity were cross-linked to 125I-insulin with the bifunctional cross-linker disuccinimidyl suberate. With cytosols from either rat muscle, liver, kidney or brain or human erythrocytes, only a single protein (Mr = 110,000) was specifically labeled. Three different lines of evidence indicated that this labeled protein is insulin-degrading enzyme, a cysteine protease which accounts for most of the insulin-degrading activity in cell extracts. Firstly, the cross-linking of 125I-insulin to this protein is inhibited by unlabeled insulin over the same concentration range of insulin which inhibits degradation. Separated insulin A and B chain were less potent at inhibiting cross-linking, whereas bovine serum albumin and cytochrome c were without effect. Secondly, antibodies to purified insulin-degrading enzyme precipitated the labeled protein in parallel with their ability to precipitate the insulin-degrading activity of the extracts. Thirdly, when the insulin-degrading activity was purified 40,000-fold from erythrocytes, this Mr 110,000 protein co-purified. These results indicate that cross-linking 125I-insulin may be a convenient method for labeling the insulin-degrading enzyme.     

Demonstration of a macromolecule cross-reacting with antibodies to luteinizing hormone releasing hormone and its tissue distribution

Hypothalamic cytosol contains a macromolecule which cross-reacts with antibodies to luteinizing hormone releasing hormone (LRH). This cross-reacting material (macro-CRM) is insoluble in methanol or acid ethanol, and its molecular weight is about 70,000. Macro-CRM is also found in cytosols of extra-hypothalamic regions of the brain, liver, kidney, spleen, and skeletal muscle. Plasma contains only marginal amounts of macro-CRM. This substance inhibits the binding of [¹²⁵I]LRH to LRH antibodies in a reversible, competitive manner.     

A comparative study of the composition of the microsomal membranes of the liver, brain and skeletal muscles in vertebrates]

Phospholipid and cholesterol amounts, intrinsic protein/lipid ratios in liver, brain and skeletal muscle microsomal membranes of 14 species of vertebrate animals have been studied. No significant differences between phospholipid amounts in tissues as well as vertebrate classes have been discovered. The highest cholesterol amount has been found in brain microsomes, the smallest one in sarcoplasmic reticulum membranes. In reptile brain and muscle microsomes a higher amount of cholesterol compared to that in species of other vertebrate classes has been found. In brain membranes intrinsic protein and lipid amounts are approximately equal, while in liver and muscle microsomes a protein component predominates. Phospholipid/protein ratio is larger in brain membranes than in liver and muscle ones. Cholesterol/protein ratio reaches the highest values in microsomal membranes of reptile tissues. Brain membranes of vertebrate animals are characterized by a greater stability of protein-lipid composition than liver and muscle ones.     

Androgen binding in peripheral tissues of fetal rhesus macaques: effects of androgen metabolism in liver

In rhesus monkeys sexual differentiation of the brain and reproductive tract (RT) is androgen-dependent. Presumably these effects are mediated through the androgen receptor (AR). The AR has not been characterized in fetal tissues such as liver, kidney, heart, spinal cord and RT in this species. We characterized AR binding using [3H]R1881 as the ligand in cytosols from tissues obtained on days 100-138 of gestation. Scatchard analyses revealed a single, saturable, high affinity AR in liver, kidney, heart, spinal cord and RT. The apparent dissociation constant (Kd) ranged from 0.52 to 0.85 nM with no significant tissue differences. The number of AR (Bmax; fmol/mg protein) differed significantly (P less than 0.01) between tissues (liver greater than RT much greater than kidney greater than or equal to heart greater than or equal to spinal cord). Radioinert testosterone (T) and 5 alpha-dihydrotestosterone (DHT) but not androstenedione, progesterone, estradiol-17 beta, estrone or cortisol in a 50-fold molar excess inhibited [3H]R1881 binding to the AR in spinal cord, heart, kidney and RT. However, in liver only DHT competed significantly (P less than 0.01) for binding. This difference in binding of DHT vs T in the liver was further investigated by incubating liver and kidney cytosols with [3H]DHT and [3H]T at 4 degrees C. We identified the metabolic products by mobility on Sephadex LH-20 columns and reverse isotope dilution. Liver cytosols metabolized [3H]DHT to 5 alpha-androstane- 3 alpha,17 beta-diol (5 alpha-diol) and [3H]T to 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-diol) at 4 degrees C. In contrast, kidney cytosols metabolized [3H]DHT while [3H]T remained unchanged. Further studies indicated that a 50-fold molar excess of 5 alpha-diol inhibited the binding of [3H]R1881 in liver cytosols by about 50% whereas the same molar concentration of 5 beta-diol had no effect. These data demonstrate the presence of AR in peripheral tissues of fetal rhesus monkeys and suggest that androgens through their receptors may affect development of these tissues. Liver cytosols are capable of metabolizing T and DHT at 4 degrees C at conditions similar to those used for measuring cytosolic AR. However, T and DHT are metabolized differently, generating different isomers which have different affinities for hepatic AR.     

