Importance of glucose-6-phosphate dehydrogenase activity in cell death

The intracellular redox potential plays an important role incell survival. The principal intracellular reductant NADPH is mainlyproduced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by6-phosphogluconate dehydrogenase. Considering the importance of NADPH,we hypothesized that G6PDH plays a critical role in cell death. Ourresults show that 1) G6PDHinhibitors potentiatedH₂O₂-inducedcell death; 2) overexpression ofG6PDH increased resistance toH₂O₂-induced cell death; 3) serum deprivation, astimulator of cell death, was associated with decreased G6PDH activityand resulted in elevated reactive oxygen species (ROS);4) additions of substrates for G6PDHto serum-deprived cells almost completely abrogated the serumdeprivation-induced rise in ROS; 5)consequences of G6PDH inhibition included a significant increase inapoptosis, loss of protein thiols, and degradation of G6PDH; and6) G6PDH inhibition caused changesin mitogen-activated protein kinase phosphorylation that were similarto the changes seen withH₂O₂.We conclude that G6PDH plays a critical role in cell death by affectingthe redox potential.     

Studies in the Nitrogen Metabolism of Apple Fruits: THE CLIMACTERIC RISE IN RESPIRATION IN RELATION TO CHANGES IN THE EQUILIBRIUM BETWEEN PROTEIN SYNTHESIS AND BREAKDOWN

1. A close parallelism in the drift of the rate of respirationand the protein-N/ non-protein-N ratio is shown to occur bothin apple fruits attached to the tree and when detached fromthe tree at various stages of development and stored for severalmonths at 12 C.
2. In detached fruits the fall in respirationwhich occurs immediately(during the first 48 hours) after pickingis only accompaniedby a concomitant fall in net protein invery young fruits inwhich active cell division is taking place.Subsequently, infruit of all ages when a climacteric rise inrespiration occursit is accompanied by a net increase in protein.
3. It is argued that the climacteric rise in respiration is aresultof increase in the level of protein which will be expectedtoreduce the ATP/ADP ratio.
4. Over the climacteric period,although rate of respiration andnet protein content both rise,R rises more rapidly than proteinand, subsequently, falls ata faster rate than P. It is suggestedthat this may be due tothe ‘new’ protein containinga higher proportionof enzyme(s) directly involved in respirationand leading, forexample, to a reduction in the ATP/ADP ratio.

     

Egg-Mass Size and Cell Size: Effects of Temperature on Oxygen Distribution

Two processes strongly influence the distribution of oxygenwithin egg masses and cells: the supply of oxygen by diffusionand the consumption of oxygen by embryos and mitochondria. Theseprocesses are differentially sensitive to temperature. The diffusioncoefficient of oxygen depends only weakly on temperature, havinga Q₁₀ of approximately 1.4. In contrast, the consumption ofoxygen depends strongly on temperature, having a Q₁₀ between1.5 and 4.0. Thus, at higher temperatures, the ratio of oxygensupply to demand decreases. I show, by extending a model ofoxygen distribution within metabolizing spheres, that maximalegg-mass sizes and cell sizes are predicted to be smaller athigher temperatures. For egg masses, definitive data are notyet available. For ectothermic cells, this prediction appearsto be supported; cells from a variety of ectothermic organisms,unicellular and multicellular, are smaller when the cells areproduced at warmer temperatures. Establishing a specific connectionbetween this pattern and oxygen distributions requires demonstrationof (1) oxygen concentration gradients within metabolizing spheresand (2) central oxygen concentrations low enough to affect function.Egg masses from a variety of taxa show steep oxygen concentrationgradients and often are severely hypoxic or anoxic in centrallocations. Severe hypoxia appears capable of retarding developmentor killing embryos. Similar kinds of data for ectothermic cellshave not yet been collected, but the literature on oxygen gradientswithin mammalian cells suggests that intracellular gradientsmay be important.     

