The steroidal glycoalkaloid α-tomatine

The literature relating to chemical, biochemical and biological aspects of the steroidal glycoalkaloid, α-tomatine, is reviewed. The alkaloid, which can be used as a starting compound for the synthesis of steroidal hormones, is toxic to a wide range of living organisms. The significance of tomatine to plants which elaborate it is discussed and some possible uses of the compound are mentioned.     

Conversion of phosphatidylglycerol to lyso(bis)phosphatidic acid by alveolar macrophages

We report here studies of the synthesis of lyso(bis)phosphatidic acid [L(b)PA] by normal and BCG-elicited rabbit alveolar macrophages. This study was prompted by our earlier observations that 1) alveolar macrophages did not synthesize L(b)PA de novo despite its abundance in these cells, 2) BCG-elicited cells contained only one-quarter the amount of L(b)PA as normal cells, and 3) the turnover of arachidonate in L(b)PA led to hydroxyeicosatetraenoic acid and leukotriene synthesis. We found that exogenous phosphatidylglycerol (PG) was specifically converted to L(b)PA by both types of cells although BCG-elicited cells had only one-quarter the synthetic capacity of normal cells. Other phospholipids were found to become cell associated but were not significantly metabolized. Both glycerol moieties and the phosphate were incorporated into the product L(b)PA. However, substitution of the ester with an alkyl linkage in position 1 blocked the conversion of PG to L(b)PA. Most of the alkylphosphatidylglycerol was converted to phosphatidylcholine and phosphatidylethanolamine. This result implied that catabolism of the acyl group in position 1 was essential for L(b)PA synthesis. Because alveolar macrophages are present in a surfactant-rich milieu, we suggest that surfactant provides a source of PG for macrophage synthesis of L(b)PA in situ. It is interesting that the surfactants from rabbits challenged with BCG have a significant decrease in PG content.     

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.      

Biosynthesis and degradation of α-tomatine in developing tomato fruits

Fruits of tomato incorporated [2-¹⁴C]mevalonic acid lactone into the steroidal glycoalkaloid α-tomatine. Young fruits showed the greatest alkaloid-synthesizing ability but this decreased as the fruits developed. Analysis of sap exuded from fruit stalks and also application of[4-¹⁴C]cholesterol to leaves confirmed that tomatine is not transported into fruits from vegetative organs. Accumulation of this alkaloid in fruits thus appears entirely due to synthesis. Excised fruits of all developmental stages degraded injected [¹⁴C]tomatine and rates were directly related to fruit age. The pattern of accumulation/decline in fruit tomatine may be explicable on the basis of changing capacity for synthesis/degradation during development. Label from injected [¹⁴C]tomatine was present mainly in chlorophylls and carotenoids where it increased with time as that in tomatine decreased. The significance of the relationship between tomatine disappearance and carotenoid development is briefly discussed. The aglycone tomatidine was not detected in green fruits but a Δ¹⁶-5α-pregnenolone-like compound was.     

Effects of the steroidal alkaloid tomatine in auxin bioassays and its interaction with indole-3-acetic acid

Summary The steroidal alkaloid tomatine did not enhance elongation of oat coleoptile and first internode sections, or of wheat coleoptile sections. Higher concentrations of the alkaloid inhibited elongation and interacted antagonistically with IAA. Although 10^-4 M tomatine alone did not influence elongation of oat coleoptile sections, it did reduce growth response to exogenous IAA. Tomatine concentrations less than 10^-4 M did not influence response to IAA. The auxin activity of tomatine, reported by Vendrig, was therefore not confirmed.     

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.     

Effects of light and photoperiodic conditions on abscisic acid in leaves and roots of Acer pseudoplatanus L.

Roots of Acer pseudoplatanus seedlings grown in liquid nutrient medium contained much lower levels of both free and bound abscisic acid than did leaves. The levels of free abscisic acid were similar in young expanding and of mature leaves, but lower in older senscing leaves. Growing plants under long days or short days did not influence the levels of free and bound abscisic acid in leaves. However, under both long days and short days, levels of bound abscisic acid were lower at the end of the dark period than 8 h later during the light period. Phaseic acid was also detected during the light period but never at the end of the dark period.Abbreviations ABA abscisic acid - PA phaseic acid - SD short day - GLC gas-liquid chromatography - LD long day     

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.