bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nicotinamide Riboside, an Unusual, Non‐Typical, Substrate of Purified Purine‐Nucleoside Phosphorylases
Authors: Wielgus‐Kutrowska Beata , Kulikowska Ewa , Wierzchowski Jacek , Bzowska Agnieszka , Shugar David 
Published: July 2004

Nicotinamide 1‐β‐d‐riboside (Nir), the cationic, reducible moiety of the coenzyme NAD+, has been confirmed as an unusual substrate for purified purine‐nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The Km, values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1–2 orders of magnitude higher than for the natural substrate inosine, but the Vmax values were comparable, 96% and 35% that for Ino. The pseudo first‐order rate constants, Vmax/Km, were 1.1% and 2.5% for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP.Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with K1 values close to the Km for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7‐methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with Km values similar to those for inhibition of other natural substrates.The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates.The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme‐inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.
Sequence Specificity of C‐Terminal Src Kinase (Csk)
Authors: Ruzzene Maria , Songyang Zhou , Marin Oriano , Donella‐Deana Arianna , Brunati Anna Maria , Guerra Barbara , Agostinis Patrizia , Cantley Lewis C. , Pinna Lorenzo A. 
Published: July 2004

An eicosapeptide encompassing the C‐terminal tail of c‐Src (Tyr527) which is conserved in most Src‐ related protein kinases, is phosphorylated by C‐terminal Src kinase (CSK) and by the two Src‐related protein kinases c‐Fgr and Lyn, with similar kinetic constants. Two related peptides reproducing the C‐terminal segments of c‐Src mutants defective in CSK phosphorylation [MacAuley, A., Okada, M., Nada, S., Nakagawa, H. & Cooper, J. A. (1993) Oncogene 8, 117–124] AFLEDSCTGTEPLYQRGENL (mutant number 28) and AFLEDNFTGTKPQYHPGENL (mutant number 29), proved a better and a much worse substrates, respectively than the wild‐type peptide, with either CSK or the two Src kinases. By changing individual residues in the best peptide substrate, it was shown that the main element responsible for its improved phosphorylation is leucine at position –1 (instead of glutamine), while lysine at position –3 (instead of glutamate) has a detrimental effect, possibly accounting for the negligible phosphorylation of peptide derived from mutant number 29. By contrast to most peptide substrates, including the Src C‐terminal peptides, which exhibit relatively high Km values, a polyoma‐virus‐middle‐T‐antigen‐(mT)‐derived peptide with tyrosine embedded in a highly hydrophobic sequence (EEEPQFEEIPIYLELLP) exhibits with CSK a quite low Km value (63 μM). Consistent with this, the optimal sequence selected by CSK in an oriented peptide library is XXXIYMFFF. This is different from sequences selected by Lyn (DEEIYEELX) and c‐Fgr (XEEIYGIFF), although they all share a high selection for a hydrophobic residue at n–1. In sharp contrast, TPKIIB/p38Syk, related to the catalytic domain of p72syk, selects acidic residues at nearly all positions, n–1 included.These data support the notion that the features determining the specific phosphorylation of the C‐terminal tyrosine residue of Src do not reside in the primary structure surrounding the target tyrosine. They also show that this site does not entirely fulfil the optimal consensus sequence recognized by CSK, disclosing the possibility that as yet unrecognized CSK targets structurally unrelated to the C‐terminal tyrosine residue of Src kinases may exist.
The Effect of High Pressure on Thermolysin
Authors: Kunugi Shigeru , Kitayaki Moto , Yanagi Yuuichi , Tanaka Naoki , Lange Reinhard , Balny Claude 
Published: July 2004

