Characteristics of Cl−-dependentl-[35S]cysteic acid transport into rat brain synaptic membrane vesicles

Uptake ofl-[³⁵S]cysteic acid (L-CA) in rat synaptic membrane vesicles was investigated. Preincubation with either 10 mMl-glutamic acid (L-Glu), 25 mM L-CA, 10 mMdl-homocysteic acid, or 25 mMdl-2-amino-4-phosphonobutyrate on membrane vesicles enhanced L-[³⁵S]CA and L-[³H]Glu uptake. Na⁺ (5 mM) and omission of Cl^– from the assay medium decreased L-[³⁵S]CA uptake into both 10 mM L-Glu-loaded and non-loaded membrane vesicles. The anion transport blockers, 4-acetamide-4-isothiocyano-2,2-disulfonic acid stibene (SITS) and 4,4-diisothiocyano-2,2-disulfonic acid stilbene (DIDS), inhibited L-[³⁵S]CA uptake in a dose-dependent manner. The maximal uptake rate for L-[³⁵S]CA was decreased by 50 M SITS, while the apparent Km value of L-CA was not changed. SITS increased the EC₅₀ value of Cl^– for L-[³⁵S]CA uptake from 5 mM to 10 mM with reduction of the maximal effect. These results suggested that L-[³⁵S]CA uptake into synaptic membrane vesicles was mediated by a SITS-sensitive hetero-exchange transport with non-labeled substrates.Abbreviations SITS 4-Acetamide-4-isothiocyano-2,2-disulfonic acid stilbene - DIDS 4,4-Diisothiocyano-2,2-disulfonic acid stilbene - CA Cysteic acid - APB 2-Amino-4-phosphonobutyrate - CSA Cysteine sulfinic acid - EGTA Ethyleneglycol bis(aminoethylether) tetraacetate - GABA -Aminobutyric acid     

Microtubule cytoskeleton in intact and wounded coenocytic green algae

Microtubule (MT) arrangements were investigated, with immunofluorescence and electron microscopy, in two related species of coenocytic green algae. Intact cells of both Ernodesmis verticillata (Kützing) Boergesen and Boergesenia forbesii (Harvey) Feldmann have two morphologically distinct populations of MTs: a highly regular cortical array consisting of a single layer of parallel, longitudinal MTs; and perinuclear MTs radiating from the surface of the envelope of each interphase nucleus. In both algae, mitotic figures lack perinuclear MTs around them. Pre-incubation with taxol does not alter the appearance of these arrays. The cortical and nuclear MTs appear to coexist throughout the nuclear cycle, unlike the condition in most plant cells. At the cut/contracting ends of wounded Ernodesmis cells, cortical MTs exhibit bundling and marked convolution, with some curvature and slight bundling of MTs throughout the cell cortices. In Boergesenia, wound-induced reticulation and separation of the protoplasm into numerous spheres also involves a fasciation of MTs within the attenuating regions of the cytoplasm. Although some cortical MTs are fairly resistant to cold and amiprophos-methyl-induced depolymerization, the perinuclear ones are very labile, depolymerizing in 5–10 min in the cold. The MT cytoskeleton is not believed to be directly involved in wound-induced motility in these plants because amiprophos-methyl and cold depolymerize most cortical MTs without inhibiting motility. Also, the identical MT distributions in intact cells of these two algae belie the very different patterns of cytoplasmic motility. Although certain roles of the MT arrays may be ruled out, their exact functions in these plants are not known.Abbreviations APM amiprophos-methyl - DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MT(s) microtubule(s) - PBS phosphate-buffered saline     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

The action of specific inhibitors of auxin transport on uptake of auxin and binding of N-1-naphthylphthalamic acid to a membrane site in maize coleoptiles

