Partial Purification and Characterization of the Maize Mitochondrial Pyruvate Dehydrogenase Complex1

The pyruvate dehydrogenase complex was partially purified and characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD. Immunoblot analyses identified these proteins as the E1β-, E1α-, E2-, and E3-subunits, respectively. The molecular mass of maize E2 is considerably smaller than that of other plant E2 subunits (76 kD). The activity of the maize mitochondrial complex has a pH optimum of 7.5 and a divalent cation requirement best satisfied by Mg2+. Michaelis constants for the substrates were 47, 3, 77, and 1 μm for pyruvate, coenzyme A (CoA), NAD+, and thiamine pyrophosphate, respectively. The products NADH and acetyl-CoA were competitive inhibitors with respect to NAD+ and CoA, and the inhibition constants were 15 and 47 μm, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of ATP and the addition of Mg2+.

In vivo suppression of the renal Na+/Pi cotransporter by antisense oligonucleotides.

A 20-mer phosphorothioate oligonucleotide (AS1) was designed to hybridize to the message for the rat kidney sodium phosphate cotransporter NaPi-2 close to the translation initiation site. Single intravenous doses of this oligonucleotide were given to rats maintained on a low phosphorus diet to increase NaPi-2 expression. At 3 days after oligonucleotide infusion, rats receiving 2.5 micromol of AS1 exhibited a reduction in renal NaPi-2 to cyclophilin mRNA ratio by 40% +/- 17%, and rats receiving 7.5 micromol of AS1 exhibited a reduction in NaPi-2 to cyclophilin mRNA ratio by 46% +/- 21%. Reversed-sequence AS1 was without effect. The higher dose of 7.5 micromol of AS1 also reduced the rate of phosphate uptake into renal brush border membrane vesicles and the expression of NaPi-2 protein detected by Western blotting in these vesicles. Reversed sequence AS1 was again without effect on these parameters. These results suggest that systemically infused oligonucleotides can exert antisense effects in the renal proximal tubule.

Avaliação do antagonismo do efeito do sódio sobre o tratamento de água residuária de Charqueada com elevada salinidade em reator anaeróbio de manta de lodo (UASB)

As águas residuárias provenientes da indústria do charque são conhecidas por apresentarem elevado teor de cloreto de sódio, aliado a grandes concentrações de matéria orgânica proveniente do sangue liberado ao longo do processo industrial. Esse tipo de água residuária apresenta potencial para degradação biológica, contudo, o cloreto de sódio, em concentração elevada, pode inibir a atividade dos microrganismos e, em alguns casos, levar sistemas biológicos à falência. No presente trabalho, foi avaliada a viabilidade de degradação anaeróbia de efluente sintético de Charqueada contendo elevado teor de cloreto de sódio, em reator anaeróbio tipo UASB (Upflow Anaerobic Sludge Blanket), em escala de laboratório. Foram utilizados 4 reatores, alimentados com água residuária sintética com características similares à água residuária de Charqueada. O reator 1 foi utilizado como controle, o reator 2 recebeu NaCl e os demais (3 e 4) foram operados na presença de NaCl acrescidos de: betaína e potássio com cálcio, respectivamente. Os compostos citados são conhecidos como antagonizantes, por possuirem capacidade de minimizar o efeito inibitório do sódio sobre o processo de digestão anaeróbia. Os reatores foram inoculados com lodo de reator UASB e submetidos à concentração de 5000 mg/L de matéria orgânica, como DQO. A carga orgânica aplicada foi de 5 Kg/m3.d e os reatores não suportaram tal carga. Reiniciou-se a operação com aumento progressivo da DQO de 500 a 2000 mg/L resultando em cargas orgânicas de 0,5 a 2,0 Kg/m3.d, respectivamente. Após estabilização dos reatores, iniciou-se a fase de introdução de cloreto de sódio (1.500 a 13.500 mg/L) e antagonizantes com aumento progressivo a cada fase. Na presença ou ausência de antagonizantes, os reatores 2, 3 e 4 não tiveram o desempenho alterado até a concentração de NaCl de 6000 mg/L. Na presença de 9000 mg/L de NaCl, a betaína se mostrou pouco efetiva como soluto compatível no reator 3 e os antagonizantes do reator 4, potássio e cálcio, apresentaram efeitos estimulatórios. As morfologias encontradas ao longo do experimento foram cocos, víbrios, bacilos, sarcinas, além de morfologias semelhantes a Methanosarcina sp. e Methanosaeta sp. O aumento da concentração de cloreto de sódio provocou a redição da população de Arqueas.

