P-chloromercuribenzenesulfonic Acid Research Articles

Low Nitrogen Enhances Apoplastic Phloem Loading and Improves the Translocation of Photoassimilates in Rice Leaves and Stems.

The grain filling of rice depends on photoassimilates from leaves and stems. Phloem loading is the first crucial step for the transportation of sucrose to grains. However, phloem loading mechanisms in rice leaves and stems and their response to nitrogen (N) remain unclear. Here, using a combination of electron microscopy, transportation of a phloem tracer and 13C labeling, phloem loading was studied in rice leaves and stems. The results showed that the sieve element-companion cell complex lacked a symplastic connection with surrounding parenchyma cells in leaves and stems. The genes expression and protein levels of sucrose transporter (SUTs) and sugars will eventually be exported transporters (SWEETs) were detected in the vascular bundle of leaves and stems. A decrease in the 13C isotope remobilization from leaves to stems and panicles following treatment with p-chloromercuribenzenesulfonic acid indicated that rice leaves and stems actively transport sucrose into the phloem. Under low-N (LN) treatment, the activities of α-amylase, β-amylase and sucrose phosphate synthase (SPS) in stems and activity of SPS in leaves increased; genes expression and protein levels of SUTs and SWEETs in leaves and stems increased; the 13C isotope reallocation in panicles increased. These indicated that LN enhanced apoplastic phloem loading in stems and leaves. This improved the translocation of photoassimilates and consequently increased grain filling percentage, grain weight and harvest index. This study provides evidence that rice leaves and stems utilize an apoplastic loading strategy and respond to N stimuli by regulating the genes expression and protein levels of SUTs and SWEETs.

Covid-19 vaccination should be mandatory for healthcare workers

Transport characteristics of benzylpenicillin in the central nervous system was examined using ATP-depleted rat choroid plexus. In the presence of an outwardly directed Cl- gradient, accumulation of benzylpenicillin was stimulated...

Phloem loading in rice leaves depends strongly on the apoplastic pathway.

Phloem loading is the first step in sucrose transport from source leaves to sink organs. The phloem loading strategy in rice remains unclear. To determine the potential phloem loading mechanism in rice, yeast invertase (INV) was overexpressed by a 35S promoter specifically in the cell wall to block sugar transmembrane loading in rice. The transgenic lines exhibited obvious phloem loading suppression characteristics accompanied by the accumulation of sucrose and starch, restricted vegetative growth and decreased grain yields. The decreased sucrose exudation rate with p-chloromercuribenzenesulfonic acid (PCMBS) treatment also indicated that rice actively transported sucrose into the phloem. OsSUT1 (SUCROSE TRANSPORTER 1) showed the highest mRNA levels of the plasma membrane-localized OsSUTs in source leaves. Cross sections of the OsSUT::GUS transgenic plants showed that the expression of OsSUT1 and OsSUT5 occurred in the phloem companion cells. Rice ossut1 mutants showed reduced growth and grain yield, supporting the hypothesis of OsSUT1 acting in phloem loading. Based on these results, we conclude that apoplastic phloem loading plays a major role in the export of sugar from rice leaves.

The Hexose efflux from the peeled grape berry

Aim: The transport of sugars into grape berry mesocarp cells across the plasma and vacuolar membranes after onset of ripening is a complex process. Elements of the sugar transport mechanism may be assessed by exposing the mesocarp cells and investigating sugar movement across the membranes. The purpose of this study was to gain insights into the nature of the transport mechanism by creating conditions conducive to hexose efflux from the peeled berry.Methods and results: The experimental technique employed was a derivate of the ‘berry-cup’ technique. The skin of ripening cv. Shiraz berries was peeled in situ and, after an initial wash, hexose efflux into a collection medium (MES buffer) was monitored. Additionally, during the period of intensive sugar accumulation (one week after veraison) and two weeks later, hexose efflux was assessed following three modifications: (i) using berries excised from the vine, (ii) using MES buffer (2-(N-morpholino)ethanesulfonic acid, pH 5.5) containing PCMBS (p-chloromercuribenzenesulfonic acid, 1mM), and (iii) using cold (10°C) or warm (40°C) MES buffer. Hexose quantities collected into the buffer were dependent on ripening stage and buffer temperature, but they were not dependent on an intact berry-to-cluster connection. The inhibitory effect of PCMBS was observed early in ripening, but not two weeks later.Conclusions: These results lead us to the conclusion that the origin of the collected hexoses was vacuolar as opposed to vascular, and that the hexose efflux mechanism is differently sensitive to PCMBS at the two stages of ripening.Significance and impact of the study: This simple technique was effective at providing insights into hexose transport within the grape berry at the cellular level.