Using both 1-mm segments of corn (Zea mays L.) coleoptiles and a preparation of membranes isolated from the same source, we have compared the effectiveness of several inhibitors of geotropism and polar transport in stimulating uptake of auxin (indole-3-acetic acid, IAA) into the tissue and in competing with N-1-naphthylphthalamic acid (NPA) for a membrane-bound site. Low concentrations of 2,3,5-triiodobenzoic acid (TIBA), NPA, 2-chloro-9-hydroxyfluorene-9-carboxylic acid (morphactin), and fluorescein, eosin, and mercurochrome all stimulated net uptake of [³H]IAA by corn coleoptile tissues while higher concentrations reduced the uptake of both [³H]IAA and another lipophilic weak acid, [¹⁴C]benzoic acid. Since low concentrations of fluorescein and its derivatives competed for the same membrane-bound site in vitro as did morphactin and NPA, the basis for both the specific stimulation of auxin accumulation and the inhibition of polar auxin transport by all these compounds may be their ability to interfere with the carrier-mediated efflux of auxin anions from cells. At higher concentrations, the decrease in accumulation of weak acids was nonspecific and thus may be the result of acidification of the cytoplasm and a general decrease in the driving force for uptake of the weak acids. Triiodobenzoic acid was an exception. Low concentration of TIBA (0.1–1 M) were much less effective than NPA in competing for the NPA receptor in vitro, but little different from NPA in ability to stimulate auxin uptake. One possibility is that TIBA, a substance which is polarly transported, may compete with auxin for the polar transport site while NPA, morphactin, and the fluorescein derivatives may render this site inactive.Abbreviations C1-NPA 2,3,4,5-tetrachloro-N-1-naphthylphthalamic acid - IAA indole-3-acetic acid - -NAA -naphthaleneacetic acid - -NAA -naphthalenacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

Immunofluorescence microscopy of microtubule arrangement in Closterium acerosum (Schrank) Ehrenberg

The microtubule (MT) arrangement in Closterium acerosum cells was observed by indirect immunofluorescence microscopy both during and following cell division, and during cell expansion without cell division. (During the division period, some cells of this alga divide whereas other cells expand in their middle region without division.) Before septum formation, all cells had a ring-like MT bundle (MT ring) in their middle. Both septum formation and expansion without cell division occurred at the position of this ring. During the periods of division, short, hair-like MTs appeared around the nucleus in some of the cells, in addition to the MT ring. In dividing cells, spindle MTs appeared as the chromosomes were condensed. During the early stages of expansion of the semicells, after cell division, the spindle MTs assumed a radial arrangement, moved, and settled in a position between the daughter chloroplasts. These MTs disappeared about 1.5 h after septum formation. As the new semicells were growing, wall MTs appeared, arranged transversely along the expanding wall. These transverse MTs disappeared gradually 4–5 h after septum formation, and only an MT ring remained near the boundary between the new and old semicells. The MT ring was present until the next cell division or expansion without cell division. During the latter course of development, transverse wall MTs were present only at the band-like expanding region. At the earlier stage of expansion without cell division, the short, hair-like MTs remained around the nucleus, but as time passed, both the hair-like MTs and, somewhat later, the transverse ones disappeared and only the MT rings remained. The remaining MT ring was not always positioned at the boundary between the expanding and the old cell region. The temporal relationships between the changes in MT arrangement, and the orientation and localization of cellulose-microfibril deposition are discussed.Abbreviations DAPI 46-diamino-2-phenylindole - EGTA ethyleneglycol-bis-(-aminoethylether)-N, N, N, N-tetraacetic acid - MT mierotubule - PMSF phenylmethylsulfonyl fruoride     

Immunoaffinity purification using monoclonal antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown Alaska peas