Sobrecarga e restrição de cloreto de sódio durante a gestação: repercussão sobre a estrutura cardíaca e renal no neonato

Introdução: Diversos estudos indicaram consequências de alterações na nutrição materna durante a gestação sobre a saúde da prole adulta, tais como: hipertensão, doenças cardiovasculares, resistência à insulina, diabete melito e doença renal. No entanto, a literatura é pobre em avaliações decorrentes de modificações nutricionais maternas sobre a prole logo após o nascimento. Métodos: Ratas Wistar durante o período gestacional foram alimentadas com dieta hipossódica (HO - 0,15% de NaCl), normossódica (NR - 1,3% de NaCl) ou hipersódica (HR - 8% de Na Cl). Após o nascimento, nas primeiras vinte e quatro horas foram coletados rins e coração dos neonatos machos e fêmeas (n=6- 8/grupo) para verificar as possíveis alterações na estrutura cardíaca e renal pelo método de estereologia. Também foi avaliada a expressão proteica e gênica dos componentes do sistema renina angiotensina (SRA) no coração e rins através do método ELISA indireto e RT-qPCR. Resultados: O peso ao nascimento foi menor em machos e fêmeas da prole de mães alimentadas com dieta hipossódica durante a gestação quando comparado NR e HR. Não houve diferença no volume renal, volume de seus compartimentos (córtex, medula e pelve) e número de glomérulos entre os grupos experimentais (HO, NR e HR). No entanto, o número de glomérulos foi maior em fêmeas comparado aos machos nos três grupos experimentais. O diâmetro transverso do núcleo dos cardiomiócitos no ventrículo esquerdo e no ventrículo direito de machos da prole HR foi maior do que na prole NR. A expressão proteica do receptor AT1 no rim de machos da prole foi menor no grupo HO do que no grupo NR e HR. A expressão proteica do receptor AT2 também foi menor em machos do grupo HO do que no grupo NR. Não houve diferença entre os grupos na expressão proteica dos receptores AT1 e AT2 no rim das fêmeas. Conclusão: O presente estudo detectou alterações na estrutura cardíaca de neonatos machos, mas não em neonatos fêmeas decorrentes de sobrecarga de sal durante a gravidez. As alterações observadas na expressão dos receptores AT1 e AT2 no rim de neonatos machos podem ser responsáveis por alterações na função renal

