bluuebunny/crossref_metadata_2025 huggingface dataset:
PCR amplification of a polymorphic minisatellite VNTR locus in whiting [Merlangius merlangus L.)
Authors: McGregor D. , Galvin P. , Sadusky T. , Cross T.
Published: July 2010

An approach has been developed for the screening of allelic variation at minisatellite DNA loci that substantially reduces the time and hazards involved. Primers were designed for a minisatellite region isolated from a gadoid fish species [Merlangius merlangus L.), enabling amplification by polymerase chain reaction, so that differences in the number of minisatellite repeat units (allelic variability) were detectable by ethidium bromide fluorescence (over UV light) following separation by agarose gel electrophoresis. This amplifiable minisatellite variable number tandem repeat region, the first non‐primate marker of its kind, can be used successfully with DNA extracted by a rapid Chelex® protocol. From a sample of 97 individuals, 24 alleles were resolved (750–2200 kb) and heterozygosity was estimated at 0–94.
A chicken linkage map based on microsatellite markers genotyped on a Japanese Large Game and White Leghorn cross
Authors: Takahashi H. , Tsudzuki M. , Sasaki O. , Niikura J. , Inoue‐Murayama M. , Minezawa M.
Published: July 2010

A detailed linkage map is necessary for efficient detection of quantitative trait loci (QTL) in chicken resource populations. In this study, microsatellite markers isolated from a (CA)n‐enriched library (designated as ABR Markers) were mapped using a population developed from a cross between Japanese Game and White Leghorn chickens. In total, 296 markers including 193 ABR, 43 MCW, 31 ADL, 22 LEI, 3 HUJ, 2 GCT, 1 UMA and 1 ROS were mapped by linkage to chicken chromosomes 1–14, 17–21, 23, 24, 26–28 and Z. In addition, five markers were assigned to the map based on the chicken draft genomic sequence, bringing the total number of markers on the map to 301. The resulting linkage map will contribute to QTL mapping in chicken.
Estimation of dispersal distances of the obligately plant‐associated ant Crematogaster decamera
Authors: TÜRKE MANFRED , FIALA BRIGITTE , LINSENMAIR KARL EDUARD , FELDHAAR HEIKE
Published: July 2010

1. In obligate symbioses with horizontal transmission, the population dynamics of the partner organisms are highly interdependent. Host population size limits symbiont number, and distribution of partners is restricted by the presence and thus dispersal abilities of their respective partner. The Crematogaster decamera–Macaranga hypoleuca ant–plant symbiosis is obligate for both partners. Host survival depends on colonisation by its ant partner while foundress queens require hosts for colony establishment.

2. An experimental approach and population genetic analyses were combined to estimate dispersal distances of foundresses in their natural habitat in a Bornean primary rainforest.

3. Colonisation frequency was significantly negatively correlated with distance to potential reproductive colonies. Results were similar for seedlings at natural densities as well as for seedlings brought out in the area experimentally. Population genetic analysis revealed significant population differentiation with an FST of 0.041 among foundresses (n = 157) located at maximum 2280 m apart. In genetic spatial autocorrelation, genotypes of foundresses were significantly more similar than expected at random below 550 m and less similar above 620 m. Direct estimation of dispersal distances by pedigree analysis yielded an average dispersal distance of 468 m (maximum 1103 m).

4. For ants that disperse on the wing, genetic differentiation at such small spatial scales is unusual. The specific nesting requirements of the queens and the necessity for queens to find a host quickly could lead to colonisation of the first suitable seedling encountered, promoting short dispersal distances. Nonetheless, dispersal distances of C. decamera queens may vary with habitat or host spatial distribution.
EPR study of thermally generated free radicals in nuts
Authors: Yordanov Nicola D. , Mladenova Ralitsa
Published: July 2010

EPR studies of free radicals generated in roasting process of peanut, almond, walnut and apricot are reported separately for the kernels and flakes. The raw flakes exhibit a weak singlet EPR signal, whereas raw kernels are EPR silent. Two different EPR signals are recorded in the course of roasting. One in flakes with g = 2.0040, which is independent of temperature and time of roasting and is attributed to C‐centered free radical. The same C‐centered (g = 2.0040) free radical is recorded at high temperature (>140 °C) and for extended time (>20 min) of roasted kernels connected with burning of the material. However, an O‐centered (g = 2.0048) free radical of ‘lipid’ type appears in kernels at roasting temperature of 100–140 °C and short time (5–20 min). On the contrary, free radical concentration in kernels and flakes increases with increase in roasting temperature and decreases with the time after that. The kinetics of the decay of free radicals concentration in flakes and kernels is followed at room temperature under the storage environment (air or argon).
Fusarium DNA traceability along the bread production chain
Authors: Terzi Valeria , Morcia Caterina , Faccioli Primetta , Faccini Nadia , Rossi Vittorio , Cigolini Manuela , Corbellini Maria , Scudellari Diego , Delogu Giovanni
Published: July 2010

Wheat grain contamination with toxigenic Fusarium spp. is of great economic concern to cereal producers and to the grain processing industry and is of great relevance for the quality and safety of the final products. In particular, the bread production chain can potentially be a vehicle for mycotoxin ingestion above the tolerable total daily intake. A quantitative polymerase chain reaction‐based analytical approach has been developed as a possible tool to estimate and control the risk of mycotoxins, particularly deoxynivalenol (DON). This DNA‐based analytical method has been applied to detect and quantify contamination levels of Fusarium graminearum and Fusarium culmorum in naturally infected wheat grain samples. The persistence of Fusarium contamination was also detected along the bread production chain in wholemeal, flour and bread. A significant correlation was found between Fusarium DNA and DON concentration in all samples.
Characteristics of some nutritional composition of melon (Cucumis melo hybrid ‘ChunLi’) seeds
Authors: Mian‐hao Hu , Yansong Ao
Published: July 2010

