Interactions of syndecan-1 and heparin with human collagens

Glycosaminoglycan (GAG)–collagen interactions play importantroles in cell adhesion and extracellular matrix assembly; however,the chemical bases for these interactions are not fully understood.We have used affinity co-electrophoresis (ACE) (Lee,M.K. andLander,A.D., Proc. Natl. Acad. Sci. USA, 88, 2768–2772,1991) to study the binding of the heparan sulphate proteoglycansyndecan-1 and heparin to human collagens. [³⁵S]Syndecan-1 [fromnormal murine mammary gland (NMuMG) epithelial cells] and low-M_r({small tilde}6 kDa) [¹²⁵I]heparin were subjected to electrophoresisthrough agarose gel lanes containing human collagens at variousconcentrations, and binding affinities were measured from shiftsin migration of the labelled materials. Results demonstratethat the affinities of each collagen for syndecan-1 and low-M_rheparin were similar, and followed the order: type V> >type IV     

Sulphate release from keratan sulphate and heparan sulphate by bovine N-acetylglucosamine-6-sulphate sulphatase

The functions of sulphated monosaccharides within glycosaminoglycans(GAGs) and glycoproteins are being studied intensely, but progressis hindered by an inability to selectively desulphate glycoconjugates.We recently identified an N-acetylglucosamine-6-sulphate sulphatase(NG6SS) from bovine kidney that can remove sulphate from N-acetylglucosamine-6-sulphate(GlcNAc-6-SO₄) within oligosaccharides and glycoproteins. However,the potential ‘endosulphatase’ activity of the NG6SStoward GAGs is not known. To test for this possibility, [³H]glucosamine-,[³H]galactose- and ³⁵SO_4- labelled keratan sulphate (KS) wereseparately prepared by metabolic radiolabelling of bovine cornea.NG6SS quantitatively removed sulphate from KS without releaseof sugar fragments. The enzyme had a K_m of 4.7 mM toward freeGlcNAc-6-SO₄, but its K_m for commercially available bovine cornealKS was found to be 9.1 µM. Analyses of both KS and heparansulphate after treatment with NG6SS demonstrated significantloss of sulphate from GlcNAc-6-SO₄ in both GAGs. These findingsmay be relevant for future studies aimed at defining the function(s)of GlcNAc-6-SO₄ residues in GAGs and understanding the catabolismof GAGs, especially in regard to sulphatidoses, such as SanfilippoD syndrome in humans, which involves a deficiency of NG6SS activity catabolism endosulphatase glycosaminoglycans sulphation     

Translocation Profiles of [11C] and [13N]-Labelled Metabolites after Assimilation of 11CO2 and [13N]-Labelled Ammonia Gas by Leaves of Helianthus annuus L. and Lupinus albus L.

Tracer amounts of atmospheric [¹³N]-Iabelled ammonia gas, wereabsorbed by leaves of Lupinus albus and Helianthus annuus inboth the light and the dark. Exogenous [¹³N]-ammonia was onlyabsorbed in the dark when the feeding occurred shortly aftera period of illumination and the tissue was not depleted ofits carbohydrate reserves (e.g. starch). Incorporation of the[¹³N]-ammonia appeared to occur via the leaf glutamine synthetase/glutamatesynthase (GS/GOGAT) cycle since 2.0 mol m^–3 MSX, an inhibitorof the GS reduced uptake in both the light and dark. Photosyntheticincorporation of ¹¹CO₂ was not affected by this treatment Therate of movement of [¹³N]-assimilates in the petiole of attachedleaves of Helianthus and Lupinus was similar to that of the¹¹Cl-photo assimilates. Export of both [¹³N] and [¹¹C]-Iabelledassimilates from the leaf and movement in the petiole in boththe light and the dark was inhibited by source leaf anoxia (i.e.nitrogen gas). Translocation was re-established at the samerate when the feed leaf was exposed to gas containing more than2% O₂ which permitted dark respiration to proceed. After aninitial feeding of either ¹¹CO₂ or [¹³N]-ammonia at ambient(21%) O₂ exposure of the source leaf to 2% O2, or 50% O² didnot alter the rates of translocation, indicating that changesin photosynthetic activity in the source leaf due to photorespiratoryactivity need not markedly alter, at least during the shortperiod, the loading and translocation of either [¹¹C ] or [¹³N]-labelledleaf products. Key words: Translocation, CO₂, NH₃, Leaves, Helianthus annuus, Lupinus albus     