Sedimentology and age determinations on core PS2813-1

Die Rekonstruktion des Einflusses von Strömungen und glazialmarinen Prozessen auf das Sedimentationsgeschehen am Kontinentalhang der Antarktischen Halbinsel im westlichen Weddellmeer basiert auf sedimentologischen und geophysikalischen Daten eines Kolbenlotkerns. Der Sedimentkern wurde während des Fahrtabschnitts ANT-XIV/3 mit dem FS "Polarstern" aus einer mächtigen Levee-Struktur eines Rinnen-Rückensystems gewonnen. Es wurden sedimentologische sowie sedimentphysikalische Untersuchungen an dem Kernmaterial durchgeführt. Die texturellen Änderungen im Kern und die Variationen der gemessenen Parameter ermöglichen eine lithofazielle Gliederung und stratigraphische Einstufung der Sedimentabfolge. Die untersuchten Sedimente umfassen den Zeitraum der vier letzten Klimazyklen bis heute und repräsentieren die Ablagerungsbedingungen von mehr als 340 000 Jahren. Vier Faziestypen wurden unterschieden, die sowohl glaziale als auch interglaziale Ablagerungsräume charakterisieren. (1) Die überwiegend groblaminierten Sedimentabfolgen wurden der Laminitfazies zugeordnet. Unter glazialen Umweltbedingungen kam es infolge schwacher Bodenströmungen zur Ablagerung feinkörniger, laminierter, strömungsbetonter Sedimente. (2) Strukturlose, sehr homogene Sedimentabfolgen des Kems beschreiben einen weiteren, den Kaltzeiten zugeordneten, Faziestyp, der durch geringe Variationen in den Sedimenteigenschaften charakterisiert ist. (3) Kernabschnitte, die weitgehend strukturlos sind bzw. leichte Bioturbationen und relativ viel eistransportiertes Material aufweisen, wurden als IRD-Fazies bezeichnet. Sie repräsentiert den Übergang vom Glazial zum Interglazial, in dem sich das Schelfeis und die Meereisbeckung zurückzogen. In den Sedimenten kam es infolge der gesteigerten Kalbungsrate zur Anreicherung der Eisfracht. (4) Die relativ biogenreichen, hellen Ablagerungen wurden der interglazialzeitlichen Karbonatfazies zugeteilt. Der signifikant erhöhte Anteil planktischer Foraminiferen weist auf eine gesteigerte Bioproduktivität im Oberflächenwasser hin, die aus verstärkten jahreszeitlichen Schwankungen der Meereisbedeckung resultiert. Die betrachteten Sedimentationsprozesse, wie biologische Produktivität, Umlagerungsprozesse durch Meeresströmungen, gravitativer Sedimenttransport und Eistransport, sind das Abbild komplexer Wechselwirkungen aus Meeresspiegelschwankungen, Änderungen ozeanographischer Bedingungen und der Vereisungsdynamik. Das Sedimentationsgeschehen im Untersuchungsgebiet wurde folglich durch die Variationen der vorherrschenden Umweltbedingungen bestimmt. Im Glazial kam es unter einer geschlossenen Meereisbedeckung zur Ablagerung feinkörniger, geschichteter Sedimente. Vorwiegend Turbiditströmungen kontrollierten das Sedimentationsgeschehen innerhalb des betrachteten Rinnen-Rückensystems. Unter dem Einfluß der Coriolis-Kraft und wahrscheinlich einer Konturströmung wurden die suspendierten, feinkörnigen Partikel aus dem zentralen Bereich der Rinne verdriftet und über dem nördlichen Uferwall abgelagert. Höherenergetische gravitative Prozesse beeinflußten das Sedimentationsgeschehen episodisch und sind durch gut sortierte Ablagerungen mit erhöhten Gehalten im Mittel- bis Grobsiltbereich dokumentiert. Höhere Sedimentationsraten in den Glazialen trugen verstärkt zur Bildung des Uferwalls bei. Die Ablagerungen der ebenfalls glazialzeitlichen homogenen Fazies belegen unterschiedliche Ablagerungsbedingungen und eine Verschiebung der dominierenden Prozesse. Während des Übergangs vom Glazial zum Interglazial nahm die Bodenwasserbildungsrate durch das Aufschwimmen des Schelfeises zu, wodurch die Strömungsintensität gesteigert wurde. Eine verstärkte Eisbergaktivität wird durch die Anreichung des IRD-Materials dokumentiert. Während interglazialer Zeiten ermöglichten offen-marine Bedingungen im Südsommer eine leicht erhöhte biologische Produktivität, so daß der Ablagerungsraum durch die Sedimentation biogener Komponenten verstärkt beeinflußt wurde.

