Fluid pressure in human dermal fibroblast aggregates measured with micropipettes

Previous studies indicated that connective tissue cells in dermis are involved in control of interstitial fluid pressure (P_if). We wanted to develop and characterize an in vitro model representative of loose connective tissue to study dynamic changes in fluid pressure (P_f) over a time course of a few minutes. P_f was measured with micropipettes in human dermal fibroblast cell aggregates of varying size (<100- and >100-µm diameter) and age (days 1-4) kept at different temperatures (15, 25, and 35°C). Pressures were measured at different depths of micropipette penetration and after treatment with prostaglandin E₁ isopropyl ester (PGE₁), latanoprost (PGF₂), and ouabain. P_f was positive (more than +2 mmHg) during control conditions and increased with increasing aggregate size (day 2), age (day 4 vs. day 1), temperature, and depth of micropipette penetration. P_f decreased from 2.9 to 2.0 mmHg during the first 10 min after application of 10 µl of 1 mM PGE₁ (P < 0.001). P_f increased from 3.0 to 4.8 mmHg (P < 0.01) after administration of 10 µl of 1.4 µM ouabain and from 3.1 to 4.4 mmHg after addition of 5 µl of 1.42 mM PGF₂ (P > 0.05). In conclusion, we have developed and validated a new in vitro method for studying fluid pressure in loose connective tissue elements with the advantage of allowing reliable and rapid screening of substances that have a potential to modify P_f and studying in more detail specific cell types involved in control of P_f. This study also provides evidence that fibroblasts in the connective tissue can actively modulate P_f. micropuncture; prostaglandin E₁; prostaglandin F₂; ouabain; integrins     

Effect of Abscisic Acid on the K+ Efflux and Membrane Potential of Nicotiana tabacum L. Leaf Cells

K⁺ efflux from tobacco (Nicotiana tabacum L, cv. Samsun NN)leaf discs into the external medium was increased and the membranepotential (Em) changed in the positive direction with a changein pH from 8.0 to 4.0. E_m was affected by the external concentrationof KCl, greatly decreasing with a change in concentration from1 mM to 100 mM. The equilibrium potential of the membrane forK⁺ (E_k) was decreased in a Nernst fashion with increasing externalconcentrations of KCl. E_k is more positive than E_m above ca.50 µM KCl. Most of the experiments were carried out underconditions in which the difference between the electrochemicalpotential for K⁺ on the inside to the outside of the cell (µ_kis positive. Thus, K⁺ may passively flow to the outside of thecells accompanied by the depolarization of the membrane. Abscisic acid (ABA) increased the K⁺ efflux under conditionsof passive transport. K⁺ efflux was accelerated with an increasingconcentration of ABA, being maximal at 10^–4 M–10^–3M. This acceleration was due to the enhancement of the potassiummotive force (µ_k/F) which is the force causing the netpassive transport of K⁺. The membrane potential was decreasedfrom –205 mV to –170 mV by 2 x 10^–4 M ABAwithin 10 min. The depolarization was not transient, being lostfor at least 3 hr. These results show that ABA accelerated passive K⁺ efflux, whichaccompanied depolarization of the membrane. (Received June 22, 1981; Accepted August 24, 1981)     

Catecholaminergic modulation of respiratory rhythm in an in vitro turtle brain stem preparation