The effects of high pressure on thermolysin activity and spectroscopic properties were studied. Thermolysin showed distinct pressure‐induced activation with a maximum observed at 200–250 MPa for a dipeptide amide substrate and at 100–120 MPa for a heptapeptide substrate. By examining the pressure dependence of the hydrolytic rate for the former substrate using a high pressure stopped‐flow apparatus as a mixing device under elevated pressures, the activation volume of the reaction was ‐71 ml mol−1 at 25°C. ΔV‡ was accompanied by a negative activation expansibility and a value of ‐95 ml mol−1 was obtained at 45°C. A prolonged incubation of thermolysin under high pressure, however, caused a time‐dependent deactivation.These changes due to pressure were monitored by several spectroscopic methods. The fourth‐derivative absorbance spectrum showed an irreversible change, mostly in the tyrosine and tryptophan regions, at a pressure higher than 300 MPa. Intrinsic fluorescence and circular dichroism measurements of thermolysin in solution also detected irreversible changes. All these measurements indicated that a change occurred at higher pressures and are explained by a simple two‐state transition model accompanied by a large, negative change in the volume of reaction.
Molecular Cloning and Nucleotide Sequence of an Endo‐1,5‐α‐L‐Arabinase Gene from Bacillus Subtilis
Authors: Sakamoto Tatsuji , Yamada Masako , Kawasaki Haruhiko , Sakai Takuo 
Published: July 2004

The nucleotide sequence of the gene encoding an endo‐1,5‐α–arabinase (protopectinase C) of Bacillus subtilis was determined by sequencing fragments amplified by the cassette‐ligation‐mediated PCR (CLM‐PCR). The gene covering the start and stop codon was amplified by PCR with two specific primers, which were designed from the sequence data determined by CLM‐PCR. An approximately 1.5‐kb amplification product was cloned into the vector pUC119, forming a plasmid termed pPPC. An ORF that encodes the arabinase composed of 324 amino acids including a 33‐amino‐acid signal peptide was assigned. Comparison of the deduced amino acid sequence of the enzyme with that of an Aspergillus niger endoarabinase showed 37% identity in a 207‐amino‐acid overlap. The optimal nucleotide sequence for catabolite repression of B. subtilis was found upstream of the structural gene. In a culture of Escherichia coli DH5u cells harboring pPPC, no arabinase activity was detected, either intracellularly or extracellularly, suggesting that the B. subtilis promotor is not functional in this transformant.In B. subtilis IFO 3134 strain, production of protopectinase C was repressed by readily metabolizable carbohydrates. In contrast, productivity (total enzyme activityhacterial growth) of the enzyme was increased about fourfold in the presence of 0.75 M potassium phosphate in the culture medium. The phosphate anion seemed to be involved in the stimulation of protopectinase C production in this stain.
Expression of Natural and Synthetic Genes Encoding Herpes Simplex Virus 1 Protease in Escherichia Coli and Purification of the Protein
Authors: Apeler Heiner , Gottschalk Uwe , Guntermann Diana , Hansen Jutta , Mässen Jutta , Schmidt Elisabeth , Schneider Karl‐Heinz , Schneidereit Marcus , Rübsamen‐Waigmann Helga 
Published: July 2004

An attractive target for anti‐herpes chemotherapy is the herpes simplex virus 1 (HSV‐1) protease encoded by the UL26 gene. Studies with HSV‐1 strains that harbour mutations in the protease gene have demonstrated that the protease is essential for DNA packaging and virus maturation.The UL26 translation product is 635 amino acids long and undergoes autoproteolytic processing between residues Ala247/Ser248 and Ala610/Ser611. The N‐terminal processing product (amino acids 1–247) contains the protease domain.To perform crystallization studies and high throughput screening for potent inhibitors, large amounts of the HSV‐1 protease are required. However, expression of the natural HSV‐1 protease gene in Escherichia coli using a T7‐promoter‐regulated system is low and does not allow for the efficient production of larger amounts of highly purified enzyme. In this report, we describe the use of a synthetic protease gene with optimized E. coli codon usage. The level of protease expression was at least 20 times higher with the synthetic gene as compared to the natural UL26 gene. The HSV‐1 protease was purified to homogeneity in three steps using mixed‐bed ion‐exchange chromatography, affinity chromatography, and hydroxyapatite chromatography.
Memory and Imprinting Effects in Multienzyme Complexes
Authors: Lebreton Sandrine , Gontero Brigitte , Avilan Luisana , Ricard Jacques 
Published: July 2004