Ratio of sugar concentrations in the phloem sap and the cytosol of mesophyll cells in different tree species as an indicator of the phloem loading mechanism

Sucrose concentration in phloem sap was several times higher than in the cytosol of mesophyll cells. The results suggest that phloem loading involves active steps in the analyzed tree species. Phloem loading in source leaves is a key step for carbon partitioning and passive symplastic loading has been proposed for several tree species. However, experimental evidence to prove the potential for sucrose diffusion from mesophyll to phloem is rare. Here, we analyzed three tree species (two angiosperms, Fagus sylvatica, Magnolia kobus, and one gymnosperm, Gnetum gnemon) to investigate the proposed phloem loading mechanism. For this purpose, the minor vein structure and the sugar concentrations in phloem sap as well as in the subcellular compartments of mesophyll cells were investigated. The analyzed tree species belong to the open type minor vein subcategory. The sucrose concentration in the cytosol of mesophyll cells ranged between 75 and 165mM and was almost equal to the vacuolar concentration. Phloem sap could be collected from F.sylvatica and M.kobus and the concentration of sucrose in phloem sap was about five- and 11-fold higher, respectively, than in the cytosol of mesophyll cells. Sugar exudation of cut leaves was decreased by p-chloromercuribenzenesulfonic acid, an inhibitor of sucrose-proton transporter. The results suggest that phloem loading of sucrose in the analyzed tree species involves active steps, and apoplastic phloem loading seems more likely.

Use of D-glucose-fenpiclonil conjugate as a potent and specific inhibitor of sucrose carriers.

Until now, specific inhibitors of sucrose carriers were not available. This led us to study the properties of the recently synthesized D-glucose-fenpiclonil conjugate (D-GFC). This large amphiphilic glucoside exhibited an extremely low phloem systemicity in contrast to L-amino acid-fenpiclonil conjugates. Using Ricinus seedlings, the effect of D-GFC on 0.5 mM [14C]sucrose (Suc), 3-O-[3H]methylglucose, and [3H]glutamine uptake by cotyledon tissues was compared with that of p-chloromercuribenzenesulfonic acid (PCMBS). D-GFC dramatically inhibited H+-Suc symport at the same concentrations as PCMBS (0.5 and 1 mM), but in contrast to the thiol reagent, it did not affect 3-O-methylglucose and glutamine transport, nor the acidification of the incubation medium by cotyledon tissues. Similarly, 0.5 mM D-GFC inhibited active Suc uptake by Vicia faba leaf tissues and by Saccharomyces cerevisiae cells transformed with AtSUC2, a gene involved in Suc phloem loading in Arabidopsis, by approximately 80%. The data indicated that D-GFC was a potent inhibitor of Suc uptake from the endosperm and of Suc phloem loading. It is the first chemical known to exhibit such specificity, at least in Ricinus, and this property permitted the quantification of the two routes involved in phloem loading of endogenous sugars after endosperm removal.

Glycinergic-Fipronil Uptake Is Mediated by an Amino Acid Carrier System and Induces the Expression of Amino Acid Transporter Genes in Ricinus communis Seedlings.