An in vitro brainstem preparation from adult turtles was used to determine effects ofdopamine (DA) and norepinephrine (NE) on the pattern of respiratorymotor output recorded from hypoglossal nerve roots (XII). Bath-appliedDA (10-200 µM) increased the frequency of respiratory bursts(peaks) from 0.9 ± 0.2 to 2.4 ± 0.3 (SE) peaks/min, resultingin a 99 ± 9% increase in neural minute activity. R[+]-SCH-23390 (10 µM,D₁ antagonist) and eticlopride (20 µM, D₂ antagonist) attenuatedthe DA-mediated increase in peak frequency by 52 and 59%,respectively. On the other hand, the DA-receptor agonists apomorphine(D₁,D₂), quinelorane(D₂), and SKF-38393 (D₁) had no effect on peakfrequency. Prazosin, an₁-adrenergic antagonist (250 nM) abolished the DA-mediated frequency increase. Although NE(10-200 µM) and phenylephrine (10-200 µM,₁-adrenergic agonist) increasedpeak frequency from 0.5 ± 0.1 to 1.2 ± 0.3 peaks/min and from0.6 ± 0.1 to 1.0 ± 0.2 peaks/min, respectively, these effectswere not as large as that with DA alone. The data suggest that bothdopaminergic and adrenergic receptor activation in the brain stemincrease respiratory frequency in turtles, but the DA receptor-mediatedincrease is dependent on coactivation of₁-adrenergic receptors.

     

Stimulation of RNA Synthesis in Isolated Nuclei by Auxin-Binding Proteins-I and -II

The auxin-binding proteins (ABP-I and ABP-II) purified frometiolated mung bean seedlings stimulated RNA synthesis in isolatednuclei both in the presence and absence of (NH₄)₂SO₄. In theabsence of (NH₄)₂SO₄, maximum stimulation of RNA synthesis occurredat 100 µg ABP-I (25%) and 300 µg ABP-II (60%), whereasin the presence of 50 mM (NH₄)₂SO₄ maximum stimulation occurredat 60 µg ABP-I (10%) and 100 µg ABP-II (40%). Thesestimulatory effects on RNA synthesis by ABP-I and ABP-II werecompletely abolished by the addition of -amanitin (4 µg/0.5ml reaction mixture). IAA had no effect on the stimulation ofRNA synthesis by ABP-I and ABP-II. (Received September 30, 1985; Accepted March 5, 1986)     

CYP450 dietary inhibitors attenuate TNF-alpha-stimulated endothelial molecule expression and leukocyte adhesion

Enhanced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and other endothelial cell adhesion molecules (ECAMs) are associated with the onset and progression of inflammatory bowel disease (IBD). We show in this study that two cytochrome P-450 (CYP450) inhibitors from Citrus paradis (grapefruit), bergamottin, and 6',7'-dihydroxybergamottin (DHB) block tumor necrosis factor (TNF)--stimulated expression of MAdCAM-1 in cultured endothelial cells and also reduce ₄₇-dependent lymphocyte adhesion. Bergamottin (20–50 µM) or DHB (10–30 µM) pretreatment dose-dependently reduced TNF--mediated expression of MAdCAM-1 and lymphocyte adhesion. Bergamottin and DHB also prevented expression of two other ECAMs, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (but not E-selectin). SKF-525a, a specific CYP450 inhibitor, also blocked the expression of MAdCAM-1 mediated by TNF-. Similar to SKF-525a (20 µM), bergamottin (20 µM) and DHB (20 µM) directly inhibited the activity of CYP450 3A4. These results suggest that natural CYP450 inhibitors may be effective in reducing ECAM expression and leukocyte adhesion and therefore be useful in the clinical treatment of inflammatory states like IBD. cytochrome P-450; inflammatory bowel disease; lymphocytes; mucosal adhesion cell adhesion molecule-1     

Multivalent sialyl-LeX: potent inhibitors of E-selectin-mediated cell adhesion; reagent for staining activated endothelial cells

