Electrochemical potential of protons in vesicles reconstituted from purified,proton-translocating adenosine triphosphatase

Summary Measurements were made of the difference in the electrochemical potential of protons ( ) across the membrane of vesicles reconstituted from the ATPase complex (TF ₀ ·F ₁) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential () and pH difference across the membrane ( pH), respectively.In the presence of Tris buffer the maximal and no pH were produced, while in the presence of the permeant anion NO ₃ ^– the maximal pH and a low were produced by the addition of ATP. When the ATP concentration was 0.24mm, the was 140–150 mV (positive inside) in Tris buffer, and the pH was 2.9–3.5 units (acidic inside) in the presence of NO ₃ ^– . Addition of a saturating amount of ATP produced somewhat larger and pH values, and the attained was about 310 mV.By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4–5 during ATP hydrolysis.The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

Basis of the Relationship between Ash Content in the Flag Leaf and Carbon Isotope Discrimination in Kernels of Durum Wheat

The relationship between ash content and carbon isotope discrimination () was studied in durum wheat (Triticum durum Desf.) grown in a Mediterranean region (Northwest Syria) under three different water regimes (hereafter referred to as environments). In two of these environments, 144 genotypes were cultivated under rain-fed conditions. In the third environment, 125 genotypes were cultivated under irrigation. Ash content was measured in the flag leaf about 3 weeks after anthesis, whereas was analysed in mature kernels. Total transpiration of the photosynthetic tissues of the culm contributing, from heading to maturity, to the filling of kernels was also estimated. Leaf ash content, expressed either on dry matter or leaf area basis or as total ash per blade, correlated positively (p< 0.001) with in the three environments. However, this relationship was not the result of a positive correlation across genotypes between and tissue water content. Moreover, only a small part of the variation in across genotypes was explained by concomitant changes in ash content. When all genotypes across the three environments were plotted, and ash content followed a non-linear relationship (r ² = 74), with tending to a plateau as the ash content increased. However, for the set of genotypes and environments combined, total ash content per leaf blade was positively and linearly related (r ² = 0.76) with the accumulated culm transpiration. The non-linear nature of the relationship between ash content and is sustained by the fact that culm transpiration also showed a non-linear relationship with kernel . Therefore, differences in leaf ash content between environments, and to a lesser extent between genotypes, seem to be brought about by variations in accumulated transpiration during grain formation.     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Anionic Tetrahedral Complexes as Serine Protease Inhibitors

Potent inhibitors of proteases are constantly sought because of their potential as new therapeutic lead compounds. In this paper we report a simple computational methodology for obtaining new ideas for functional groups that may act as effective inhibitors. We relate this study to serine proteases. We have analyzed all of the factors that operate in the enzyme-substrate interactions and govern the free energy for the transformation of the Michaelis complex (MC) to the anionic covalent tetrahedral complex (TC). The free energy of this transformation ( G_MC-TC ) is the quantitative criterion that differentiates between the catalytic and inhibitory processes in proteases. The catalytic TC is shifted upwards (G_MC-TC > 0) relative to the MC in the free energy profile of the reaction, whereas the inhibitory tetrahedral species is shifted downward (G_MC-TC < 0). Therefore, the more stable the TC, the more effective it should be as an inhibitor. We conclude that the dominant contribution to the superstabilization of an anionic TC for transition state analog inhibitors originates from the formation of a -covalent bond between the reactive centers of the enzyme and its inhibitor. This energetic effect is a quantitative value obtained in ab initio calculations and provides an estimate as to whether a functional group is feasible as potent inhibitor or not. To support our methodology, we describe several examples where good agreement is shown between modeled ab initio quantum chemical calculations and experimental results extracted from the literature.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

Relationships among violaxanthin deepoxidation,thylakoid membrane conformation,and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (Gossypium hirsutum L.)