A Prolyl Endoproteinase that Acts Specifically on the Extrinsic 18-kDa Protein of Photosystem II: Purification and Further Characterization

An endoproteinase, which specifically cleaves the Pro12-Leu13bond of the extrinsic 18-kDa protein of PSII, was purified fromPSII membranes of spinach. The presence of 0.05% (w/v) Tween20 and 1 M NaCl was essential for maintenance of proteolyticactivity during the purification. The molecular mass of theenzyme was estimated to be 95 kDa by gel-filtration chromatography.Active fractions contained a polypeptide of 165 kDa that wasconverted into diffusely stained polypeptides of 54 kDa uponreduction with dithiothreitol. The K_m of the 18-kDa proteinin the proteolytic reaction was 0.3 µM. Inhibition ofthe proteolysis by compounds that contain prolyl bonds revealedthat both a prolyl bond and a positive charge are necessaryfor interaction with the proteinase, but some other structuralfactor(s) must also be involved in the high-affinity interactionbetween the proteinase and the 18-kDa protein. Reconstitutionof NaCl-treated PSII membranes with the 23-kDa protein and/orthe 18-kDa protein revealed that the 18-kDa protein was notcleaved by the proteinase when the substrate protein was functionallyassociated with the membranes. A comparison of the propertiesof the proteinase with those of a proline-specific endopeptidasefrom Flavobacterium suggests that these enzymes are quite differentin terms of substrate specificity. (Received December 13, 1993; Accepted March 24, 1994)     

Decreased affinity for oxygen of cytochrome-c oxidase in Leigh syndrome caused by SURF1 mutations

Mutations in the gene SURF1 prevent synthesis of cytochrome-c oxidase (COX)-specific assembly protein and result in a fatal neurological disorder, Leigh syndrome. Because this severe COX deficiency presents with barely detectable changes of cellular respiratory rates under normoxic conditions, we analyzed the respiratory response to low oxygen in cultured fibroblasts harboring SURF1 mutations with high-resolution respirometry. The oxygen kinetics was quantified by the partial pressure of oxygen (PO₂) at half-maximal respiration rate (P₅₀) in intact coupled cells and in digitonin-permeabilized uncoupled cells. In both cases, the P₅₀ in patients was elevated 2.1- and 3.3-fold, respectively, indicating decreased affinity of COX for oxygen. These results suggest that at physiologically low intracellular PO₂, the depressed oxygen affinity may lead in vivo to limitations of respiration, resulting in impaired energy provision in Leigh syndrome patients. oxygen kinetics; mitochondrial disease     

Evidence for Physically Distinct Systemic Signalling Pathways in the Wounded Tomato Plant

The physical pathway of a systemic signal linking local woundingand systemic synthesis of proteinase inhibitors was investigatedin tomato (Lycopersicon esculentum Mill. ‘Moneymaker’)plants. Lucifer Yellow was used to visualize wound induced flowin the xylem. Cuts under water or severe wounds (heat or largecrushing wounds) induced flows in the xylem to other parts ofthe plant in a pattern determined by the vascular architecture.The detailed distribution of systemic proteinase inhibitor activityfollowing these wounds was similar to the pattern of wound inducedflow in the xylem. Steaming the petiole of the wounded organdid not prevent the systemic induction of proteinase inhibitorby a severe wound. It was concluded that elicitors releasedby a severe wound were distributed systemically in the xylem.Small crushing wounds did not induce systemic flow in the xylembut did induce proteinase inhibitor activity in organs importingvia the phloem. Steaming the petiole of the wounded leaf preventedsystemic induction of proteinase inhibitor by small crushingwounds, a result which is consistent with the translocationof elicitors in the phloem. These results indicate the participationof more than one signalling pathway in the systemic inductionof proteinase inhibitor synthesis by wounding. Copyright 1999Annals of Botany Company Elicitors, proteinase inhibitors, Lycopersicon esculentum, signal pathway, vascular anatomy, wound response.     

Action of Sorghum Proteinase on the Protein Bodies of Sorghum Starchy Endosperm

Protein bodies isolated from the starchy endosperm of ungerminatedsorghum exhibited some autolytic activity but seemed incapableof significant self-hydrolysis. Enzyme assay, transmission electronmicroscopy, sodium dodecyl sulphate—polyacrylamide gelelectrophoresis and amino acid analysis revealed that a proteinaseextract from germinated sorghum could degrade the protein bodiesin a manner resembling that which takes place in vivo. The proteinbodies were degraded mainly from the periphery. Glutelin (matrixprotein) was first hydrolysed, followed by the prolamin proteinbody protein. Proteinase extracts from both the germ and endospermof germinated sorghum were capable of degrading the proteinbodies. This finding is consistent with the concept that theproteinase is synthesized in the germ and then secreted intothe starchy endosperm during germination. Key words: Sorghum bicolor, protein body degradation, proteinase.     

