The inheritance of salt exclusion in woody perennial fruit species

Citrus and grapevines are salt-sensitive perennial crops. Damage caused by salinity is due mostly to accumulation of excessive concentrations of salt (Na- and Cl ions) in shoot tissues. In both crops, however, some rootstock varieties can restrict the accumulation of salt in scion leaves and stems. Salt-excluding rootstocks, however, are often deficient with regard to other desirable characteristics and as such their use is limited. As a consequence, we have conducted a range of crosses within both crops to select new salt-excluding hybrids which may have potential as new rootstocks and also to investigate the inheritance of salt exclusion in these woody perennials.In citrus, both Cl-ion and Na-ion exclusion has been observed as a continuous character and progenies segregate widely for their ability to restrict the accumulation of these ions in shoot tissues. The ability to exclude Cl ions appears to be independent of the ability to exclude Na ions. Thus a good Cl-ion excluder is not necessarily a good Na-ion excluder and vice versa. It has been possible, however, to select new salt-excluding citrus hybrids which perform as well as and sometimes better than parent varieties when grafted with a common scion and grown in artificially salinised field plots.In grapevines, the research has concentrated on the ability for Cl-ion exclusion and depending on the Cl-ion-excluding parent chosen this is inherited as either a polygenic or monogenic trait. In crosses between Vitis champini (Cl-ion excluder) and Vitis vinifera (Cl-ion accumulator), the ability to restrict Cl-ion accumulation in shoot tissues segregates widely with some offspring transgressing the performance of either parent. In crosses and backcrosses involving V. berlandieri and V. vinifera, however, hybrids segregate as either Cl-ion excluders or accumulators suggesting the effect of a major dominant gene for Cl-ion exclusion from V. berlandieri. This was evident from both field and glasshouse experiments although possible modifying genes from V. vinifera may affect the expression of this gene under glasshouse conditions in some crosses.     

Modification of Leaf Formation by Cytokinin and Chlormequat (CCC) in Vitis

Application of the cytokinin, 6-(benzylamino)-9-(2-tetrahydropyrany1)-9H-purine(PBA, 1mM) and 2-chloroethyl trimethyl ammonium chloride (chlormequat;3 mM), a growth retardant, to grapevines induced the formationof fused leaves (two laminae and two petioles) and double leaves(two separate petioles each with a single lamina). Double leaveswere found in Vitis vinifera L. seedlings, in Vitis ripariaand in Muscadinia (Vitis) rotundifolia. In some of the treatedvines, leaves arose with opposite phyllotaxy. Other anomaliesincluded production of two opposite axillary buds in axils ofopposite leaves and production of two tendrils per node. Sometendrils grew into shoots. In Muscat of Alexandria, applicationof GA₃ (3 µM, 15 µM), followed by an applicationof chlormequat (0.4 mM, 1.2 mM), led to a marked reduction inthe extension growth of axillary shoots. Vitis vinifera L., Vitis riparia, Muscadinia (Vitis) rotundifolia, grapevines, leaf formation, cytokinin, chloromequat     

Influence of saline irrigation on growth,ion accumulation and partitioning,and leaf gas exchange of carrot (Daucus carota L.)

