Repression of Asolectin-Dependent Activation of Partially Lipid Depleted ATPase Prepared from the Plasma Membrane-Enriched Fraction of Cucumber Roots due to Ca2+ Starvation

The ATPase activity of the plasma membrane-enriched fractionwas severely inhibited by withdrawal of Ca²⁺ from the mediumfor 5 days, although the root system appeared to be unaffectedto visual inspection. Partially lipid-depleted ATPases withsimilar ratios of phospholipid to protein were prepared fromthe plasma membrane-enriched fraction of cucumber roots culturedwith control medium and one lacking Ca²⁺, and their propertieswere compared. SDS disc polyacrylamide gel electrophoresis showedthat the polypeptide components were essentially similar betweencontrol and Ca²⁺-starved roots. Partially lipid-depleted ATPasereassociated with asolectin, the lecithin from soybean, showedtypical characteristics of plasma membrane type ATPase; pH optimumat 6.5, high specificity for ATP as substrate and strong inhibitionby vanadate but not nitrate. The activity of reassociated ATPaseobtained from the control roots was apparently higher than theactivity obtained from Ca²⁺-starved roots. The amount of asolectinrequired for maximum activation of the partially lipid-depletedATPase prepared from control roots was much lower than thatprepared from Ca²⁺-starved roots. Reassociation of partiallylipid-depleted ATPase with asolectin produced higher ATPaseactivity than that with individual phospholipids. The activationof partially lipid-depleted ATPase prepared from control rootswith asolectin was not inhibited by addition of a sample preparedfrom Ca²⁺-starved roots. Thus, a decrease in the functionalassociation of ATPase with phospholipids might be one of thephysiological injuries in root cell membranes of cucumber causedby Ca²⁺ starvation. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received February 23, 1988; Accepted August 18, 1988)     

Repression of Proton extrusion from Intact Cucumber Roots and the Proton Transport Rate of Microsomal Membrane Vesicles of the Roots due to Ca2+ Starvation

Proton extrusion from cucumber roots decreased markedly duringCa²⁺ starvation in the presence of KC1. Vesicles with ATP-dependentproton transport activity were prepared from the microsomalmembrane fraction of control and Ca²⁺-starved roots. The protontransport rate of the vesicles from Ca²⁺-starved roots was repressedto less than half of the vesicles prepared from the controlroots. K⁺-Mg²⁺-ATPase activity associated with the vesiclesprepared from Ca²⁺-starved roots was approximately one-thirdof the activity associated with those prepared from controlroots. K_m values of the proton transport rate and K⁺-Mg²⁺-ATPasefor ATP were much higher in vesicles prepared from Ca²⁺-starvedroots. The repression of proton extrusion linked with K⁺ uptake inthe Ca²⁺-starved roots could be largely caused by the reducedproton pumping activity associated with microsomal membranesin the roots. (Received May 25, 1987; Accepted October 14, 1987)     

Increase in Proton-Transport Activity of Tonoplast Vesicles as an Adaptive Response of Barley Roots to NaCl Stress

H⁺-Transport activity of the vesicles prepared from barley rootswas studied at the early phase after application of NaCl stress.The activity reached maximal level at 3 days after the treatmentwith 200 mM NaCl which moderately reduced the growth. This activityincrease could be suppressed in the presence of cycloheximideand actinomycin D. The properties of the membrane vesicles associated with H⁺-transportactivity prepared from both control and NaCl-stressed rootssuggested that it was of tonoplast origin based on the followingfindings: optimal pH at 7.5, strong inhibition by nitrate butnot by vanadate, and stimulation by chloride. The density gradient centrifugation of vesicles with DextranT70 did not show any detectable difference in the distributionpatterns of H⁺-transport activities between control and NaClstressedroots. Furthermore, K_m values for ATP of the H⁺-transport activityof vesicles prepared from control and NaCl-stressed roots werethe same. Therefore, H⁺-transport activity with properties similarto those of the control roots was increased by NaCl stress.The results are discussed in terms of an adaptive mechanismof barley against salt stress. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received April 18, 1988; Accepted July 20, 1988)     

Morphological differentiation of endothelial cells co-cultured with astrocytes on type-I or type-IV collagen

Summary In this study bovine aortic endothelial cells were co-cultured with astrocytes from fetal Wistar Kyoto rats. Endothelial cells growing on type-I collagen, development. Although some cells appeared to be mature, horseradish peroxidase penetrated within 1 min of incubation through the intercellular junctions of these endothelial elements maintained on type-I collagen. In contrast, endothelial cells on type-IV collagen, co-cultured with astrocytes, were well developed; their intercellular junctions were well established, and plasmalemmal vesicles reduced in number. As a result, horseradish peroxidase was unable to penetrate through the endothelial cells grown on type-IV collagen and co-cultured with astrocytes because of the reduced extent of the junctional and vesicular transport. These findings reveal that (1) type-IV collagen is essential for the differentiation of endothelial cells, (2) endothelial cell-astrocyte interactions occur during co-culture, and (3) endothelial permeability depends on astrocyte-produced factors, in addition to type-IV collagen.     

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells

We have isolated and characterized the genomic clone CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspensioncultured cells, the chimeric gene consisting of 1.1 kb CHN50 5 upstream region fused to the coding sequence of -glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5 deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

Antibiotic production by the marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: localization of activity to the chromatophores

Abstract Over 200 strains of marine purple photosynthetic bacteria were isolated. Two strains showed antibiotic activity towards Saccharomyces cerevisiae and were tentatively identified as Chromatium purpuratum . Crude antibiotic, prepared by solvent extraction, showed a broad antimicrobial spectrum. The highest activity was found in the chromatophore fraction. Chromatographic separation of purified light harvesting complex from one strain, NKPB 031704, showed the presence of two separate pigmented compounds which were responsible for antimicrobial activity. Our findings reveal the unexpected ability of photosynthetic bacteria to produce broad spectrum antibiotics. In addition, this is the first example of intracellular localization of antibiotic activity in a marine bacterium.     

Effect of Calcium on the Structure and Template Activity of Pea Chromatin

The absorption spectrum of native pea chromatin solubilizedunder minimal shearing conditions changed with increasing Ca²⁺concentration; the ratio of maximum to minimum absorption decreasedand the maximum absorption peak shifted to a longer wavelength.The concentration of Ca²⁺ to cause half complete sedimentationof chromatin was much lower for the solubilized native chromatin(more condensed and larger in size) than for the sonicated chromatin(less condensed and smaller in size). Solubilized native chromatinshowed a two-step melting profile in the absence of Ca²⁺. In the presence of Ca²⁺ the two T_ms disappeared and a new higherT_m appeared. Template activity of solubilized native chromatinincreased 3-fold upon dispersion and fragmentation by sonication.Addition of a small amount of ethylene glycol-bis (ß-aminoethylether)-N, N'-tetraacetic acid (EGTA) promoted the template activityof solubilized native chromatin, but not that of sonicated ordenatured DNA. The effect of EGTA was reversed by Ca²⁺. Thechromatin reconstituted in the presence of EGTA showed a lowerT_m than the chromatin reconstituted in the presence of Ca²⁺.The relationship between chromatin structure and its templateactivity is discussed in relation to Ca²⁺. (Received August 12, 1985; Accepted December 7, 1985)     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)