The kinetics and temperature dependencies of development and relaxation of light-induced absorbance changes caused by deepoxidation of violaxanthin to antheraxanthin and zeaxanthin (Z; peak at 506 nm) and by light scattering (S; peak around 540 nm) as well as of nonphotochemical quenching of chlorophyll fluorescence (NPQ) were followed in cotton leaves. Measurements were made in the absence and the presence of dithiothreitol (DTT), an inhibitor of violaxanthin deepoxidase. The amount of NPQ was calculated from the Stern-Volmer equation. A procedure was developed to correct gross AS (S_g) for absorbance changes around 540 nm that are due to a spectral overlap with Z. This protocol isolated the component which is caused by light-scattering changes alone (S_n). In control leaves, the kinetics and temperature dependence of the initial rate of rise in S_n that takes place upon illumination, closely matched that of Z. Application of DTT to leaves, containing little zeaxanthin or antheraxanthin, strongly inhibited both S_n and NPQ, but DTT had no inhibitory effect in leaves in which these xanthophylls had already been preformed, showing that the effect of DTT on A_n and NPQ results solely from the inhibition of violaxanthin deepoxidation. The rates and maximum extents of S_n and NPQ therefore depend on the amount of zeaxanthin (and/or antheraxanthin) present in the leaf. In contrast to the situation during induction, relaxation of Z upon darkening was much slower than the relaxation of S_n and NPQ. The relaxation of S_n and NPQ showed quantitatively similar kinetics and temperature dependencies (Q₁₀=2.4). These results are consistent with the following hypotheses: The increase in lumen-proton concentration resulting from thylakoid membrane energization causes deepoxidation of violaxanthin to antheraxanthin and zeaxanthin. The presence of these xanthophylls is not sufficient to cause S_n or NPQ but, together with an increased lumen-proton concentration, these xanthophylls cause a conformational change, reflected by S_n. The conformational change facilititates nonradiative energy dissipation, thereby causing NPQ. Membrane energization is prerequisite to conformational changes in the thylakoid membrane and resultant nonradiative energy dissipation but the capacity for such changes in intact leaves is quite limited unless zeaxanthin (and/or antheraxanthin) is present in the membrane. The sustained S_n and NPQ levels that remain after darkening may be attributable to a sustained high lumen-proton concentration.Abbreviations A antheraxanthin - DTT dithiothreitol - F, F_m chlorophyll fluorescence yield at actual, full closure of the PSII centers - NPQ nonphotochemical chlorophyll fluorescence quenching - PFD photon flux density - PSII photosystem II - V violaxanthin - Z zeaxanthin - S_n, Z spectral absorbance change caused by light-scattering, violaxanthin deepoxidation We thank Connie Shih for skillful assistance in growing the plants, and for conducting HPLC analyses. A Carnegie Institution Fellowship and a Feodor-Lynen-Fellowship by the Alexander von Humboldt-Foundation to W. B. is gratefully acknowledged. This work was supported in part by Grant No. 89-37-280-4902 of the Competitive Grants Program of the U.S. Department of Agriculture to O.B. This is C. I. W. — D. P. B. Publication No. 1094.     

ATP-induced ΔpH formation in chloroplast ATP synthase proteoliposomes

Summary A procedure to reconstitute CF₀CF₁ proteoliposomes by gel filtration through a Sephadex-column pre-equilibrated with valinomycin and potassium is described. Proteoliposomes reconstituted by this procedure catalyze an ATP-induced pH of 2.5 to 3.5 units. pH was measured with either 9-aminoacridine or with the pH indicator pyranine trapped inside the proteoliposomes. CF₀CF₁ proteoliposomes prepared by conventional techniques catalyzed an ATP-induced formation, but were unable to catalyze an ATP-induced pH even in the presence of valinomycin.The ATP-induced pH was sensitive to uncouplers and energy transfer inhibitors and was increased at low temperatures. It is suggested that ATP-induced pH was observed in these proteoliposomes due to the efficient removal of intravesicular ammonium introduced with the CF₀CF₁ preparation. The ammonium acted as an internal buffer, and thus prevented an observable pH formation.     