Immunological quantitation and immunohistochemical localization of leukotriene A4 hydrolase in guinea pig tissues

We prepared a highly specific polyclonal antibody against leukotriene (LT) A4 hydrolase using a recombinant human enzyme. Using this antibody, we quantified LTA4 hydrolase protein content in the cytosols of guinea pig tissues. The enzyme protein content correlated well with the enzyme activity with a correlation coefficient of 0.87. However, the enzyme activity per mg of the enzyme in the cytosols was low, particularly in the liver and adrenal gland, compared with the specific activity of the purified enzyme. These observations suggest the presence of inhibitory substances and/or inactive enzymes in the cytosols of these tissues. To determine the cellular localization of LTA4 hydrolase in tissues other than blood cells, we carried out immunohistochemical examinations of guinea pig tissues. We identified epithelial cells in the tracheobronchial system and gastrointestinal tract, smooth muscle cells in the bronchi and aorta, vascular endothelial cells, and the intestinal plexus as novel cellular sources of the enzyme in the parenchyme of the tissue. Thus, LTA4 hydrolase was widely distributed in various types of parenchymal cells in the tissues, and this observation warrants further investigations on the biological activities of LTB4 in these cells and tissues.     

Effects of stress on carbohydrate metabolism in the teleost Notopterus notopterus (Pallas)*

The effects of different types of stre35 on carbohydrate metabolism in N. notopterus were investigated. Starvation alone brings about a signifiant increse in the glycogen content of the saccus vasculosus and a significant decrease in the brain glycogen concentration. The increased glycogen concentration in the saccus vasculosus may be a device to safeguard the brain against glucose deficiency during starvation stress. Rapid depletion of the muscle glycogen following fasting shows that the muscle glycogen IS the readily utilizable source of energy during starvation. Exposure of N. noropierus to air brought about an increaSe in the liver glycogen and blood glucose levels but did not affect other paramcters studied. Physical exhaustion of N. notopierus is noticed within 1–2 min of exercise. The readily available source of energy for exercise is the muscle glycogen. and the lactic acid produced is probably metabolized in the muscle itself. Saccus vasculosus glycogen. though inde-pendent of changes in the blood glucose levels, may possibly be controlled by variations in the brain glycogen.     

Tissue-specific changes in protein synthesis associated with seasonal metabolic depression and recovery in the north temperate labrid, Tautogolabrus adspersus

The tissue-specific changes in protein synthesis were tracked in relation to the seasonal metabolic depression in cunner (Tautogolabrus adsperus). In vivo protein synthesis rate and total RNA content were determined in liver, white muscle, brain, heart, and gill during periods of normal activity before metabolic depression, entrance into and during winter dormancy, and during the recovery period. The decrease in water temperature from 8 degrees C to 4 degrees C was accompanied by a 55% depression of protein synthesis in liver, brain, and heart and a 66% depression in gill. Protein synthesis in white muscle fell below detectable levels at this temperature. The depression of protein synthesis is an active process (Q(10) = 6-21 between 8 degrees C and 4 degrees C) that occurs in advance of the behavioral and physiological depression at the whole animal level. Protein synthesis was maintained at these depressed levels in white muscle, brain, heart, and gill until water temperature returned to 4 degrees C in the spring. Liver underwent a hyperactivation in the synthesis of proteins at 0 degrees C, which may be linked to antifreeze production. During the recovery period, a hyperactivation of protein synthesis occurred in white muscle, which is suggestive of compensatory growth, as well as in heart and liver, which is considered to be linked to increased activity and feeding. Seasonal changes in total RNA content demonstrate the depression of protein synthesis with decreasing temperature to be closely associated with translational capacity, but the stimulation of protein synthesis during recovery appears to be associated with increased translational efficiency.     

