Inhibition of stomatal opening during uptake of carbohydrates by guard cells in isolated epidermal tissues

The uptake of glucose and other carbohydrates into the guard cells of Commelina communis L. was found to inhibit the opening of the stomata. The concentration of glucose necessary to achieve about 50% inhibition was of the same order of magnitude as the potassium concentration required for opening; the uptake systems for potassium and glucose appear to be competitive and to exhibit the same degree of affinity. It is suggested that the uptake of glucose occurs via a proton cotransport, which, depolarizing the membrane potential, slows down the electrogenic import of potassium ions. The process of stomatal closure, in contrast, appears not to be affected by carbohydrate uptake. In guard cells of Tulipa gesneriana L. and Vicia faba L., which do not possess subsidiary cells, import of glucose or other carbohydrates did not interfere with the regulation of stomatal movements.     

An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection.

A human mAb (HmAb) termed F105 was obtained by fusion of antibody-producing EBV-transformed cells with the HMMA2.11TG/O cell line. F105 is an IgG1 kappa antibody that binds to the surfaces of cells infected with all HIV-1 strains tested: MN, RF, IIIB, and SF2, but not uninfected cells. The HmAb immunoprecipitates GP120 from all four strains. F105 does not react with denatured GP120 on Western blots, but does react with viral lysates and purified GP120 dotted onto nitrocellulose filter paper under nondenaturing conditions. rGP120 from SF2 and soluble rCD4 inhibit antibody binding to infected cells in a dose-dependent manner. F105 inhibits the binding of free, infectious virions to uninfected HT-H9 cells with 50% of maximal (100%) inhibition at approximately 1 microgram/ml. F105 inhibits infection of HT-H9 cells by 100 tissue culture infective dose 50% units of MN and IIIB strains with 50% inhibition at concentrations of HmAb readily achievable in man. It appears that the F105 HmAb reacts with a conformationally defined epitope on HIV-1/GP120 that is exposed on the free virion and is important for binding to the cell surface by the virion. The epitope, which is immunogenic in humans, appears to be within, or topographically near, the CD4-binding site. F105 and the F105 epitope are potentially useful in therapy and in the design of peptide or anti-Id based vaccines; monitoring of the expression of the Id may prove useful in evaluating immune responses in infected individuals or vaccinated volunteers.     

Continuous culture system for production of biopolymer levan using erwinia herbicola

The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and (13)C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low-angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer.     

Stimulation of human nitric oxide synthase by tetrahydrobiopterin and selective binding of the cofactor.

To check the stimulatory potency of the tetrahydro forms of the two major pteridines occurring in human tissues, neopterin and biopterin, NO synthase was purified 6000-fold from human cerebellum. Tetrahydrobiopterin stimulated the activity up to 4.5-fold in a concentration dependent manner with a maximum above 1 microM, whereas tetrahydroneopterin was completely inactive in concentrations up to 100 microM. Tetrahydrobiopterin, but not neopterin derivatives, were copurified with the NO synthase activity. Our results demonstrate that human cerebellum contains a tetrahydrobiopterin dependent NO synthase activity.     

Spatial and temporal patterns of intracellular calcium in colonic smooth muscle

Summary Intracellular calcium [Ca²⁺] _i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca²⁺] _i transient followed by a steady-state increase as the characteristic [Ca²⁺] _i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca²⁺] _i signal in single cells. The distribution of [Ca²⁺] _i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca²⁺] _i present in the subplasmalemmal space and in one cell pole. [Ca²⁺] _i gradients within these regions were not constant but showed temporal changes in the form of [Ca²⁺] _i oscillations and spatial changes in the form of [Ca²⁺] _i waves. [Ca²⁺] _i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca²⁺] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca²⁺], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca²⁺] _i waves was also independent of influx of extracellular Ca²⁺. [Ca²⁺] _i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca²⁺] _i induced by different agonists were encoded into changes of baseline [Ca²⁺] _i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca²⁺] _i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca²⁺] _i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca²⁺] _i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar⁹,Met(O₂)¹¹]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.     

Effects of prolonged estrogen-progesterone treatment and hypophysectomy on the stimulation of short-latency maternal behavior and aggression in female rats

Two experiments were undertaken to examine the stimulation of home-cage and/or maternal aggressiveness by a hormonal treatment stimulating short-latency maternal behavior. Nonpregnant ovariectomized rats were treated with a 16-day regimen providing pregnancy levels of estrogen (E, 5-mm Silastic capsule) and progesterone (P, daily injection of 4 mg) followed by E and P withdrawal, with or without a terminal injection of estradiol benzoate (EB, 5 micrograms/kg). In Experiment 1, hormonally treated and control females were exposed continuously to pups and tested for aggression toward male intruders on the fifth day of pup exposure. Females receiving E/P/Oil and E/P/EB were highly aggressive whether or not they had yet shown maternal behavior, whereas vehicle-treated females were nonaggressive. In Experiment 2, hypophysectomized (HYPX) and Sham-HYPX females received either E/P/EB or a control treatment and were tested with male intruders (a) immediately preceding and (b) on the fifth day of continuous pup exposure. HYPX and Sham-HYPX females treated with E/P/EB were almost equally aggressive both preceding and following pup exposure (during which they initiated maternal care), whereas HYPX and Sham-HYPX vehicle-treated females were nonaggressive at both tests. In contrast, maternal behavior latencies were reduced by E/P/EB only among Sham-HYPX females. The results establish that an E/P/EB-treatment which elicits short-latency maternal responses also increases aggressiveness toward intruders. Pituitary products, although involved in the mediation of maternal responsiveness, do not contribute significantly to the stimulation of female aggressiveness by ovarian hormones.     

The cyclic diguanylic acid regulatory system of cellulose synthesis in Acetobacter xylinum. Chemical synthesis and biological activity of cyclic nucleotide dimer, trimer, and phosphothioate derivatives

An unusual compound, cyclic-bis(3'----5') diguanylic acid (c-di-GMP or cGpGp), is involved in the regulation of cellulose synthesis in the bacterium Acetobacter xylinum. This cyclic dinucleotide acts as an allosteric, positive effector of cellulose synthase activity in vitro (Ka = 0.31 microM) and is inactivated via degradation by a Ca2(+)-sensitive phosphodiesterase, PDE-A (Km = 0.25 microM). A series of 13 analogs cyclic dimer and trimer nucleotides were synthesized, employing a phosphotriester approach, and tested for the ability to mimick c-di-GMP as activators of cellulose synthase and as substrates for PDE-A. Seven of the synthetic compounds stimulate cellulose synthase activity and all of these activators undergo the Ca2(+)-inhibited degradation reaction. The order of affinities for synthase activators is cGpGp approximately cdGpGp approximately cGp(S)Gp (S-diastereomer) greater than cIpGp greater than cdGpdGp greater than cXpGp greater than cIpIp greater than cGp(S)Gp (R-diastereomer). Three cyclic dinucleotides of negligible affinity for either enzyme are cApAp, cUpUp, and cCpCp. This same order of affinities essentially pertains to the analogs as inhibitors of PDE-A activity, but at least one cyclic dinucleotide, cXpXp, which does not bind to cellulose synthase, is also a substrate for the degradation reaction, demonstrating that although the two enzymes share a similar, high degree of specificity for c-di-GMP, their cyclic dinucleotide binding sites are not identical. Phosphodiester bonds of activators in which an exocyclic oxygen is replaced with an atom of sulfur (cGp(S)Gp isomers) resist the action of PDE-A, and such derivatives may be prototypes for synthetic non-hydrolyzable c-di-GMP analogs.