Thermal Stability of Glucose Oxidase from Penicillium adametzii

The thermal stability of glucose oxidase was studied at temperatures between 50 and 70°C by kinetic and spectroscopic (circular dichroism) methods. The stability of glucose oxidase was shown to depend on the medium pH, protein concentration, and the presence of protectors in the solution. At low protein concentrations (<15 g/ml) and pH > 5.5, the rate constants k _in, s^–1, for thermal inactivation of glucose oxidase were high. Circular dichroic spectra suggested an essential role of structures in stabilizing the protein globule. At a concentration of 15 g protein/ml, the activation energy E _Aof thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal/mol. Other thermodynamic activation parameters estimated at 60°C had the following values: H= 78.4 kcal/mol, G= 25.5 kcal/mol, and S= 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCl, polyethylene glycols, and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol, and citrate exerted destabilizing effects.     

Desaturation of oxygenated fatty acids in Lesquerella and other oil seeds

Species of the genus Lesquerella, within the Brassicaceae family, have seed oils containing hydroxy fatty acids. In most Lesquerella species, either lesquerolic (14-hydroxy-eicosa-11-enoic), auricolic (14-hydroxy-eicosa-11,17-dienoic) or densipolic (12-hydroxy-octadeca-9,15-dienoic) acid dominates in the seed oils. Incubations of developing seed from Lesquerella species with 1-¹⁴C-fatty acids were conducted in order to study the biosynthetic pathways of these hydroxylated fatty acids. [¹⁴C]Oleic (octadeca-9-enoic) acid, but not [¹⁴C]linoleic (octadeca-9,12-dienoic) acid, was converted into the hydroxy fatty acid, ricinoleic (12-hydroxy-octadeca-9-enoic) acid, which was rapidly desaturated to densipolic (12-hydroxy-octadeca-9,15-dienoic) acid. In addition, [¹⁴C] ricinoleic acid added to Lesquerella seeds was efficiently desaturated at the 15 carbon. A pathway for the biosynthesis of the various hydroxylated fatty acids in Lesquerella seeds is proposed. The demonstration of desaturation at position 15 of a fatty acid with a hydroxy group at position 12 in Lesquerella prompted a comparison of the substrate recognition of the desaturases from Lesquerella and linseed. It was demonstrated that developing linseed also was able to desaturate ricinoleate at position 15 into densipolic acid. In addition, the linseed 15 desaturase was able to desaturate vernolic (12,13-epoxy-octadeca-9-enoic) acid and safflower microsomal 12 desaturase was able to desaturate 9-hydroxy-stearate. Thus, hydroxy and epoxy groups may substitute for double bonds in substrate recognition for oil-seed 12 and 15 desaturases.Abbreviations GLC gas-liquid chromatography - lysoPC palmitoyl-lysophosphatidylcholine - PC phosphatidylcholine This work was supported by grants from Stifteisen Svensk Oljeväxtforskning, Skanska Lantmännen Foundation, Swedish Farmers Foundation for Agricultural research, The Swedish Natural Science Research Council and The Swedish Council for Forestry and Agricultural Research. Nicki Engeseth was supported by the National Science Foundation under a grant award in 1992.     

Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of<Emphasis Type="Italic"> PHGPX</Emphasis> and<Emphasis Type="Italic"> URE2</Emphasis> genes in aluminum resistance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 , phgpx2 , and phgpx3), were tested for their sensitivity to Al. The triple mutant, phgpx1 /2/3, was more sensitive to Al (55% reduction in growth at 300 M Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3 to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3 and phgpx1/2/3 mutants were sensitive to oxidative stress (exposure to H₂O₂ or diamide). Lipid peroxidation was also increased in the phgpx1/2/3 mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1 and gtt2), glutathione biosynthesis (gsh1 and gsh2), glutathione reductase (glr1) and a glutathione transporter (opt1) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2 mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 M Al. Intracellular oxidation levels in the ure2 mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H₂O₂) increase compared to BY4741; however, the ure2 mutant showed no change in lipid peroxidation compared to the control. The phgpx1/2/3 and ure2 mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.Communicated by D.Y. Thomas     

Ultrastructure des cellules de Leydig et des cellules de Sertoli au cours du cycle testiculaire du Canard Pékin