Free, monovalent SLeX (Neu5Ac2-3GalßI-4(Fucl-3)-GlcNAc),SLn (Neu5Ac2-3Galß1-4GlcNAc) and corresponding BSA-conjugatedforms—displaying different ratios of SLeX and SLn to protein—weretested for their ability to inhibit binding of HL-60 cells toImmobilized E-selectin. Free SLeX and conjugated SLeX-BSA inhibitedcell binding in a dose-dependent manner. SLn and SLn-BSA didnot inhibit binding. SLeX₁₆BSA (16 mol tetrasaccharide/mol BSA)and monovalent SLeX inhibited cell binding with measured inhibitoryconcentrations (IC₅₀s) of 1 µM and 1 mM, respectively,demonstrating a three-order-of-magnitude enhancement of inhibitoryactivity with the multivalent form of SLeX. A SLex₇BSA conjugatewas 10-fold less potent than those with 11 or 16 mol SLeX/molBSA. An assay which measured neutrophil rolling on interleukin(IL)-1ß-activated human umbilical vein endothelialcells (HUVECs) showed 50% reduction in the number of rollingneutrophils in the presence of 1 µM SLeX₁₆BSA, whereasthe level of free, monovalent SLeX oligosaccharide requiredto produce the same effect was {small tilde}0.3 mM. SLeX–BSAwas found to be an excellent reagent for staining endothelialcells expressing E-selectin. Biotinylated SLeX–BSA inconjunction with Texas red avidin-stained lipopolysaccharide(LPS)-activated HUVECs, and co-incubation of activated cellswith anti-E-selectin, specifically blocked staining. The distributionof E-selectin, as determined by binding of SLeX–BSA, wasvirtually identical with that obtained by binding of anti-E-selectinantibody. The pattern was punctate in nature, rather than beingdiffuse, suggesting that E-selectin may be organized as clusterswithin the plasma membrane. The results suggest that multivalentforms of SLeX bind to E-selectin with higher affinity than domonovalent glycans. Clustering of E-selectin in the membranemay be important for binding to counter-receptors on leukocytecell surfaces. cell adhesion E-selectin glycoconjugate leukocyte receptors sialyl-LeX     

Effects of External Potassium Supply on Compartmentation and Flux Characteristics of Radiocaesium in Intact Spring Wheat Roots

Originally published in Annals of Botany84: 639–644 1999.For technical reasons beyond our control the flux symbol wasomitted from this paper. The paper is reprinted here in itsentirety. Short term experiments investigated the effects of potassiumsupply on radiocaesium influx/efflux and the radiocaesium compartmentationin intact spring wheat roots. Short term (24–72 h) influxanalysis showed that net influxes of radiocaesium to both rootand xylem were reduced approximately ten-times by increasingexternal potassium concentration from 50 µM to 200 µM.Efflux analysis distinguished three components for radiocaesium(namely cell wall+free space, cytoplasm and vacuole) and showedthat the rates of Cs⁺efflux at an external potassium concentrationof 100 µM (19.16 and 1.70 Bq g^-1min^-1for _coand _vo, respectively)were about three-times faster than those at 50 µM (7.24and to 0.41 Bq g^-1min^-1for _coand _vo, respectively). The resultsalso showed that external potassium concentration did not havea significant effect on the distribution of¹³⁷Cs between cytoplasmand vacuole, as indicated by the ratio of¹³⁷Cs in the two compartments.Results obtained in this study suggested that the inhibitoryeffect of potassium on the net uptake of radiocaesium by theplant root may be partially ascribed to the fact that at higherexternal potassium concentrations Cs⁺efflux rates were muchhigher. The mechanisms involved are discussed. Copyright 2000Annals of Botany Company Compartmentation, efflux analysis, potassium, radiocaesium, Triticum aestivum, wheat.     

Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions

Phosphatidylinositol4,5-bisphosphate (PIP₂) affects profoundly several cardiacion channels and transporters, and studies ofPIP₂-sensitive currents in excised patches suggest thatPIP₂ can be synthesized and broken down within 30 s.To test when, and if, total phosphatidylinositol 4-phosphate (PIP) andPIP₂ levels actually change in intact heart, we used a new,nonradioactive HPLC method to quantify anionic phospholipids. Total PIPand PIP₂ levels (10-30 µmol/kg wet weight) do notchange, or even increase, with activation ofG_q/phospholipase C (PLC)-dependent pathways by carbachol(50 µM), phenylephrine (50 µM), and endothelin-1 (0.3 µM).Adenosine (0.2 mM) and phorbol 12-myristate 13-acetate (1µM) bothcause 30% reduction of PIP₂ in ventricles, suggesting thatdiacylglycerol (DAG)-dependent mechanisms negatively regulate cardiacPIP₂. PIP₂, but not PIP, increases reversiblyby 30% during electrical stimulation (2 Hz for 5 min) in guinea pigleft atria; the increase is blocked by nickel (2 mM). Both PIP andPIP₂ increase within 3 min in hypertonic solutions, roughlyin proportion to osmolarity, and similar effects occur in multiple celllines. Inhibitors of several volume-sensitive signaling mechanisms do not affect these responses, suggesting that PIP₂ metabolismmight be sensitive to membrane tension, per se.