Seeds from a melon hybrid ‘ChunLi’ were analysed for their proximate composition. The seeds contained high percentages of lipids (35.36%) and proteins (29.90%). Hexane‐extracted oil had acid, peroxide, iodine and saponification values of 1.51, 3.95, 89.5 and 226.73, respectively. Gas chromatographic analysis of the oil revealed the presence of twenty‐five fatty acids varying from C4 to C24 with the exception of C5, C7, C11 and C19. The concentrations of individual fatty acids varied from trace quantities to about 54.8%. Linoleic, oleic, palmitic and stearic acids were the principal fatty acids contributing to 53.9%, 12.1%, 23.9% and 5.7%, respectively, of the total fatty acids which had a relatively high percentage (67.5%) of unsaturated fatty acids. Seed proteins were rich in arginine, aspartic and glutamic acids while limiting amino acids were methionine and lysine.
Canonical correlation: a multivariate technique to determine the contribution of various dependent and independent variables
Authors: Butt Masood S. , Shahzadi Naureen , Sharif Mian K. , Nasir Muhammad
Published: July 2010

Composite flour chapaties were prepared by blending wheat, guar gum, lentil and chickpea flours in different proportions to evaluate their hypoglycaemic and hypocholesterolaemic worth because of their fibre content. Stepwise regression analysis revealed that dietary fibre and intestine length contributed negatively, while peroxide value (POV) contributed positively to the serum cholesterol and glucose. Likewise, for total chapati scores, dough stability and phytic acid showed inverse contribution, while folding ability, dough development time, dietary fibre, POV, crude fibre and texture performed positively. The canonical correlation was applied to develop a model between two groups of variables, where the first group comprised three dependent variables, i.e. total chapati scores, cholesterol and glucose and the second set comprised thirteen independent variables, i.e. crude fibre, POV, phytic acid, dietary fibre, dough development time, dough stability, feed intake, intestine length, low‐density lipoprotein, triglycerides, albumin, texture and folding ability. The selection of independent variables was based on the significant chemical, rheological, sensory and efficacy characteristics in a multiple stepwise regression. The value of coefficient of determination for dietary fibre of the flour samples was observed to be 0.99749 indicating that 99.74% of the variation in dietary fibre can be explained by all other variables in the second set. However, 17.35% and 4.83% of variation in total chapati scores and cholesterol levels, respectively, can be explained by the entire variables in the first group, while 17.20% of variation in glucose can be explained by all other variables of the first group. The squared multiple regression of each variable in the first set with all the variables in the second set indicated that a 99.97% variation in total chapati scores, cholesterol and glucose is explained by the variables in the second set. The F‐values were significant for total chapati scores, cholesterol and glucose. The results substantiated that all the variables in the second set are important to account for the significant variation for the variables tested in the first set. Canonical analysis revealed the existence of a high correlation between dependent variables and other independent variables.
Effect of using interesterified and non‐interesterified corn and palm oil blends on quality and fatty acid composition of Turkish White cheese
Authors: Javidipour Issa , Tunçtürk Yusuf
Published: July 2010

Chemically interesterified blends (CIB) and non‐interesterified blends (NIB) of corn and palm oils (75%w/25%w) were studied in the production of Turkish White cheese (TWC) to modify the fatty acid composition of traditional product. Milk fat (3%) was replaced by CIB and NIB for 25%, 50%, 75% and 100%. All cheese groups were ripened at 5 °C for 90 days. Samples were taken from each group after 3, 30, 60 and 90 days and analysed for their basic composition, lipolysis and proteolysis. CIB‐incorporated cheeses showed a higher degree of lipolysis than the control sample and the NIB‐incorporated counterparts. Fatty acid composition and sensory properties of the final product showed that the incorporation of CIB and NIB in TWC improved the nutritional content of the product because it altered the fatty acid composition without any adverse effect on sensorial quality. We concluded that in production of TWC, 50% of milk fat could be successfully replaced with CIB and NIB, preferably CIB, because of its superior sensory quality.
Prevention of enzymatic browning of apple cylinders using different solutions
Authors: Tortoe Charles , Orchard John , Beezer Anthony
Published: July 2010

Inhibition of enzymatic browning and decay on cut surfaces of Golden Delicious apple using ascorbic acid, cysteine, sodium chloride, calcium chloride, citric acid and sodium ascorbate alone or in combinations was investigated at 4 and 10 °C for a storage period of 0, 7 and 14 days, in an attempt to find the most effective treatment. Apple segments immersed in ascorbic acid and citric acid alone showed visual traces of browning after 7 days storage at 4 °C. After 14 days storage, only ascorbic acid and ascorbic acid plus sodium chloride had moderate browning, while all other treatments were severely affected. However, at 10 °C, only ascorbic acid was effective in reducing the level of browning, although its effect was minimal after 14 days storage. Browning was more severe at 10 °C than 4 °C in all solutions. The browning measurement (a* value) became increasingly positive from 7‐ to 14‐day storage. Microbial decay was absent in all treatments within 7 days at 4 and 10 °C. However, three test solutions showed microbial decay after 14 days storage at 10 °C in addition to the control solution, which showed decay at both 4 and 10 °C storage temperatures.
Anti‐oxidant content of different coloured sweet peppers, white, green, yellow, orange and red (Capsicum annuum L.)
Authors: Matsufuji Hiroshi , Ishikawa Keiko , Nunomura Osamu , Chino Makoto , Takeda Mitsuharu
Published: July 2010