Like those of many horticultural crop species, the growth and leaf gas exchange responses of carrot (Daucus carota L.) to salinity are poorly understood. In this study ion accumulation in root tissues (periderm, xylem and phloem tissues) and in leaves of different ages was assessed for carrot plants grown in the field with a low level of salinity (5.8 mM Na(+) and 7.5 mM Cl(-)) and in a glasshouse with salinity ranging from 1-80 mM. At low levels of salinity (1-7.5 mM), in both the field and glasshouse, carrot leaves accumulated high concentrations of Cl(-) (140-200 mM); these appear to be the result of a high affinity for Cl(-) uptake and a low retention of Cl(-) in the root system. However, Cl(-) uptake is under tight control, with an 80-fold increase in external salinity resulting in only a 1.5-fold change in the Cl(-) concentration of the shoot and no increase in the Cl(-) concentration of the root xylem tissue. In contrast to Cl(-), shoot Na(+) concentrations were comparatively low (30-40 mM) but increased by seven-fold when salinity was increased by 80-fold. Growth over the 56-d treatment period in the glasshouse was insensitive to salinity less than 20 mM, but at higher concentrations the yield of carrot tap roots declined by 7 % for each 10 mM increase in salinity. At low levels of salinity the accumulation of high concentrations of Cl(-) (150 mM) in carrot laminae did not appear to limit leaf gas exchange. However, photosynthesis and stomatal conductance were reduced by 38 and 53 %, respectively, for plants grown at a salinity of 80 mM compared with those grown at 1 mM. Salinity-induced reductions in both p(i) and carbon isotope discrimination (delta) were small (2.5 Pa and 1.4 per thousand, respectively, at 80 mM) indicating that the reduction in photosynthesis was only marginally influenced by CO(2) supply. At a salinity of 80 mM the photosynthetic capacity was reduced, with a 30 % reduction in the CO(2)-saturated rate of photosynthesis (A(max)) and a 40 % reduction in both the apparent rate of RuBP-carboxylase-limited CO(2) fixation (V(cmax)) and the electron transport rate limiting RuBP regeneration (J(max)). This study has shown that carrot growth and leaf gas exchange are insensitive to the high leaf Cl(-) concentrations that occur at low levels (1-7 mM) of salinity. However, growth is limited at salinity levels above 20 mM and leaf gas exchange is limited at salinity levels above 8 mM.     

Effects of resting membrane potential and intactness of the T-tubules on caffeine contractures in rat skeletal muscle

We have studied the effects of changes in the resting membrane potential (Vm) and T-tubules on caffeine contracture (25 mM) elicited in rat soleus muscle in vitro at 34 degrees C. In high [K]o (30-140 mM, [K]o X [Cl]o constant) caffeine contractures were reduced by about 40-50% and had a faster time course than in normal Krebs ([K]o = 5 mM). Detubulation of the muscles by an osmotic treatment produces a reduction of about 30% in the caffeine contracture tension. Our results with high K solutions suggest a reduced sensitivity of the myofibrils to calcium released by caffeine. The effects of detubulation on caffeine contracture suggest that caffeine may have a direct effect on sarcolemma in addition to its well known action on the sarcoplasmic reticulum (SR). However, a depletion of the calcium content in the SR of depolarized muscle fibres as well as an anatomical damage produced by the osmotic treatment can not be ruled out as an explanation for the reduced caffeine contracture.     

Anion/anion exchange in human neutrophils

Of the total one-way chloride fluxes (approximately 1.4 meq/liter cell water X min) in steady state human polymorphonuclear leukocytes bathed in 148 mM Cl media, approximately 70% behaves as self-exchange mediated by a nonselective anion carrier that is not inhibited by stilbene disulfonates. Five properties of this carrier-mediated exchange were investigated: substrate saturation is seen with respect to 36Cl influx as a function of the external Cl concentration [for normal-Cl cells, the apparent Km(Cl) is approximately 22 mM when Cl replaces para-amino- hippurate (PAH) and approximately 5 mM when Cl replaces glucuronate], and with respect to 36Cl efflux as a function of the concentration of internal Cl replacing PAH [apparent Km(Cl) congruent to 35 mM for cells bathed in 148 mM Cl]; there is trans stimulation of 36Cl influx by internal Cl (replacing PAH) with an apparent Km(Cl) congruent to 35 mM, and of 36Cl efflux by external Cl with an apparent Km(Cl) congruent to 22 mM (Cl replacing PAH) or approximately 5 mM (Cl replacing glucuronate); there is substrate competition between Cl and PAH, but the carrier appears devoid of affinity for glucuronate; influxes and effluxes mediated by the carrier are subject to competitive inhibition by extracellular alpha-cyano-4-hydroxycinnamate (CHC), with an apparent Ki congruent to 9 mM in Cl medium or approximately 1 mM in PAH medium (transport of the inhibitor itself is very slow); and internal Cl and external Cl or PAH undergo 1:1 countertransport, which is CHC sensitive. A simple equilibrium-competition model is proposed that accounts for all the extracellular ligand interactions presented for normal-Cl cells. Least-squares values of the carrier's true Michaelis constants for extracellular Cl, PAH, and CHC are 5.03 +/- 0.83, 50.3 +/- 14.9, and 0.29 +/- 0.09 mM, respectively.     