棉铃虫卵内蛋白酶性质研究

在棉铃虫Helicoverpa armigera卵母细胞内检测到蛋白酶活性,其作用Ph在酸性范围,酶活性受E-64、Pepstatin和iPr₂P-F等多种抑制剂抑制。在Ph4.0时蛋白酶对牛血红蛋白有较高水解率。抗蓖麻蚕Philosamia cynthia ricini卵半胱氨酸蛋白酶血清和抗蓖麻蚕卵天冬氨酸蛋白酶血清可以识别棉铃虫卵内成分。实验结果表明;棉铃虫卵内可能存在半胱氨酸蛋白酶类、丝氨酸蛋白酶类和天冬氨酸蛋白酶类,并且与蓖麻蚕卵内蛋白酶有一定的相似性。     

Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types

The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC). HUVEC and HAEC were subjected to cyclic strain (10% or 20%, 1 Hz), to a motion control that simulated fluid agitation over the cells without strain, or to static conditions for 24 h. We measured H₂O₂ production with dichlorodihydrofluorescein acetate and superoxide with dihydroethidium fluorescence changes; superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities spectrophotometrically; and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 protein expression with Western blot analyses. HUVEC under cyclic strain showed 1) higher intracellular H₂O₂ levels, 2) increased SOD, catalase, and GPx activities, and 3) greater VCAM-1 and ICAM-1 protein expression, compared with motion control or static conditions. However, in HAEC, motion control induced higher levels of ROS, enzyme activities associated with ROS defense, and VCAM-1 and ICAM-1 expression than cyclic strain. The opposite responses obtained with these two human EC types may reflect their vessels of origin, in that HAEC are subjected to higher cyclic strain deformations in vivo than HUVEC. phenotype; reactive oxygen species; inflammation; shear stress     

Mechanisms of Na+-K+ pump regulation in cardiac myocytes during hyposmolar swelling

We have previously demonstrated that the sarcolemmalNa⁺-K⁺pump current(I_p) in cardiacmyocytes is stimulated by cell swelling induced by exposure tohyposmolar solutions. However, the underlying mechanism has not beenexamined. Because cell swelling activates stretch-sensitive ionchannels and intracellular messenger pathways, we examined their rolein mediating I_pstimulation during exposure of rabbit ventricular myocytes to ahyposmolar solution.I_p was measuredby the whole cell patch-clamp technique. Swelling-induced pumpstimulation altered the voltage dependence ofI_p. Pumpstimulation persisted in the absence of extracellularNa⁺ and under conditions designedto minimize changes in intracellular Ca²⁺, excluding an indirectinfluence on I_pmediated via fluxes through stretch-activated channels. Pumpstimulation was protein kinase C independent. The tyrosine kinaseinhibitor tyrphostin A25, the phosphatidylinositol 3-kinase inhibitorLY-294002, and the protein phosphatase-1 and -2A inhibitor okadaic acidabolished I_pstimulation. Our findings suggest that swelling-induced pumpstimulation involves the activation of tyrosine kinase,phosphatidylinositol 3-kinase, and a serine/threonine proteinphosphatase. Activation of this messenger cascade maycause activation by the dephosphorylation of pump units.     

Physiological Ecology of Riverside Species: Adaptive Responses of Plants to Submergence

In river floodplains, variation in flooding conditions resultsin successional stages in colonization ranging from annual pioneersto long-lived perennials. Reactions to submergence of speciesfrom the mid-successional zone are compared with adaptive responsesof species from other zones. Presence and abundance are relatedto elevation and can be explained by characteristics of biomassproduction, and recovery in response to various submergenceintensities. Rumex species, from early to late successional stages, serveas models to elucidate, in more detail, mechanisms of adaptation.Flooding-resistant species develop large numbers of adventitiousroots upon submergence and exposure to low oxygen conditions.Due to internal oxygen transport through aerenchyma, soil aroundthese roots is reoxidized, which stimulates bacterial nitrification.Ethylene and auxin promote adventitious rooting. Increased petioleelongation is also an adaptive feature of submergence-resistantRumex species. Differences between species in submergence-inducedgrowth are not only controlled by variation in endogenous levelsof ethylene but also by different sensitivities to this hormone.Auxin does not affect Rumex petiole elongation, but a clearpositive effect of gibberellin is demonstrated. Apparently,submergence induces a higher sensitivity to gibberellin andethylene in the petioles of flooding-resistant Rumex. Many ofthe submergence reactions can also be induced by restrictingthe oxygen supply, suggesting that low-oxygen might be a triggeringfactor. The Rumex species we study represent various distinctcommunities. Thus, the ecophysiological phenomena observed inthese model plants may explain processes and patterns in otherspecies too and thus are interpretable at the riverside communitylevel.Copyright 1994, 1999 Academic Press Ecophysiology, submergence, flooding, hormones, adaptation, nitrification, depth accommodation, adventitious rooting, Rumex     