Oxalate inhibits renal proximal tubule cell proliferation via oxidative stress, p38 MAPK/JNK, and cPLA2 signaling pathways

Exposure of renal proximal tubule cells to oxalate may play an important role in cell proliferation, but the signaling pathways involved in this effect have not been elucidated. Thus the present study was performed to examine the effect of oxalate on ³H-labeled thymidine incorporation and its related signal pathway in primary cultured rabbit renal proximal tubule cells (PTCs). The effects of oxalate on [³H]thymidine incorporation, lactate dehydrogenase (LDH) release, Trypan blue exclusion, H₂O₂ release, activation of mitogen-activated protein kinases (MAPKs), and ³H-labeled arachidonic acid (AA) release were examined in primary cultured renal PTCs. Oxalate inhibited [³H]thymidine incorporation in a time- and dose-dependent manner. However, its analogs did not affect [³H]thymidine incorporation. Oxalate (1 mM) significantly increased H₂O₂ release, which was blocked by N-acetyl-L-cysteine (NAC) and catalase (antioxidants). Oxalate significantly increased p38 MAPK and stress-activated protein kinase (SAPK)/c-Jun NH₂-terminal kinase (JNK) activity, not p44/42 MAPK. Oxalate stimulated [³H]AA release and translocation of cytosolic phospholipase A₂ (cPLA₂) from the cytosolic fraction to the membrane fraction. Indeed, oxalate significantly increased prostaglandin E₂ (PGE₂) production compared with control. Oxalate-induced inhibition of [³H]thymidine incorporation and increase of [³H]AA release were prevented by antioxidants (NAC), a p38 MAPK inhibitor (SB-203580), a SAPK/JNK inhibitor (SP-600125), or PLA₂ inhibitors [mepacrine and arachidonyl trifluoromethyl ketone (AACOCF₃)], but not by a p44/42 MAPK inhibitor (PD-98059). These findings suggest that oxalate inhibits renal PTC proliferation via oxidative stress, p38 MAPK/JNK, and cPLA₂ signaling pathways. kidney; mitogen-activated protein kinase; phospholipase A₂     

Partial Purification and Characterization of Gibberellin 3{beta}-hydroxylase from Immature Seeds of Phaseolus vulgaris L.

Gibberellin 3/ß-hydroxylase,a 2-oxoglutarate-dependentdioxygenase that catalyzes the hydroxylation of GA₂₀ to GA₁,was purified 313-fold from immature seeds of Phaseolus vulgarisL. The mol wt of the enzyme was estimated to be 42,000 by gelfiltration HPLC and SDS-polyacrylamide gel electrophoresis.The enzyme exhibited maximum activity at pH 7.7. The Km valuesfor [2,3-³H]GA20 and [2,3-³H]GA, were 0.29µu and 0.33µm, respectively. The enzyme requires 2-oxoglutarate asa cosubstrate; the K_m value for 2-oxoglutarate was 250µMusing [³H]- GA₂₀ as a substrate. Fe²⁺ and ascorbate significantlyactivated the enzyme at all purification steps, while catalaseand BSA activated the purified enzyme only. The enzyme was inhibitedby divalent cations Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Hg²⁺.3ß-Hydroxylation of [³H]- GA₂₀ was also inhibitedby non-radioactive GA₅, GA₉,GA₁₅, GA₂₀ and GA₄₄. The possiblesite of 3ß-hydroxylation in gibberellin biosynthesisis discussed in terms of the substrate specificity of partiallypurified gibberellin 3ß-hydroxylase. (Received February 29, 1988; Accepted June 3, 1988)     

Removal of a dimeric form of surfactant protein C from mouse lungs: its acceleration by reduction