Sulfate and chloride transport in Caco-2 cells: differential regulation by thyroxine and the possible role of DRA gene

The current studies were undertaken to establish an in vitro cellular model to study the transport of SO and Cl(-) and hormonal regulation and to define the possible function of the downregulated in adenoma (DRA) gene. Utilizing a postconfluent Caco-2 cell line, we studied the OH(-) gradient-driven (35)SO and (36)Cl(-) uptake. Our findings consistent with the presence of an apical carrier-mediated (35)SO/OH(-) exchange process in Caco-2 cells include: 1) demonstration of saturation kinetics [Michaelis-Menten constant (K(m)) of 0.2 +/- 0.08 mM for SO and maximum velocity of 1.1 +/- 0.2 pmol x mg protein(-1) x 2 min(-1)]; 2) sensitivity to inhibition by DIDS (K(i) = 0.9 +/- 0.3 microM); and 3) competitive inhibition by oxalate and Cl(-) but not by nitrate and short chain fatty acids, with a higher K(i) (5.95 +/- 1 mM) for Cl(-) compared with oxalate (K(i) = 0.2 +/- 0.03 mM). Our results also suggested that the SO/OH(-) and Cl(-)/OH(-) exchange processes in Caco-2 cells are distinct based on the following: 1) the SO/OH(-) exchange was highly sensitive to inhibition by DIDS compared with Cl(-)/OH(-) exchange activity (K(i) for DIDS of 0.3 +/- 0.1 mM); 2) Cl(-) competitively inhibited the SO/OH(-) exchange activity with a high K(i) compared with the K(m) for SO, indicating a lower affinity for Cl(-); 3) DIDS competitively inhibited the Cl(-)/OH(-) exchange process, whereas it inhibited the SO/OH(-) exchange activity in a mixed-type manner; and 4) utilizing the RNase protection assay, our results showed that 24-h incubation with 100 nM of thyroxine significantly decreased the relative abundance of DRA mRNA along with the SO/OH(-) exchange activity but without any change in Cl(-)/OH(-) exchange process. In summary, these studies demonstrated the feasibility of utilizing Caco-2 cell line as a model to study the apical SO/OH(-) and Cl(-)/OH(-) exchange processes in the human intestine and indicated that the two transporters are distinct and that DRA may be predominantly a SO transporter with a capacity to transport Cl(-) as well.     

Ionic permeability of K, Na, and Cl in potassium-depolarized nerve. Dependency on pH, cooperative effects, and action of tetrodotoxin.