Five different coloured sweet peppers (Capsicum annuum cv. Signal), white, green, yellow, orange and red were analysed for total carotenoids, α‐tocopherol, sugars (glucose, fructose and sucrose), organic acids (citric and ascorbic acids) and anti‐oxidant properties. The mature fruits, ‘Signal Red’, ‘Signal Orange’ and ‘Signal Yellow’ contained higher carotenoids, α‐tocopherol, sugars and organic acids than the immature fruits, ‘Signal Green’ and ‘Signal White’. Among the mature fruits, ‘Signal Red’ was the highest in total carotenoids [9.15 mg (100 g)−1 of fresh weight] while ‘Signal Orange’ was the highest in α‐tocopherol [5.40 mg (100 g)−1 of fresh weight]. ‘Signal Red’ and ‘Signal Orange’ contained the most sugars and organic acids. The suppression of 2,2′‐azobis (2,4‐dimethylvaleronitrile) (AMVN)‐induced oxidation of methyl linoleate by the acetone extracts from the coloured sweet peppers resulted as follows: ‘Signal Red’ > ‘Signal Orange’ ≈ ‘Signal Yellow’ > ‘Signal Green’ ≈ ‘Signal White’. The order of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activities of the acetone extracts was similar to that of suppression of methyl linoleate oxidation.
Characterisation of oat bran products with and without supercritical carbon dioxide extraction
Authors: Stevenson David G. , Eller Fred J. , Radosavljević Milica , Jane Jay‐lin , Inglett George E.
Published: July 2010

Morphology, starch structures and thermal and pasting properties of oat bran concentrate (OBC) and Nutrim‐OB (NOB), a jet‐cooked oat bran product, with or without supercritical carbon dioxide extraction (SCD) were studied. Oil composition was analysed. OBC had three thermal transitions, starch gelatinisation and two amylose–lipid complex transitions. NOB only had amorphous amylose–lipid transition. SCD significantly decreased gelatinisation temperatures of OBC. Pasting properties, showed NOB had significantly higher peak viscosity and breakdown, with significantly lower final viscosity, setback and pasting temperature compared with OBC. SCD increased peak viscosity for NOB and final viscosity for OBC. Thermogravimetric analysis showed NOB had higher water‐holding capacity (WHC) than OBC, while SCD decreased WHC. Amylopectin molecular weight was 3.4 × 108 for OBC and was significantly lower for NOB (1.4 × 108). Predominant fatty acids were oleic (41%), linoleic (38%) and palmitic (16%). Study showed NOB had different properties from OBC thereby expanding industrial applications.
Medication therapy management services in community pharmacy: a pilot programme in HIV specialty pharmacies
Authors: Rosenquist Ashley , Best Brookie M. , Miller Teresa A. , Gilmer Todd P. , Hirsch Jan D.
Published: July 2010

Rationale, aims and objectives  Pharmacist‐provided medication therapy management services (MTMS) have been shown to increase patient's adherence to medications, improve health outcomes and reduce overall medical costs. The purpose of this study was to describe a pilot programme that provided pharmacy‐based MTMS for patients with HIV/AIDS in the state of California, USA.

Method  Pharmacists from the 10 pilot pharmacies were surveyed using an online data collection tool. Information was collected to describe the types of MTMS offered, proportion of patients actively using specific MTMS, pharmacist beliefs regarding effect on patient outcomes and barriers to providing MTMS, ability to offer MTMS without pilot programme funding and specialized pharmacist or staff training.

Results  Each responding pharmacy (7 of 10) varied in the number of HIV/AIDS patients served and prescription volume. All pharmacists had completed HIV/AIDS‐related continuing education programmes, and some had other advanced training. The type of MTMS being offered varied at each pharmacy with ‘individualized counselling by a pharmacist when overuse or underuse was detected’ and ‘refill reminders by telephone’ being actively used by the largest proportion of patients. Most, but not all, pharmacists cited reimbursement as a barrier to MTMS provision. Pharmacists believed the MTMS they provide resulted in improved satisfaction (patient and provider), medication usage, therapeutics response and patient quality of life.

Conclusion  The type of MTMS offered, and proportion of patients actively using, varied among participating pilot pharmacies.

Pharmacists believed MTMS improved medication usage and patient outcomes, although availability of reimbursement to sustain MTMS was a concern. Further study of objective outcomes data is ongoing.
Kinetic Mechanism of the Exchanges Catalysed by the Adenine‐Nucleotide Carrier
Authors: DUYCKAERTS Claire , SLUSE‐GOFFART Claudine M. , FUX Jean‐Pierre , SLUSE Francis E. , LIEBECQ Claude
Published: July 2010

Initial rates of the exchange ADPin/ADPoutcatalysed by the adenine‐nucleotide carrier of rat‐heart mitochondria have been studied under conditions where internal and external ADP may be varied. The initial rate was measured within 1 s by the carboxyatractyloside‐stop method, using a rapid‐mixing technique.

The double‐reciprocal plotsV0−1versus [ADP]out−1at different internal‐ADP concentrations andV0−1versus [ADP]in−1at different external‐ADP concentrations exhibit straight‐line relationships having a common point of intersection on the axis of ordinates.

These results demonstrate the essential role of a ternary complex and thus exclude the ping‐pong mechanism generally accepted. The kinetic equation implies a strong positive cooperativity in the binding of the two substrates.

