Mischarging mutants of Su+2 glutamine tRNA in E. coli. I. Mutations near the anticodon cause mischarging

In order to select the mischarging mutants of Su+2 glutamine tRNA, auxotrophic amber mutants of E. coli K12 which cannot be suppressed particularly by Su+2 were screened. By utilizing these mutants, cysam235 and metam3, several tens of mischarging mutants of Su+2 were isolated, as those conferring altered suppression patterns for a set of tester amber mutants of bacteria and phages. Nucleotide sequence analysis revealed that the mutation sites were found to be exclusively at psi 37 residue located at the 3'-end of anticodon loop, changing it to either A37 or C37. These mutants were obtained as those suppressing cysam235, and not metam3. From these, secondary mutants were selected. In these mutants suppression patterns were further altered by the additional base substitutions, capable of suppressing metam3. Such mutants were obtained exclusively from A37 and not from C37 mutant tRNA. Additional mutations to A37 were found to be either A29 or C38, which are located at the lowermost two base pairs in anticodon stem. The mischarging sites in Su+2 glutamine tRNA locate in the newly detected region of tRNA, differing from the previous case of Su+3 tyrosine or Su+7 tryptophan tRNAs. Implication of this finding is discussed on L-shaped tRNA molecule in relation to aminoacyl-tRNA synthetase recognition. Suppression patterns given by the double-mutants, A37A29 and A37C38, were consistent with the observation that the mutant tRNAs interact with tryptophanyl-tRNA synthetase.     

The membrane components of crustacean neuromuscular systems. I. Immunity of different electrogenic components to tetrodotoxin and saxitoxin

Axon spikes in crayfish and lobster neuromuscular preparations were blocked by tetrodotoxin or saxitoxin (concentration 10⁻⁹ to 10⁻⁸ g/ml). Responses evoked in the excitatory synaptic membrane by ionophoretically applied glutamate, or in the inhibitory by GABA were unaffected by concentrations of the poisons up to 10⁻⁵ g/ml. These confirm other findings that the poisons do not affect electrically inexcitable membrane components. “Miniature” p.s.p.’s, which indicate local secretory activity in the presynaptic terminals were unaffected by the poisons. Electrical stimuli applied to the axon terminals elicited localized p.s.p.’s after spike electrogenesis of the axons was blocked. Thus, persistence of secretory activity may be linked to persistence of depolarizing K activation in the axons. Spikes induced in the muscle fibers by procaine were not affected by the poisons. In correlation with other data this finding indicates that the depolarizing electrogenic element, which does not depend upon Na activation in the normally gradedly responsive muscles, differs chemically from the Na activation component which is present in the conductile membrane of various cells. Three other varieties of electrically excitable response which are present in crayfish muscle fibers (hyperpolarizing Cl activation, depolarizing K inactivation, and K activation) were, likewise, immune to the toxin.     

Isolation of an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase from the monocot Agapanthus africanus

A cDNA encoding an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase (AaAA7GT) was isolated from Agapanthus africanus petals; this is the first AAGT identified in a monocot. Peak expression of AaAA7GT in developing A. africanus petals occurred before the flowering stage, and was later than found previously for other anthocyanin biosynthetic genes. Analysis of recombinant proteins showed AaAA7GT had strict substrate preference for anthocyanidin 3-O-glycosides. The AaAA7GT amino acid had high sequence similarity to glycoside hydrolase family 1 (GH1) proteins, which typically act as β-glycosidases. A phylogenetic analysis of amino acid sequences suggested that AAGTs were derived from glycosidase early in the angiosperm lineage.     

alpha7 integrin expressing human fetal myogenic progenitors have stem cell-like properties and are capable of osteogenic differentiation

During muscle development, precursor cells fuse to form myofibers. Following injury in adult muscle, quiescent satellite cells become activated to regenerate muscle in a fashion similar to fetal development. Recent studies indicate that murine skeletal myoblasts can differentiate along multiple cell lineages including the osteoblastic pathway. However, little is known about the multipotency of human myogenic cells. Here, we isolate myogenic precursor cells from human fetal and adult muscle by sorting for the laminin-binding alpha7 integrin and demonstrate their differentiation potential and alteration in adhesive behavior. The alpha7-positive human fetal progenitors were efficient at forming myotubes and a majority expressed known muscle markers including M-cadherin and c-Met, but were heterogeneous for desmin and MyoD expression. To test their pluripotent differentiation potential, enriched populations of alpha7-positive fetal cells were subjected to inductive protocols. Although the myoblasts appeared committed to a muscle lineage, they could be converted to differentiate along the osteoblastic pathway in the presence of BMP-2. Interestingly, osteogenic cells showed altered adhesion and migratory activity that reflected growth factor-induced changes in integrin expression. These results indicate that alpha7-expressing fetal myoblasts are capable of differentiation to osteoblast lineage with a coordinated switch in integrin profiles and may represent a mechanism that promotes homing and recruitment of myogenic stem cells for tissue repair and remodeling.     

Identification and characterization of genes induced for anthocyanin synthesis and chlorophyll degradation in regenerated torenia shoots using suppression subtractive hybridization, cDNA microarrays, and RNAi techniques

Anthocyanin synthesis and chlorophyll degradation in regenerated torenia (Torenia fournieri Linden ex Fourn.) shoots induced by osmotic stress with 7% sucrose were examined to identify the genes regulating the underlying molecular mechanism. To achieve this, suppression subtractive hybridization was performed to enrich the cDNAs of genes induced in anthocyanin-synthesizing and chlorophyll-degrading regenerated shoots. The nucleotide sequences of 1,388 random cDNAs were determined, and these were used in the preparation of cDNA microarrays for high-throughput screening. From 1,056 cDNAs analyzed in the microarrays, 116 nonredundant genes were identified, which were up regulated by 7% sucrose to induce anthocyanin synthesis and chlorophyll degradation in regenerated shoots. Of these, eight genes were selected and RNAi transformants prepared, six of which exhibited anthocyanin synthesis inhibition and/or chlorophyll degradation in their leaf discs. Notably, the RNAi transformants of the glucose 6-phosphate/phosphate translocator gene displayed inhibition both of anthocyanin synthesis and chlorophyll degradation in both leaf discs and regenerated shoots. There was also less accumulation of anthocyanin in the petals, and flowering time was shortened. The genes we identified as being up-regulated in the regenerated torenia shoots may help further elucidate the molecular mechanism underlying the induction of anthocyanin synthesis and chlorophyll degradation.