The passive ionic membrane conductances (gj) and permeabilities (Pj) of K, Na, and Cl of crayfish (Procambarus clarkii) medial giant axons were determined in the potassium-depolarized axon and compared with that of the resting axon. Passive ionic conductances and permeabilities were found to be potassium dependent with a major conductance transition occurring around an external K concentration of 12-15 mM (Vm = -60 to -65 mV). The results showed that K, Na, and Cl conductances increased by 6.2, 6.9, and 27-fold, respectively, when external K was elevated from 5.4 to 40 mM. Permeability measurements indicated that K changed minimally with K depolarization while Na and Cl underwent an order increase in permeability. In the resting axon (K0 = 5.4 mM, pH = 7.0) PK = 1.33 X 10(-5), PCl = 1.99 X 10(-6), PNa = 1.92 X 10(-8) while in elevated potassium (K0 = 40 mM, pH 7.0), PK = 1.9 X 10(-5), PCl = 1.2 X 10(-5), and PNa = 2.7 X 10(-7) cm/s. When membrane potential is reduced to 40 mV by changes in internal ions, the conductance changes are initially small. This suggests that resting channel conductances depend also on ion environments seen by each membrane surface in addition to membrane potential. In elevated potassium, K, Na, and Cl conductances and permeabilities were measured from pH 3.8 to 11 in 0.2 pH increments. Here a cooperative transition in membrane conductance or permeability occurs when pH is altered through the imidazole pK (approximately pH 6.3) region. This cooperative conductance transition involves changes in Na and Cl but not K permeabilities. A Hill coefficient n of near 4 was found for the cooperative conductance transition of both the Na and Cl ionic channel which could be interpreted as resulting from 4 protein molecules forming each of the Na and Cl ionic channels. Tetrodotoxin reduces the Hill coefficient n to near 2 for the Na channel but does not affect the Cl channel. In the resting or depolarized axon, crosslinking membrane amino groups with DIDS reduces Cl and Na permeability. Following potassium depolarization, buried amino groups appear to be uncovered. The data here suggest that potassium depolarization produces a membrane conformation change in these ionic permeability regulatory components. A model is proposed where membrane protein, which forms the membrane ionic channels, is oriented with an accessible amino terminal group on the axon exterior. In this model the ionizable groups on protein and phospholipid have varied associations with the different ionic channel access sites for K, Na, and Cl, and these groups exert considerable control over ion permeation through their surface potentials.     

Use of in vitro method to evaluate some grapevine varieties for tolerance and susceptibility to sodium bicarbonate-induced chlorosis

Nodal shoot segments of four grapevine genotypes well known for their Fe-chlorosis characteristic [Vitis vinifera × Vitis berlandieri Fercal, resistant; V. berlandieri × Vitis rupestris 1103 P, mid-resistant; Solonis (Vitis riparia × V. rupestris × Vitis candicans) × Othello (Vitis labrusca × V. riparia × V. vinifera) 1613 C, susceptible; V. vinifera L. cv. Perlette, resistant] were cultured in vitro. The effects of three levels of iron sodium ethylene-diaminotetraacetate (FeNaEDTA; 9, 18, 36 mg l⁻¹) and three mixtures of iron and 840 mg l⁻¹ NaHCO₃ (sodium bicarbonate) in the Murashige and Skoog (MS) medium supplemented 4.9 μM indole-3-butyric acid (IBA) were compared. We assayed the chlorosis rating of leaves, total chlorophyll of leaves, dry shoot weights of the plantlets, and active and total Fe content of the leaves. The most suitable concentration in determining the reaction of genotypes to iron chlorosis was 9 mg l⁻¹ FeNaEDTA. Bicarbonate addition had negative effects on the iron intake and development of plants. While all genotypes were affected by non-ferrous conditions, Fercal and Perlette were found to be the most resistant genotypes and 1613 C rootstock as the most susceptible. The chlorosis rating of the tested genotypes ranked according to their known degree of tolerance and susceptibility to lime-induced chlorosis. The results of this study showed that the in vitro technique could successfully be used in viticulture to get results in shorter times in the studies, aiming at breeding new rootstocks and varieties suitable to calcareous soil conditions and determining the reactions of existing genotypes to Fe chlorosis.     