Two models are proposed: (a) the ternary complex performs the exchange and the transport of the substrates in a single step; (b) the carrier is mobile and transports the substrates one by one, the formation of a ternary complex being needed to release the first product.
Ca2+ Transport by Intact Synaptosomes: the Voltage‐Dependent Ca2+ Channel and a Re‐evaluation of the Role of Sodium/Calcium Exchange
Authors: AKERMAN Karl E. O. , NICHOLLS David G.
Published: July 2010

The verapamil‐sensitive Ca2+ channel in the synaptosomal plasma membrane is investigated. Verapamil is without effect on Ca2+ uptake or steady‐state content in synaptosomes with a polarized plasma membrane, but completely inhibits the additional Ca2+ uptake following plasma‐membrane depolarization by high [K+], by veratridine plus ouabain or by high concentrations of the permeant cation tetraphenylphosphonium. Verapamilinsensitive Ca2+ influx and steady‐state content are identical in polarized and depolarized synaptosomes, even though the Na+ electrochemical potential is greatly decreased in the latter, indicating that Na+/Ca2+ exchange is not a significant mechanism for Ca2+ efflux under these conditions. A transient Na+‐dependent Ca2+ efflux can only be observed on addition of Na+ to Na+‐depleted, depolarized synaptosomes. While 0.2 mM verapamil decreases the rate of 86Rb+ efflux and 22Na+ entry during depolarization induced by veratridine plus ouabain, the final steady‐state Nat accumulation is not inhibited. Ca2+ efflux from synaptosomes following mitochondrial depolarization does not occur by a verapamil‐sensitive pathway.
Competition of Rifampicin with Binding of Substrate and RNA to RNA Polymerase
Authors: KESSLER Christoph , Huaifeng MI , HARTMANN Gsuido R.
Published: July 2010

The rate of formation of dinucleoside tetraphosphate, pppApU, from ATP and UTP by RNA polymerase on the A 1 promoter of the mutant Dl11 of bacteriophage T7 is distinctly and specifically reduced not only by the third template‐directed nucleotide, CTP, but also by CMP. The inhibitory effect of CMP is not changed when the enzyme contains prebound rifampicin. The synthesis of pppApU is also strongly reduced after preincubation of thc enzyme with RNA. This inhibitory effect of RNA is, however, distinctly diminished by rifampicin bound to the enzyme prior to the addition of RNA. On the other hand RNA can suppress the specific binding of the antibiotic to the RNA polymerase subassembly α2β.
One‐Electron Photoreduction of Bacterial Cytochrome P‐450 by Ultraviolet Light
Authors: PIERRE Jacqueline , BAZIN Marc , DEBEY Pascale , SANTUS René
Published: July 2010

Bacterial ferric cytochrome P‐450 can be photoreduced by ultraviolet light in presence of camphor as shown by the formation of the ferrous cytochrome P‐450–CO complex absorbing at 446 nm in C'O‐saturated solutions. This photoreduction can only be performed with wavelengths (λ< 315 nm) corresponding to the absorption of light by aromatic residues and in particular, tryptophan. The primary process leading to the photoreduction is thus the photoionization of tryptophan residue(s) near the active site of the protein as deduced from competitive electron scavenging with N2O. In agreement with results obtained with chemical reduction, the quantum yield of photoreduction is proportional to the content of the high‐spin state of the ferric cytochrome P‐450 solution. It is also shown that the camphor concentration affects the CO binding.
Reconstitution of Liver NADH: Cytochromeb5Oxidoreductase and ofDesulfovibrio vulgarisFlavodoxin with 1‐Carba‐1‐deazaflavin
Authors: POMPON Denis , GUIARD Bernard , LEDERER Florence
Published: July 2010

Flavin‐free cytochromeb5reductase was reconstituted with 1‐deazaflavin and 5‐deazaflavin mononucleotides and dinucleotides. The 5‐deazaenzyme functioned in transhydrogenation reactions but lacked electron transferase activity. The 1‐deazaenzyme was fully competent for both input and output reactions. The flavin reduction rate was lowered about sevenfold upon N‐1 substitution of FAD, but hydrogen abstraction from NADH remained the limiting step. Autoxidation of the reduced enzyme was more rapid than with the normal cofactor. Oxidation was accompanied by appearance of a transient blue‐type semiquinone and superoxide ion production.

Flavin‐free apoflavodoxin was reconstituted with 1‐deaza‐1‐carbaflavin mononucleotide (1‐deaza‐FMN). Its behaviour toward dithionite and oxygen was qualitatively highly similar to that of native flavodoxin.

These observations contrast with the fact that apoflavocytochromeb2could not be reconstituted with 1‐deaza‐FMN [Pompon, D. and Lederer, F. (1979)Eur. J. Biochem. 96, 571–579]. These results, as well as other data from the literature, are discussed in the light of existing hypotheses, which try to correlate flavin protein interactions and flavoprotein function.
Oxene Donors Yield Low‐Level Chemiluminescence with Microsomes and Isolated Cytochrome P‐450
Authors: CADENAS Enrique , SIES Helmut , GRAF Hermann , ULLRICH Volker
Published: July 2010

Microsomes supplemented with different oxene donors yield light emission. The ability of oxene donors to support monooxygenation paralleled the observed chemiluminescence intensities, and the following order was observed: iodosobenzene > cumene hydroperoxide >t‐butyl hydroperoxide > H2O2. Pyridine‐N‐oxide, which was reported to be effective in the bacterial camphor monooxygenase, showed neitherO‐dealkylation of ethoxycoumarin nor chemiluminescence activity.

Iodosobenzene‐induced chemiluminescence was not associated with high malondialdehyde accumulation, at variance witht‐butyl hydroperoxide, the lower light emission intensity of which was largely accompanied by lipid peroxidation, predominantly in a second phase of chemiluminescence. The ratio of maximal malondial‐dehyde formed/maximal chemiluminescence intensity was 30‐fold higher fort‐butyl hydroperoxide than for iodosobenzene.

Low‐level chemiluminescence elicited by iodosobenzene was identified spectrally to be due, at least in part, to singlet oxygen, because of similar photoemission intensities at 634 nm and 703 nm (singlet oxygen dimol emission) and low intensity at 668 nm.