Résumé L'ultrastructure des cellules de Leydig et des cellules de Sertoli du testicule du Canard Pékin a été étudiée au cours de la phase printanière du cycle sexuel, soit de janvier à juillet. Parallèlement on a effectué chez les mêemes animaux la recherche histochimique de la ⁵-3 -hydroxystéroïdedeshydrogénase (⁵-3 -HSDH) ainsi que le dosage, par chromatographie en phase gazeuse des stéroïdes androgènes dans le plasma veineux périphérique et dans le testicule.Les cellules de Leydig du Canard possèdent les organites cytoplasmiques spécifiques des cellules stéroïdogènes (reticulum lisse, mitochondries à crêtes tubulaires) ainsi que d'autres structures souvent rencontrées dans ce type cellulaire (microfilaments, vacuoles, granules denses). Les cellules de Sertoli contiennent un reticulum agranulaire moins développé que celui des cellules de Leydig et, très rarement, des mitochondries à crêtes tubulaires. Ces divers organites cytoplasmiques subissent un cycle saisonnier. La différenciation du reticulum lisse et des crêtes mitochondriales tubulaires commence en janvier et atteint son optimum en mars. Leur régression s'amorce en avril; d'abord accompagnée de structures dégénératives transitoires; elle conduit à la dispartion totale de ces organites en mait. Aucun indice de nécrose n'est observé dans ces cellules. Histochimiquement, une activité ⁵-3 -HSDH est présente dans les cellules de Leydig et, à un degré moindre, dans les tubes séminifères. Son intensité varie au cours du cycle.La confrontation de l'étude morphologique avec les résultats des dosages hormonaux montre qu'il existe une bonne corrélation entre le développement puis la régression du reticulum lisse et des crêtes tubulaires des mitochondries ainsi que des critères histochimiques de la ⁵-3 -HSDH d'une part et l'évolution de la testostérone plasmatique et testiculaire d'autre part. De plus on observe une augmentation du rapport testostérone/⁴-androstènedione testiculaire parallèlement au développement des organites cytoplasmiques. Ces organites semblent donc bien impliqués dans la synthèse et la sécrétion de la testostérone chez le Canard.

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Ultrastructure of Leydig and Sertoli cells in the testicular cycle of the Pekin duckBiochemical and cytoenzymological correlations

Summary Leydig and Sertoli cells of the testis of the Pekin duck were studied ultrastructurally during the spring phase of the sexual cycle, from January to July. Simultaneously, in the same animals, ⁵-3 -hydroxysteroiddehydrogenase (⁵-3 -HSDH) activity was ascertained histochemically and androgenic steroids of the plasma and testes were assayed by gas-liquid chromatography.The Leydig cells of the duck possess cytoplasmic organelles specific to steroidogenic cells (smooth reticulum, tubular mitochondria) as well as other structures often found in this cell type (microfilaments, vacuoles, denses bodies). The Sertoli cells contain an agranular reticulum that is less developed than that of the Leydig cells, and rarely show mitochondria with tubular cristae. These various cytoplasmic organelles undergo a seasonal cycle. The differentiation of the smooth reticulum and the mitochondrial tubular cristae begins in January and reaches a maximum in March. They begin to regress in April, at first with transitory degenerative structures, and then by total disappearance of these organelles by May. No indication of necrosis is observed in the cells. Histochemically ⁵-3 -HSDH activity is present in the Leydig cells, and to a slightly lesser degree in the seminiferous tubules. The intensity varies during the cycle.The comparison of the results of the morphological study with the hormone assays shows that a good correlation exists with the development and regression of the smooth endoplasmic reticulum and tubular cristae in the mitochondria, as well as the histochemical criteria of the ⁵-3 -HSDH on one hand, and the levels of plasma and testicular testosterone on the other hand. In addition there is an increase in the ratio of testicular testosterone to ⁴-androstenedione which parallels the development of the cytoplasmic organelles. These organelles thus seem to be implicated in the synthesis and secretion of testosterone in the duck.

Nous tenons à remercier très vivement Mme G. Collenot qui nous a initiées aux techniques d'histoenzymologie et nous a très gentiment permis de faire cette partie de nos recherches dans son laboratoire. Nous remerçions également M. Claude Pennarun, photographe, pour son excellente collaboration.     