Li, Zhong-Yuan, Yasuhiro Suzuki, Mafumi Kurozumi, Hui-QingShen, and Chen-Xia Duan. Removal of a dimeric form of surfactant protein C from mouse lungs: its acceleration by reduction.J. Appl. Physiol. 84(2): 471-478, 1998.Clearance of hydrophobic surfactant-associated protein C (SP-C)and its dimeric form([SP-C]₂) wasinvestigated. SP-C and[SP-C]₂ obtained fromproteinosis patients were fluorescently labeled and were instilled intomouse lungs as lipid-protein complexes.[SP-C]₂ was removedmore slowly than SP-C, with apparent half-lives of 30 and 18 h,respectively. A significant amount of[SP-C]₂ was removed asSP-C, and the conversion rate was 0.22 µg · h¹ · mouse¹.By correcting the removal as SP-C, we obtained 38 h for a possible half-life of [SP-C]₂.Conversion from SP-C to[SP-C]₂ seemed very slow. Decrease in glutathione (GSH) in the lung inhibited the conversion of [SP-C]₂to SP-C and GSH-treatment of liposomes accelerated clearance of[SP-C]₂. These resultssuggest that the removal of [SP-C]₂ from lung isaccelerated by reduction and that GSH acts as a reducing agent in thelung.

     

Abscisic acid and the accumulation, biological activity and metabolism of four derivatives of [3H]gibberellin A1 in barley aleurone layers

Tritiated GA₁ and four of its synthetic derivatives were studiedin relation to their biological activity, uptake and metabolismby barley aleurone layers. Incubation was done in the presenceand absence of ABA. Tentative identification of some of themetabolites was made by TLC and GLC radiocounting of the metaboliteand its acid hydrolyzed derivative. Only GA₁ promoted -amylase synthesis. Uptake ranged from 20to 42%, varying with the derivative. ABA enhanced uptake of[³H]GA₁ and [³H]pseudoGA₁ and inhibited uptake of [³H]ketoGA₁the Wagner-Meerwein rearrangement product of [³H]GA₁ Uptakeof [³H]GA₁ methyl ester ([³H]GA₁-Me) and [³H]dihydroGA₁ wasunaffected by ABA. [³H]GA₁ was converted to an amphoteric GA₁ derivative ([³H]amphoGA₁)and [³H]GA₁-glycosyl ester. GA₁-Me was metabolized to four products,all of them GA₁ derivatives, including an apparent amphotericGA₁ derivative. DihydroGA₁ was quite stable; only one metabolitewas produced in sufficient yield to analyze. This product didnot cochromatograph with either of the expected acid hydrolyzedepimers of [³H]dihydroGA₁. [³H]ketoGA₁ was readily metabolizedto one product, probably the glycoside. [³H]pseudoGA₁ remainedessentially unmetabolized. Metabolism of all compounds testedwas not dramatically affected by ABA. Surprisingly, no metabolitesfrom hydroxylation at the 2-position were found. ¹ Present address: Monsanto Agricultural Co., 800 N. LindberghBlvd., St. Louis, MO 63166, U.S.A. (Received January 31, 1977; )     

Trehalose-P synthase of mycobacteria: its substrate specificity is affected by polyanions

The trehalose-P synthase was purified to near homogeneity fromthe cytoplasmic fraction of Mycobacterium smegmatis. At thefinal stage of purification, the enzyme preparation showed onemajor band of 59 kDa on SDS gels. The 59 kDa band became labeledwith N₃-UDP[³²P]-glucose, and this labeling was inhibited ina concentration-dependent manner by either unlabeled UDP-glucoseor GDP-glucose. The native enzyme also had a molecular weightof about 60 kDa by gel filtration, indicating that the activeenzyme is a monomer. The 59 kDa protein was subjected to endoproteinaseLys-C digestion, and three peptides isolated by HPLC were sequenced.The sequences of 56 amino acids in these three peptides showed60% identity to the trehalose-P synthases of Saccharomyces cerevesiaeand Schizosaccharomyces pombe. The purified mycobacterial enzymecatalyzed the synthesis of trehalose-P from glucose-6-P anda variety of nucleoside diphosphate glucose derivatives, dependingon whether a polyanion was absent or present. Thus, UDP-glucoseand GDP-glucose were the best glucosyl donors, but maximum activitywith UDP-glucose required the presence of a polyanion such asheparin, whereas activity with GDP-glucose was relatively independentof polyanion. The presence of heparin in the incubation mixtureincreased the affinity of the enzyme for UDP-glucose by a factorof 100, or more. However, the affinity for GDP-glucose was onlytwofold better in the presence of heparin. The purified synthasealso utilized ADP-glucose and CDP-glucose, but the K_m for theseglucosyl donors was quite high even in the presence of polyanion.The effect of heparin on UDP-glucose activity was dose-dependentand maximum at about 1–2 µ;g of heparin/incubation.However, the size of the heparin molecule (i.e., the numberof monosaccharide residues) was critical for activation, andonly those heparins with 18 or more monosaccharide units wereeffective in stimulating activity. trehalose polyanions mycobacteria GDP-glucose heparin     