Phloem-mobile insecticides are efficient for piercing and sucking insect control. Introduction of sugar or amino acid groups to the parent compound can improve the phloem mobility of insecticides, so a glycinergic-fipronil conjugate (GlyF), 2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)ureido) acetic acid, was designed and synthesized. Although the "Kleier model" predicted that this conjugate is not phloem mobile, GlyF can be continually detected during a 5 h collection of Ricinus communis phloem sap. Furthermore, an R. communis seedling cotyledon disk uptake experiment demonstrates that the uptake of GlyF is sensitive to pH, carbonyl cyanide m-chlorophenylhydrazone (CCCP), temperature, and p-chloromercuribenzenesulfonic acid (pCMBS) and is likely mediated by amino acid carrier system. To explore the roles of amino acid transporters (AATs) in GlyF uptake, a total of 62 AAT genes were identified from the R. communis genome in silico. Phylogenetic analysis revealed that AATs in R. communis were organized into the ATF (amino acid transporter) and APC (amino acid, polyaminem and choline transporter) superfamilies, with five subfamilies in ATF and two in APC. Furthermore, the expression profiles of 20 abundantly expressed AATs (cycle threshold (Ct) values <27) were analyzed at 1, 3, and 6 h after GlyF treatment by RT-qPCR. The results demonstrated that expression levels of four AAT genes, RcLHT6, RcANT15, RcProT2, and RcCAT2, were induced by the GlyF treatment in R. communis seedlings. On the basis of the observation that the expression profile of the four candidate genes is similar to the time course observation for GlyF foliar disk uptake, it is suggested that those four genes are possible candidates involved in the uptake of GlyF. These results contribute to a better understanding of the mechanism of GlyF uptake as well as phloem loading from a molecular biology perspective and facilitate functional characterization of candidate AAT genes in future studies.

Sulfite inhibition of sucrose transport into vacuoles of cottonwood leaves

The mechanism of sucrose transport and the influence of various concentrations of sulfite on its activity was studied in the mesophyll vacuoles isolated from cottonwood (&lt;em&gt;Populus deltoides&lt;/em&gt; Bartr. ex Marsh) leaves. Kinetic analysis of [&lt;sup&gt;14&lt;/sup&gt;C] sucrose uptake by isolated vacuoles revealed the presence of a saturable component with K&lt;sub&gt;m&lt;/sub&gt; for sucrose uptake = 23 mM and Vmax = 21.2 nmol sucrose 10&lt;sup&gt;-6&lt;/sup&gt; vacuoles min&lt;sup&gt;-1&lt;/sup&gt;. The saturable component was inhibited by SH-blocker, p-chloromercuribenzenesulfonic acid (PCMBS) and was substrate specific. The transport of sucrose was not changed by ATP with or without Mg&lt;sup&gt;2+&lt;/sup&gt;, by the ionophore, valinomycin and by protonophores, carbonylcyanide m-chlorophenyl hydrazone (CCCP) or dinitrophenol (DNP). Sulfite at concentrations of 0.01-2.5 mM inhibited the uptake of [&lt;sup&gt;14&lt;/sup&gt;C] sucrose by isolated vacuoles. Inhibition was of the noncompetitive type with K; = 2.48 mM of sulfite (22&lt;sup&gt;o&lt;/sup&gt;C).

Uptake and metabolic effects of salicylic acid on the pulvinar motor cells of Mimosa pudica L.

In this paper, the salicylic acid (o-hydroxy benzoic acid) (SA) uptake by the pulvinar tissues of Mimosa pudica L. pulvini was shown to be strongly pH-dependent, increasing with acidity of the assay medium. This uptake was performed according to a unique affinity system (Km = 5.9 mM, Vm = 526 pmol mgDW−1) in the concentration range of 0.1–5 mM. The uptake rate increased with increasing temperature (5–35 °C) and was inhibited following treatment with sodium azide (NaN3) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), suggesting the involvement of an active component. Treatment with p-chloromercuribenzenesulfonic acid (PCMBS) did not modify the uptake, indicating that external thiol groups were not necessary. KCl, which induced membrane depolarization had no significant effect, and fusicoccin (FC), which hyperpolarized cell membrane, stimulated the uptake, suggesting that the pH component of the proton motive force was likely a driving force. These data suggest that the SA uptake by the pulvinar tissues may be driven by two components: an ion-trap mechanism playing a pivotal role and a putative carrier-mediated mechanism. Unlike other benzoic acid derivatives acting as classical respiration inhibitors (NaN3 and KCN), SA modified the pulvinar cell metabolism by increasing the respiration rate similar to CCCP and 2,4-dinitrophenol (DNP). Furthermore, SA inhibited the osmoregulated seismonastic reaction in a pH dependent manner and induced characteristic damage to the ultrastructural features of the pulvinar motor cells, particularly at the mitochondrial level.