(Table I, page 1348) Composition of ferromanganese oxide concretions from lake Ontario (Extract)

A large deposit of ferromanganese oxide concretions has been found in the northern portion of Lake Ontario. The concretions occur mainly in the form of coatings on sand grains but manganese nodules are present at several localities. Mineralogically, the ferromanganese oxide phases are amorphous, and their Fe and Mn contents are similar to those in concretions from other environments. However, their Ni, Co, and Cu contents are significantly higher than those reported in previously described North American lacustrine ferromanganese concretions, and this may, in part, be a reflection of their probable low rates of accumulation.

Chemistry and fluid inclusions of the sheeted dike comples of ODP Holes 137-504B and 140-504B

Fluid inclusions in variably altered diabase recovered from Ocean Drilling Program Legs 137 and 140 at Hole 504B, Costa Rica Rift, exhibit fluid salinities up to 3.7 times that of seawater values (11.7 wt% NaCl equivalent) and exhibit uncorrected homogenization temperatures of 125°C to 202°C. The liquid-dominated inclusions commonly are entrapped in zones of secondary plagioclase and may be primary in origin. Fluid salinities are similar to compositions of fluids venting on the seafloor (0.4-7.0 wt% NaCl) and overlap with those measured in metabasalt samples recovered from near the Kane Fracture Zone on the Mid-Atlantic Ridge and from the Troodos ophiolite, Cyprus. The salinity variations may reflect hydration reactions involving formation of secondary mineral assemblages under rock-dominated conditions, which modify the ionic strength of hydrothermal fluids by consuming or liberating water and chloride ion. Rare CO2-CH4-bearing inclusions, subjacent to zones where talc after olivine becomes an important secondary mineral phase (1700 mbsf), may have formed due to local interaction of seawater and olivine at low water to rock ratios. Corrected average fluid inclusion homogenization temperatures exhibit a gradient from 159°C at a depth of 1370 mbsf to 183°C at a depth of 1992 mbsf and are in apparent equilibrium with the present conductive downhole temperatures. These data indicate that fluid inclusions may be used to estimate downhole temperatures if logging data are unavailable. The compositional and thermal evolution of the diabase-hosted fluids may reflect late-stage, off-axis circulation and conductive heating of compositionally modified seawater in the sheeted dike complex at Hole 504B.

Analysis of Mn nodules from cruise GH72-2

The GH72-2 shipborne survey was carried out in the northwest Pacific during 31 days under the 'Basic investigations for exploration of deep sea mineral resources' program. The sediments encountered could be classified as follows: 1) Most of the brown clays occur on the abyssal plain of the basins at depth over 4500m. 2) Calcareous oozes are predominant at the top, slope and foot of seamounts and guyots. 3) Terrigeneous sediments are distributed near islands. The concentrated zone of ferromanganese nodules was located in the Magellan seamounts area. However, the metal contents in Mn, Cu, Ni and Co for these nodules are relatively poor, and these ferromanganese deposits occur at a depth over 5000m. It is interesting to note that the shape of the nodules is sometimes nearly spherical, and that the chemical composition of the nodules is characterized by the low ratio Mn/Fe and Co/Ni.

Plasma osmolyte concentrations and rectal gland mass of bull sharks Carcharhinus leucas, captured along a salinity gradient

