Sequence homologies of glucose-dehydrogenases of Bacillus megaterium and Bacillus subtilis

The sequence homologies of the glucose dehydrogenase subunits of B. megaterium and B. subtilis are compared. From the known B. megaterium aminoacid sequence and the base sequence of the cloned B. subtilis structural gene we predict the B. megaterium structural glucose dehydrogenase gene. Assuming the minimal mutational changes to convert one gene into the other 23 transitions, 30 transversions, 1 inversion, 3 insertion-deletions, but no frameshifts are postulated necessary to interconvert the structural genes. The homology of both enzyme subunits of 85% reflects the close evolutionary distance between B. subtilis and B. megaterium.     

Protein Synthesis Linked to Respiration and Phosphorylation in Intact Euglena Mitochondria

Highly purified condensed mitochondria obtained from bleachedmutant. W₁₀BSmL of Euglena gracilis Klebs var bacillaris Coriincorporate [³⁵S]methionine into protein when fortified withmalate, ADP, Mg²⁺, phosphate and a sucrose osmoticum. Twentyto twenty-five polypeptide bands were found to be labeled inorganello when the labeled protein was subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis. Methionine incorporation,but not respiration or oxidative phosphorylation, was blockedby chloramphenicol and other 70S ribosomal translation inhibitorsbut cycloheximide and ribonuclease were without effect. Inhibitorsof electron transport and uncouplers of oxidative phosphorylationwere excellent inhibitors of protein synthesis. Thus, thesemitochondrial preparations carry out protein synthesis in organellothat is linked to respiration and oxidative phosphorylation. ¹Present address: VA Hospital Outpatient Clinic, 17 Court St.,Boston, MA 02115, U.S.A. ²Present address: Laboratories de Microbiologia e Inmunologia,Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile. ³Present address: Botany Department, University of Massachusetts,Amherst, MA 01003, U.S.A. (Received June 17, 1985; Accepted October 28, 1985)     

Polyamine Metabolism and Osmotic Stress : II. Improvement of Oat Protoplasts by an Inhibitor of Arginine Decarboxylase

We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with dl-α-difluoromethylarginine (DFMA), a specific `suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.     

A comparison of the submergence response of deepwater and non-deepwater rice

Twelve cultivars of rice (Oryza sativa L.), representing deepwater, short-statured, and semidwarf types, were tested for their response to submergence. The magnitude of the response varied between cultivars; however, all cultivars responded to submergence by rapid growth once internodal elongation had started. Three of these cultivars were tested for elongation capacity at four ages. The deepwater rice was capable of rapid internodal elongation in response to submergence at 4 weeks of age. Growth of the short-statured and semidwarf cultivars was not stimulated by submergence until about 10 weeks of age. In air, the internodes of deepwater rice grew slower than did those of the short-statured and semidwarf cultivars. We also investigated the elongation response of stem sections of all 12 cultivars to an atmosphere containing 3% O₂, 6% CO₂, 91% N₂ (all by volume), and 1 microliter per liter ethylene. We found that the response of each of the non-deepwater cultivars was qualitatively and quantitatively similar to that of the deepwater rice.     

Transport of gibberellin a(1) in cowpea membrane vesicles

The permeability properties of gibberellin A₁ (GA₁) were examined in membrane vesicles isolated from cowpea hypocotyls. The rate of GA₁ uptake was progressively greater as pH decreased, indicating that the neutral molecule is more permeable than anionic GA₁. Membrane vesicles used in this study possessed a tonoplast-type H⁺-translocating ATPase as assayed by MgATP-dependent quenching of acridine orange fluorescence and methylamine uptake. However, GA₁ uptake was not stimulated by MgATP. At concentrations in excess of 1 micromolar, GA₁, GA₅, and GA, collapsed both MgATP-generated and artifically imposed pH gradients, apparently by shuttling H⁺ across the membrane as neutral GA. The relatively high permeability of neutral GA and the potentially detrimental effects of GA in uncoupling pH gradients across intracellular membranes supports the view that GA₁ accumulation and compartmentation must occur by conversion of GA₁ to more polar metabolites.     