The endogenous indole auxins of red-light grown pea (Pisum sativum L.) epicotyls were investigated. Immunoaffinity purification of indole-3-acetic acid (IAA) and its methylester was achieved using two monoclonal antibodies. Antibodies against free IAA were raised against IAA-C5-BSA, a hapten-carrier-conjugate giving rise to highly specific antibodies for indole auxins with a free acetic-acid group at position 3. Immunoaffinity adsorbents prepared with these antibodies were used for single-step purification of extracts of Alaska pea epicotylar tissue prior to quantification by high-performance liquid chromatography (HPLC) with on-line fluorescence detection. Monoclonal antibodies against a hapten-carrier-conjugate with IAA linked to bovine serum albumin through the carboxyl group (IAA-C1-BSA) were used for the isolation of IAA esters. Indol-3-acetic acid was identified in the elongation zone of the third internode of red-light-grown Alaska pea. 4-Chloro-indole-3-acetic acid, a constituent of immature pea seeds which is considered to be a very active auxin, was absent from the elongation zone. Several compounds were retained by the column based on antibodies against IAA-C1-BSA. Of these the methylester of IAA was identified by HPLC with on-line fluorescence detection, by co-migration in thin-layer chromatography and by gas chromatography-mass spectrometry. The methyl ester of IAA was very active in promoting elongation of pea third-internode segments. When fed to the epicotylar segments the IAA methylester was rapidly metabolized with IAA being the major metabolite. The methylester of IAA should therefore be classified as a labile auxin conjugate.Abbreviations 4Cl-IAA 4-chloro-indole-3-acetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA Indole-3-acetic acid - IAA-C5-BSA, IAA-C1-BSA, IAA-NI-BSA hapten-carrier-conjugates with IAA linked to bovine serum albumin through the C5-position, the carboxyl group, and the indole nitrogen, respectively - IAA-Me the methylester of IAA This study was supported by the Danish Research Council (SJVF 13-4148 and 13-4547 to P.U.) and by The Research Center for Plant Biotechnology.     

Auxin uptake and action of N-1-naphthylphthalamic acid in corn coleoptiles

The validity of a chemiosmotic hypothesis for uptake of weak acids as an explanation for the accumulation of auxin by cells has been explored further by comparing the uptake of indole-3-acetic acid (IAA) by 1-mm segments of corn (Zea mays L.) coleoptiles with that of benzoic acid and two neutral indoles, indoleethanol and indoleacetonitrile, which do not ionize. These substances, while structurally related to IAA lack both auxin activity and polar transport. Uptake of IAA and benzoic acid increase with decreasing external pH, whereas the uptake of the two neutral indoles is independent of external pH.Although metabolism of IAA, during 90 min or less, is minimal and without significant effect on its uptake, metabolism of benzoic acid appears responsible for the apparent saturation of benzoic acid uptake at high concentrations. An inhibitor of auxin transport, N-1-naphthylphathalamic acid (NPA), stimulates uptake of IAA but has no effect on uptake of either benzoic acid or the two neutral indoles. Thus, NPA does not affect the driving forces for accumulation of weak acids but probably specifically decreases the flux of the auxin anions relative to undissociated auxin. Since the electrochemical potential of auxin anions is usually higher in than outside cells, blocking the anion flux with NPA would enhance auxin uptake. Azide, which abolishes accumulation of both IAA and benzoic acid, may simply collapse the pH gradient across the plasma membrane.In the absence of NPA, increasing concentrations of auxins or the analogoue -naphthaleneacetic acid (-NAA) exert two opposing effects on the uptake of IAA-depression and stimulation. Stimulation results from saturating the anion flux. With uptake fully stimulated by NPA, however, increasing concentrations of auxins or analogues only depress uptake of [³H]IAA. These results are consistent with more than one path for auxin transport each with a different dependence on concentration. In depressing NPA-stimulated IAA uptake, the effectiveness of -NAAIAA-NAA benzoic acid, a specificity similar to that of an auxin binding site in vitro that has been implicated by others in auxin transport. The results support the general hypothesis that cellular auxin uptake and polar transport through tissues are chemiosmotically coupled to the electrochemical potential of auxin and protons.Abbreviations IAA indole-3-acetic acid - -NAA -naphthaleneacetic acid - -NAA -naphthaleneacetic acid - NPA N-1-naphthylphthalamic acid     