Genetic variability of proteins from mitochondria and mitochondrial fractions of mouse organs

Proteins of whole mitochondria from mouse liver and brain and proteins of liver mitochondrial fractions (plasma and rough membrane fraction) were separated by two-dimensional electrophoresis. Protein patterns of two inbred strains of mouse, C57BL/6J and DBA/2J, and of F₁ mice of these two strains were studied. The protein patterns obtained from the different mitochondrial materials were analyzed with regard to their protein composition and the genetic variability of proteins (qualitative and quantitative protein variants). Included in this analysis are data previously obtained from the cytosols and plasma membranes of the same organs and mouse strains. The results showed the following. (1) Mitochondria and organelle-free cell components (cytosol and plasma membranes) have only a few percent of their proteins in common, while two organs, liver and brain, reveal up to approximately 50% organ-nonspecific proteins. The frequency of proteins common to solubilized and structure-bound proteins ranges below 20%. (2) Genetic variability in protein amount occurs much more frequently than genetic variability in protein structure. Liver proteins reveal more genetic variants than brain proteins. Proteins solubilized in the cell show more genetic variation than structure-bound proteins. Furthermore, the results show that with regard to the composition and the genetic variability of proteins, liver and brain differ more in their mitochondria than in their cytosol and plasma membranes.This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to Sonderforschungsbereich 29.     

砷对兰州鲇组织中代谢酶活性及RNA和蛋白质含量影响

采用毒性试验方法,研究了安全浓度（1.288 mg/L）条件下亚砷酸钠（NaAsO2）对兰州鲇（Silurus lanzhouensis）脑、鳃、肝脏、肌肉4种组织中6-磷酸葡萄糖脱氢酶（G-6-PDH）和乳酸脱氢酶（LDH）活性,以及RNA和蛋白质含量的影响。结果表明,染毒21d时,As（Ⅲ）可显著降低4种组织中G-6-PDH和LDH活性、RNA和蛋白质含量（P0.05）。撤毒后21d,除脑和肝组织中蛋白质含量未恢复到对照组水平（P0.05）,肝脏中G-6-PDH活性超过了对照组水平（P0.05）外,其余各组织中G-6-PDH和LDH活性、RNA和蛋白质含量均可恢复到对照组水平（P0.05）。以上结果表明,As（Ⅲ）对兰州鲇组织中代谢酶活性具有明显的抑制作用,可致组织细胞RNA损伤和可溶性蛋白质减少,但这种影响是可逆的,撤毒后一定时间内可恢复到正常水平。       

Human skeletal muscle cytosols are refractory to cytochrome c-dependent activation of type-II caspases and lack APAF-1

Apoptotic regulatory mechanisms in skeletal muscle have not been revealed. This is despite indications that remnant apoptotic events are detected following exercise, muscle injury and the progression of dystrophinopathies. The recent elicitation of a cytochrome c-mediated induction of caspases has led to speculation regarding a cytochrome c mechanism in muscle. We demonstrate that cytosols from skeletal muscle biopsies from healthy human volunteers lack the ability to activate type-II caspases by a cytochrome c-mediated pathway despite the confirmed presence of both procaspase-3 and -9. This was not due to the presence of an endogenous inhibitor, as the muscle cytosols enhanced caspase activity when added to a control cytosol, subsequently activated by cytochrome c and dATP. In addition, we demonstrate that muscle cytosols lack the apoptosis protease activator protein-1 (APAF-1), both at the protein and mRNA levels. These data indicate that human skeletal muscle cells will be refractory to mitochondrial-mediated events leading to apoptosis and thus can escape a major pro-apoptotic regulatory mechanism. This may reflect an evolutionary adaptation of cell survival in the presence of the profusion of mitochondria required for energy generation in motility.