Bull sharks (Carcharhinus leucas) were captured across a salinity gradient from freshwater (FW) to seawater (SW). Across all salinities, C leucas were hyperosmotic to the environment. Plasma osmolarity in FW-captured animals (642 +/- 7 mosM) was significantly reduced compared to SW-captured animals (1067 +/- 21 mosM). In FW animals, sodium, chloride and urea were 208 +/- 3, 203 +/- 3 and 192 +/- 2 mmol l(-1), respectively. Plasma sodium, chloride and urea in SW-captured C leucas were 289 +/- 3, 296 +/- 6 and 370 +/- 10 mmol l(-1), respectively. The increase in plasma osmolarity between FW and SW was not linear. Between FW (3 mosM) and 24%o SW (676 mosM), plasma osmolarity increased by 22% or 0.92% per 1parts per thousand rise in salinity. Between 24%o and 33parts per thousand, plasma osmolarity increased by 33% or 4.7% per 1 parts per thousand rise in salinity, largely due to a sharp increase in plasma urea between 28parts per thousand and 33parts per thousand. C. leucas moving between FW and SW appear to be faced with three major osmoregulatory challenges, these occur between 0-10parts per thousand, 11-20parts per thousand and 21-33parts per thousand. A comparison between C leucas captured in FW and estuarine environments (20-28%o) in the Brisbane River revealed no difference in the mass of rectal glands between these animals. However, a comparison of rectal gland mass between FW animals captured in the Brisbane River and Rio San Juan/Lake Nicaragua showed that animals in the latter system had a significantly smaller rectal gland mass at a given length than animals in the Brisbane River. The physiological challenges and mechanisms required for C leucas moving between FW and SW, as well as the ecological implications of these data are discussed. (C) 2004 Elsevier Inc. All rights reserved.

Gelatinisation of starch in mixtures of sugars. II. Application of differential scanning calorimetry

Differential scanning calorimetry was used to investigate the effect of mixtures of glucose and fructose, and five types of honeys on starch gelatinisation. At a 1:1 starch:water ratio, glucose generally increased the enthalpy (DeltaH(gel)) and temperatures (T-onset, T-peak and T-end) of gelatinisation more than fructose. Upon mixing, DeltaH(gel) of the low-temperature endotherm decreased in comparison to the sole sugars, but was fairly constant (7.7 +/- 0.33 J/g dry starch). DeltaH(gel) of the high-temperature endotherm increased with the fructose content. For both endotherms, the gelatinisation temperatures were unchanged (CV less than or equal to 3%) for the mixtures. With the honeys (moisture, 14.9-18.0%; fructose, 37.2-44.0%; glucose, 28.3-31.9%) added at 1.1-4.4 g per g dry starch, the enthalpy and temperatures of gelatinisation did not vary significantly (CV less than or equal to 6%). Typical thermograms are presented, and the results are interpreted in the light of the various proposed mechanisms for starch gelatinisation in sugar-water systems, total sugar content and possible sugar-sugar interactions. The thermograms were broader in the presence of the sugars and honeys, and a biphasic character was consistently exhibited. The application of an exponential equation to the gelatinisation temperatures of the starch-honey mixtures revealed an opposing influence of fructose and glucose during gelatinisation. The mechanism of starch gelatinisation may be better understood if techniques could be perfected to quantify breakage and formation of hydrogen bonds in the starch granules, and suggested techniques are discussed. (C) 2004 Elsevier Ltd. All rights reserved.

Folate supplementation fails to affect vascular function and carotid artery intima media thickness in cyclosporin A-treated renal transplant recipients