S-acylation regulates Kv1.5 channel surface expression

The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. An imbalance in the ratio of inward and outward conducting channels is unfavorable and often detrimental. For example, over- or underexpression of voltage-gated K⁺ (Kv) channels can be cytotoxic and in some cases lead to disease. In this study, we demonstrated a novel role for S-acylation in Kv1.5 cell surface expression. In transfected fibroblasts, biochemical evidence showed that Kv1.5 is posttranslationally modified on both the NH₂ and COOH termini via hydroxylamine-sensitive thioester bonds. Pharmacological inhibition of S-acylation, but not myristoylation, significantly decreased Kv1.5 expression and resulted in accumulation of channel protein in intracellular compartments and targeting for degradation. Channel protein degradation was rescued by treatment with proteasome inhibitors. Time course experiments revealed that S-acylation occurred in the biosynthetic pathway of nascent channel protein and showed that newly synthesized Kv1.5 protein, but not protein expressed on the cell surface, is sensitive to inhibitors of thioacylation. Sensitivity to inhibitors of S-acylation was governed by COOH-terminal, but not NH₂-terminal, cysteines. Surprisingly, although intracellular cysteines were required for S-acylation, mutation of these residues resulted in an increase in Kv1.5 cell surface channel expression, suggesting that screening of free cysteines by fatty acylation is an important regulatory step in the quality control pathway. Together, these results show that S-acylation can regulate steady-state expression of Kv1.5. quality control; potassium; channels; palmitoylation; posttranslational     

Staphylococcal serine proteinase increases intracellular free calcium concentration in human polymorphonuclear leukocytes

Staphylococcal serine proteinase (SSP) can influence various functions of human polymorphonuclear leukocytes (PMNL) including chemotaxis and phagocytosis. Since the rise in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation, we tested the ability of SSP to increase the intracellular free calcium concentration in human PMNL using the fluorescent calcium indicator Fura-2AM. PMNL isolated from healthy donors responded to SSP in the concentration range of 10 to 100 µg/ml. The highest Ca²⁺ rise (104 ± 47 nM) was observed for 10 µg/ml SSP. It was mainly dependent (81 ± 11%) on extracellular calcium influx, however, SSP mobilized 68 ± 7% of Ca²⁺ from intracellular calcium stores. Boiling of SSP or preincubation with phenylmethylsulphonylfluoride (an serine proteinase inhibitor) did not change its ability to increase intracellular free calcium concentration in PMNL. It suggests that active center of SSP is not responsible for Ca²⁺ mobilization. Finally, PMNL responded to each of three consecutive stimulations with SSP independently of the presence of high or low extracellular Ca² concentration. This may be an additional mechanism responsible for activation of human PMNL and degradation of alveolar walls during the staphylococcal infection in the lower airways.     

Apolipoprotein A-IV attenuates oxidant-induced apoptosis in mitotic competent, undifferentiated cells by modulating intracellular glutathione redox balance

Oxidant-mediated modulation of the intracellular redox state affects the apoptotic cascade by altering the balance between cellular signals for survival and suicide. Apolipoprotein A-IV (Apo A-IV) is known to possess antioxidant-like activity. In the present study, we tested 1) whether Apo A-IV could influence redox-dependent apoptosis and, if so, 2) whether such an effect could be mediated by modulation of intracellular redox balance. Mitotic competent, undifferentiated PC-12 cells were incubated with either tert-butyl hydroperoxide (TBH) or diamide with or without preincubation with human Apo A-IV. Apo A-IV significantly decreased apoptosis produced by both TBH and diamide, and washout of A-IV before incubation with TBH and diamide did not eliminate its protective effect. Apo A-I had no such protective effect. The Apo A-IV effect was not blocked by D,L-buthionine-[S,R]-sulfoximine, but it was reversed by both dehydroisoandrosterone and transfection with an antisense oligodeoxynucleotide to glucose-6-phosphate dehydrogenase (G6PD). Apo A-IV abolished the transient, oxidant-induced rise in glutathione disulfide (GSSG) and cellular redox imbalance previously shown to initiate the apoptotic cascade. Apo A-IV had no effect on GSSG reductase activity, but it stimulated G6PD activity 10-fold. These results suggest a novel role for Apo A-IV in the regulation of intracellular glutathione redox balance and the modulation of redox-dependent apoptosis via stimulation of G6PD activity. tert-butyl hydroperoxide; diamide; dehydroisoandrosterone; glucose-6-phosphate dehydrogenase; antisense     