Ultracytochemical localization of adenylate cyclase activity in rat thymocytes

Summary Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO₄ as an activator, 5 mM theophylline as an inhibitor of 3,5-AMP-phosphodiesterase and 2 mM lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10^–4 M adrenalin in the presence of 5-guanylylimido-diphosphate (GMP-PNP) as well as with 10^–2 M NaF. In the cells incubated in a medium devoid of theophylline and containing 5-AMP instead of AMP-PNP, 5-nucleotidase activity was observed in the same cell structures as AC activity. Hydrolysis of 5-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5-AMP in all cell structures. No staining was observed with 2 mM -glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5-AMP or p-nitrophenyl phosphate, but not -glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 30 nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.     

The microtubular cytoskeleton in cells of cold-treated roots of maize (Zea mays L.) shows tissue-specific responses

Summary Microtubules (MTs) in cells of various tissues at different distances from the apex of the maize root exhibited different sensitivities to cold (5 °C), as judged by MT reorientation and tendency to depolymerization. Their responses seem to be related to their initial intracellular arrangements. Generally, MTs in cells which were ceasing elongation were the least sensitive during the early stages (6–24 h) of cold treatment, but during the later stages (5–7 d) MTs in most of these cells eventually depolymerized. Pericycle cells showed a unique cold response. Here the MTs were conspicuously cold-labile and quickly depolymerized near the root-tip. However, after 1 d many pericycle cells in more proximal regions had repolymerized their MTs as dense, randomly organized arrays. These persisted for the remainder of the cold treatment. A similar resistance to longterm chilling, by means of MT repolymerization, was found in cells of the root cap, quiescent centre and cells of the distal part of the former meristem. MT repolymerization in the cold may enable the apex to resume growth when more favourable (warmer) conditions return.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DMSO dimethylsulfoxide - EGTA ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - IgG immunoglobulin G - MT microtubule - PEG polyethylene glycol - PIPES piperazine-N,N-bis(diethanesulfonic acid)     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol-3yl-acetic acid

Phenylacetic acid (PAA), a naturally-occurring acidic plant growth substance, was readily taken up by pea (Pisum sativum L. cv. Alderman) stem segments from buffered external solutions by a pH-dependent, non-mediated diffusion. Net uptake from a 0.2 M solution at pH 4.5 proceeded at a constant rate for at least 60 min and, up to approx. 100 M, the rate of uptake was directly proportional to the external concentration of the compound. The net rate of uptake of PAA was not affected by the inclusion of indol-3yl-acetic acid (IAA) in the uptake medium (up to approx. 30 M) and, unlike the net uptake of IAA, was not stimulated by N-1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid. At an external concentration of 0.2 M and pH 4.5, the net rate of uptake of PAA was about twice that of IAA. It was concluded that the uptake of PAA did not involve the participation of carriers and that PAA was not a transported substrate for the carriers involved in the uptake and polar transport of IAA. Nevertheless, the inclusion of 3–100 M unlabelled PAA in the external medium greatly stimulated the uptake by pea stem segments of [1-¹⁴C]IAA (external concentration 0.2 M). It was concluded that whilst PAA was not a transported substrate for the NPA-sensitive IAA efflux carrier, it interacted with this carrier to inhibit IAA efflux from cells. Over the concentration range 3–100 M, PAA progressively reduced the stimulatory effect of NPA on IAA uptake, indicating that PAA also inhibited carrier-mediated uptake of IAA. The consequences of these observations for the regulation of polar auxin transport are discussed.Abbreviations IAA indol-3yl-acetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

RecA-ssDNA interaction: Induced strand cleavage by hydroxyl radical at a defined distance from the 5′ end