Activation of specific membrane mechanisms in the myocardium cells on early stages of ischemia

Electron probe microanalysis was employed to determine the elemental concentration (K,Na,Cl) in a myocyte on cryosections of the papillary muscle of the isolated rat (Wistar) heart. Protocols of global ischemia and ischemic conditions under glucose-free anoxic perfusion were applied. It was shown that global ischemia induces potassium deficiency (94 +/- 2 mM) in the myocyte and an increase in the level of sodium (72 +/- 4 mM) and chlorine (42 +/- 1 mM) in the cytoplasm compared with intact cell (122 +/- 2; 36 +/- 1; 24 +/- 1 mM). Glucose-free anoxic perfusion leads to a smooth fall of potassium concentration in the cell up to 54 +/- 2 mM with the retention of intracellular sodium (40 +/- 1 mM) and chlorine (26 +/- 1 mM) level. The present finding suggest that, in early ischemia, specific membrane mechanisms of ion transport are activated. Among these are KNa channel, Hi(+)-Nao+ exchange, KATP channel, lactate transport from the cell, associated either with potassium efflux to the extracellular space or chlorine influx into the myocyte. It is assumed that Na/K-ATPase is also activated under ischemic conditions.     

Root apoplastic transport and water relations cannot account for differences in Cl− transport and Cl−/NO3 − interactions of two grapevine rootstocks differing in salt tolerance

Grapevine is moderately sensitive to salinity and accumulation of toxic levels of Cl^? in leaves is the major reason for salt-induced symptoms. In this study, apoplastic Cl^? uptake and transport mechanism(s) were investigated in two grapevine (Vitis sp.) rootstock hybrids differing in salt tolerance; 1103 Paulsen (salt tolerant) and K 51–40 (salt sensitive). Increased external salinity caused high Cl^? accumulation in shoots of the salt sensitive K 51–40 in comparison to Paulsen. Measurement of ¹⁵NO₃ ^? net fluxes under high salinity showed that by increasing external Cl^? concentrations K 51–40 roots showed reduced NO₃ ^? accumulation. This was associated with increased accumulation of Cl^?. In comparison to Paulsen, K 51–40 showed reduced NO₃ ^?/Cl^? root selectivity with increased salinity, but Paulsen had lower selectivity over the whole salinity range (0–45 mM). To examine if root hydraulic and permeability characterisations accounted for differences between varieties, the root pressure probe was used on excised roots. This showed that the osmotic Lp_r was significantly smaller than hydrostatic Lp_r, but no obvious difference was observed between the rootstocks. The reflection coefficient (σ) values (0.48–0.59) were the same for both rootstocks, and root anatomical studies showed no obvious difference in apoplastic barriers of the main and lateral roots. Comparing the uptake of Cl^? with an apoplastic tracer, PTS (3-hydroxy-5,8,10-pyrentrisulphonic acid), showed that there was no correlation between Cl^? and PTS transport. These results indicated that bypass flow of salts to the xylem is the same for both rootstocks (0.77 ± 0.2 and 1.05 ± 0.12 %) and hence pointed to differences in membrane transport to explain difference in Cl^? transport to the shoot.     

Translocation efficiency of apolipoprotein B is determined by the presence of beta-sheet domains, not pause transfer sequences

Cotranslational translocation of apoB100 across the endoplasmic reticulum (ER) membrane is inefficient, resulting in exposure of nascent apoB on the cytosolic surface of the ER. This predisposes apoB100 to ubiquitinylation and targeting for proteasomal degradation. It has been suggested that pause transfer sequences (PTS) present throughout apoB cause inefficient translocation. On the other hand, our previous study demonstrated that the translocation efficiency of apoB100 is dependent on the presence of a beta-sheet domain between 29 and 34% of full-length apoB100 (Liang, J.-S., Wu, X., Jiang, H., Zhou, M., Yang, H., Angkeow, P., Huang, L.-S., Sturley, S. L., and Ginsberg, H. N. (1998) J. Biol. Chem. 273, 35216-35221); this region of apoB has no PTS. However, the effects of the beta-sheet domain may require the presence of PTS elsewhere in the N-terminal region of apoB100. To further investigate the roles of PTS and beta-sheet domains in the translocation of apoB100 across the ER, we transfected McArdle RH7777, HepG2, or Chinese hamster ovary cells with human albumin (ALB)/human apoB chimeric cDNA constructs: ALB/B12-17 (two PTS but no beta-sheet), ALB/B29-34 (beta-sheet but no PTS), ALB/B36-41 (two PTS and a beta-sheet), and ALB/B49-54 (neither PTS nor a beta-sheet). ALB/ALB1-40 served as a control. Compared with ALB/ALB1-40, secretion rates of ALB/B12-17, ALB/B29-34, and ALB/B36-41 were reduced. Secretion of ALB/B49-54 was similar to that of ALB/ALB1-40. However, only ALB/B29-34 and ALB/B36-41 had increased proteinase K sensitivity, ubiquitinylation, and increased physical interaction with Sec61alpha. These results indicate that the translocation efficiency of apoB100 is determined mainly by the presence of beta-sheet domains. PTS do not appear to affect translocation, but may affect secretion by other mechanisms.     