Oxidized, free, stable phosphoribuiokinase from Chlamydomonas reinhardtii was almost completely devoid of catalytic activity (0.06 s−1site). However, when it was bound to glyceraldehyde‐3‐phosphate dehydrogenase from the same organism, it displayed significant activity (3.25 s−1/site). Moreover, this complex tended to spontaneously dissociate upon dilution; the isolated phosphoribulokinase activity increased up to 56 s−1site, subsequently decreased, and finally became almost completely inactive. Its intrinsic kinetic properties (Km and kcat) changed with the variation of the overall activity. These effects were paralleled by changes of conformation of the enzyme as revealed by fluorescence analysis. A model is proposed that allows quantitative expression of the dynamics of the dissociation of the oxidized bienzyme complex and the effects of either of the two substrates, ATP and ribulose 5‐phosphate, on this dissociation process. Whereas ATP destabilized the complex and promoted its dissociation, ribulose 5‐phosphate tended to stabilize this complex.Inactive, stable, oxidized phosphoribulokinase may form a complex with glyceraldehyde‐3‐phosphate dehydrogenase regaining its catalytic activity. In this case, glyceraldehyde‐3‐phosphate dehydrogenase acts in a manner similar, but not identical to a chaperonin. The information content of the phosphoribulokinase gene, as defined by the sequence of its base pairs, was therefore not sufficient to specify full enzyme activity. It needed the presence of glyceraldehyde‐3‐phosphate dehydrogenase to give the oxidized phosphoribulokinase a conformation competent for its activity.The potential biological significance of these effects remains to be discovered.
Growth of Differentiated Ovine Tracheal Epithelial Cells In Vitro
Authors: Radi Z. A. , Ackermann M. R. 
Published: July 2004

SummaryCulture of ovine tracheal epithelial cells is a useful tool for conducting various in vitro studies. We describe herein an in vitro technique and the conditions for culturing primary epithelial cells derived from tracheas of adult sheep. Ovine tracheas were surgically removed from 2‐ to 3‐month‐old healthy sheep and tracheal epithelial cells were isolated by 0.15% pronase digestion. After epithelial cells isolation, a Millicell insert with porous membrane was coated with 0.05% human placental collagen and the epithelial cells were added to the membrane. To create an air–liquid interface environment for the cells, the apical compartment of the membrane containing the tracheal epithelial cells was left exposed to 5% CO2 at 37°C for 2 days then increased to 9% CO2 while cells in the basolateral compartment underneath the membrane contained the growth medium necessary for cells nourishment. Pepsin digestion was more effective in reducing the number of fibroblasts than other procedures. Cells were allowed to grow for 6–7 days to form a confluent monolayer and nearly 21 days for cilia formation on the apical surface as determined by light microscopy of haematoxylin and eosin‐stained sections of membranes. In order to further confirm the epithelial origin of cells, cells were stained for cytokeratin antigen by immunohistochemistry. Most ciliated epithelial cells were immunoreactive for cytokeratin. This is the first report of differentiated ovine tracheal epithelial cells growth and isolation. This technique can be used in numerous in vitro investigative studies in ovine species as an animal model for human disease.
The Effect of Parentage on the Prevalence, Severity and Location of Lesions of Osteochondrosis in Swine
Authors: Ytrehus B. , Grindflek E. , Teige J. , Stubsjøen E. , Grøndalen T. , Carlson C. S. , Ekman S. 
Published: July 2004

SummaryThe aims of this study were to determine the effects of parentage and gender on the prevalence, severity and location of lesions of osteochondrosis manifesta (OCM) and osteochondrosis dissecans (OCD) in offspring from different Norwegian Landrace boars and to examine the relationships between lesion characteristics and selected growth parameters. Fifteen sires were selected based on their high breeding value for osteochondrosis. Seven locations in the distal humerus and the distal femur from 1680 offspring of these animals were evaluated for severity of OCM and presence of OCD by gross examination of serially sectioned humeri and femora. Osteochondrosis manifesta was most prevalent in the trochlea of humerus, the sagittal ridge of humerus, the medial condyle of femur and the medial sulcus obliquus of femur. The severity of the lesions and the prevalence of OCD were highest in the trochlea and the sagittal ridge of humerus. Castrates had significantly higher OCM scores than sows. There were significant effects of both sire and dam on the OCM scores of the offspring in most locations; however, growth rate and weight at slaughter did not influence the OCM score.
Evaluation of Stratification Factors and Score‐scales in Clinical Trials of Treatment of Clinical Mastitis in Dairy Cows
Authors: Hektoen L. , Ødegaard S. A. , Løken T. , Larsen S. 
Published: July 2004