Summary Interaction of the RecA protein with single-stranded DNA (ssDNA) was analyzed by challenge with the hydroxyl radical, which can cleave the DNA backbone. We found that RecA protein induces cleavage by the radical at a defined distance from the 5 end. The cleavage was at the 11th nucleotide in many oligodeoxynucleotides. Cleavage may be intermittent since a second cleavage was induced at the 22nd or 21st site. This specific cleavage was observed under optimal conditions for filament formation, homologous pairing and strand exchange. Specificity in cleavage was, however, decreased by replacement of ATP by adenosine 5-(-thio)triphosphate (ATPS), replacement of RecA protein by a mutant (RecAl) protein, or an increase in Mg^2– concentration. We propose that RecA protein induces a special structural alteration, such as bending, perhaps sequentially, on ssDNA and that this altered site plays an important role in homologous pairing and strand exchange.Abbreviations ssDNA single-stranded DNA - dsDNA doublestranded DNA - ATPS adenosine 5-(-thio)triphosphate Deceased     

Synthesis of oligoguanylates on oligocytidylate templates

Summary Short oligocytidylates can act as templates for the self-condensation of guanosine 5-phosphorimidazolide. In the absence of a catalytic metal ion or in the presence of Pb²⁺ a noticeable template effect is already observed with the dimer and the yield of long oligomers reaches a plateau with a hexamer template. Short templates give oligomers longers than the template length. The products are predominantly 2-5 linked for the Pb²⁺-catalyzed reaction while mixed linkages are observed in the uncatalyzed reaction.In the presence of Zn²⁺, a template effect is first observed with the pentamer and is maximal by the heptamer. The products are predominantly 3-5 linked. Oligomers shorter than or as long as the template are obtained in substantial yield, and longer products in much lower yields.Abbreviations G Guanosine - Gp guanosine 2(3)-phosphate - pG guanosine 5-phosphate - Gp! guanosine cyclic 2,3-phosphate - ImpG guanosine 5-phosphorimidazolide - ImpG^* [8-¹⁴C]-guanosine 5-phosphorimidazolide - pGp 5-phosphoguanosine 2(3)-phosphate - G²pG guanylyl-[2-5]-guanosine - G³pG guanylyl-[3-5]-guanosine - ImpGpG 5-phosphorimidazolide of GpG - (pG)_n (n = 2,3) oligomers of pG - GppG P₁, P₂-diguanosine 5-diphosphate - GppGpG 5-[guanosine 5-pyrophosphate] of GpG - NH₂pG guanosine 5-phosphoramidate - (pG)₄₊ tetramer and higher oligoguanylates with 5 terminal phosphate - oligo(G) oligoguanylate - Cp cytidine 2(3)-phosphate - Cp! cytidine cyclic 2,3-phosphate - (Cp)_n–1 Cp! (n= 2,3,4) oligocytidylates terminated by 5-OH groups and 2,3-cyclic phosphates - oligo(C) oligocytidylate - poly(C) polycytidylic acid - poly(U) polyuridylic acid - poly(C,G) random copolymer of C and G - BAP bacterial alkaline phosphatase (E. coli) - EDTA ethylenediaminetetraacetic acid - R_f chromatographic mobility     

Hormones andCuscuta development: interaction of cytokinin and indole-3-acetic acid in the growth and curvature of subapical stem segments