Waterlogging and salinity effects on two Suaeda salsa populations

Adaptations to combined salinity and waterlogging stress were evaluated in two Suaeda salsa populations from different saline environments. Seedlings were exposed to 1, 200 and 600 mM NaCl in drained or waterlogged sand for 22 days in a glasshouse. Waterlogging did not significantly affect the K(+) /Na(+) ratio or Cl(-) concentration in leaves of either population. Adventitious roots were produced only by the inland population and under the waterlogged condition. X-ray microanalysis showed that S. salsa roots of the intertidal population accumulated more [Na(+) ] and [Cl(-) ] in both the cortex and stele than the roots of the inland population. The ability of roots to exclude Na(+) and Cl(-) was greater in the intertidal population than in the inland population, which may explain why leaves of the intertidal population accumulated less Na(+) and Cl(-) than the leaves of the inland population. The lower level of Cl(-) than Na(+) in leaves of both populations may result from the greater ability of roots to exclude Cl(-) than Na(+) . These traits may help the two S. salsa populations adapt to their different saline environments.     

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

The secretin-stimulated human pancreatic duct secretes HCO(3)(-)-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO(3)(-) secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl(-)/HCO(3)(-) exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ～140 mM HCO(3)(-) or more, mouse and rat ducts secrete ～40-70 mM HCO(3)(-). Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO(3)(-) secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl(-)/Cl(-) exchange and electroneutral Cl(-)/HCO(3)(-) exchange. gpSlc26a6 in Xenopus oocytes mediated Cl(-)/Cl(-) exchange and bidirectional exchange of Cl(-) for oxalate and sulfate, but Cl(-)/HCO(3)(-) exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl(-), oxalate, and sulfate transport but no detectable Cl(-)/HCO(3)(-) exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of (36)Cl(-) influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO(3)(-) secretion in species that share a high HCO(3)(-) secretory output.     

Sodium-dependent chloride transport in basolateral membrane vesicles isolated from rabbit proximal tubule

The mechanisms for Cl transport across basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were examined by using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). The transporters studied included Cl/base exchange, Cl/base/Na cotransport, K/Cl cotransport, and Cl conductance. Initial rates of chloride influx (JCl) were determined from the measured time course of SPQ fluorescence in BLMV following inwardly directed gradients of Cl and gradients of other ions and/or pH. For a 50 mM inwardly directed Cl gradient in BLMV which were voltage and pH clamped (7.0) using K/valinomycin and nigericin, JCl was 0.80 +/- 0.14 nmol S-1 (mg of vesicle protein)-1 (mean +/- SD, n = 8 separate preparations). In the absence of Na and CO2/HCO3 in voltage-clamped BLMV, JCl increased 56% +/- 5% in response to a 1.9 pH unit inwardly directed H gradient; the increase was further enhanced by 40% +/- 3% in the presence of CO2/HCO3 and inhibited 30% +/- 8% by 100 microM dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Na gradients did not increase JCl in the absence of CO2/HCO3; however, an outwardly directed Na gradient in the presence of CO2/HCO3 increased JCl by 31% +/- 8% with a Na KD of 7 +/- 2 mM. These results indicate the presence of Cl/OH and Cl/HCO3 exchange, and Cl/HCO3 exchange trans-stimulated by Na. There was no significant effect of K gradients in the presence or absence of valinomycin, suggesting lack of significant K/Cl cotransport and Cl conductance under experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-acetyl alpha-D-glucosaminidase from the venom of African puff adder (Bitis arietans).

