Possible Involvement of Inhibitory GTP Binding Regulatory Protein in α2-Adrenoceptor-Mediated Inhibition of Adenylate Cyclase Activity in Cerebral Cortical Membranes of Rats

Influences of alpha 2-adrenoceptor stimulation on adenylate cyclase activity were investigated in cerebral cortical membranes of rats. Pretreatment of the membranes with islet-activating protein and NAD resulted in a significant increase in basal activity as well as in GTP- or forskolin/GTP-induced elevation of adenylate cyclase activity. Strong activation of adenylate cyclase was also caused in membranes pretreated with cholera toxin together with NAD in comparison to that in control membranes, suggesting that adenylate cyclase activity is perhaps regulated by stimulatory and inhibitory GTP binding regulatory protein existing in synaptic membranes. In addition, adrenaline (with propranolol) or clonidine significantly reduced adenylate cyclase activity stimulated by pretreatment with forskolin and GTP. The inhibitory effects of adrenaline were also observed in membranes pretreated with cholera toxin and NAD. Moreover, the inhibition by adrenaline or clonidine was completely abolished by treatment with (a) yohimbine or (b) islet-activating protein and NAD. It is suggested that alpha 2-receptor stimulation causes inhibitory influences on adenylate cyclase activity mediated by the inhibitory GTP binding regulatory protein in synaptic membranes of rat cerebral cortex.     

Construction and characterization of a cosmid of Streptomyces lividans

Summary We constructed a plasmid pR4C1 in which a DNA fragment containing the cohesive ends of an actinophage, R4 was inserted into the ClaI site of plasmid pIJ365. The plasmid pR4C1 was packaged efficiently into R4 phage particles in vivo and therefore transferred by transduction. Southern hybridization analysis of DNA from pR4C1-transducing R4 phage particles indicated that the plasmid DNA was encapsidated as head-to-tail concatemers with the cohesive ends in the termini. We designated the pR4C1 plasmid a cosmid, following the termination of Collins and Hohn (1978).     

Temperature-dependent QTLs in <Emphasis Type="Italic">indica</Emphasis> alleles for improving grain quality in rice: increased prominence of QTLs responsible for reduced chalkiness under high-temperature conditions

Quantitative trait loci (QTLs) for the apparent quality of brown rice under high temperatures during ripening were analyzed using chromosomal segment substitution lines. Segments from the indica cultivar Habataki were substituted into a japonica cultivar with a Sasanishiki background. We found the following two QTLs for increasing grain quality in the Habataki allele on chromosome 3: (1) qTW3-2, located near the marker RM14702, decreased the percentage of total white immature (TWI) grains, and (2) qRG3-2, located near RM3766, increased the percentage of regular grains. The effects of these two QTLs were more obvious under high-temperature ripening conditions; hence, these loci are considered QTLs not only for reducing TWI grains but also for increasing high-temperature tolerance. Additionally, we found two QTLs, i.e., qTW3-1 and qRG3-1, responsible for reduced grain quality near RM14314 on chromosome 3. Although the QTL for narrow grains in the Habataki allele qNG3 was genetically linked to qTW3-2, the effect was only slightly significant, and the length/width ratio of qNG3-carrying grains was within the range observed in widely grown japonica cultivars. Incorporating the Habataki region, including qRG3-2 and qTW3-2 but not qTW3-1 and qRG3-1, in addition to previously reported grain quality QTLs in breeding japonica cultivars will improve high-temperature tolerance and grain quality.     

Seasonal differences in arbuscular mycorrhizal fungal communities in two woody species dominating semiarid caatinga forests

Tropical dry forests are strongly affected by seasonality, but its effects on belowground communities are poorly studied. Thus, the objective of this study was to reveal the effect of the season (dry versus wet) on the mycorrhizal status of roots and their potential colonization, and to determine the composition and abundance of spore-based communities of arbuscular mycorrhizal fungi (AMF) in rhizospheric soil of two dominant woody species in caatinga communities (tropical dry forest of the Brazilian Northeast). Soil and root samples were taken four times in each season (dry and wet). In the cases of the number of glomerospores and the number of infective propagules of AMF, there were significant differences between the hosts, with greater values observed in the rhizosphere of Commiphora leptophloeos than Mimosa tenuiflora. Mycorrhizal colonization and the number of infective propagules of AMF differed also between the seasons, being higher in the dry than the wet season. In total, fourteen AMF species were found in the rhizosphere of C. leptophloeos and twelve species were associated with M. tenuiflora. There was a predominance of the fungal genus Acaulospora, with seven species, followed by Gigaspora and Glomus. The species studied and the seasons differ in the composition and structure of the AMF community in the rhizosphere of the plants. The ecological significance of those differences needs to be examined further.     

