Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

Correlation of tumor metastasis with sterol carrier protein and plasma membrane sterol levels

Squalene and sterol carrier protein (SCP) levels and sterol/phospholipid molar ratios of whole cells and plasma membranes were measured in cultured primary tumor and metastatic cell lines. SCP is abundant in all cell lines. However, metastatic lines have significantly lower SCP levels and plasma membrane sterol/phospholipid ratios than do primary lines. The results indicate that extremely malignant, metastatic cells are unable to produce or maintain adequate levels of both SCP and plasma membrane sterols when grown in lipoprotein deficient media. This defect, in vivo, probably causes excess uptake of SCP and lipid.     

Comparison of gravimetry and planimetry in determining dry matter budgets for three species of phytophagous lepidopteran larvae

Gravimetric and a combination areal-gravimetric methods for determining dry matter budgets for leaf eating Lepidoptera were compared. The gravimetric method is based on dry weight/live weight ratios of the leaves fed to the larvae. In the areal-gravimetric method, the quantity of food offered to the larvae is determined from the area of leaf tracings and the dry matter content per unit area of the leaves. The areal-gravimetric method permits the use of larger leaf sprays and an open, gauze enclosed rearing chamber. There were no consistent differences in budget factors (growth, ingestion or egestion), nor were there any differences in the observed variability of the data attributable to the method used. However, the expected variability based on instrument precision for the gravimetric method is less than for the areal-gravimetric method. Experimental factors inherent in the gravimetric method introduce variability to the measurements that are not present in the areal method. Thirty to 60% of the variability in budget factors was attributed to intrinsic properties of the larvae, even though the larvae were taken from the same egg masses.

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Résumé Les budgets en matière sèche consommée par des lépidoptères ont été comparés par les méthodes gravimétrique et planimétrique. La méthode gravimétrique est basée sur le rapport poids sec/poids frais de feuilles consommées par les chenilles. Avec la méthode planimétrique, la quantité d'aliment proposée aux chenilles est déterminée par les tracés de la surface des feuilles et le contenu de matière sèche par unité de surface des feuilles. La méthode de planimétrie permet l'utilisation de plus grands rameaux de feuilles et de cages d'élevage extérieures en gaze. Il n'y avait pas de différence appréciable dans les éléments du budget (croissance, ingestion et déjection), ni aucune différence dans la variabilité observée des données attribuable à la méthode utilisée. Cependant, la variabilité attendue d'après la précision des mesures avec la méthode gravimétrique est inférieure à celle de la méthode planimétrique. est inférieure à celle de la méthode planimétrique. Des éléments expérimentaux, inhérents à la méthode gravimétrique, introduisent une variabilité dans les mesures que l'on n'a pas avec la méthode planimétrique. 30–60% de la variabilité dans la consommation ont été attribués à des paramètres internes à la chenille, même quand elles provenaient toutes de la même ooplaque.

     

Microtubule arrays in the cortex and near the germinal vesicle of immature starfish oocytes

An extensive array of long, crisscrossing microtubules has been discovered in the cortex of oocytes of the starfish Pisaster ochraceus. The microtubules were visualized in cortex preparations by indirect immunofluorescence microscopy using antibodies to tubulin. The cortical array of microtubules is present in all oocytes before and for about 30 min after the application of 1-methyladenine, the hormone that induces oocyte maturation. The presence of microtubules was confirmed by electron microscopy. The microtubules in this array are depolymerized when oocytes are treated with colchicine or nocodozole and are augmented when oocytes are treated with taxol. Dihydrocytochalasin B treatment of the oocytes causes the microtubules to aggregate, presumably by altering a microfilament network also found in the cortex. The distribution of microtubules was also explored in whole oocytes stained with antitubulin. One or two aster-like structures were observed adjacent to the germinal vesicle of each oocyte.     