Sucrose transporter plays a role in phloem loading in CMV‐infected melon plants that are defined as symplastic loaders

Based on the high density of plasmodesmata interconnecting the intermediary cells and their neighboring phloem parenchyma or bundle-sheath cells, and based on the insensitivity to the sucrose transport inhibitor p-chloromercuribenzenesulfonic acid (PCMBS), cucurbits have been concluded to be symplastic loaders. In the present study, we identified and characterized the full-length sequence of sucrose transporter gene (CmSUT1) from melon (Cucumis melo L. cv. Hale's best jumbo). In vitro experiments confirmed that the identified gene product has sucrose transporter activity in baker's yeast. Healthy and cucumber mosaic virus (CMV)-infected melon plants were employed to examine sucrose transporter activity in planta. Pretreatment with PCMBS inhibited loading of newly fixed ¹⁴CO₂ into minor veins of CMV-infected plants. Moreover, CMV infection caused significant increase in CmSUT1 transcripts expression, mainly in vascular bundles of minor veins, which was associated with elevated sucrose content in phloem sap collected from source-leaf petioles. We propose that cucurbit plants contain the machinery for apoplastic phloem loading and that CMV infection causes a quantitative shift in the mode by which photoassimilates are loaded into the sieve tube.

Salicylic Acid Transport in Ricinus communis Involves a pH-Dependent Carrier System in Addition to Diffusion

Despite its important functions in plant physiology and defense, the membrane transport mechanism of salicylic acid (SA) is poorly documented due to the general assumption that SA is taken up by plant cells via the ion trap mechanism. Using Ricinus communis seedlings and modeling tools (ACD LogD and Vega ZZ softwares), we show that phloem accumulation of SA and hydroxylated analogs is completely uncorrelated with the physicochemical parameters suitable for diffusion (number of hydrogen bond donors, polar surface area, and, especially, LogD values at apoplastic pHs and Delta LogD between apoplast and phloem sap pH values). These and other data (such as accumulation in phloem sap of the poorly permeant dissociated form of monohalogen derivatives from apoplast and inhibition of SA transport by the thiol reagent p-chloromercuribenzenesulfonic acid [pCMBS]) lead to the following conclusions. As in intestinal cells, SA transport in Ricinus involves a pH-dependent carrier system sensitive to pCMBS; this carrier can translocate monohalogen analogs in the anionic form; the efficiency of phloem transport of hydroxylated benzoic acid derivatives is tightly dependent on the position of the hydroxyl group on the aromatic ring (SA corresponds to the optimal position) but moderately affected by halogen addition in position 5, which is known to increase plant defense. Furthermore, combining time-course experiments and pCMBS used as a tool, we give information about the localization of the SA carrier. SA uptake by epidermal cells (i.e. the step preceding the symplastic transport to veins) insensitive to pCMBS occurs via the ion-trap mechanism, whereas apoplastic vein loading involves a carrier-mediated mechanism (which is targeted by pCMBS) in addition to diffusion.