Novel Drought-Inducible Genes in the Highly Drought-Tolerant Cowpea: Cloning of cDNAs and Analysis of the Expression of the Corresponding Genes

Ten cDNAs of genes that were induced by dehydration stress werecloned by differential screening from the highly drought-tolerantlegume, cowpea (Vigna unguiculata), a major crop in West Africa.The clones were collectively named CPRD (cowpea clones responsiveto dehydration). Northern blot analysis revealed that nine ofthe CPRD genes were induced by dehydration stress, but the timingof induction of mRNA synthesis varied among the CPRD genes.We analyzed the effects of other environmental stresses on theexpression of the CPRD8, CPRD14 and CPRD22 genes, and we foundthat these genes were strongly induced by high-salinity stressbut not by cold or heat stress. Drought-stressed cowpea plantsaccumulated abscisic acid (ABA) to a level that was 160 timeshigher than that in unstressed plants. The CPRD8 and CPRD22genes were induced to a significant extent by the applicationof exogenous ABA but the CPRD14 gene was not. These resultsindicate the existence of at least two signal-transduction pathwaysbetween the detection of water stress and the expression ofCPRD genes in cowpea. Sequence analysis of CPRD8 and CPRD22cDNAs revealed that they encoded putative proteins that wererelated to old yellow enzyme and group 2 LEA proteins, respectively.The protein encoded by CPRD14 exhibited sequence homology todihydroflavonol-4-reductase (DFR) and vestitone reductase (VR).Old yellow enzyme, DFR and VR have not been identified as drought-inducibleproteins in other plants, whereas LEA genes have been well characterizedas drought-inducible genes. The various gene products mightfunction to protect cells from environmental stress. (Received April 17, 1996; Accepted August 28, 1996)     

Involvement of protein phsophatase-1 in cytoskeletal organization of cultured endothelial cells

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effect of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vien endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton. © 1995 Wiley-Liss, Inc.     

Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies

The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle-shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers.     

Nested Deletions from a Fixed Site as an Aid to Nucleotide Sequencing: an in vitro System Using Tn3 Transposase

We have previously constructed a cloning/sequencing vector,with an in vivo system capable of creating nested deletionsfrom the end of transposon Tn3, which is useful for sequencinglarge DNAs. Here we report an in vitro system which uses anammonium sulfate fraction of extract from E. coli cells harboringa Tn3 transposase overproducer plasmid to generate nested deletions.A key feature of the procedure is exhaustive digestion of thereaction products with a restriction enzyme that cleaves onlybetween the Tn3 "right" terminus and the cloned fragment. Thisstep reduces the noise level due to mechanisms other than deletionsfrom the Tn3 terminus, and facilitates detection and isolationof the desired deletion products. This system enables us tosave at least 2 days' time when obtaining the necessary deletionscompared with the in vivo system.     

Growth inhibition of a human oral bacterium Porphyromonas gingivalis by rat cysteine proteinase inhibitor cystatin S

T. UMEMOTO, Y. NAITO, M. LI, I. SUZUKI AND I. NAMIKAWA. 1996. Agar diffusion analysis demonstrated that rat cystatin S, a cysteine proteinase inhibitor, inhibited the growth of all tested strains of a human oral, Gram-negative anaerobic periodontopathogen Porphyromonas gingivalis. Its specific inhibitory activity against this tissue-invasive bacterium but not against other tested oral bacterial species emphasized the importance of specific cysteine proteinases for growth of P. gingivalis.     

Life history of amphibians and gravity.

Anurans hold a unique position in vertebrate phylogeny, as they made the major transition from water to land. Through evolution they have acquired fundamental mechanisms to adapt to terrestrial gravity. Such mechanisms are now shared among other terrestrial vertebrates derived from ancestral amphibians. Space research, using amphibians as a model animal, is significant based on the following aspects: (1) Anuran amphibians show drastic changes in their living niche during their metamorphosis. Environments for tadpoles and for terrestrial life of frogs are quite different in terms of gravity and its associated factors. (2) Certain tadpoles, such as Rhacophorus viridis amamiensis, have a transparent abdominal wall. Thus visceral organs and their motion can be observed in these animals in non-invasive manner through their transparent abdominal skin. This feature enables biologists to evaluate the physiological state of these amphibians and study the autonomic control of visceral organs. It is also feasible for space biologists to examine how such autonomic regulation could be altered by microgravity and exposure to the space environment.