Organization of microtubules in stabilized meristematic plant cells revealed by a rat monoclonal antibody reacting only with the tyrosinated form of alpha-tubulin

A rat monoclonal antibody against yeast tubulin (clone YL 1/2; Kilmartin et al., 1982) that reacts specifically with mammalian alpha-tubulin carrying a carboxyterminal tyrosine residue (Wehland et al., 1983) was used to localize microtubules in plant cells derived from onion root apices (Allium cepa L.). YL 1/2 reacted with all types of microtubular arrays known to occur in higher plant meristematic cells such as interphase cortical microtubules, pre-prophase bands, the mitotic spindle and phragmoplast microtubules. The specific labeling of microtubules in isolated cells from onion root tips by YL 1/2 indicates that plant cells like animal cells contain tubulin tyrosine ligase, the enzyme which posttranslationally modifies alpha-tubulin. This enzyme could be involved in the dynamic regulation of microtubular arrays in all eukaryotic cells.     

Physical map of the bacteriophage L (Salmonella typhimurium)

Abstract A circular restriction map of the genome of the phage L ( Salmonella typhimurium ) has been constructed with five restriction endonucleases, Eca I, Eco RI, Bam HI, Bgl I, and Pst I. The Eco RI fragments of phage-L DNA were cloned into pACYC184, and the resulting recombinant plasmids pL1, pL2,…,pL7 were introduced into Salmonella typhimurium . The genes present on the fragments cloned were identified by the marker rescue experiments with the L-phage amber mutants. A physical gene map of the L genome obtained in this way was compared with that of P22.     

Topo-optische Färbung der Erythrocytenmembran mit Thiazin- und Chinolinfarbstoffen

Zusammenfassung Die Untersuchungen erwiesen, daß die Farbstoffe 1:9-Dimethyl-Methylenblau, Azur A und N,N-Diäthylpseudoisozyaninchlorid für topo-optische Reaktionen an der Membran von Erythrocyten geeignet sind. Die Farbstoffmoleküle werden an der Membran orientiert gebunden. Ihre Bindung kann durch Behandlung mit Präzipitationslösungen stabilisiert werden, und zugleich wird die Anisotropie verstärkt. Die optische Analyse ergab, daß 1:9-Dimethyl-Methylenblau und Azur A radiär zur Membran ausgerichtet sind, während sich N,N-Diäthylpseudoisozyaninchlorid membranparallel anlagert.

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Tope-optical staining with thiazin and quinolin dyestuffs of the erythrocyte membrane

Summary The present studies prove the dyestuffs 1.9-dimethyl methyleneblue, azure A and N,N-diethylpseudoisocyanine chloride suitable for topo-optical reactions with the membrane of the red blood cell. The dye molecules are bound in orientated fashion. Treatment with precipitants stabilizes the binding of dye molecules and, in addition, it enhances the birefringence. Optical analysis revealed 1.9-dimethyl methyleneblue and azure A bound in radial position, however, N,N-diethylpseudoisocyanine chloride was bound parallel to the membrane's plane.

     

Influence of phage T3 and T7 gene functions on a type III(EcoP1) DNA restriction-modification system in vivo

Summary The ocr ⁺ gene function (gp 0.3) of bacteriophages T3 and T7 not only counteracts type I (EcoB, EcoK) but also type III restriction endonucleases (EcoP1). Despite the presence of recognition sites, phage DNA as well as simultaneously introduced plasmid DNA are protected by ocr ⁺ expression against both the endonucleolytic and the methylating activities of the EcoP1 enzyme. Nevertheless, the EcoP1 protein causes the exclusion of T3 and T7 in P1-lysogenic cells, apparently by exerting a repressor-like effect on phage gene expression. T3 which induces an S-adenosylmethionine hydrolase is less susceptible to the repressor effect of the SAM-stimulated EcoP1 enzyme. The abundance of EcoP1 recognition sites in the T7 genome is explained by their near identity with the T7 DNA primase recognition site.Abbreviations d.p.m. decompositions per min - EcoB, EcoK, EcoP1, EcoP15, EcoRII, EcoR124, HinfIII restriction endonucleases coded by Escherichia coli strains B or K, E. coli plasmids P1, P15, RII or R124, and Haemophilus influenzae Rf 232, resp. - e.o.p. efficiency of plating - gp gene product (in the sense of protein) - m.o.i. multiplicity of infection (phage/cell) - ocr ⁺ gene function which overcomes classical restriction - p.f.u. plaque-forming units - SAM S-adenosylmethionine - sam ⁺ gene function with S-adenosylmethionine-cleaving enzyme (SAMase) activity - UV ultraviolet light Dedicated to Professor Konstantin Spies on the occasion of his sixtieth birthday     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.