Reducing equivalents to cytochrome P‐450, NADPH or NADH, exerted an inhibitory effect on iodosobenzene‐induced chemiluminescence. Aminopyrine, a substrate for microsomal monooxyganases, inhibited chemiluminescence, probably by favoring hydroxylation.

Oxene donor‐supplemented isolated cytochrome P‐450 yielded low‐level chemiluminescence and the order of efficiency of the oxene donors mirrored that observed with intact microsomes.

The fact that microsomes and ferricytochrome P‐450 supplemented with oxene donors yield chemiluminescence (identified partly as singlet oxygen dimol emission) is discussed in terms of heterolytic versus homolytic cleavage of theO‐Obond of hydroperoxides.
A Comparative Study of Citrate Efflux from Mitochondria of Oleaginous and Non‐oleaginous Yeasts
Authors: EVANS Christopher T. , SCRAGG Alan H. , RATLEDGE Colin
Published: July 2010

Citrate efflux from mitochondria of 10 different yeasts was investigated.

Isolated mitochondria of all the yeasts show the possession of a carrier system for dicarboxylic and tricarboxylic anions, phosphate and pyruvate.

The tricarboxylate carrier is also specific for l‐malate in all 10 yeasts.

In citrate‐loaded mitochondria, citrate efflux and l‐malate uptake are directly proportional to the concentration of extramitochondrial l‐malate until the translocator becomes saturated (> 8 mM l‐malate in Candida curvata D).

Rates of citrate efflux in the presence of l‐malate are linear for a period of 2 min and are approximately 2.5‐times greater in oleaginous than in non‐oleaginous yeasts.

The malate‐citrate translocator has a significantly lower Km for l‐malate in oleaginous yeasts (∼ 4.0 mM) than in non‐oleaginous yeasts (∼ 8.4 mM).

Both pyruvate and phosphate appear to stimulate the malate‐citrate exchange in mitochondria of C. curvata D.

Oleginous yeast mitochondria contain 3—4‐times higher intramitochondrial citrate levels than non‐oleaginous yeast. In all these yeasts the enzymes for synthesis and metabolism of citrate were exclusively mitochondrial, the only exception being the possession of a cytosolic ATP; citrate lyase by oleaginous yeasts.

In metabolically active mitochondria, once the malate‐citrate carrie is saturated with l‐malate, efflux of citrate is directly proportional to the concentration of extramitochondrial pyruvate (as acetyl donor).

The rates of efflux of newly synthesised citrate from metabolically active mitochondria, in the presence of saturating l‐malate and pyruvate, are 6–8‐times greater in oleaginous yeasts and the citric‐acid‐producing yeast.

The inhibition of aconitase by fluorocitrate produced a ninefold increase in citrate efflux from non‐oleaginous yeast mitochondria but only a slight increase in oleaginous yeasts.

It is concluded that in fully functional yeast mitochondria, citrate efflux is limited by the amount of intra‐mitochondrial citrate made available to the citrate translocator for exchange.
Ca2+requirement of prostanoid but not of superoxide production by rat Kupffer cells
Authors: DIETER Peter , SCHULZE‐SPECKING Agnes , DECKER Karl
Published: July 2010

The release of the prostanoids prostaglandin D2(PGD2), prostaglandin E2(PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half‐maximal rate of PGE2release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6–0.7 μM free extracellular Ca2+and ≥ 100 μM free Ca2+was required to stimulate PGE2formation maximally.

The calmodulin antagonist R24571 partially inhibited the release of PGE2elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2irrespective of the stimulus. TMB 8 [3,4,5‐trimethoxybenzoic acid 8‐(diethylamino)‐octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2induced by zymosan and phorbol ester.

The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+‐dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+and mobilization of Ca2+from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.
Human Antioxidant‐Response‐Element‐Mediated Regulation of Type 1 NAD(P)H:quinone Oxidoreductase Gene Expression
Authors: Li Ying , Jaiswal Anil K.
Published: July 2010

Human antioxidant‐response element (hARE) containing two copies of the AP1/AP1‐like elements arranged as inverse repeat is known to mediate basal and β‐naphthoflavone‐induced transcription of the type 1 NAD(P)H:quinone oxidoreductase (NQO1) gene. Band‐shift assays revealed that β‐naphthoflavone increased binding of nuclear proteins at the hARE. Super shift assays identified Jun‐D and c‐Fos proteins in the band‐shift complexes observed with control and β‐naphthoflavone‐treated Hepa‐1 nuclear extracts. Hepa‐1 cells stably transformed with hARE‐tk‐chloramphenicol acetyl transferase (CAT) recombinant plasmid were used to demonstrate that, in addition to β‐naphthoflavone, a variety of antioxidants, tumor promoters and hydrogen peroxide (H2O2) also increased expression of hARE‐mediated CAT gene. β‐naphthoflavone induction of the CAT gene expression in Hepa‐1 cells was found insensitive to inhibitors of protein kinase C and tyrosine kinases. However, binding of regulatory proteins at the hARE and the CAT gene expression in Hepa‐1 cells were increased by dithiothreitol, 2‐mercaptoethanol and diamide. Treatment of the Hepa‐1 cells with N‐ethylmaleimide reduced binding of proteins at the hARE and interfered with expression and β‐naphthoflavone induction of the CAT gene. These results suggested a role of sulfhydryl modification of hARE binding (Jun and Fos) proteins which mediate basal and induced expression of the NQO1 gene. We also report that in‐vitro ‐translated products of the proto‐oncogenes, Jun and Fos, bind to the hARE in band‐shift assays. The incubation of Jun and Fos proteins with small amounts of nuclear extract from dimethylsulfoxide‐treated (control) or β‐naphthoflavone treated Hepa‐1 cells prior to band‐shift assays increased the binding of Jun and Fos proteins to the hARE. Interestingly, the increase in binding of Jun and Fos proteins to the hARE was more prominent with β‐naphthoflavone treated nuclear extract as compared to the control nuclear extract. In addition, incubation of control nuclear extract with β‐naphthoflavone, microsomes and NADPH increased the binding of Jun and Fos proteins to the hARE. Evidence from in vitro studies indicate the presence of unknown nuclear factor(s) that receive signals from metabolites of β‐naphthoflavone and modulate Jun and Fos binding to the AP1 site contained within the hARE.
A Segment Corresponding to Amino Acids Val170‐Arg182 of Bovine Arrestin is Capable of Binding to Phosphorylated Rhodopsin
Authors: Kieselbach Thomas , Irrgang Klaus‐D. , Rüppel Hartmann
Published: July 2010