Background: Cyclosporin A (CsA)-treated renal transplant recipients (RTR) exhibit relative hyperhomocystinemia and vascular dysfunction. Folate supplementation lowers homocysteine and has been shown to improve vascular function in healthy subjects and patients with coronary artery disease. The aim of this study was to assess the effects of 3 months of folate supplementation (5 mg/day) on vascular function and structure in RTR. Methods: A double-blind, placebo-controlled crossover study was conducted in 10 CsA-treated RTR. Vascular structure was measured as carotid artery intima media thickness (IMT) and function was assessed as changes in brachial artery diameter during reactive hyperemia (RE) and in response to glyceryl trinitrate (GTN). Function data were analyzed as absolute and percent change from baseline and area under the diameter/time curve. Blood samples were collected before and after supplementation and analyzed for total plasma homocysteine, folate, vitamin B-12 and asymmetric dimethyl arginine (ADMA) in addition to regular measures of hemoglobin, hematocrit, mean corpuscular volume (MCV) and serum creatinine. Results: Folate supplementation significantly increased plasma folate by 687% (p < 0.005) and decreased homocysteine by 37% (p < 0.05) with no changes (p > 0.05) in vitamin B 12 or ADMA. There were no significant (p > 0.05) changes in vascular structure or function during the placebo or the folate supplementation phases; IMT; placebo pre mean +/- SD, 0.52 +/- 0.12, post 0.50 +/- 0.11; folate pre 0.55 +/- 0.17, post 0.49 +/- 10.20 mm 5% change in brachial artery diameter (RH, placebo pre 10 +/- 8, post 6 +/- 5; folate pre 9 +/- 7, post 7 +/- 5; GTN, placebo pre 18 +/- 10, post 17 +/- 9, folate pre 16 +/- 9, post-supplementation 18 +/- 8). Conclusion: Three months of folate supplementation decreases plasma homocysteine but has no effect on endothelial function or carotid artery IMT in RTR.

Physical and chemical factors influencing the germination of Clostridium difficile spores

AIMS: To investigate the influence of chemical and physical factors on the rate and extent of germination of Clostridium difficile spores. METHODS AND RESULTS: Germination of C. difficile spores following exposure to chemical and physical germinants was measured by loss of either heat or ethanol resistance. Sodium taurocholate and chenodeoxycholate initiated germination together with thioglycollate medium at concentrations of 0.1-100 mmol l(-1) and 10-100 mmol l(-1) respectively. Glycine (0.2% w/v) was a co-factor required for germination with sodium taurocholate. There was no significant difference in the rate of germination of C. difficile spores in aerobic and anaerobic conditions (P > 0.05) however, the initial rate of germination was significantly increased at 37 degrees C compared to 20 degrees C (P < 0.05). The optimum pH range for germination was 6.5-7.5, with a decreased rate and extent of germination occurring at pH 5.5 and 8.5. CONCLUSIONS: This study demonstrates that sodium taurocholate and chenodeoxycholate initiate germination of C. difficile spores and is concentration dependant. Temperature and pH influence the rate and extent of germination. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript enhances the knowledge of the factors influencing the germination of C. difficile spores. This may be applied to the development of potential novel strategies for the prevention of C. difficile infection.

Chloride resistance and durability of cement paste and concrete containing metakaolin

Metakaolin (MK), a calcined clay, was included as a partial cement replacement material, at up to 20% by weight of binder, in cement pastes and concrete, and its influence on the resistance to chloride ingress investigated. Reductions in effective chloride diffusion coefficients through hardened cement paste were obtained for binary blends and by combining OPC, MK and a second cement replacement material of pulverised fuel ash or ground granulated blast furnace slag. Steady state oxygen diffusion measurements through hardened cement pastes measured using an electrochemical cell showed that the interaction between charged species and the pore surfaces is a major factor in determining chloride diffusion rate. Rheology of the binder, particularly at high MK replacement levels, was found to have a dramatic influence on the diffusion performance of cement pastes. It was concluded that plasticising admixtures are essential for adequate dispersion of MK in cement pastes. Chloride concentration profile analysis of the concrete cylinders, exposed to sodium chloride solution for one year, was employed to obtain apparent chloride diffusion coefficients for concrete specimens. MK was found to reduce the depth of chloride penetration into concrete when compared with that of unblended mixes. Corrosion rate and corrosion potential measurements were taken on steel bars embedded in concrete exposed to a saline environment under conditions of cyclic wetting and drying. The initiation time for corrosion was found to be significantly longer for MK blended mixes than for plain OPC systems. The aggregate-paste interfacial zone of MK blended systems was investigated by steady state diffusion of chloride ions through mortar containing glass beads as model aggregate. For the model aggregate specimens tested the work confirmed the hypothesis that properties of the bulk paste are the controlling factors in ionic diffusion through mortar.