Bicarbonate-dependent and bicarbonate-independent mechanisms contribute to nondiffusive uptake of acetate in the ruminal epithelium of sheep

The present study investigated the significance of apical transport proteins for ruminal acetate absorption and their interaction with different anions. In anion competition experiments in the washed reticulorumen, chloride disappearance rate (initial concentration, 28 mM) was inhibited by the presence of a short-chain fatty acid mixture (15 or 30 mM of each acetate, propionate, and butyrate). Disappearance rates of acetate and propionate, but not butyrate (initial concentration, 25 mM each) were diminished by 40 or 80 mM chloride. In isolated ovine ruminal epithelia mounted in Ussing chambers, an increase in chloride concentration from 4.5 to 90 mM led to a decrease of apical acetate uptake at a concentration of 0.5 mM. Mucosal nitrate inhibited acetate uptake most potently whereas sulfate had no effect. Decreasing mucosal pH from 7.4 to 6.1 approximately doubled uptake of acetate both at 0.5 and 10 mM, but this doubling was almost abolished when HCO(3)(-) was absent. The stimulated uptake at mucosal pH 6.1 consisted of a bicarbonate-dependent, nitrate-inhibitable part (K(m) = 54 mM) and a bicarbonate-independent component (K(m) = 12 mM) that was also sensitive to nitrate inhibition. Maximal uptake was three times larger for bicarbonate-dependent vs. bicarbonate-independent uptake. Mucosal addition of 200 microM DIDS, 400 microM p-chloromercuribenzene sulfonic acid, 800 microM p-hydroxymercuribenzoic acid, or 100 microM phloretin had no effects on acetate uptake although the latter two inhibited l-lactate uptake. Our data conclusively show a dominant involvement of proteins in apical acetate uptake. Previously described pH effects on acetate absorption originate mainly from modulation of acetate/bicarbonate exchange. Additionally, there is bicarbonate-independent uptake of acetate anions that is protein coupled but not via monocarboxylate cotransporter.

Permeability of rat and rabbit erythrocyte membranes for a series of amides

Permeability coefficients of rat and rabbit erythrocyte membranes for a series of amides, as well as for erythrocytes treated with p-chloromercuribenzenesulfonic acid monosodium salt (pCMBS) have been determined at 25 and 37 °C. Directly proportional dependence of the pCMBS treated erythrocyte permeability for investigated substances and their partition coefficients between the hydrophobic phase and water as well as the values of activation energy of this process indicate that penetration of small hydrophilic molecules is realized by passive diffusion through the lipid bilayer. The results obtained indicate that penetration of small hydrophilic molecules of formamide through lipids is determined by the existence of a free space between hydrocarbon chains that arises from kink formation. The differences in permeability between rat and rabbit erythrocyte membranes could arise in particular as a result of the differences in lipid composition.

Molecular characterization of the mercurial sensitivity of a frog urea transporter (fUT)

The amphibian urea transporter (fUT) shares many properties with the mammalian urea transporters (UT) derived from UT-A and UT-B genes. The transport of urea by fUT is inhibited by the mercurial agent p-chloromercuribenzenesulfonic acid (pCMBS). We found that in oocytes expressing cRNA encoding fUT, a 5-min preincubation in 0.5 mM mercury chloride (HgCl2) also significantly reduced urea uptake. The transport of urea by fUT was rendered mercury (Hg2+) insensitive by mutating either of the residues C185 or H187, both of which lie within the M-I region (close to the hypothetical UT pore). In oocytes expressing a mixture of the C185 and H187 mutants, Hg2+ sensitivity was reestablished. The transport of urea by the mouse UTs mUT-A2 and mUT-A3 was not sensitive to Hg2+. Introducing cysteine residues analogous to that mutated in fUT into mUT-A2 or mUT-A3 did not induce Hg2+ sensitivity. Additionally, introducing the double cysteine, histidine mutations into mUT-A2 or mUT-A3 still did not induce Hg2+ sensitivity, indicating that a region outside of the M-I region also contributes to the Hg2+-induced block of fUT. Using a series of chimeras formed between UT-A3 and fUT, we found that as well as C185 and H187, residues within the COOH terminal of fUT determine Hg2+ sensitivity, and we propose that differences in the folding of this region between fUT and mUT-A2/mUT-A3 allow access of Hg2+ to the fUT channel pore.