Concentration/response effect of 2,2′, 4,4′, 5,5′-hexabromobiphenyl on cell-cell communication in vitro: assessment by fluorescence redistribution after photobleaching (“FRAP”)

Inhibition of gap junction-mediated cell-cell communication might be a mechanism for several types of cellular dysfunctions, including tumor promotion. Although many different assays have been designed to measure gap junction-mediated intercellular communication, we applied a new technique, termed Fluorescence Redistribution After Photobleaching (FRAP), to assess the ability of a known tumor promoter, 2,2, 4,4, 5,5-hexabromobiphenyl (245-HBB), to inhibit cell-cell communication in a concentration-dependent manner. WB-F344 (rat epithelial) cells were plated at low density, exposed to noncytotoxic concentrations of 1, 5, or 20 µg 245-HBB/ml medium, and stained with 6-carboxyfluorescein diacetate. Single cells in pairs or clusters of touching cells in each exposure group were examined with FRAP. The results revealed an inverse correlation between the degree offluorescence redistribution in photobleached cells and the concentration of 245-HBB. Therefore, FRAP appears to be a sensitive and rapid technique for determining complete or partial inhibition of chemically induced intercellular communication in vitro. These results also provide further evidence for the ability of 245-HBB to inhibit gap junction-mediated cell-cell communication in a concentration-dependent manner.Abbreviations 6-CFDA 6-carboxyfluorescein diacetate - FRAP fluorescence redistribution after photobleaching - 245-HBB 2,2, 4,4, 5,5-hexabromobiphenyl Michigan Agricultural Experiment Station journal article No. 12531.     

D-Gluconate is an alternative growth substrate for cultivation of Schizosaccharomyces pombe mutants

Schizosaccharomyces pombe cells grow on d-gluconate as the sole carbon and energy source. d-Gluconate is taken up in symport with protons by a specific symporter, pH being the sole driving force. d-Gluconate uptake is independent of the sugar transporting system (e.g. for d-glucose) and of . The carrier is expressed constitutively, and its activity is not subject to glucose repression. Hence, d-gluconate is a suitable carbon and energy source for growth, when d-glucose or other hexoses have to be eliminated e.g. for selection of mutants deficient in hexose transport.Abbreviations 2-DG 2-deoxy-d-glucose - CCCP carbonylcyanide m-chlorophenylhydrazone - pH pH-gradient - electrical potential difference across the plasma membrane - SD standard deviation - SEM standard error of the mean - TPP⁺ tetraphenylphosphonium     

Ecosystem Modulation of Dissolved Carbon Age in a Temperate Marsh-Dominated Estuary

We measured the concentrations and isotopic values (¹⁴C and ¹³C) of dissolved inorganic, dissolved organic, and particulate organic carbon (DIC, DOC, and POC, respectively) in the Parker River watershed and estuary in Massachusetts, USA, to determine the age of carbon (C) entering the estuary and how estuarine processing affects the quantity and apparent age of C transported to the Gulf of Maine. The watershed measurements indicated the transport of ¹⁴C-enriched modern DIC and DOC and variably aged POC from the watershed to the estuary. The transport of organic matter from the watershed was dominated by DOC transport, with POC making up less than 10% of the total. Surveys within the watershed aimed at determining which land-use type dominated the DOC export indicated that wetlands, although they made up only around 20% of the land use, could be responsible for approximately 75% of the DOC export. We therefore conclude that the wetland land uses of the Parker River watershed are exporting mainly ¹⁴C-enriched modern DOC. DIC isotopes indicate that the source of DIC in the Parker River watershed is dominated by the weathering of noncarbonate parent material by ¹⁴C-enriched carbon dioxide (CO₂) originating from the respiration of young organic matter in soils. Transects in the estuary displayed net additions of all C species. For DOC and DIC, the export of this internally added DOC and DIC was approximately equal to the amount being exported from the watershed, showing the importance of focusing on estuaries when estimating the export of C to the coastal ocean. With respect to DIC, the total input is even larger when the atmospheric exchange of excess pCO₂ is calculated. The ¹⁴C-DOC and ¹⁴C-DIC transects indicate that the internally added DOC and DIC is ¹⁴C-enriched modern material. The source of this material is the fringing marshes and estuarine phytoplankton, with the relative importance of these two sources changing over time. Taken together, the bulk C and ¹⁴C measurements show that the estuary is adding significant quantities of young DOC despite the presence of vast quantities of old marsh peat flanking the entire estuary. Furthermore, the DIC data indicate that ¹⁴C-enriched modern material is what is fueling the majority of heterotrophic respiration within the system.     

