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.     

Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.     

Alterations of glial tumor cell Ca2+ metabolism and Ca2+-dependent cAMP accumulation by phorbol myristate acetate

C6 glial tumor cells exposed to phorbol myristate acetate (PMA) possessed lowered cAMP content, reduced ability to accumulate cAMP in response to norepinephrine or cholera toxin, and a 3-fold increase in the concentration of norepinephrine producing 50% of the maximal rate of cAMP accumulation. Detectable effects on cAMP accumulation occurred within 10 min of exposure to PMA, and prominent effects by 2 h. PMA similarly affected cells pretreated with cycloheximide. In contrast, Ca2+-depleted preparations of control and PMA-treated cells accumulated cAMP identically in response to norepinephrine or cholera toxin. Ca2+ restoration, which increased the rate of cAMP accumulation in control cells severalfold, did not enhance cAMP accumulation in PMA-treated cells. Neither high catecholamine nor high extracellular Ca2+ concentrations reversed the suppression of cAMP accumulation by PMA. Trifluoperazine, which inhibited the Ca2+-dependent component of norepinephrine-stimulated cAMP accumulation in control cells, did not significantly reduce norepinephrine-stimulated cAMP accumulation in PMA-treated cells. Cell free preparations of control and PMA-treated cultures did not differ significantly in calmodulin content or in Ca2+-stimulated adenylate cyclase, Ca2+-dependent cAMP phosphodiesterase, and (Ca2+-Mg2+)-ATPase activities. The Ca2+ content, however, of intact cells decreased with time of PMA treatment. Within minutes after exposure to PMA, the ability of Ca2+-depleted cells to take up 45Ca was significantly reduced. Both 45Ca uptake and Ca2+-dependent cAMP accumulation were reduced over the same PMA concentration range.     

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.     

Model‐assisted identification of metabolic engineering strategies for Jatropha curcas lipid pathways

Efficient approaches to increase plant lipid production are necessary to meet current industrial demands for this important resource. While Jatropha curcas cell culture can be used for in vitro lipid production, scaling up the system for industrial applications requires an understanding of how growth conditions affect lipid metabolism and yield. Here we present a bottom‐up metabolic reconstruction of J. curcas supported with labeling experiments and biomass characterization under three growth conditions. We show that the metabolic model can accurately predict growth and distribution of fluxes in cell cultures and use these findings to pinpoint energy expenditures that affect lipid biosynthesis and metabolism. In addition, by using constraint‐based modeling approaches we identify network reactions whose joint manipulation optimizes lipid production. The proposed model and computational analyses provide a stepping stone for future rational optimization of other agronomically relevant traits in J. curcas.     

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.