臭氧对水稻根系活力、可溶性蛋白含量与抗氧化系统的影响

采用旋转布气法开顶式气室 (Open top chambers, OTCs) 装置, 研究4种臭氧 (O3) 浓度水平 (过滤大气, O3浓度20nl·L-1;环境大气, O3浓度40nl·L-1;中等O3浓度处理, O3浓度为75nl·L-1;高浓度处理, O3浓度为150nl·L-1) 下水稻 (Oryzasativa) 根系中根系活力、可溶性蛋白含量、膜脂过氧化程度与抗氧化系统的变化差异。主要结果表明与过滤大气处理相比, O3浓度升高 (75和150nl·L-1) 使植株根系活力显著降低, 根系大幅度、过早地衰退;根系可溶性蛋白质含量显著下降;根系MDA含量显著升高, 膜脂过氧化程度加剧;SOD活性呈先升高后下降的变化趋势根系中H2O2含量大幅度显著上升, 并随着O3处理浓度升高和暴露时间延长变化幅度增大;CAT与POD活性则表现出升高趋势, 但处理后期升高幅度略微降低;整个处理期间根系ASA含量无显著变化。环境大气处理与过滤大气处理植株各个指标变化趋势基本一致并略微下降, 随着处理时间延长根系活力与蛋白质含量出现显著下降, 其他指标无显著差异。试验结果表明O3浓度升高会对植物地下部分根系产生影响;随着O3胁迫时间的延长, 植物将面临着缺乏强有力的根系生理代谢活力支持。     

H(+)-linked transport of salicylic acid, an NSAID, in the human trophoblast cell line BeWo

We investigated thetransport of salicylic acid and L-lactic acid across theplacenta using the human trophoblast cell line BeWo. We performeduptake experiments and measured the change in intracellular pH(pH_i). The uptakes of [¹⁴C]salicylic acid andL-[¹⁴C]lactic acid were temperature- andextracellular pH-dependent and saturable at higher concentrations. Bothuptakes were also reduced by FCCP, nigericin, and NaN₃.Various nonsteroidal anti-inflammatory drugs (NSAIDs) stronglyinhibited the uptake of L-[¹⁴C]lactic acid.Salicylic acid and ibuprofen noncompetitively inhibited the uptake ofL-[¹⁴C]lactic acid.-Cyano-4-hydroxycinnamate (CHC), a monocarboxylate transporterinhibitor, suppressed the uptake ofL-[¹⁴C]lactic acid but not that of[¹⁴C]salicylic acid. CHC also suppressed the decrease ofpH_i induced by L-lactic acid but had littleeffect on that induced by salicylic acid or diclofenac. These resultssuggest that NSAIDs are potent inhibitors of lactate transporters,although they are transported mainly by a transport system distinctfrom that for L-lactic acid.

