Starch-binding domain shuffling in Aspergillus niger glucoamylase

Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N-terminus, catalytic domain + linker + starch-binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIDeltaL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII approximately GAII approximately GAI > RGAIDeltaL approximately RGAI approximately GAE. Insoluble starch hydrolysis rates were GAI > RGAIDeltaL > RGAI > GAE approximately RGAII > GAII, while insoluble starch-binding capacities were GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE. These results indicate that: (i) moving the SBD to the N-terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.macerans cyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch.     

Caenorhabditis elegans EVL-14/PDS-5 and SCC-3 are essential for sister chromatid cohesion in meiosis and mitosis

Sister chromatid cohesion is fundamental for the faithful transmission of chromosomes during both meiosis and mitosis. Proteins involved in this process are highly conserved from yeasts to humans. In screenings for sterile animals with abnormal vulval morphology, mutations in the Caenorhabditis elegans evl-14 and scc-3 genes were isolated. Defects in cell divisions were observed in germ line as well as in vulval and somatic gonad lineages. Through positional cloning of these genes, we have shown that EVL-14 and SCC-3 are likely the only C. elegans homologs of the yeast sister chromatid cohesion proteins Pds5 and Scc3, respectively. Both evl-14 and scc-3 mutants displayed defects in the meiotic germ line. In evl-14 mutants, synaptonemal complexes (SCs) were detectable but more than the usual six DAPI (4',6'-diamidino-2-phenylindole)-positive structures were seen at diakinesis, suggesting that EVL-14/PDS-5 is important for the maintenance of sister chromatid cohesion in late prophase. In scc-3 mutant animals, normal SCs were not visible and approximately 24 DAPI-positive structures were seen at diakinesis, indicating that SCC-3 is necessary for sister chromatid cohesion. Immunostaining revealed that localization of REC-8, a homolog of the yeast meiotic cohesin subunit Rec8, to the chromosomes depends on the presence of SCC-3 but not that of EVL-14/PDS-5. scc-3 RNA interference (RNAi)-treated embryos were 100% lethal and displayed defects in cell divisions. evl-14 RNAi caused a range of phenotypes. These results indicate that EVL-14/PDS-5 and SCC-3 have functions in both mitosis and meiosis.     

Histidine-rich glycoprotein plus zinc reverses growth inhibition of vascular smooth muscle cells by heparin

Vascular smooth muscle cell (SMC) hyperplasia is known to be an important component in the pathogenesis of arteriosclerosis and restenosis. Although heparin has been well recognized as the representative molecule suppressing SMC growth in vitro, attempts to use heparin as a therapeutic anti-restenosis drug have not favorably influenced the angiographic or clinical outcome after angioplasty in some clinical trials. In this study, we have examined the effect of histidine-rich glycoprotein (HRG), a relatively abundant serum glycoprotein (~100 micrograms/ml in human serum), on the growth inhibition of cultured vascular SMC by heparin. Vascular SMC growth was significantly inhibited by heparin, giving nearly 85% inhibition with 100 micrograms/ml heparin. HRG reversed heparin-induced SMC growth inhibition in a dose dependent manner; 75% restoration of cell growth was observed when 100 micrograms/ml of HRG was co-added with 100 micrograms/ml heparin. Interestingly, micromolar concentrations of the zinc ion (0-10 microM), compatible with concentrations released from activated platelets, were found to enhance the restorative action of HRG. Western blot experiment demonstrated no significant amounts of the HRG moiety in fetal bovine serum, eliminating the possible contribution of contaminant HRG from culture media. These findings indicate that HRG, in combination with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explain the lack of success of heparin as a therapeutic anti-restenosis drug in clinical trials.     

Synthesis of beta-substituted cationic porphyrins and their interactions with DNA

The beta-substituted cationic porphyrins (7, 8 and 10) have been synthesized and their interactions with plasmid DNA investigated. We found that substituents at the beta-position of porphyrins (7 and 8) have apparently affected their interactions with DNA compared with non-beta-substituted porphyrins (10).     

Frogs without polliwogs: evolution of anuran direct development

Direct development is the assumption of the adult morphology without progression through an intervening, morphologically distinct, free-living larval phase. We discuss the ecological factors contributing to the evolution of this derived life-history strategy in frogs, and the developmental modifications that facilitate such an unusual mode of embryogenesis. Studies on the Puerto Rican tree frog, Eleutherodactylus coqui, have identified several such modifications, including developmental adaptations for dealing with increased egg size, and loss of tadpole structures. Surprisingly, this direct developer still undergoes a thyroid hormone-dependent metamorphosis, which occurs before hatching. We suggest how the ancestral biphasic developmental pattern may have been rearranged during the evolution of direct development.     