The activity of N-acetyl-alpha-D-glucosaminidase from venom of the African puff adder (Bitis arietans) has been detected. The enzyme from the venom was purified by chromatography on Q-sepharose, CM-cellulose, and N-acetyl-alpha-D-glucosamine-agarose affinity column. The enzyme has a molecular weight of 102 kDa determined by size exclusion chromatography on Sephacryl 200. It migrated as a 51-kDa band on SDS polyacrylamide gels. The enzyme is maximally active at pH 5.5 and 40 degrees C. The B. arietans NAGase hydrolyzed exclusively terminally linked alpha-(1-4) GlcNAc residues from nonreducing ends of oligosaccharides. It hydrolysed chito-oligosaccharide, MU-GlcNAc and chitobiose with K(M) values of 0.15 mM and 1.22 mM, respectively. Swollen chitin and oligosaccharide above (GlcNAc)(4) were not hydrolysed by the enzyme. B. arietans NAGase was strongly inhibited noncompetitively by Hg(2+), competitively by 1-thio-beta-D-GlcNAc and N-acetyl glucosamine (NAG) with K(i) of 0.55, 0.25 and 8 mM, respectively. Colombin the active component of antivenom preparation from Aristolodia albida inhibited the enzyme competitively with K(i) of 0.6 mM. Delineation of the active site by chemical modification revealed the involvement of His and Trp in the catalysis of the enzyme.     

Extracellular chloride regulation of Kv2.1, contributor to the major outward Kv current in mammalian outer hair cells

Outer hair cells (OHC) function as both receptors and effectors in providing a boost to auditory reception. Amplification is driven by the motor protein prestin, which is under anionic control. Interestingly, we now find that the major, 4-AP-sensitive, outward K(+) current of the OHC (I(K)) is also sensitive to Cl(-), although, in contrast to prestin, extracellularly. I(K) is inhibited by reducing extracellular Cl(-) levels, with a linear dependence of 0.4%/mM. Other voltage-dependent K(+) (Kv) channel conductances in supporting cells, such as Hensen and Deiters' cells, are not affected by reduced extracellular Cl(-). To elucidate the molecular basis of this Cl(-)-sensitive I(K), we looked at potential molecular candidates based on Cl(-) sensitivity and/or similarities in kinetics. For I(K), we identified three different Ca(2+)-independent components of I(K) based on the time constant of inactivation: a fast, transient outward current, a rapidly activating, slowly inactivating current (Ik(1)), and a slowly inactivating current (Ik(2)). Extracellular Cl(-) differentially affects these components. Because the inactivation time constants of Ik(1) and Ik(2) are similar to those of Kv1.5 and Kv2.1, we transiently transfected these constructs into CHO cells and found that low extracellular Cl(-) inhibited both channels with linear current reductions of 0.38%/mM and 0.49%/mM, respectively. We also tested heterologously expressed Slick and Slack conductances, two intracellularly Cl(-)-sensitive K(+) channels, but found no extracellular Cl(-) sensitivity. The Cl(-) sensitivity of Kv2.1 and its robust expression within OHCs verified by single-cell RT-PCR indicate that these channels underlie the OHC's extracellular Cl(-) sensitivity.     