Phloem Loading. A Reevaluation of the Relationship between Plasmodesmatal Frequencies and Loading Strategies

The incidence of plasmodesmata in the minor vein phloem of leaves varies widely between species. On this basis, two pathways of phloem loading have been proposed: symplastic where frequencies are high, and apoplastic where they are low. However, putative symplastic-loading species fall into at least two categories. In one, the plants translocate raffinose-family oligosaccharides (RFOs). In the other, the primary sugar in the phloem sap is sucrose (Suc). While a thermodynamically feasible mechanism of symplastic loading has been postulated for species that transport RFOs, no such mechanism is known for Suc transporters. We used p-chloromercuribenzenesulfonic acid inhibition of apoplastic loading to distinguish between the two pathways in three species that have abundant minor vein plasmodesmata and are therefore putative symplastic loaders. Clethra barbinervis and Liquidambar styraciflua transport Suc, while Catalpa speciosa transports RFOs. The results indicate that, contrary to the hypothesis that all species with abundant minor vein plasmodesmata load symplastically, C. barbinervis and L. styraciflua load from the apoplast. C. speciosa, being an RFO transporter, loads from the symplast, as expected. Data from these three species, and from the literature, also indicate that plants with abundant plasmodesmata in the minor vein phloem have abundant plasmodesmata between mesophyll cells. Thus, plasmodesmatal frequencies in the minor veins may be a reflection of overall frequencies in the lamina and may have limited relevance to phloem loading. We suggest that symplastic loading is restricted to plants that translocate oligosaccharides larger than Suc, such as RFOs, and that other plants, no matter how many plasmodesmata they have in the minor vein phloem, load via the apoplast.

Activation of mitochondrial lactate uptake by flavone induces apoptosis in human colon cancer cells.

Lactate production from glucose even in the presence of oxygen is a characteristic of cancer cell metabolism and an important feature for tumor progression. Here, we describe that an increased uptake of lactate into mitochondria of HT-29 human colon cancer cells by treatment of cells with the flavonoid flavone is associated with an increased production of mitochondrial superoxide anions and apoptotic cell death. In search of the mitochondrial transporter that could promote enhanced lactate uptake and energetic flow through the electron transport chain, we used fluorescein as a model substrate. Flavone increased fluorescein uptake at pH 7.4 into mitochondria of HT-29 cells almost tenfold while lactate inhibited uptake significantly. Uptake of fluorescein in the absence or presence of flavone was strongly increased by lowering pH from 7.4 to 6.0 and almost abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). The lactate-sensitive part of fluorescein transport was completely blocked by p-chloromercuribenzenesulfonic acid (pCMBS), a specific inhibitor of the monocarboxylate transporter-1 (MCT-1) that by Western blotting and immunofluorescence was identified in mitochondria of HT-29 cells. Finally, lactate increased and pCMBS inhibited the flavone-induced generation of mitochondrial O2-* radicals and in turn blunted the apoptotic response. In conclusion, our studies provide evidence that flavone reverts the metabolic phenotype of transformed colonocytes towards a phenotype characteristic for normal cells. Transformed colonocytes, however, seem especially vulnerable to O2-*, produced in mitochondria as a consequence of these metabolic alterations, and respond with the induction of apoptosis.