Interleukin-18 promotes sleep in rabbits and rats

Interleukin (IL)-1beta is involved in physiological sleep regulation. IL-18 is a member of the IL-1 family, and its signal-transduction mechanism is similar to that of IL-1. Therefore, we hypothesized that IL-18 might also be involved in sleep regulation. Three doses of IL-18 (10, 100, and 500 ng) were injected intracerebroventricularly (icv) into rabbits at the onset of the dark period. The two higher doses of IL-18 markedly increased non-rapid eye movement sleep (NREMS), accompanied by increases in brain temperature (Tbr). These effects were lost after the heat inactivation of IL-18. The 500 ng of IL-18 injection during the light period also increased NREMS and Tbr. Similar results were obtained after icv injection of 100 ng of IL-18 into rats. Furthermore, intraperitoneal injection of 30 microg/kg of IL-18 slightly, but significantly, increased NREMS, whereas it significantly decreased electroencephalogram slow-wave activity in rats. Intraperitoneal IL-18 failed to induce fever. An anti-human IL-18 antibody had little effect on spontaneous sleep in rabbits, although the anti-IL-18 antibody significantly attenuated muramyl dipeptide-induced sleep. These data suggest that IL-18 is involved in mechanisms of sleep responses to infection.     

Conditions that affect sleep alter the expression of molecules associated with synaptic plasticity

Many theories propose that sleep serves a purpose in synaptic plasticity. We tested the hypothesis, therefore, that manipulation of sleep would affect the expression of molecules known to be involved in synaptic plasticity. mRNA expression of four molecules [brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeleton-associated protein (Arc), matrix metalloproteinase-9 (MMP-9), and tissue plasminogen activator (tPA)] was determined after 8 h of sleep deprivation and after 6 h of a mild increase in ambient temperature, a condition that enhances sleep in rats. After sleep deprivation, BDNF, Arc, and tPA mRNAs in the cerebral cortex increased while MMP-9 mRNA levels decreased. Conversely, after enhanced ambient temperature, BDNF, Arc, and tPA mRNAs decreased while MMP-9 mRNA increased. In the hippocampus, sleep deprivation did not significantly affect BDNF and tPA expression, although Arc mRNA increased and MMP-9 mRNA decreased. Brain temperature enhancement decreased Arc mRNA levels in the hippocampus but did not affect BDNF, MMP-9, or tPA in this area. Results are consistent with the notion that sleep plays a role in synaptic plasticity.     

Interleukin-15 and interleukin-2 enhance non-REM sleep in rabbits

Interleukin (IL)-15 and -2 share receptor- and signal-transduction pathway (Jak-STAT pathway) components. IL-2 is somnogenic in rats but has not been tested in other species. Furthermore, the effects of IL-15 on sleep have not heretofore been described. We investigated the somnogenic actions of IL-15 in rabbits and compared them with those of IL-2. Three doses of IL-15 or -2 (10, 100, and 500 ng) were injected intracerebroventriculary at the onset of the dark period. In addition, 500 ng of IL-15 and -2 were injected 3 h after the beginning of the light period. IL-15 dose dependently increased non-rapid eye movement sleep (NREMS) and induced fever. IL-15 inhibited rapid eye movement sleep (REMS) after its administration during the light period; however, all doses of IL-15 failed to affect REMS if given at dark onset. IL-2 also dose dependently increased NREMS and fever. IL-2 inhibited REMS, and this effect was observed only in the light period. IL-15 and -2 enhanced electroencephalographic (EEG) slow waves during the initial 9-h postinjection period, then, during hours 10-23 postinjection, reduced EEG slow-wave activity. Current data support the notion that the brain cytokine network is involved in the regulation of sleep.     

Growth of Streptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production

Growth of Streptomyces hygroscopicus under conditions of simulated microgravity in a rotating-wall bioreactor resulted in a pellet form of growth, lowered dry cell weight, and inhibition of rapamycin production. With the addition of Teflon beads to the bioreactor, growth became much less pelleted, dry cell weight increased but rapamycin production was still markedly inhibited. Growth under simulated microgravity favored extracellular production of rapamycin, in contrast to a greater percentage of cell-bound rapamycin observed under normal gravity conditions. Received: 20 September 1999 / Received revision: 18 November 1999 / Accepted: 19 November 1999     

Dexras1: a G protein specifically coupled to neuronal nitric oxide synthase via CAPON

Because nitric oxide (NO) is a highly reactive signaling molecule, chemical inactivation by reaction with oxygen, superoxide, and glutathione competes with specific interactions with target proteins. NO signaling may be enhanced by adaptor proteins that couple neuronal NO synthase (nNOS) to specific target proteins. Here we identify a selective interaction of the nNOS adaptor protein CAPON with Dexras1, a brain-enriched member of the Ras family of small monomeric G proteins. We find that Dexras1 is activated by NO donors as well as by NMDA receptor-stimulated NO synthesis in cortical neurons. The importance of Dexras1 as a physiologic target of nNOS is established by the selective decrease of Dexras1 activation, but not H-Ras or four other Ras family members, in the brains of mice harboring a targeted genomic deletion of nNOS (nNOS-/-). We also find that nNOS, CAPON, and Dexras1 form a ternary complex that enhances the ability of nNOS to activate Dexras1. These findings identify Dexras1 as a novel physiologic NO effector and suggest that anchoring of nNOS to specific targets is a mechanism by which NO signaling is enhanced.