     

Metabolism and Translocation of Gibberellins in the Seedlings of Pharbitis nil (II). Photoperiodic Effects on Metabolism and Translocation of Gibberellins Applied to Cotyledons

We investigated the metabolism and translocation of two gibberellins(GAs), [³H]GA₂₀ and [³H]GA₁, which were applied at low concentrationto the cotyledons of Pharbitis nil (cv. Violet). Seedlings weregrown under three different photoperiodic conditions: continuouslight (CL-CL), continous light followed by short day conditions(CL-DT) and long day conditions followed by short day conditions(DT-DT). Translocation of the applied [³H]GAs from cotyledonsto hypocotyls was promoted by DT for all GAs examined. Whilethe conversion of the translocated [³H]GA₁ to [³H]GA₈ and itsconjugates was rapid in hypocotyl, the conversion of translocated[³H]GA₂₀ to [³H]GA₂₉ was slow. Radioactivity in epicotyls wasdetected much more rapidly on application of [³H]GA₂₀ than of[³H]GA₁, [³H]GA₈ and [³H]GA₂₉ and their conjugates. The conversionof [³H]GA₂₀ to [³H]GA₁ in the epicotyl was more rapid underCL-CL conditions. This result in consistent with the higherlevel of endogenous GA₁ existing in epicotyls under CL-DT thanDT-DT conditions. However, when [³H]GA₁ was applied to the cotyledon,only small amounts of [³H]GA₈ and its conjugates were detectedin the epicotyl regardless of the photoperiodic conditions.This result may suggest that the translocation and metabolismof [³H]GA₂₀ from cotyledons to epicotyl was faster under CL-CLthan DT-DT conditions and may correlate with the increased epicotylelongation of GA₂₀ treated plants under CL-DT than DT-DT conditions. (Received June 28, 1995; Accepted November 2, 1995)     

Regulation of Ribulose-l,5-bisphosphate Carboxylase Activity in Response to Changes in the Source/Sink Balance in Single-Rooted Soybean Leaves: The Role of Inorganic Orthophosphate in Activation of the Enzyme

The results of our previous study [Sawada et al. (1989) PlantCell Physiol. 30: 691] implied that, under sink-limited conditions,a decrease in the activity of ribulose-l,5-bisphosphate carboxylase(EC 4.1.1.39 [EC] ) caused a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted leaves of soybean (Glycinemax L. Merr. cv. Tsurunoko). This reduction in the rate of photosynthesisin source leaves seemed to correspond to a decrease in the demandby sink tissues for photoassimilates. In the present study,the activity of RuBPcase in vivo was estimated by measuringthe "initial" activity immediately after extraction from standardleaves (grown under a regime of 10 h of light and 14 h of darkness)and from sink-limited leaves (exposed for 6 or 7 d to continuouslight to alter the source/sink balance). The rate of photosynthesisin the sink-limited leaves decreased to 47% of that in the standardleaves. The "initial" activity of RuBPcase was 4.3 in the standardleaves but only 1.6 µmole CO₂.(mg Chl)^–1.min^–1in the sink-limited leaves. These results appear to indicatethat the reduction in photosynthetic activity under sink-limitedconditions was mostly due to a deactivation of RuBPcase. Theactivity of deactivated RuBPcase in the sink-limited leaveswas restored to 4.1 µmole CO₂.(mg Chl)^–1.min^–1by incubation of the enzyme in a medium that contained onlyNa₂HPO₄. This result suggests that free P_i in chloro-plastsplays an important role in the activation of the enzyme. Thelevel of P_i in the sink-limited leaves was 62% of that in thestandard leaves. On the basis of these various results, it appearsthat the deactivation of RuBPcase in the sink-limited leavesis the result of a decrease in the level of P_i. The role offree P_i in the activation of RuBPcase, in particular at atmosphericconcentrations of CO₂, was also investigated. (Received November 30, 1989; Accepted May 11, 1990)     

Enhanced response to caffeine and 4-chloro-m-cresol in malignant hyperthermia-susceptible muscle is related in part to chronically elevated resting [Ca2+]i

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. We have measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) using double-barreled, Ca²⁺-selective microelectrodes in myoballs prepared from skeletal muscle of MH-susceptible (MHS) and MH-nonsusceptible (MHN) swine. Resting [Ca²⁺]_i was approximately twofold in MHS compared with MHN quiescent myoballs (232 ± 35 vs. 112 ± 11 nM). Treatment of myoballs with caffeine or 4-chloro-m-cresol (4-CmC) produced an elevation in [Ca²⁺]_i in both groups; however, the concentration required to cause a rise in [Ca²⁺]_i elevation was four times lower in MHS than in MHN skeletal muscle cells. Incubation of MHS cells with the fast-complexing Ca²⁺ buffer BAPTA reduced [Ca²⁺]_i, raised the concentration of caffeine and 4-CmC required to cause an elevation of [Ca²⁺]_i, and reduced the amount of Ca²⁺ release associated with exposure to any given concentration of caffeine or 4-CmC to MHN levels. These results suggest that the differences in the response of MHS skeletal myoballs to caffeine and 4-CmC may be mediated at least in part by the chronic high resting [Ca²⁺]_i levels in these cells. calcium homeostasis; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid     