Cuscuta stem (vines) exhibits two modes of growth—longitudinal elongation forming free-hanging vines, or coiling growth to twine around the host. The elongation zone of free-hanging vine extended up to 160 mm from the stem apex and in vivo growth rate (during 8 h of growth) was maximal in the 20-to-40-mm region. While gibberellic acid (GA₃) or fusicoccin (FC) could maintain (GA₃) or enhance (FC) the growth rate of apical (10 or 25 mm) segments, indole-3-acetic acid (IAA) (10 M) induced growth only in subapical (5–160 mm) segments. In vitro growth rate induced by IAA (10 M) was similar to the in vivo growth rate up to 40 mm. Thereafter, up to 100 mm, IAA induced growth rate exceeded in vivo growth. p ]Subapical segments (13 mm) from 5- to 40-mm regions responded to a cytokinin (BA, Z, or iP) or to low IAA (0.1 M) with curved growth, whereas the segments grew straight in the presence of high IAA (10 M). Curvature (measured as the angle subtended at the center of the circle of which the segment formed an arc) induced by BA and low (0.1 M) IAA was greater than either added separately. Besides, segments induced to curve in BA + low-IAA solution could be made to straighten out by transferring to a solution containing high IAA (10 M) with or without BA. Thus in vivo patterns of straight and coiling growth could be mimicked reversibly in vitro by adjusting the relative concentrations of cytokinin and auxin; low auxin and cytokinin induced coiling growth, whereas high auxin and cytokinin induced straight growth. p ]Beyond 40 mm, BA had no growth-promoting or curvative-inducing effect.Cuscuta vine segments thus showed sequential sensitivity to applied hormones, the apical region (0–25 mm) to GA₃, the subapical (5–40 mm) region to BA and IAA and the region beyond (40–160 mm) to IAA alone.     

Modeling the cucumber fermentation: Growth ofLactobacillus plantarum

Summary Specific growth rate models of product-inhibited cell growth exist but are rarely applied to fermentations beyond ethanol and large-scale antibiotic production. The present paper summarizes experimental data and the development of a model for growth of the commercially important bacterium,Lactobacillus plantarum, in cucumber juice. The model provides an excellent correlation of data for the influence on bacterial growth rate of NaCl, protons (H⁺), and the neutral, inhibitory forms of acetic acid and the fermentation product, lactic acid. The effects of each of the variables are first modeled separately using established functional forms and then combined in the final model formulation.Nomenclature [C] inhibitory component concentration, mM - [C]_max concentration of the inhibitory component where the specific growth rate is zero, mM, determined by model fitting - [H⁺] hydrogen ion concentration, mM - [HLa] undissociated lactic acid concentration, mM - [La^–] dissociated lactic acid concentration, mM - [La_t] total lactic acid ([HLa]+[La^–]) concentration, mM - [HAc] undissociated acetic acid concentration, mM - [Ac^–] dissociated acetic acid concentration, mM - [Ac_t] total acetic acid ([HAc]+[Ac^–]) concentration, mM - [NaCl] sodium chloride concentration, %, w/v - specific growth rate, h^–1 - _max maximum specific growth rate, h^–1 - ₀ specific growth rate, h^–1, at 0 concentration of additive - K _ij inhibition coefficient - , ,K _m coefficients determined by model fitting Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture or North Carolina Agricultural Research Service, nor does it imply approval to the exclusion of other products that may be suitable.     

A human metallothionein pseudogene containing AG/CT repetitive elements

Summary A lambda phage recombinant clone, 25 S, which contains a 15.5-kb EcoRI human genomic DNA fragment, has been characterized. Restriction mapping and Southern blot hybridization indicated a 3.0-kb HindIII fragment containing metallothionein (MT)-like sequences. Several interesting features were found upon comparison of this nucleotide sequence with that of other human MT genes: (1) sequences representing the 5 regulatory region, the 5 untranslated region, and the first exon are not contained in the 3.0-kb HindIII fragment; (2) the coding sequence of the second exon (amino acids 10–31 encoding a portion of the -domain of the MT protein) has 11 amino acid changes out of a total of 21, whereas, the third exon (amino acids 32–61, representing the complete -domain of the MT protein) has only 4 amino acid substitutions; however, all cysteine residues are conserved; (3) this MT-like gene retains intron sequences and processing signals; (4) Southern blot analysis of human genomic DNA indicated this MT-like gene is located on a 10.5-kb EcoRI genomic DNA fragment; and (5) unusual AG/CT-rich repetitive elements are located within the second intron and upstream of the second exon of this MT-like gene. This gene is not expressed in response to metal induction in two human cell lines, as shown by northern blot analyses. Based on these observations, this MT-like gene represents a unique nonprocessed pseudogene of the human MT multigene family.     

