Plant regeneration from hypocotyl and petiole callus of Trifolium pratense L.

Red clover (Trifolium pratense L.) seedlings were screened for the ability to regenerate plantlets from hypocotyl-derived callus tissue. Media sequences described by Beach and Smith (1979) and Collins and Phillips (1982) and a variation using media from both sequences were tested. Plantlets were regenerated from three out of 642 genotypes. In all three cases, callus was initiated on B5C medium and regeneration was accomplished on SPL medium. Attempts to regenerate plants from petiole-derived callus tissue have so far been successful only with regenerants of clone F49. Petiole callus from epicotyl-derived F49 plants proved to be non-regenerative. Pollen viability varied significantly among individuals regenerated from callus cultures of clone F49. Root tip squashes from F49 regenerants revealed the normal diploid chromosome number (2n=14). The frequency of regeneration within progeny from reciprocal crosses between F49 regenerants and several non-regenerative genotypes was 29%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - KN kinetin - NAA -naphthaleneacetic acid     

Limiting concentrations of activated mononucleotides necessary for poly(C)-directed elongation of oligoguanylates

Summary Selected imidazolide-activated nucleotides have been subjected to hydrolysis under conditions similar to those that favor their template-directed oligomerization. Rate constants of hydrolysis of the P–N bond in guanosine 5-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5-monophosphate imidazolide (ImpG), k_h, have been determined in the presence/absence of magnesium ion as a function of temperature and polycytidylate [poly(C)] concentration. Using the rate constant of hydrolysis of 2-MeImpG and the rate constant of elongation, i.e., the reaction of an oligoguanylate with 2-MeImpG in the presence of poly(C) acting as template, the limiting concentration of 2-MeImpG necessary for oligonucleotide elongation to compete with hydrolysis can be calculated. The limiting concentration is defined as the initial concentration of monomer that results in its equal consumption by hydrolysis and by elongation. These limiting concentrations of 2-MeImpG are found to be 1.7 mM at 37°C and 0.36 mM at 1°C. Boundary conditions in the form of limiting concentration of activated nucleotide may be used to evaluate a prebiotic model for chemical synthesis of biopolymers. For instance, the limiting concentration of monomer can be used as a basis of comparison among catalytic, but nonenzymatic, RNA-type systems.We also determined the rate constant of dimerization of 2-MeImpG, k₂=0.45±0.06 M^–1 h^–1 in the absence of poly(C), and 0.45±0.06k₂0.97±0.13 M^–1 h^–1 in its presence at 37°C and pH 7.95. This dimerization, as well as the trimerization of 2-MeImpG, which represent the first steps in the oligomerization reaction, are markedly slower than the elongation of longer oligoguanylates, (pG) _n n>6. This means that in the presence of low concentrations of 2-MeImpG (1.7 mM) the system directs the elongation of longer oligomers more efficiently than the formation of short oligomers such as dimers and trimers. These results will be discussed as a possible example of chemical selection in template-directed reactions of nucleotides.     

Calcium-binding proteins in the parathyroid gland. Detailed studies of parathyroid secretory protein

In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondenaturing/denaturing gel system and incubated them with 45Ca2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca2+ in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca2+. In the presence of 5 mM MgCl2 and 60 mM KCl, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent Kd of 1.1 mM. Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evidence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells.     

Growth versus molting time of caterpillars as a function of temperature,nutrient concentration and the phenolic rutin

Summary A factorial experiment tested the effects of varying nutrient concentration (normal versus diluted), presence or absence of the phenolic allelochemical rutin and daytime temperature (20, 25 and 30° C) on growth, molting and food utilization efficiencies of tobacco hornworms (Manduca sexta). Two of the utilization efficiencies (approximate digestibility and efficiency of conversion of ingested food) were unaffected by temperature; the third one, efficiency of conversion of digested food, was affected by temperature but there was no consistent effect. Lower temperatures significantly increased the proportion of the stadium spent molting, with larvae at a daytime temperature of 20° C spending 9% more of the stadium in molting than larvae at 30° C. Growth time was not influenced by nutrient concentration. When temperature was low and rutin absent, molt time and the proportion of the stadium spent molting were affected by nutrient concentration. Addition of the phenolic rutin did not have an appreciable effect on growth time or digestive processes. However, it increased molting time by 7 to 14% and thus increased the duration of the stadium and reduced relative growth rate. These results indicate that the effect of food quality on growth rate is a function of the thermal conditions of insect herbivores. At cooler temperatures, a disproportionate increase in time spent molting, rather than altered food utilization efficiencies, contributed to lower growth rates. The consequences for larval growth of fluctuating temperatures due to diurnal cycles and the presence of predators forcing larvae into thermally suboptimal microhabitats are discussed.     