Molecular-weight-dependent effects of nonanticoagulant heparins on allergic airway responses

We have hypothesized that antiallergic activity of inhaledheparin is molecular weight dependent and mediated by"nonanticoagulant fractions" (NAF-heparin). Therefore, we studiedcomparative effects of high-, medium-, and ultralow-molecular-weight(HMW, MMW, and ULMW, respectively) NAF-heparins on acutebronchoconstrictor response (ABR) and airway hyperresponsiveness (AHR)in allergic sheep. Specific lung resistance was measured in 23 allergicsheep, before and immediately after challenge withAscaris suum antigen, without andafter pretreatment with inhaled NAF-heparins. Airwayresponsiveness was estimated before and 2 h postantigen as thecumulative provocating dose of carbachol in breath units, whichincreased specific lung resistance by 400%. NAF-heparins attenuatedABR and AHR in a molecular-weight-dependent fashion. HMW NAF-heparin(n = 8) was the least effective agent: it attenuated ABR [inhibitory dose causing 50% protection(ID₅₀) = 4 mg/kg] but hadno effect on AHR. MMW NAF-heparin (n = 8) showed intermediate efficacy (ABRID₅₀ = 0.8 mg/kg, AHRID₅₀ = 1.4 mg/kg), whereas ULMWNAF-heparin (n = 7) was the mosteffective agent (ABR ID₅₀ = 0.4 mg/kg, AHR ID₅₀ = 0.2 mg/kg). ULMWNAF-heparin was 3.5 times more potent in attenuating antigen-inducedAHR when administered "after" antigen challenge and failed toinhibit the bronchoconstrictor response to carbachol and histamine. In15 additional sheep, segmental antigen challenge caused a marked increase in histamine in bronchoalveolar lavage fluid that was notprevented by any of the inhaled NAF-heparins. These data indicate thatantiallergic activity of inhaled heparin is independent of itsanticoagulant action and resides in the <2,500 ULMW chains. Theantiallergic activity of NAF-heparins is mediated by an unknown biological action and may have therapeutic potential.     

Contractile activity is required for sarcomeric assembly in phenylephrine-induced cardiac myocyte hypertrophy

Agonist-inducedhypertrophy of cultured neonatal rat ventricular myocytes (NRVM) hasbeen attributed to biochemical signals generated during receptoractivation. However, NRVM hypertrophy can also be induced byspontaneous or electrically stimulated contractile activity in theabsence of exogenous neurohormonal stimuli. Using single-cell imagingof fura 2-loaded myocytes, we found that low-density, noncontractingNRVM begin to generate intracellularCa²⁺ concentration([Ca²⁺]_i)transients and contractile activity within minutes of exposure to the₁-adrenergic agonistphenylephrine (PE; 50 µM). However, NRVM pretreated with verapamiland then stimulated with PE failed to elicit[Ca²⁺]_itransients and beating. We therefore examined whether PE-induced [Ca²⁺]_itransients and contractile activity were required to elicit specificaspects of the hypertrophic phenotype. PE treatment (48-72 h)increased cell size, total protein content, total protein-to-DNA ratio,and myosin heavy chain (MHC) isoenzyme content. PE also stimulatedsarcomeric protein assembly and prolonged MHC half-life. However,blockade of voltage-gated L-typeCa²⁺ channels with verapamil,diltiazem, or nifedipine (10 µM) blocked PE-induced total protein andMHC accumulation and prevented the time-dependent assembly ofmyofibrillar proteins into sarcomeres. Inhibition of actin-myosincross-bridge cycling with 2,3-butanedione monoxime (7.5 mM) alsoprevented PE-induced total protein and MHC accumulation, indicatingthat mechanical activity, rather than[Ca²⁺]_itransients per se, was required. In contrast, blockade of[Ca²⁺]_itransients and contractile activity did not prevent the PE-induced increase in cell surface area, activation of the mitogen-activated protein kinases ERK1 and ERK2, or upregulation of atrial natriuretic factor gene expression. Thus contractile activity is required to elicitsome but not all aspects of the the hypertrophic phenotype induced by₁-adrenergic receptoractivation.