     

Hypertrophic effect of selective beta(1)-adrenoceptor stimulation on ventricular cardiomyocytes from adult rat

Weinvestigated whether selective ₁-adrenoceptorstimulation causes hypertrophic growth on isolated ventricularcardiomyocytes from adult rat. As parameters for the induction ofhypertrophic growth, the increases of [¹⁴C]phenylalanineincorporation, protein and RNA mass, and cell size weredetermined. Isoproterenol (Iso, 10 µM) alone had no growtheffect. In the presence of the ₂-adrenoceptor antagonist ICI-118551 (ICI, 10 µM), Iso caused an increase in[¹⁴C]phenylalanine incorporation, protein and RNA mass,cell volume, and cross-sectional area. We showed for phenylalanineincorporation that the growth effect of Iso+ICI could be antagonized by₁-adrenoceptor blockade with atenolol (10 µM) ormetoprolol (10 µM), indicating that it was caused by selective₁-adrenoceptor stimulation. The growth response toIso+ICI was accompanied by an increase in ornithine decarboxylase (ODC)activity and expression. Inhibition of ODC by the ODC antagonistdifluoromethylornithine (1 mM) attenuated this hypertrophic response,indicating that ODC induction is causally involved. The growth responseto Iso+ICI was found to be cAMP independent but was sensitive togenistein (100 µM) or rapamycin (0.1 µM). The reaction was enhancedin the presence of pertussis toxin (10 µM). We conclude thatselective ₁-adrenoceptor stimulation causes hypertrophicgrowth of ventricular cardiomyocytes by a mechanism that is independentof cAMP but dependent on a tyrosine kinase and ODC.

     

Protein kinase C mediates the inhibitory effect of substance P on HCO3- secretion from guinea pig pancreatic ducts

The inhibitory control of pancreatic ductal HCO₃^– secretion may be physiologically important in terms of limiting the hydrostatic pressure developed within the ducts and in terms of switching off pancreatic secretion after a meal. Substance P (SP) inhibits secretin-stimulated HCO₃^– secretion by modulating a Cl^–-dependent HCO₃^– efflux step at the apical membrane of the duct cell (Hegyi P, Gray MA, and Argent BE. Am J Physiol Cell Physiol 285: C268–C276, 2003). In the present study, we have shown that SP is present in periductal nerves within the guinea pig pancreas, that PKC mediates the effect of SP, and that SP inhibits an anion exchanger on the luminal membrane of the duct cell. Secretin (10 nM) stimulated HCO₃^– secretion by sealed, nonperfused, ducts about threefold, and this effect was totally inhibited by SP (20 nM). Phorbol 12,13-dibutyrate (PDBu; 100 nM), an activator of PKC, reduced basal HCO₃^– secretion by 40% and totally blocked secretin-stimulated secretion. In addition, bisindolylmaleimide I (1 nM to 1 µM), an inhibitor of PKC, relieved the inhibitory effect of SP on secretin-stimulated HCO₃^– secretion and also reversed the inhibitory effect of PDBu. Western blot analysis revealed that guinea pig pancreatic ducts express the -, _I-, -, -, -, -, -, and µ-isoforms of PKC. In microperfused ducts, luminal H₂DIDS (0.5 mM) caused intracellular pH to alkalinize and, like SP, inhibited basal and secretin-stimulated HCO₃^– secretion. SP did not inhibit secretion further when H₂DIDS was present in the lumen, suggesting that SP and H₂DIDS both inhibit the activity of an anion exchanger on the luminal membrane of the duct cell. pancreas; Cl^–/HCO₃^– exchanger; inhibition; epithelium     