Short-term nitrogen-induced modulation of phosphoenolpyruvate carboxylase in tobacco and maize leaves

Untransformed maize and tobacco plants and tobacco plants constitutively expressing nitrate reductase were grown with sufficient NO(3)- to support maximal growth. Four days prior to treatment the tobacco plants were deprived of nitrogen. Excised maize leaves and tobacco leaf discs were fed with either 40 mM KNO(3) or 40 mM KCl (control) in the light. Phosphoenolpyruvate (PEP) carboxylase (Case) activity was measured at 0.3 mM and 3 mM PEP. The light- induced increase in PEPCase V(max) was greater in maize than tobacco. Furthermore light decreased malate sensitivity in maize (which was N-replete) but not in N-deficient tobacco. NO(3)- treatment increased PEPCase V:(max) values in both species and decreased the sensitivity to inhibition by malate, but effects of NO(3)- were much more pronounced in tobacco than maize. PEPCase kinase activity was, however, greater in maize leaves NO(3)- than in the Cl(-)-treated controls, suggesting that it is responsive to leaf nitrogen supply. A correlation between foliar glutamine content and PEPCase activity was observed. It is concluded that PEPCase is sensitive to N metabolites which favour increased flow through the anapleurotic pathway in both C(3) and C(4) plants.     

Acceleration of superoxide generation in polymorphonuclear leukocytes and inhibition of platelet aggregation by alk(en)yl thiosulfates derived from onion and garlic in dogs and humans

We recently identified sodium n-propyl thiosulfate (NPTS) and sodium 2-propenyl thiosulfate (2PTS) from boiled onion and garlic, respectively, as causative agents of hemolytic anemia in dogs. We present here data concerning the effects of these alk(en)yl thiosulfates on superoxide (O(2)(-)) generation in peripheral polymorphonuclear leukocytes (PMNs) and on adenosine 5'-diphosphate (ADP)-induced platelet aggregation in dogs and humans in vitro. Both NPTS and 2PTS increased O(2)(-) generation significantly (P<0.05 at 1mM NPTS, P<0.005 at 0.1 and 1mM 2PTS) and reduced its reaction time significantly (P<0.05 between 0.01 and 1mM NPTS and at 1mM 2PTS) in canine PMNs stimulated by phorbol 12-myristate 13-acetate, compared with the control without alk(en)yl thiosulfates. However, a tendency to return to the control level was observed at 10mM of the alk(en)yl thiosulfates in both O(2)(-) generation and its reaction time. Although NPTS and 2PTS did not exert any significant effect on the O(2)(-) generation in human PMNs, 2PTS reduced its reaction time significantly (P<0.05) at 1 and 10mM compared with the control, showing that 2PTS accelerated O(2)(-) generation in human PMNs. The difference in effects on O(2)(-) generation may be due to that in susceptibility to alk(en)yl thiosulfates between canine and human PMNs. On the other hand, NPTS and 2PTS were shown to significantly inhibit ADP-induced platelet aggregation at 0.01mM (P<0.01) in canine platelets and at 0.001-0.1mM (P<0.05) in human platelets. In contrast, the maximal aggregation percentage returned to the control level at 1mM of alk(en)yl thiosulfates in both canine and human platelets. From these results, we conclude that NPTS and 2PTS have the potential to promote immune functions and prevent cardiovascular diseases.     

IONIC COMPOSITION OF THE CYTOPLASM OF NITELLA FLEXILIS

The K, Na and Cl concentrations of the chloroplast layer andthe flowing cytoplasm of Nitella flexilis have been determinedby applying an internal perfusion technique, which enabled usto avoid contamination of ions from the cell sap. K, Na andCl concentrations of the chloroplast layer are 110, 26 and 136mM and those of the flowing cytoplasm are 125, 5 and 36 mM respectively.The cell sap contains 80 mM K, 28 mM Na and 136 mM Cl. Althoughthere are some variations in these values among samples, theflowing cytoplasm is rich in K and poor in Cl and especiallyin Na. The exchange of K and Na across the tonoplasl occursfairly easily (half-time, a few hours), while that of Cl occursextremely slowly (half-time, a few days). ¹This work was supported by Research Grants from the Ministryof Education of Japan     

Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.