     

Chloride Accumulation as a Homeostatic System: Set Points and Perturbations: THE PHYSIOLOGICAL SIGNIFICANCE OF INFLUX ISOTHERMS, TEMPERATURE EFFECTS AND THE INFLUENCE OF PLANT GROWTH SUBSTANCES

It has previously been observed that Cl^–influx falls withincreasing internal CI^–concentration. This paper attemptsto show that this is a bona fide negative feedback involvedin a homeostatic CI^–accumulation system. First, predictions are made of how the final steady concentrationto which Cl^–is accumulated ([Cl]₁) would be expected tovary as external Cl^–concentration ([Cl]₀) or temperature,change. This is done for the cases where influx has (a) no feedbackcontrol, or is (b) under error-actuated or (c) under reciprocalfeedback control. Secondly, Cl^–influx and [Cl]₁are measured in carrot andmaize root tissue over a range of [Cl]_o and temperatures forcomparison with the prediction. [Cl)₁varies by only about 20%over a range of [Cl]₀ and temperatures which have large immediateeffects on Cl^–influx. This is consistent only with error-actuatedfeedback control. Changes in [Cl]₀ and temperature are bestregarded simply as perturbations of Cl^–influx since theyhave no marked effect on [Cl)₁. In such a homeostatic system the feedback signal is a functionof (C_R – [Cl], ), where C_R represents the value of aninbuilt set point. C_R must be constant with [Cl]_o and temperature,and its nature is briefly discussed. It is further predictedthat the accumulated level can only be altered in a controlledmanner by a change in C_R, and that this would alter influx bythe same proportion. The effects of abscisic acid in increasingboth [Cl]₁ and Cl^–influx are consistent with an effectprimarily on C^R. These conclusions imply that when varieties or species differin levels accumulated, they do so not because influx isothermsdiffer but rather because their set points differ. Key words: Homeostasis, Chloride, Accumulation     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

Effects of osmotic shrinkage on voltage-gated Ca2+ channel currents in rat anterior pituitary cells

Increased extracellular osmolarity ([Os]_e) suppresses stimulated hormone secretion from anterior pituitary cells. Ca²⁺ influx may mediate this effect. We show that increase in [Os]_e (by 18–125%) differentially suppresses L-type and T-type Ca²⁺ channel currents (I_L and I_T, respectively); I_L was more sensitive than I_T. Hyperosmotic suppression of I_L depended on the magnitude of increase in [Os]_e and was correlated with the percent decrease in pituitary cell volume, suggesting that pituitary cell shrinkage can modulate L-type currents. The hyperosmotic suppression of I_L and I_T persisted after incubation of pituitary cells either with the actin-disrupter cytochalasin D or with the actin stabilizer phalloidin, suggesting that the actin cytoskeleton is not involved in this modulation. The hyperosmotic suppression of Ca²⁺ influx was not correlated with changes in reversal potential, membrane capacitance, and access resistance. Together, these results suggest that the hyperosmotic suppression of Ca²⁺ influx involves Ca²⁺ channel proteins. We therefore recorded the activity of L-type Ca²⁺ channels from cell-attached patches while exposing the cell outside the patch pipette to hyperosmotic media. Increased [Os]_e reduced the activity of Ca²⁺ channels but did not change single-channel conductance. This hyperosmotic suppression of Ca²⁺ currents may therefore contribute to the previously reported hyperosmotic suppression of hormone secretion. L-type Ca²⁺ channels; osmosensitivity; mechanosensitivity; osmolarity; hyperosmolarity     

Effects of altered tonicity by sodium chloride on L-tryptophan binding to hepatic nuclei