Changes in Adenosine Pyrophosphates in Cantaloupe Fruit Ripening Normally and after Treatment with Ethylene

During the climacteric rise in respiration of cantaloupe fruit(Cucumis melo L., var. reticulatus Naud.) the concentrationper gramme fresh weight of adenosine triphosphate (ATP) increasedand that of adenosine diphosphate (ADP) did not change; thusa net synthesis of adenosine pyrophosphate occurs during therespiratory climacteric. A net synthesis of protein which wasobserved was positively correlated with the concentration ofATP. Ethylene treatment stimulated a climacteric-like rise inthe respiration and in the rate of ripening in fruit harvestedat 9 to 32 days after anthesis. The ratio ATP/ADP increasedin fruit ripened with ethylene only when harvested 20 days ormore after anthesis.     

Studies on the Growth in Culture of Plant Cells: II. CHANGES IN RESPIRATION RATE AND NITROGEN CONTENT ASSOCIATED WITH THE GROWTH OF ACER PSEUDOPLATANUS L. CELLS IN SUSPENSION CULTURE

Changes in nitrogen content and in respiration rate have beeninvestigated in cell suspension cultures of Acer pseudoplatanus.Nitrogen content and rate of oxygen uptake rise sharply earlyin the period of culture, during which there is no significantincrease in dry weight and only a small increase in cell number.During the subsequent period of rapid cell division there isa decline in both respiration rate and nitrogen content permg dry weight or per cell. Pronounced rises in respiration rateand cell nitrogen therefore occur prior to the period of rapidcell division. The strong correlation between nitrogen contentand oxygen consumption suggests that the respiration rate ismuch more closely related to changes in protein content thanto changes in cell number, dry weight, or packed-cell volume.     

Getting out of a Tight Squeeze--Enzymatic Regulation of Claw Muscle Atrophy in Molting

The claw closer muscle of the land crab, Gecarcinus lateralis,undergoes a cyclical atrophy and restoration during the intervalbetween ecdyses. During proecdysis (stage D₀), 30–60%of the muscle protein is degraded, which reduces tissue massand facilitates withdrawal of the propodus at ecdysis. Proteinis resynthesized as the muscle grows back to its previous sizeduring metecdysis. This atrophy is specific to the claws andcan be accentuated by multiple limb autotomy. Crustacean musclescontain five cytosolic proteinases that degrade myofibrillarproteins. Four of these constitute a family of enzymes requiringCa²⁺ for activity. These calcium-dependent proteinases (CDPs)hydrolyze myofibrillar proteins in vitro and in situ and showincreased activity in atrophic claw muscles, which suggeststhat CDPs play an important role in myofibrillar protein metabolism.The fifth enzyme is a multicatalytic proteinase (MCP), a multisubunitproteolytic complex that degrades a wide range of peptide andprotein substrates. The catalytic properties of the complexare altered with low concentrations of sodium dodecyl sulfateor by brief heating at 60°C. Only the heat-activated formdegrades myofibrillar proteins. Since the CDPs hydrolyze contractileproteins about 30-fold more rapidly than the heat-activatedMCP, the MCP probably has a more limited or specialized functionin molt-induced claw muscle atrophy.     

Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca²⁺]_i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca²⁺ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor     

PKA induces Ca2+ release and enhances ciliary beat frequency in a Ca2+-dependent and -independent manner

The intent of this work was to evaluate the role of cAMP inregulation of ciliary activity in frog mucociliary epithelium and toexamine the possibility of cross talk between the cAMP- andCa²⁺-dependent pathways in thatregulation. Forskolin and dibutyryl cAMP induced strong transientintracellular Ca²⁺ concentration([Ca²⁺]_i)elevation and strong ciliary beat frequency enhancement with prolongedstabilization at an elevated plateau. The response was not affected byreduction of extracellular Ca²⁺concentration. The elevation in[Ca²⁺]_iwas canceled by pretreatment with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Underthose experimental conditions, forskolin raised the beat frequency to amoderately elevated plateau, whereas the initial strong rise infrequency was completely abolished. All effects were canceled by H-89,a selective protein kinase A (PKA) inhibitor. The results suggest adual role for PKA in ciliary regulation. PKA releasesCa²⁺ from intracellular stores,strongly activating ciliary beating, and, concurrently, producesmoderate prolonged enhancement of the beat frequency by aCa²⁺-independent mechanism.