Processing of acidic proline-rich proprotein by human salivary gland convertase

Previously it was found that proproteins for basic and glycosylated salivary proline-rich proteins (PRP) were cleaved prior to secretion from cells by furin, a well-known convertase. In contrast proproteins for acidic PRPs are not cleaved by furin or other convertases. To investigate the convertase responsible for in vivo processing of acidic PRP proproteins, homogenates of human sublingual glands were fractionated by centrifugation at 10,000× g and 100,000× g and activity demonstrated in all fractions. The 100,000× g pellet was fractionated into Golgi, smooth endoplasmic reticulum and microsomal fractions with the latter containing the enzyme. Subfractionation of the microsomes revealed that the activity was located in the membrane proteins. Since the microsomes contain components of the secretory pathway the enzyme in this fraction may be responsible for intracellular cleavage of the acidic PRP proprotein. The enzyme was active at alkaline pH. It was strongly inhibited by metal chelators indicating that it is a metalloprotease. It was not inhibited by an acid protease inhibitor, but partly inhibited by some serine protease inhibitors indicating that serine proteases may play a role in degradation. Co 2+ and to some extent Zn 2+ activated the enzyme, but it was strongly inhibited by Hg 2+ and Cu 2+ as well as the organomercurial p-chloromercuribenzenesulfonic acid. Thus it appears that the enzyme contains an important SH group. These characteristics indicate that the convertase is related to a group of metal- and thiol-dependent proteases known as thimet oligopeptidases, but in contrast to the latter enzymes the sublingual convertase was not inhibited by angiotensin antagonists.

Redox activity associated with the maturation and capacitation of mammalian spermatozoa

As rat spermatozoa undergo epididymal maturation, they acquire the ability to exhibit a spontaneous burst of luminol–peroxidase-dependent chemiluminescence when released into a simple, defined culture medium. This activity was suppressed by inhibitors of plasma membrane redox systems such as diphenylene iodonium, p-chloromercuribenzenesulfonic acid, and capsaicin, but was resistant to inhibition by resiniferatoxin and rotenone. The luminol–peroxidase signal was dependent on the presence of bicarbonate, enhanced by the substitution of fructose for glucose, and severely suppressed by desferoxamine, superoxide dimutase, and catalase. Both l- and d-arginine were stimulatory, suggesting the involvement of NO in this spontaneous chemiluminescence activity. The l-arginine-dependent, but not the d-arginine-dependent, activity was significantly suppressed by an inhibitor of nitric oxide synthase ( N G-nitro- l-arginine methyl ester). l- and d-arginine could also stimulate redox activity observed in immature caput epididymal cells, but only after prolonged incubation. The inhibitory effects of uric acid and ascorbate suggested the chemiluminescence signal might be induced by peroxynitrite. This conclusion was supported by confocal imaging of the cells following treatment with 4-amino-5-methylamino-2′,7′-difluorofluorescein. Stimulation or suppression of the redox activity detected by luminol–peroxidase led to corresponding changes in the ability of the spermatozoa to exhibit acrosomal exocytosis, indicating that this pathway is of fundamental biological significance.

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn2+, Hg2+, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K m and V max of the enzyme for CMC were 3.4 mg ml−1 and 44.7 μmol min−1 (mg protein)−1, respectively.

Redox regulation of ascorbic acid transport: Role of transporter and intracellular sulfhydryls

AbstractAscorbic acid is one of the most sensitive cellular defenses against oxidant damage. However, it requires a sodium‐ and energy‐dependent transporter to enter cells against a concentration gradient. To test the hypothesis that ascorbate transport is sensitive to redox stress, we studied changes in transport of the vitamin in response to sulfhydryl modification of the protein and to GSH depletion in cultured endothelial cells. Transport of ascorbic acid, measured as the uptake of radiolabeled ascorbate, was inhibited by the membrane‐impermeant sulfhydryl reagents thorin, p‐chloromercuribenzene sulfonic acid, and 5,5′‐dithiobis‐(2‐nitrobenzoic acid) in a dose‐dependent manner without significant depletion of intracellular GSH. Sulfhydryl reagents capable of penetrating the plasma membrane, including phenylarsine oxide, p‐chloromercuribenzoic acid, and N‐ethylmaleimide, inhibited transport and lowered cellular GSH. Diamide, which induces disulfide formation, increased ascorbate transport over a narrow concentration range under conditions in which GSH was not depleted. On the other hand, specific depletion of intracellular GSH by several different mechanisms did inhibit transport. Together, these results suggest that the ascorbate transporter is sensitive to redox modulation. This relates in part to sulfhydryl groups exposed on the exofacial ascorbate transporter, and to sulfhydryl groups that are sensitive to changes in the redox state of intracellular GSH.