On a relation between interfacial free energy-dependent and noninterfacial free energy-dependent adherence of oral streptococci to solid substrata

In several systems bacterial adhesion occurs despite a positive interfacial free energy of adhesion, F_adh; this implies that other interactions are involved. We hypothesize that the number of bacteria adhering at F_adh=0 represents their ability to adhere by other, i.e., non-F_adh-dependent interactions. Eight strains of oral streptococci were allowed to adhere to three solid substrata with different surface free energies in a flow cell system. Strain-specific linear relations were found between the numbers of bacteria adhering at saturation, n_b,s, and F_adh. When for all strains the numbers of adhering bacteria at F_adh=0 were plotted versus the slopes, denoting the sensitivity to F_adh, a linear relationship (r=0.92) was observed. It is, therefore, concluded that one strain-specific factor influences both F_adh-dependent and non-F_adh-dependent adherence. The numerical value of this factor, together with a surface energetic analysis, predicts the number of streptococci that will adhere to a given nonbiological substratum.     

Steady state changes in mitochondrial electrical potential and proton gradient in perfused liver from rats fed a high fat diet

In this work the protonmotive force (p), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non acqueous solvents.Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar p to that found in rats fed a low fat diet, but when we consider the two components of p, we find a significant decrease in mitochondrial/cytosolic pH difference (pH_m) and a significant increase in mitochondrial membrane potential (_m) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in pH_m and _m, which represent the respective driving force for their transport.The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.     

The Polymer Basis of Kinetics and Equilibria of Enzymes: The Accessible-Volume Origin of Entropy Changes in a Class Aβ-Lactamase

The occurrence of enzymatic catalysis, as for any chemical reaction, depends critically upon close contact of the reactants, since making/breaking of bonds occurs over distances of about 0.2 Å. Unlike small molecules, each enzyme molecule acts as an ordered solvent and reactant. Each group important to the enzyme reaction interacts with the substrate, then moves away, and subsequently binds another substrate. In other words, the group undergoes round trips in structure. For a round trip, the thermochemical state functions G, H, S, etc., are zero. As a consequence, control of the binding of substrate must reside in the nonbinding conformations of the polymer since they govern the different fractions of time the macromolecule is in the correct conformation for bonding. Applying standard macromolecular models to the enzymes suggests that the majority of free energy for an enzyme reaction resides in the enzyme structure as an entropic contribution. Enthalpic contributions come from bond formation with the substrates and substrate structural changes. Further, it is shown that the molecular mechanisms that can effect binding and allosteric control fall into only three classes. Three x-ray structures of class A -lactamases (native, mutant, and with substrate) show the individual binding groups at the active site change their accessible volumes depending on substrate binding and mutant form. From these volume differences, the S of reaction is calculated. The x-ray-derived G = –TS matches the G = –RT ln k₁ from changes in rate constants for the same set of -penicillinases.     

A termination mutation (2143delT) in the CFTR gene of German cystic fibrosis patients

German patients with cystic fibrosis (CF) were screened for molecular lesions in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by single strand conformation polymorphism (SSCP) and chemical cleavage of mismatch analyses. Direct sequencing of four samples that displayed the same SSCP pattern and that were susceptible to cleavage of heteroduplexes by osmium tetroxide revealed, in all cases, a deletion of a single T residue at nucleotide position 2143 within codon 671 of the CFTR gene. As a result, leucine codon 671 is changed into a termination codon. In total, the 2143delT mutation was confirmed in 6 out of 271 German non-F508 CF chromosomes by artificial restriction fragment length polymorphism analysis, indicating that this frameshift mutation accounts for about 2% of German non-508 mutations. The 6 pancreas insufficient patients who are compound heterozygous for 2143-delT suffer from the typical features of pulmonary and gastrointestinal CF disease. The 2143delT mutation completes the panel of the more frequent CFTR mutations that reside on the F508 haplotype and that contribute to its overpresentation among German non-F508 alleles that are associated with severe forms of disease.