SummaryThere is often a need to reduce sample size in clinical trials due to practical limitations and ethical considerations. Better comparability between treatment groups by use of stratification in the design, and use of continuous outcome variables in the evaluation of treatment results, are two methods that can be used in order to achieve this. In this paper the choice of stratification factors in trials of clinical mastitis in dairy cows is investigated, and two score‐scales for evaluation of clinical mastitis are introduced. The outcome in 57 dairy cows suffering from clinical mastitis and included in a clinical trial comparing homeopathic treatment, placebo and a standard antibiotic treatment is investigated. The strata of various stratification factors are compared across treatments to determine which other factors influence outcome. The two score scales, measuring acute and chronic mastitis symptoms, respectively, are evaluated on their ability to differentiate between patients classified from clinical criteria as responders or non‐responders to treatment. Differences were found between the strata of the factors severity of mastitis, lactation number, previous mastitis this lactation and bacteriological findings. These factors influence outcome of treatment and appear relevant as stratification factors in mastitis trials. Both score scales differentiated between responders and non‐responders to treatment and were found useful for evaluation of mastitis and mastitis treatment.
Effect of Vitamin E Supplementation on Various Functional Properties of Macrophages and Neutrophils Obtained from Weaned Piglets
Authors: Fragou S. , Fegeros K. , Xylouri E. , Baldi A. , Politis I. 
Published: July 2004

SummarySixteen piglets were used to determine the effect of vitamin E supplementation on several functional properties of macrophages and neutrophils obtained from weaned piglets. Piglets, immediately following weaning, were assigned to one of three experimental groups: control (no vitamin E supplementation), low level of vitamin E supplementation (100 mgdl‐α‐tocopheryl acetate/kg diet) and high level of vitamin E supplementation (300 mgdl‐α‐tocopheryl acetate/kg diet). Supplementation of vitamin E lasted for a period of 36 days, following a 3‐day adaptation period after weaning. Blood samples were collected on days 0, 12, 24 and 36 of the experimental period, monocytes/macrophages and neutrophils were isolated and the following parameters were determined in macrophages and neutrophils activated by phorbol myristate acetate: total cell‐associated and membrane‐bound urokinase plasminogen activator (u‐PA) activity and superoxide anion production. Results showed that macrophages and neutrophils isolated from piglets that received supplemental vitamin E had higher (P < 0.05) total and membrane‐bound u‐PA activities as well as higher (P < 0.05) superoxide anion production compared with the values of the corresponding cells obtained from control piglets on day 12 of the experimental period. Both levels of vitamin E supplementation (low and high) were equally effective. In contrast, vitamin E supplementation had no effect (P > 0.05) on total and membrane‐bound u‐PA activities and superoxide anion production by porcine macrophages and neutrophils on days 24 and 36 of the experimental period. In conclusion, the low level of vitamin E supplementation is recommended for piglets for the first 2 weeks after weaning.
Comparison of the Effects of Fructose and Glucose Supplementation on Metabolic Responses in Resting and Exercising Horses
Authors: Vervuert I. , Coenen M. , Bichmann M. 
Published: July 2004