In retinal rods, photoexcited rhodopsin (R*) is inactivated upon phosphorylation by rhodopsin kinase and the subsequent binding of arrestin. We have studied the structural role of a cationic region of bovine arrestin (Val170‐Arg182) using anti‐peptide IgGs specifically recognizing this segment and the corresponding oligopeptide. Our results clearly indicate that amino acids Val170‐Arg182 are shielded within the arrestin‐rhodopsin‐complex and very likely belong to a binding domain of arrestin for phosphorylated R*. The purified anti‐peptide IgGs strongly reacted with isolated arrestin but did not recognize arrestin when bound to phosphorylated R*. In agreement with these experiments, the oligopeptide Val170–Arg182 was found to compete with arrestin for binding to phosphorylated R*. Increasing concentrations of this peptide caused an oligomerization of phosphorylated rhodopsin when illuminated by white light as well as in the dark. Unphosphorylated rhodopsin did not oligomerize up to a 400‐fold molar ratio of peptide/rhodopsin. Limited proteolysis of the phosphorylated carboxy terminus of rhodopsin with endoproteinase Asp‐N caused a significant decrease in the peptide‐induced formation of oligomers. Therefore, Val170‐Arg182 of bovine arrestin probably interacts with the phosphorylated carboxy‐terminus of rhodopsin. The data presented support the proposal of Palczewski et al. (1991c) considering the region Lys163– Arg182 in bovine arrestin to be a possible binding domain for phosphorylated R*.
Purification of a Processing Enzyme (VmPE‐1) that is Involved in Post‐Translational Processing of a Plant Cysteine Endopeptidase (SH‐EP)
Authors: Okamoto Takashi , Minamikawa Takao
Published: July 2010

A cysteine endopeptidase, designated SH‐EP, occurs in the cotyledons of germinated seeds of Vigna mungo and acts to degrade the seed storage protein in protein storage vacuoles. SH‐EP is synthesized on membrane‐bound ribosomes as an inactive 45‐kDa precursor, which is cotranslationally processed to a 43‐kDa intermediate through cleavage of the signal sequence; the 43‐kDa intermediate of SH‐EP is further processed to the 33‐kDa mature enzyme via 39‐kDa and 36‐kDa intermediates [Mitsuhashi, W. & Minamikawa, T. (1989) Plant Physiol. 89, 274–279]. The present in vitro processing experiments indicated that at least two processing enzymes, designated VmPE‐1 and VmPE‐2 (V. mungo processing enzymes 1 and 2), were involved in post‐translational processing, of SH‐EP in cotyledons of V. mungo seedlings. VmPE‐1 was purified from the cotyledons as a protease that was involved in the processing of the 43‐kDa intermediate to the 36‐kDa intermediate. The enzyme has a molecular mass of 33 kDa as estimated by SDS/polyacrylamide gel electrophoresis, and showed high similarity to the jackbean asparaginyl endopeptidase in terms of the primary structure and substrate specificity. We discuss the function of VmPE‐1 in the processing of SH‐EP and related proteases in the cotyledons of germinated seeds.
Solution Structure of Porcine Pancreatic Procolipase as Determined from 1H Homonuclear Two‐Dimensional and Three‐Dimensional NMR
Authors: Breg Jan N. , Sarda Louis , Cozzone Patrick J. , Rugani Nathalie , Boelens Rolf , Kaptein Robert
Published: July 2010

Procolipase is the precursor of colipase, which acts as protein cofactor for the activity of pancreatic lipase. The solution structure of procolipase has been determined by 1H NMR using two‐ and three‐dimensional measurements. The secondary structure determination identified two separate three‐stranded β‐sheet regions with concomitant hydrogen bond patterns. The tertiary structure of the protein was determined using 863 non‐trivial proton–proton distance constraints, 14 hydrogen bond distance constraints and 55 Ø and 25 χ1 dihedral constraints. The structure that was obtained from distance geometry and energy refinement contains three highly disordered loops as well as a disordered N‐ and C‐terminal region. The remaining part of the structure is well defined with a root‐mean‐square deviation (rmsd) relative to the average of 0.09±0.02 nm for backbone atoms (residues 11–30, 37–50, 57–69, 83–89). The protein comprises two identical domains, each containing a three‐strand β‐sheet and two disulfide bonds: a 15‐residue region in each domain superimposes with 0.07 nm rmsd, measured on backbone atoms. The solution structure is nearly identical to the crystal structure. It is in agreement with previous NMR data and, in combination with these data, supports the current model of procolipase micelle interaction and the lipase activation by colipase.
Two Regions with Differential Growth‐Modulating Activity in the N‐Terminal Domain of Ras GTPase‐Activating Protein (p120GAP)
Authors: Chang Jong‐Soo , Kobayashi Mariko , Wang Dennis Z.‐M. , Maruta Hiroshi , Iwashita Shintaro
Published: July 2010