This study was concerned with theeffects of NaCl administered in vivo or added in vitro to isolatednuclei on [³H]tryptophan binding to rat hepaticnuclei assayed in vitro. Hypertonic (10.7%) NaCl administered in vivoto rats caused at 10 min a marked decrease in in vitro binding (totaland specific) of [³H]tryptophan to hepaticnuclei. In vitro incubation of isolated hepatic nuclei, but not ofisolated nuclear envelopes, with added NaCl (particularly at 0.125 × 10⁴ M and 0.25 × 10⁴ M) revealed significant inhibition of[³H]tryptophan binding. However, isolatedhepatic nuclear envelopes prepared after in vitro incubation ofisolated nuclei with added NaCl did show inhibition of[³H]tryptophan binding (total and specific)compared with controls. Other salts (KCl, MgCl₂,NaHCO₃, NaC₂H₃O₂, NaF,or Na₂SO₄), at similar concentrations to thatof NaCl except for MgCl₂, when added to isolated nuclei didnot appreciably inhibit nuclear tryptophan binding. Kinetic studies ofin vitro nuclear [³H]tryptophan binding in thepresence of 0.125 × 10⁴ M NaCl revealed thatbinding decreased at 0.5 h and continued to 2 h compared with nuclear[³H]tryptophan binding with controls (withoutNaCl addition). The results obtained in vivo in rats and those obtainedin vitro with isolated hepatic nuclei revealed NaCl-induced inhibitoryeffects on [³H]tryptophan binding to hepaticnuclei. Although the inhibitory effects were similar under the twodifferent experimental conditions, the mechanism for each may bedifferent in that the NaCl concentration in hepatic cells afteradministration of NaCl in vivo was appreciably higher than the lowlevels added in vitro to the isolated hepatic nuclei.

     

Regulation of Sulphate Transport in a Tropical Legume, Macroptilium atropurpureum, cv. Siratro

When young plants of Macroptilium atropurpureum, cv. Siratrowere deprived of external sulphate (-S plants) growth of shootsand roots continued at rates comparable to those in plants wellsupplied with sulphate (control) for 3 d and 5 d respectively.Dilution of internal sulphur therefore took place and redistributionof sulphur occurred between inorganic and organic forms andbetween roots and younger leaves. Even when S-deficiency limitedgrowth, plants contained 16% of their total sulphur as sulphate,but most of this was retained in old leaves and redistributedslowly to growing zones. The capacity for sulphate uptake increased in roots of –Splants very soon after they were deprived of external sulphate;within 24 h the absorption from 0.25 mol m^–3 SO₄^2–was more than five times that of control roots. Maximum increasedcapacity was reached after 2–3 d stress when the V_maxof system 1 was 1948 nmol h^–1g^–1root fr. wt. in–S plants and 337 nmol h^–1g^–1root fr. wt.in controls. The K^mfor system 1 did not change significantlywith S-stress being between 5–8 µM in both setsof plants. Absorption of L-cysteine was not stimulated by S-stress. There was a close, positive relationship between plant growthrate and the rate at which sulphate uptake capacity was enhancedby withholding sulphate from culture solutions. When –S plants were replaced in sulphate-containing solutiontheir capacity for SO₄^2– declined to the control levelwithin 24 h. Very marked repression of capacity was also foundwhen –S plants were treated with L-cysteine, but therewas no immediate effect with methionine. Roots of this species appear to have a very active system fordegrading L-cysteine to sulphate, 30% of the label in ³⁵S-cysteineabsorbed by roots was recovered in ³⁵SO₄^2– after 20 minor 2 h incubation. By contrast, roots had a very weak abilityto reduce sulphate. When part of the root system was in solution lacking sulphatethere was enhanced uptake of sulphate by other parts which themselveswere amply supplied with sulphate. This is seen as an exampleof compensatory absorption. The response to S-stress is specific and there were no positiveinteractions between S-stress and the absorption of phosphate,or P-stress and the uptake of sulphate. The results are discussed in relation to the close control ofsulphate uptake by internal sulphate concentration, redistributionof forms of sulphur during stress and mobility of sulphate inthe phloem. Key words: Kinetics, Amino-S, Sulpholipid, Repression;, Deficiency