AN f-TYPE CYTOCHROME FROM CHLORELLA VULGARIS

A method for isolating an f-type cytochrome (Chlorella cytochrome554) from Chlorella vulgaris var. viridis (CHODAT) utilizingN, N-diethylaminoethylcellulose is described. The spectrum ofreduced Chlorella cyt. 554 has absorption maxima at 554 mµ(-band), 524 mµ (ß-band), 417 mµ (SORETband), 352 mµ, 319 mµ and 277 mµ (proteinband). The oxidized form has absorption maxima at 554mµ530 mµ, (ß-band), 412 mµ (SORET band),360mµ 322 mµ and 275 mµ (protein band). Thespectral characteristics resembled other f-type cytochromes,e. g. in the high SORET to -extinction ratio (6.8) and an asymmetric-absorption band (especially at liquid N₂ temperature) ; butcharacteristic differences were present. Mitochondria from whitelupine seedlings and sweet potato roots reduced Chlorella cyt.554. From the effects of antimycin A and 2-heptyl-4-hydroxyquinolineN-oxide it appears that Chlorella cyt. 554 was reduced sequentiallybefore cytochrome a+a₃ and near the level of the cytochromesof the b type. Oxidation was slow using lupine mitochondriaand nil with sweet potato mitochondria. The oxidation-reductionpotential at pH 7.2 and 30? was 0.35 V. Ascorbate, cysteine,glutathione and Na₂S₂O₄ readily reduced Chlorella cyt. 554.The cytochrome was not autoxidizable and was slowly oxidizedby excess potassium ferricyanide. The reduced form did not reactwith CO and was not adsorbed by IRC-50 or Cellex-P cation exchangers. ¹ Temporary address until September 1961: Department of HorticulturalScience, University of California, Los Angeles 24, California,U. S. A. ² Present address: Plant Industry Station, Pioneering ResearchLaboratory, Marketing Quality Research Division, AgriculturalMarketing Service, Beltsville, Maryland, U. S. A. (Received January 16, 1961; )     

Characterization of norepinephrine-evoked inward currents in interstitial cells isolated from the rabbit urethra

Freshly dispersedinterstitial cells from the rabbit urethra were studied by using theperforated-patch technique. When cells were voltage clamped at 60 mVand exposed to 10 µM norepinephrine (NE) at 80-s intervals, eitherlarge single inward currents or a series of oscillatory inward currentsof diminishing amplitude were evoked. These currents were blocked byeither phentolamine (1 µM) or prazosin (1 µM), suggesting that theeffects of NE were mediated via ₁-adrenoceptors.NE-evoked currents were depressed by the blockers ofCa²⁺-activated Cl currents, niflumic acid (10 µM), and 9-anthracenecarboxylic acid (9-AC, 1 mM). The reversalpotential of the above currents changed in a predictable manner whenthe Cl equilibrium potential was altered, againsuggesting that they were due to activation of a Clconductance. NE-evoked currents were decreased by 10 µM cyclopiazonic acid, suggesting that they were dependent on store-releasedCa²⁺. Inhibition of NE-evoked currents by the phospholipaseC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate(100 µM) suggested that NE releases Ca²⁺ via an inositol1,4,5-trisphosphate (IP₃)-dependent mechanism. Theseresults support the idea that stimulation of₁-adrenoceptors releases Ca²⁺ from anIP₃-sensitive store, which in turn activatesCa²⁺-activated Cl current in freshlydispersed interstitial cells of the rabbit urethra. This elevates slowwave frequency in these cells and may underlie the mechanismresponsible for increased urethral tone during nerve stimulation.