Ras GTPase‐activating protein of 120 kDa (p120GAP) consists of a hydrophobic Gly‐Ala‐Pro‐rich stretch and src homology 2 and 3 (SH2/SH3) domains in the N‐terminal half, and a Ras GTPase‐activating domain at the C‐terminus. In order to evaluate the potential for cell‐growth regulation of the N‐terminal region of p120GAP, we isolated three distinct clones of rat 3Y1 fibroblast that express either the SH2/SH3 regions alone, the N‐terminal half, or the whole p120GAP. Clones that express the SH2‐SH3‐SH2 regions of 37 kDa (p37SH2/3) at a level of only 15–30% that of endogenous p120GAP, but not clones expressing complete p120GAP or its N‐terminal half of 55 kDa (p55GAP‐N), showed significant growth‐enhancing properties, including a higher saturation density and increased uptake of 2‐deoxyglucose. Clones expressing p37SH2/3 or p55GAP‐N maintained high levels of tyrosine‐phosphorylated p190 and p62, both of which bind the SH2 domain of p120GAP, while clones expressing the whole p120GAP showed no tyrosine phosphorylation of p62. Furthermore, in the presence of a phorbol ester, only the clones expressing p37SH2/3 showed increased tyrosine phosphorylation of p62 and c‐fos expression. These clones also showed the ability of colony formation in soft agar. These results indicate that the N‐terminal domain of p120GAP consists of two regions with differential growth‐enhancing activities and suggest that the transforming potential of SH2/SH3 regions is blocked by the N‐terminal hydrophobic Gly‐Ala‐Pro stretch.
The pH‐Dependent Changes of the Enzymic Activity and Spectroscopic Properties of Iron‐Substituted Manganese Superoxide Dismutase
Authors: Yamakura Fumiyuki , Kobayashi Kazuo , Ue Harumi , Konno Michiko
Published: July 2010

Manganese‐containing superoxide dismutases (Mn‐SODs) and iron‐containing superoxide dismutases (Fe‐SODS) from aerobic bacteria often show high metal specificity for their enzymic activities by a standard assay system using xanthine‐xanthine oxidase and cytochrome c. In this study, we have attempted to characterize the structural basis of the metal specificity of manganese‐containing SOD (Mn‐SOD) using Fe‐substituted Mn‐SOD prepared from apo‐Mn‐SOD from Serratia marcescens. The Fe3+ content of the Fe‐substituted enzyme was 1.71±0.14 mol/mol dimer and the specific activity was 34.8±4.8 units · mg protein‐1· mol Fe3+−1· mol subunit−1. Fe‐substituted Mn‐SOD was found to react with the superoxide anion at pH 8.1 with a second‐order rate constant of 6×106M−1 s−1, which is approximately 1% of that of native Mn‐SOD at the same pH. However, the rate constant increased with decreasing pH to approximately 10% (5×107M−1 s−1) that of native Mn‐SOD at pH 6.0 with a pK of 7.0. The visible absorption spectrum and EPR spectrum of Fe‐substituted Mn‐SOD also showed pH‐dependent changes with pK values of 6.6 and 7.2, respectively. Similarly, the affinity of the azide ion, an analog of the superoxide ion, for iron of Fe‐substituted Mn‐SOD increased with decreasing pH, with a pK value of 7.0 (e.g. Kd =0.1 mM at pH 6.2 and 0.9 mM at pH 8.2). The similarity of these pK values suggests that the activity, the spectral changes and the affinity of the azide ion for iron are derived from the same change in the metal environment. After comparison with the reported pK values (around 9) of similar pH‐dependent changes in the spectra, the enzymic activity and the affinity of azide for iron of Fe‐SOD from Escherichia coli, we proposed that the difference in the pK values of a hydroxide ion binding to iron between Fe‐substituted Mn‐SOD and Fe‐SOD may cause the different pH dependencies of these changes in each SOD.
Reciprocal Regulation of Prothrombin Secretion and Tyrosine Aminotransferase Induction in Hepatocytes
Authors: Yagi Kiyohito , Yamada Chikaomi , Serada Masashi , Sumiyoshi Nobuaki , Michibayashi Nobuyasu , Miura Yoshiharu , Mizoguchi Tadashi
Published: July 2010

Multicellular spheroids of hepatocytes are known to maintain liver functions for a long period. Rat hepatocytes were isolated to form spheroids by rotation culture and immobilized within calcium alginate. Immobilized spheroids had a much higher extent of tyrosine aminotransferase induction, which is one of the liver‐specific differentiated functions, than immobilized non‐aggregated cells, while the spheroids secreted significantly less prothrombin than non‐aggregated cells. Co‐culture of hepatocytes and non‐parenchymal liver cells in a monolayer enhanced tyrosine aminotransferase induction and suppressed prothrombin secretion, while conditioned medium prepared from non‐parenchymal cells greatly stimulated tyrosine aminotransferase induction and suppressed the prothrombin secretion and DNA synthesis in monolayer‐cultured hepatocytes. Prothrombin secretion in hepatocytes was subjected to cell‐density‐dependent regulation. In a similar manner to other growth‐related functions, prothrombin secretion was stimulated at low cell density. It has been reported that thrombin activates the zymogen of hepatocyte growth factor activator [Shimomura, T., Kondo, J., Ochiai, M., Naka, D., Miyazawa, K., Morimoto, Y. & Kitamura, N. (1993) J. Biol. Chem. 268, 22927–229321. Therefore, prothrombin secretion could be one of the growth‐related functions and involved in wound healing and liver regeneration.
Sequence Analysis by NMR Spectroscopy of the Peptide Lantibiotic Epilancin K7 fromStaphylococcus epidermidisK7
Authors: Van De Kamp Mart , Horstink Lennard M. , Van Den Hooven Henno W. , Konings Ruud N. H. , Hilbers Cornelis W. , Frey Angelika , Sahl Hans‐Georg , Metzger Jörg W. , Van De Ven Frank J. M.
Published: July 2010