SummaryThe aim of this study was to compare the effects of two different carbohydrate sources (fructose and glucose) on the metabolic responses in resting and exercising horses. The following regimes were fed in randomized order to five trained horses at rest and immediately before or during exercise. The resting regime comprised 0.6 kg grass meal pellets (control) or 0.6 kg grass meal pellets supplemented with either 50% glucose or 50% fructose. The exercise regime comprised 0.3 kg grass meal (control) or 0.6 kg grass meal pellets supplemented with either 50% glucose or 50% fructose fed immediately before or during simulated endurance exercise on a treadmill (30 km, total running time 120 min; 15 min rest after 60 min running time). Blood samples were collected for the analysis of glucose, insulin, free fatty acids (FFA) and lactate. In resting horses, glucose supplementation resulted in a significantly higher glycaemic and insulinaemic response than the control or fructose feedings (treatmentP < 0.05). Plasma glucose levels were significantly higher after glucose supplementation given before or during exercise. Similar plasma glucose concentrations were observed after fructose was fed before exercise, whereas fructose supplementation during exercise resulted in a lower glycaemic response than glucose feeding (P < 0.05). Plasma insulin, FFA and lactate levels showed exercise‐related changes (timeP < 0.05), but treatment did not effect these results. Plasma glucose concentrations were elevated during the simulated endurance exercise after glucose feeding, and no counter‐regulation by insulin occurred. The higher glycaemic response might be beneficial as exogenous glucose can be used as an energetic substrate during prolonged exercise. Fructose exhibited no clear advantages in comparison with glucose as a carbohydrate source for exercising horses.
Reduced ocular dominance plasticity and long‐term potentiation in the developing visual cortex of protein kinase A RIIα mutant mice
Authors: Rao Yan , Fischer Quentin S. , Yang Yupeng , McKnight G. Stanley , LaRue Adrienne , Daw Nigel W. 
Published: July 2004

AbstractThe cAMP‐dependent protein kinase (PKA) signalling pathway has been shown to play an important role in long‐term potentiation (LTP) and depression (LTD), and ocular dominance plasticity in the visual cortex. In order to investigate further the involvement of individual PKA subunits in visual cortical plasticity, LTP and LTD in vitro and ocular dominance plasticity in vivo in the developing visual cortex were examined in mice lacking the RIIα subunit of PKA. Here we show that LTP in layers II/III was decreased in RIIα knockout mice, but LTD was almost unaffected, and the ocular dominance shift induced by monocular deprivation was also partially blocked. These data provide evidence that RIIα is involved in LTP and ocular dominance plasticity, and further suggest that different afferent inputs could selectively activate particular subunits of PKA and thereby direct specific aspects of visual cortical plasticity.
Blockade of presynaptic adenosine A1 receptor responses by nitric oxide and superoxide in rat hippocampus
Authors: Shahraki Ali , Stone Trevor W. 
Published: July 2004

AbstractActivation ofN‐methyl‐d‐aspartate (NMDA) receptors prevents the neuronal responses to adenosine in hippocampal slices. As NMDA receptor activation leads to the generation of nitric oxide (NO) and superoxide, we have examined whether these can modify neuronal responses to adenosine and mediate the actions of NMDA. Field excitatory postsynaptic potentials were recorded in the CA1 region of rat hippocampal slices. Paired‐pulse interactions were studied to localize the observed interactions to presynaptic terminals. The NO donorsS‐nitroso‐N‐acetylpenicillamine and diethylamine NONOate induced a long‐lasting potentiation (NO‐induced potentiation) of field excitatory postsynaptic potential slope and significantly prevented the presynaptic inhibitory effect of adenosine or the A1 receptor agonist N6‐cyclopentyladenosine selectively with no effect on responses to baclofen. The superoxide‐generating system of xanthine/xanthine oxidase also prevented presynaptic responses to adenosine and this effect was prevented by superoxide dismutase (SOD). The guanylate cyclase inhibitor 1H‐[1,2,4]‐oxadiazolo[4,3a]quinoxalin‐1‐one (10 µm) prevented NO‐induced potentiation and the inhibitory effects ofS‐nitroso‐N‐acetylpenicillamine and xanthine/xanthine oxidase on adenosine responses. The inhibitory effect of NMDA on adenosine responses was unchanged by 1H‐[1,2,4]‐oxadiazolo[4,3a]quinoxalin‐1‐one, indicating that guanosine‐3′,5‐cyclic monophosphate does not mediate this interaction, although it was partially reduced by SOD, suggesting that superoxide might contribute. The reduction of adenosine responses by electrically‐induced long‐term potentiation was prevented by NO synthase inhibition or SOD. The results indicate that the presynaptic effects of adenosine at presynaptic sites can be prevented by NO or superoxide but that neither of these individually can fully account for the prevention of adenosine responses by NMDA.