GABAB Receptor-Mediated Enhancement of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Production in Slices of Rat Cerebral Cortex

Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose-related enhancement of the VIP response. The ability of either GABA or (-)-baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors.     

Cross-polarization/magic-angle spinning 13C-nmr of (1 → 6)-β-D-glucan (pustulan): Mechanism of gelation

¹³C-nmr has been employed to probe the molecular conformation and crystal structure of (1 → 6)-β-D -glucan (pustulan) in the solution, gel, and solid states. CP/MAS ¹³C-nmr spectra recorded for partially crystalline solid pustulan display a resonance near 82 ppm that is absent in solution spectra. The intensity and peak width of this resonance were found to depend on relative crystallinity as determined by x-ray diffraction. CP/MAS spectra of aqueous pustulan gels also exhibit the 82-ppm resonance, suggesting that the gelation mechanism may involve microcrystalline junction zones. Since the 82-ppm resonance is absent in the CP/MAS spectrum of the (1 → 6)-β-linked dimer gentiobiose, we tentatively conclude the crystal structure of this dimer does not adequately model the yet undetermined structure of pustulan.     

Calcium at the surface of cardiac plasma membrane vesicles: Cation binding,surface charge screening,and Na−Ca exchange

Summary Calcium binding and Na–Ca exchange activity were measured in isolated cardiac plasma membrane vesicles under various ionic conditions. A model was developed to describe the Ca binding characteristics of cardiac sarcolemmal vesicles using the Gouy-Chapman theory of the diffuse double layer with specific cation binding to phospholipid carboxyl and phosphate groups. The surface association constants used for Ca, Na, K and H binding to both of these groups were 7, 0.63, 0.3 and 3800m ^–1, respectively. This model allows the estimation of surface [Ca] under any specific ionic conditions. The effects of the divalent screening cation, dimethonium, on Ca binding and Na–Ca exchange were compared. Dimethonium had no significant effect on Ca binding at high ionic strength (150mm KCl), but strongly depressed Ca binding at low ionic strength. Dimethonium had no significant effect on Na–Ca exchange (Na-inside dependent Ca influx) at either high or low ionic strength. These results suggest that the Ca sites of the Na–Ca exchanger are in a physical environment where they are either not exposed to or not sensitive to surface [Ca].     

Primeval cells: Possible energy-generating and cell-division mechanisms

Summary It is proposed that the first entity capable of adaptive Darwinian evolution consisted of a liposome vesicle formed of (1) abiotically produced phospholipidlike molecules; (2) a very few informational macromolecules; and (3) some abiogenic, lipid-soluble, organic molecule serving as a symporter for phosphate and protons and as a means of high-energy-bond generation. The genetic material had functions that led to the production of phospholipidlike materials (leading to growth and division of the primitive cells) and of the carrier needed for energy transduction. It is suggested that the most primitive exploitable energy source was the donation of 2H⁺+2e^– at the external face of the primitive cell. The electrons were transferred (by metal impurities) to internal sinks of organic material, thus creating, via a deficit, a protonmotive force that could drive both the active transport of phosphate and high-energy-bond formation.This model implies that proton translocation in a closed-membrane system preceded photochemical or electron transport mechanisms and that chemically transferable metabolic energy was needed at a much earlier stage in the development of life than has usually been assumed. It provides a plausible mechanism whereby cell division of the earliest protocells could have been a spontaneous process powered by the internal development of phospholipids. The stimulus for developing this evolutionary sequence was the realization that cellular life was essential if Darwinian survival of the fittest was to direct evolution toward adaptation to the external environment.     

Superoxide dismutase and glutathione peroxidase activities in neutrophils from selenium deficient and copper deficient cattle

Oxygen consumption and the activities of the selenoenzyme glutathione peroxidase, and of the hexose monophosphate shunt were lower than normal in neutrophils from Se deficient cattle. However, these activities and the activity of Cu/zinc superoxide dismutase were unaffected in neutrophils from Cu deficient cattle. These results are discussed with reference to impaired neutrophil microbicidal activity previously demonstrated to result from Se or Cu deficiency in cattle.