Deprivation and repletion of androgen in vivo modifies triacylglycerol synthesis by rat hepatocytes

Given the same quantity of fatty acid, livers from male rats esterify less fatty acid and secrete less triacylglycerol in very-low-density lipoprotein than do livers from female animals. To elucidate the role of testosterone in maintenance of this male pattern, conversion of [1-14C]oleic acid into triacylglycerol was assessed in vitro by rat hepatocytes (male) following gonadectomy and replacement with testosterone. Following castration, incorporation of fatty acid into triacylglycerol was increased. In contrast, esterification of exogenous fatty acid into phospholipid, cholesteryl esters, and diacylglycerol was unchanged. Treatment with testosterone (75 micrograms/day) reduced incorporation of exogenous fatty acid into triacylglycerol. Higher doses of testosterone (200 or 100 micrograms/day) modified the effect, such that inhibition was observed only at low oleate (0.5 mM) concentrations. At higher substrate concentrations (1.0-2.0 mM) the inhibitory effect was no longer observed. Further, a similar dose-dependent effect of testosterone was observed following in vivo treatment of castrate females with testosterone. These data support the concept of a regulatory role of testosterone in hepatic triacylglycerol synthesis. These findings also demonstrate a biphasic effect of testosterone, an effect that is dependent not only upon the dose of testosterone administered, but also on the concentration of fatty acid to which the hepatocyte is exposed in vitro.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Differential turnover of axonally transported glycoproteins

Metabolic turnover of axonally transported glycoproteins has been examined in membranous and soluble subfractions of goldfish optic tectum following intraocular injection of [³H]fucose. Radioactivity in total transported glycoproteins reached a maximum in the tectum after 24–30 hr, then declined with a half-life of approximately 20 days. Radioactivity in the total membranous subfraction declined with a similar half-life of 20–21 days while radioactivity in the soluble fraction showed a significantly shorter half-life of approximately seven days. Various sized glycopeptides derived from the membranous subfraction showed differential rates of loss of radioactivity with the lower molecular weight nondialyzable molecules displaying the most rapid turnover. In contrast, the glycopeptides derived from the soluble fraction showed relatively uniform rates of turnover. The results are discussed in the context of metabolic compartmentalization between membranous and soluble glycoproteins and among the carbohydrate chains of the membranous molecules.Supported by NIH grant NS 11456.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.     

An alternative mechanism of bicarbonate-mediated peroxidation by copper-zinc superoxide dismutase: rates enhanced via proposed enzyme-associated peroxycarbonate intermediate

Hydrogen peroxide can interact with the active site of copper-zinc superoxide dismutase (SOD1) to generate a powerful oxidant. This oxidant can either damage amino acid residues at the active site, inactivating the enzyme (the self-oxidative pathway), or oxidize substrates exogenous to the active site, preventing inactivation (the external oxidative pathway). It is well established that the presence of bicarbonate anion dramatically enhances the rate of oxidation of exogenous substrates. Here, we show that bicarbonate also substantially enhances the rate of self-inactivation of human wild type SOD1. Together, these observations suggest that the strong oxidant formed by hydrogen peroxide and SOD1 in the presence of bicarbonate arises from a pathway mechanistically distinct from that producing the oxidant in its absence. Self-inactivation rates are further enhanced in a mutant SOD1 protein (L38V) linked to the fatal neurodegenerative disorder, familial amyotrophic lateral sclerosis. The 1.4 A resolution crystal structure of pathogenic SOD1 mutant D125H reveals the mode of oxyanion binding in the active site channel and implies that phosphate anion attenuates the bicarbonate effect by competing for binding to this site. The orientation of the enzyme-associated oxyanion suggests that both the self-oxidative and external oxidative pathways can proceed through an enzyme-associated peroxycarbonate intermediate.