The amino acid sequence of the novel lantibiotic epilancin K7 fromStaphylococcus epidermidisK7 was determined by NMR spectroscopy. NMR spectroscopy was used because sequencing by conventional Edman degradation techniques was prohibited by internal sequence blocks owing to the presence of modified residues. Epilancin K7 consists of 31 residues, including two α,β‐didehydroalanine (one‐letter code U) and two α, β‐didehydrobutyrine (O) residues, one lanthionine (A‐S‐A), two β‐methyllanthionines (A*‐S‐A), and six lysines. Epilancin K7 has a molecular mass of 3032 ± 1.5 Da. The amino acid sequence of epilancin K7 was derived from both through‐space dipolar proton‐proton interactions and through‐bond scalar proton‐carbon interactions as detected by two‐dimensional1H‐NOESY,1H‐ROESY and three‐dimensional1‐TOCSY‐NOESY, and by two‐dimensional1H,13Cheteronuclear multiple‐bond correlation spectroscopy, respectively. The sequence is as follows:imageThe N‐terminal residue X partly resembles an alanine but its exact nature is unclear. The organization of the sulfide‐bridge‐containing (β‐methyl‐) lanthionines was revealed by1H‐NMR and1H,13C‐NMR spectroscopy. Epilancin K7 has a linear structure and a high positive net charge, and therefore is classified as a type‐A lantibiotic. NMR analysis of a degraded though still active form of epilancin K7 showed that two N‐terminal residues of epilancin K7 were missing, owing to decomposition at the α,β‐didehydro alanine at position 3; it was called the epilancin K7‐(3–31)‐peptide (peptide fragment of epilancin K7 consisting of positions 3–31). The usefulness of three‐dimensional1H‐TOCSY‐NOESY, and two‐dimensional1H,13C‐heteronuclear multiple‐bond correlation spectroscopy at natural abundance for the study of (modified) polypeptides is demonstrated.
Structure/Function Relationships of Mitochondrial Monoamine Oxidase A and B Chimeric Forms
Authors: Gottowik Jürgen , Malherbe Pan , Lang Gabrielle , Prada Mosè Da , Cesura Andrea M.
Published: July 2010

Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH2‐terminus, which contains an ADP‐binding consensus sequence, little is known about their structural features or the sequences involved in the binding of substrates. In the present paper, we have studied the structure/function relationships of MAOs by constructing 18 different chimeric forms of MAO, engineered by moving progressively the junction between the NH2‐terminus of one MAO form with the COOH‐terminus of its isoenzyme. After transient expression in HEK‐293 cells, the properties of these chimeric enzymes were investigated using both selective and nonselective substrates and inhibitors. Whereas exchange of the ADP‐binding sequence did not modify the catalytic properties of either MAO isoforms, chimeras with increasing length of the NH2‐terminus of MAO‐A (up to residue 256) showed a marked decrease in affinity towards the MAO‐B substrate phenylethylamine and the inhibitorN‐(2‐aminoethyl)‐5‐chloro‐2‐pyridine carboxamide · HCl (lazabemide) when compared to wild‐type MAO‐B. No major changes were observed in thekcatvalues of these chimeras. From the data obtained, two sequences, i.e. 62–103 and 146–220, appeared of particular importance in constituting the binding site of MAO‐B. On the other hand, the catalytic properties and specificity of MAO‐A appeared to be relatively insensitive to substitution of both the NH2‐ (up to position 112) and COOH‐termini (from residue 395) of MAO‐A with the corresponding MAO‐B sequences. However, further modification of the central 283‐residue sequence of MAO‐A did not appear compatible with enzymic activity. None of the engineered chimeras showed a shift in specificity from one isoform to the other.
Fibroblast Growth Factors Decrease Inducible Nitric Oxide Synthase mRNA Accumulation in Bovine Retinal Pigmented Epithelial Cells
Authors: Goureau Olivier , Faure Violaine , Courtois Yves
Published: July 2010

Bovine retinal pigmented epithelial cells (RPE cells), after activation with interferon γ (IFNγ) and lipopolysaccharide (LPS), express an inducible nitric oxide (NO) synthase. This activity can be inhibited by the fibroblast growth factors (FGF), FGF‐1 (acidic FGF) and FGF‐2 (basic FGF). We have attempted to elucidate the molecular mechanisms involved in the FGF inhibition of NO synthase activity. Analysis by immunocytochemistry and Western blot using a polyclonal antibody against the inducible NO synthase of rat liver reveals that RPE cells co‐stimulated with FGF‐2 and LPS/IFNγ accumulate lower levels of NO synthase than in the absence of FGF‐2. Northern blot analysis by cross‐species hybridization with a mouse macrophage NO synthase cDNA probe shows that a 4.4–kb mRNA accumulates when RPE cells are activated with LPS/IFNγ. The level of inducible NO synthase mRNA in LPS/IFNγ‐activated RPE cells is markedly reduced by FGF‐1 or FGF‐2 treatment. Message stability studies revealed that the presence of FGF did not accelerate mRNA degradation, implying that FGF did not act on inducible NO synthase mRNA stability, but more probably on its expression. Furthermore an effect of FGF on IFNγ receptors was excluded, since IFNγ binding was not altered by FGF. Since NO acts as a cytostatic compound, FGF, by preventing NO synthase expression in RPE cells, may protect the retina from endotoxin and cytokine‐mediated tissue damage.