Analysis ofl-alanine:2-oxoglutarate aminotransferase isozymes in maize

Isozyme analysis ofl-alanine:2-oxoglutarate aminotransferase (ALT) in maize indicates that there are three genes encoding this enzyme activity. Two of the gene products interact with each other to form heterodimers, while the third gene product does not interact with the other two. Another isozyme that appears after gel electrophoresis and ALT staining is shown to be glutamate dehydrogenase-1. Anaerobic treatment does not result in increased ALT levels, indicating that the previously reported increase in alanine levels caused by this treatment may be due to increases in the level of pyruvate, a substrate of ALT.D. A. Russell was partially supported by a graduate student fellowship from the Division of Biology and Biomedical Sciences, Washington University. V. M. Peschke was partially supported by a postdoctoral fellowship from Monsanto. This research was supported by NIH Grant R01 GM34740.     

Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its primary oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), are mechanism-based inhibitors of monoamine oxidases A and B. The pseudo-first-order rate constants for inactivation were determined for various analogues of MPTP and MPDP+ and the concentrations in all redox states were measured throughout the reaction. Disproportionation was observed for all the dihydropyridiniums, but non-enzymic oxidation was insignificant. The dihydropyridiniums were poor substrates for monoamine oxidase A and, consequently, inactivated the enzyme only slowly, despite partition coefficients lower than those for the tetrahydropyridines. For monoamine oxidase B, the dihydropyridiniums were more effective inactivators than the tetrahydropyridines. Substitutions in the aromatic ring had no major effect on the inactivation of monoamine oxidase B, but the 2'-ethyl- and 3'-chloro-substituted compounds were very poor mechanism-based inactivators of monoamine oxidase A. It is clear that both oxidation steps can generate the reactive species responsible for inactivation.     

Regulation of Human (Caco-2) Intestinal Epithelial Cell Differentiation by Extracellular Matrix Proteins

Extracellular matrix regulation of intestinal epithelial differentiation may affect development, differentiation during migration to villus tips, healing, inflammatory bowel disease, and malignant transformation. Cell culture studies of intestinal epithelial biology may also depend on the matrix substrate used. We evaluated matrix effects on differentiation and proliferation in human intestinal Caco-2 epithelial cells, a model for intestinal epithelial differentiation. Proliferation, brush border enzyme specific activity, and spreading were compared in cells cultured on tissue culture plastic with interstitial collagen I and the basement membrane constituents collagen IV and laminin. Each matrix significantly increased alkaline phosphatase, dipeptidyl peptidase, lactase, sucrase-isomaltase, and cell spreading in comparison to plastic. However, the basement membrane proteins collagen IV and laminin further promoted all four brush border enzymes but inhibited spreading compared to collagen I. Proliferation was most rapid on type I collagen and slowest on laminin and tissue culture plastic. Basement membrane matrix proteins may promote intestinal epithelial differentiation and inhibit proliferation compared with interstitial collagen I.     

Effects of temperature, multiple allelochemicals and larval age on the performance of a specialist caterpillar

To understand the mechanisms underlying plant-insect herbivore interactions, it is necessary to examine the simultaneous effects of temperature, food quality and larval age. We examined the simultaneous effects of three allelochemicals (tomatine, rutin and chlorogenic acid) on the performance of first and second instar Manduca sexta larvae under two representative thermal regimes 21 : 10°C and 26 : 15°C for spring and summer, respectively. Thermal regime and allelochemicals interacted to influence the time from egg hatch to ecdysis to the third instar. On average, it took about half as much time to reach the third instar at 26 : 15°C as it did at 21 : 10°C. Separately, tomatine and rutin had a negative effect on developmental time from egg hatch to the third instar, but their simutaneous effects were not additive. Chlorogenic acid significantly reduced the negative effect of tomatine. The magnitude of the allelochemical effect was larger at the cooler thermal regime compared to the warmer regime. For instance, chlorogenic acid by itself had no effect at the 26 : 15°C regime, but at the 21 : 10°C regime it significantly shortened total developmental time. The effect of chlorogenic acid on stadium duration was distinctly different for the two instars. Chlorogenic acid shortened stadium duration of first instar larvae. However, depending on thermal regime and the presence of tomatine, chlorogenic acid had a negative, positive or neutral effect on stadium duration of second instar larvae. Molting duration of second instar larvae was shortened by a half day at the warmer thermal regime but was not affected by the allelochemicals. Final larval weight was influenced by rutin and chlorogenic acid. Caterpillars fed diets containing 20 moles of rutin were on average 10% lighter than those fed plain diet, whereas those fed diets containing 20 moles of chlorogenic adic were on average 7% heavier. However, the effect of chlorogenic acid depended on thermal regime. Overall, our results indicated that: 1) temperature and food quality can interact to influence insect performance and 2) these effects are influenced by larval age.