     

EFFECTS OF VARIOUS FACTORS ON THE INCREASE OF {alpha}-AMYLASE ACTIVITY IN RICE ENDOSPERM INDUCED BY GIBBERELLIN A3

The increase of -amylase activity in embryoless rice endosperminduced by the addition of gibberellin A₃ was examined undervarious conditions with an aim to establish a bioassay methodfor gibberellins. Sterilized embryoless rice endosperms were incubated in a testtube containing 0.2–1.0 ml of test solution for 4 daysat 30. -Amylase activity in the endosperm was determined bymeasuring digestion of added starch. The increase of amylaseactivity during the incubation was not affected by the additionof various vitamins, amino acids, organic acids, protease, sucrose,indoleacetic acid or kinetin. Helminthosporol, helminthosporicacid and sclerin (1–10 µg/ml) had weak promotingeffects. Under appropriate conditions, 10^–5 µg/mlof gibberellin A₃ could be detected. In double logarithmic plot,the increase in the enzyme activity was proportional to thegibberellin A₃ concentration in the range from 10^–5 to10^–2 µg/ml. (Received April 13, 1966; )     

Copper is taken up efficiently from albumin and alpha2-macroglobulin by cultured human cells by more than one mechanism

Ionic copper entering blood plasma binds tightly to albumin and the macroglobulin transcuprein. It then goes primarily to the liver and kidney except in lactation, where a large portion goes directly to the mammary gland. Little is known about how this copper is taken up from these plasma proteins. To examine this, the kinetics of uptake from purified human albumin and ₂-macroglobulin, and the effects of inhibitors, were measured using human hepatic (HepG2) and mammary epithelial (PMC42) cell lines. At physiological concentrations (3–6 µM), both cell types took up copper from these proteins independently and at rates similar to each other and to those for Cu-dihistidine or Cu-nitrilotriacetate (NTA). Uptakes from ₂-macroglobulin indicated a single saturable system in each cell type, but with different kinetics, and 65–80% inhibition by Ag(I) in HepG2 cells but not PMC42 cells. Uptake kinetics for Cu-albumin were more complex and also differed with cell type (as was the case for Cu-histidine and NTA), and there was little or no inhibition by Ag(I). High Fe(II) concentrations (100–500 µM) inhibited copper uptake from albumin by 20–30% in both cell types and that from ₂-macroglobulin by 0–30%, and there was no inhibition of the latter by Mn(II) or Zn(II). We conclude that the proteins mainly responsible for the plasma-exchangeable copper pool deliver the metal to mammalian cells efficiently and by several different mechanisms. ₂-Macroglobulin delivers it primarily to copper transporter 1 in hepatic cells but not mammary epithelial cells, and additional as-yet-unidentified copper transporters or systems for uptake from these proteins remain to be identified. transcuprein; uptake kinetics; iron competition; silver competition; HepG2 cells; PMC42 cells     

The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading

Primary open angle glaucoma (POAG) is a major blindness-causingdisease, characterized by elevated intraocular pressure dueto an insufficient outflow of aqueous humor. The trabecularmeshwork (TM) lining the aqueous outflow pathway modulates theaqueous outflow facility. TM cell adhesion, cell–matrixinteractions, and factors that influence Rho signaling in TMcells are thought to play a pivotal role in the regulation ofaqueous outflow. In a recent study, we demonstrated that galectin-8(Gal8) modulates the adhesion and cytoskeletal arrangement ofTM cells and that it does so through binding to β₁ integrinsand inducing Rho signaling. The current study is aimed at thecharacterization of the mechanism by which Gal8 mediates TMcell adhesion and spreading. We demonstrate here that TM cellsadhere to and spread on Gal8-coated wells but not on galectin-1(Gal1)- or galectin-3 (Gal3)-coated wells. The adhesion of TMcells to Gal8-coated wells was abolished by a competing sugar,β-lactose, but not by a noncompeting sugar, sucrose. Also,a trisaccharide, NeuAc2-3Galβ1-4GlcNAc, which binds specificallyto the N-CRD of Gal8, inhibited the spreading of TM cells toGal8-coated wells. In contrast, NeuAc2-6Galβ1-4GlcNAc whichlacks affinity for Gal8 had no effect. Affinity chromatographyof cell extracts on a Gal8-affinity column and binding experimentswith plant lectins, Maakia Amurensis and Sambucus Nigra, revealedthat ₃β₁, ₅β₁, and _vβ₁ integrins are major counterreceptorsof Gal8 in TM cells and that TM cell β₁ integrins carrypredominantly 2-3-sialylated glycans, which are high-affinityligands for Gal8 but not for Gal1 or Gal3. These data lead usto propose that Gal8 modulates TM cell adhesion and spreading,at least in part, by interacting with 2-3-sialylated glycanson β₁ integrins.     