Vacuolar H+-translocating ATPases from plants: Structure,function, and isoforms

The vacuolar H⁺-translocating ATPase (V-type ATPase) plays a central role in the growth and development of plant cells. In a mature cell, the vacuole is the largest intracellular compartment, occupying about 90% of the cell volume. The proton electrochemical gradient (acid inside) formed by the vacuolar ATPase provides the primary driving force for the transport of numerous ions and metabolites against their electrochemical gradients. The uptake and release of solutes across the vacuolar membrane is fundamental to many cellular processes, such as osmoregulation, signal transduction, and metabolic regulation. Vacuolar ATPases may also reside on endomembranes, such as Golgi and coated vesicles, and thus may participate in intracellular membrane traffic, sorting, and secretion.Plant vacuolar ATPases are large complexes (400–650 kDa) composed of 7–10 different subunits. The peripheral sector of 5–6 subunits includes the nucleotide-binding catalytic and regulatory subunits of 70 and 60 kDa, respectively. Six copies of the 16-kDa proteolipid together with 1–3 other subunits make up the integral sector that forms the H⁺ conducting pathway. Isoforms of plant vacuolar ATPases are suggested by the variations in subunit composition observed among and within plant species, and by the presence of a small multigene family encoding the 16-kDa and 70-kDa subunits. Multiple genes may encode isoforms with specific properties required to serve the diverse functions of vacuoles and endomembrane compartments.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - CAM Crassulacean acid metabolism - Nbd-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Bz-ATP 3-O-(4-benzoyl)benzolyadenosine 5-triphosphate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NEM N-ethylmaleimide - IP₃ inositol-1,4,5-triphosphate - H⁺-PPase H⁺-translocating pyrophosphatase - V-type vacuolar-type - P-type phosphorylated intermediate- or plasma membrane-type - F-type F₁F_o-type - V-ATPase vacuolar-type H⁺-ATPase     

Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5′-azido-salicylamido)-undecanoic acid derivatives

Summary Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5-azido-salicylamido)-undecanoic acid (5 ASU) and its acetyl ester (Ac5 ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5 ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34×10^–7 M, evidenced a slightly higher affinity than that reported for C16–C20 fatty acids. Consistent with the binding curve, 5 ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 gmM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of¹²⁵I-5 ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t_1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5 ASU and Ac5 ASU respectively. In turn, irradiation of L-FABP incubated with 5ASU or Ac5 ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid. Both results, taken together, suggested that the derivatives are linked to the protein through the sites for fatty acids. When cross-linking of¹²⁵I-5 ASU was performed after incubation with delipidated cytosol and products were analyzed by SDS-PAGE, a band was visualized in a position similar to that of purified L-FABP.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Hepatic FABP - I-FABP Intestinal FABP - C-FABP Cardiac FABP - 5 ASU-11 (5-azido-salicylamido)-undecanoic acid - Ac5 ASU-11 (O-acetyl-5-azido-salicylamido)-undecanoic acid     

Identification of oxindole-3-acetic acid,and metabolic conversion of indole-3-acetic acid to oxindole-3-acetic acid in Pinus sylvestris seeds

Oxindole-3-acetic acid (OxIAA) has been identified in germinating seeds of Scots pine (Pinus sylvestris) using gas chromatography-mass spectrometry. Seeds germinated for 5 d contained 2.7 ng OxIAA·g^-1 (dry weight) whereas ungerminated seeds contained 0.2 ng·g^-1. Isotopically labelled OxIAA was formed in seeds incubated with [1-¹⁴C]-, [2-¹⁴C]- or [²H₅]indole-3-acetic acid.Abbreviations DDC sodium diethyldithiocarbamate - GC gas chromatography - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - MS mass spectrometry - OxIAA oxindole-3-acetic acid - PVP polyvinylpyrrolidone - TMS trimethylsilyl