Inhibition of Gibberellic Acid-induced Starch Mobilization by Salicylhydroxamic acid in Avena fatua L. Seed

Inhibition of GA₃-induced endosperm mobilization in Avena fatuaL. by salicylhydroxamic acid (SHAM), a widely used alternativerespiration inhibitor, was studied. SHAM strongly inhibitedthe GA₃-induced release of reducing sugars in the incubationmedium by 3 mm de-embryonated endosperm segments; at 4 mM SHAM,GA₃-induced sugar release was inhibited by 66–79 per cent.Extracts prepared from segments incubated in 0.05 mM GA₃ with2, 5 and 10 mM SHAM showed 30, 53 and 71 per cent lower -amylaseactivity, respectively, compared to the GA₃-alone treatment.Addition of SHAM (0.5–5 mM) during the enzyme assay hadno effect on the activity of -amylase. Thus, the inhibitionof starch mobilization in endosperm by SHAM is due to inhibitionof the production and not the activity of -amylase. The inhibitionof Avena fatua seedling growth by SHAM reported earlier may,in part, be due to its effect on endosperm mobilization. Since (1) Avena fatua seeds have been shown to have little orno SHAM-sensitive respiration, and (2) concentrations of SHAMnecessary for inhibiting endosperm mobilization were significantlyhigher than those generally necessary for inhibiting alternativerespiration, the inhibition of endosperm mobilization by thiscompound does not appear to involve its effect on alternativerespiration. Avena fatua L., wild oat, -amylase, endosperm, gibberellic acid, salicylhydroxamic acid, seed     

Respiration Rate and Mitochondrial Oxidase Activity in Arum Spadix

1. Mitochondria prepared from young Arum spadix oxidise succinicand -ketoglutaric acids at about 300µl. O₂/hr./g. wetwt. but as the spadix ages the activity of the mitochondriaincreases more than tenfold. The increase results from a smallrise in the quantity of mitochondrial nitrogen brought downin the centrifuge and a larger rise in oxidase activity permg. mitochondrial nitrogen. 2. The rate of respiration of spadix slices has been measuredunder conditions in which it is not limited by slow diffusionof oxygen. Slices of young spadix respire at about 1,000–2,000µl. O₂/hr/g. but as the inflorescence opens the rate soarsto 20,000µl. O₂/hr/g. and the spadix becomes warm. 3. Cytochrome oxidase was assayed in mitochondria treated withdigitonin. There is at all times enough cytochrome oxidase toaccount for the rates of oxidation of succinate and -ketoglutarate;and enough to account for the relatively slow respiration ofthe young spadix slices but not for the fastest respirationof the mature spadix. 4. The respiration of spadix slices is found, in contradictionto an earlier report, to be sensitive to malonate.     

Analysis of the Diffusion Symmetry Developed by the Alkaline and Acid Bands which form at the Surface of Chara corallina Cells

The diffusion symmetry developed by the alkaline and aeid bandsof Chara corallina was studied. The alkaline system developeda diffusion pattern which could not be fitted to the equationfor a continuous point-source efflux. However, good correlationwas obtained between experimental data and the diffusion equationfor a hollow sphere. The calculated OH- efflux values, obtainedusing the equation of a continuous spherical-surface source,were checked against the influx values of H₁₄ obtained under the same experimental conditions. IndividualOH– band efflux values ranged from 0.07 to 5.95 pmol s^–1and total cell fluxes of 25 pmol cm^–2 s^–1 for OH^-and H were obtained (in the presence of 0.5 mM NaHCO₃). The acid system developed a cylindrical diffusion pattern, butthis could not be fitted to a mathematical equation. Numericalanalysis will have to be employed to obtain values of H⁺ efflux.     

Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphodiester {alpha}-N-acety1g1ucosaminidase

The enzyme N-acetylglucosamine-1-phosphodiester -N-acetylglucosaminidase(EC 3.1.4.45 [EC] ; uncovering enzyme) catalyzes the removal of N-acetylglucosaminefrom the N-acetylglucosamine--phospho-mannose portion of selectedlysosomal enzyme oligosaccharide chains, thereby formimg themannose 6-phosphate signal which is responsible for the targetingof these lysosomal enzymes for transport into lysosomes. Theuncovering enzyme has been purified approximately 7000-foldto electrophoretic homogeneity from Epstein-Barr virus-transformedhuman lymphoblast cells. The purification sequence involvessolubilizing this membrane-bound enzyme with Tergitol NP-10,affinity chromatography on Lentil lectin-Sepharose 4B, ion-exchangechromatography on DEAE-Sephacel, chromatography on zinc(II)-IDA-Sepharose6B, and preparative SDS-PAGE electrophoresis. The purified enzymemigrated as a single band of 114 kDa which was coincident withenzyme activity on analytical SDS-PAGE electrophoresis. Characterizationstudies of the purified enzyme demonstrated that catalytic activitywas maximal at pH 6.95 and that the enzyme retained full activityfollowing incubation for 10 min at 60°C. No requirementwas found for a divalent cation, but Zn²⁺ Hg²⁺ and Cu²⁺ werefound to reduce the enzyme's activity by 30–40%. The highestcatalytic efficiency was observed with N-acetylglucosamine phospho-methylmannosideas a substrate while uridine diphosphate-N-acetylglucosamine,N-acetylglucosamine phosphomannose-uteroferrin, and N-acetylglucosaminephosphate were also cleaved by the enzyme with decreasing efficiency.Acetamino-deoxycastanospermine was a potent inhibitor of thehuman enzyme with a K₁ of 0.35 µM, while N-acetylglucosaminephosphate (K₁ 1.58 mM) and N-acetylglucosamine (K₁ 5.1 mM) inhibitedthe enzyme to a lesser degree. N-acetylglucosamine-1-phosphodiester -N acetylglucosaminidase lymphoid cells targeting mannose-6 phosphate     

Effects of estradiol on phenylephrine contractility associated with intracellular calcium release in rat aorta

The ability of estradiol to affect phenylephrine-induced contraction and the subsequent increase in resting tone, associated with capacitative Ca²⁺ entry across the plasma membrane, was evaluated in rat aortic rings incubated in Ca²⁺-free solution. The incubation with estradiol (1–100 nM, 5 min) inhibited both the phenylephrine-induced contraction and the IRT. Neither cycloheximide (1 µM; inhibitor of protein synthesis) nor tamoxifen (1 µM; blocker of estrogenic receptors) modified the effects of estradiol. Estradiol (100 µM) also blocked the contractile response to serotonin (10 µM) but not to caffeine (10 mM). In addition, estradiol (100 µM) inhibited the contractile responses to cyclopiazonic acid (1 µM; selective Ca²⁺-ATPase inhibitor) associated with capacitative Ca²⁺ influx through non-L-type Ca²⁺ channels. Finally, estradiol inhibited the Ca²⁺-induced increases in intracellular free Ca²⁺ (after pretreatment with phenylephrine) in cultured rat aorta smooth muscle cells incubated in Ca²⁺-free solution. In conclusion, estradiol interfered in a concentration-dependent manner with Ca²⁺-dependent contractile effects mediated by the stimuli of ₁-adrenergic and serotonergic receptors and inhibited the capacitative Ca²⁺ influx through both L-type and non-L-type Ca²⁺ channels. Such effects are in essence nongenomic and not mediated by the intracellular estrogenic receptor. estrogen; ₁-adrenergic agonists