bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Cytochrome‐b5‐Containing Fusion Protein Similar to Plant Acyl Lipid Desaturases
Authors: Sperling Petra , Schmidt Hermann , Heinz Ernst 
Published: March 2005

The similarity between oleate and linoleate desaturase sequences from several plants was used lo construct degenerate oligonucleotide primers for PCR experiments with cDNA transcribed from mRNA of ripening sunflower embryos. A DNA fragment was amplified and sequenced. Specific primers derived from this partial sequence were used for rapid amplification of the 3′‐ and 5′‐ends of this cDNA. With appropriate primers derived from these sequences, a full‐length clone of 1377 bp was amplified by PCR which, after sequencing, showed an open reading frame of 458 amino acids corresponding to a putative protein of about 52 kDa.Comparison with other desaturases showed the conserved three histidine boxes and the characteristic hydropathy profile of membrane‐bound desaturases, but the amino acid identity was restricted to 18 % and the N‐terminal region was about 100 amino acids longer. This N‐terminal extension showed high similarity with cytochrome b5, and, accordingly, the whole sequence can be considered as coding for a fusion protein between cytochrome b5 and a desaturase‐like enzyme. Furthermore, we detected a similar cytochrome b5 fold in the previously sequenced Δ9 acyl‐CoA desaturase from yeast, but in this enzyme it was located at the C‐terminus. An alignment of these fusion proteins with other heme‐binding proteins revealed desaturases to be novel members of the cytochrome b5, superfamily. A truncated DNA representing 366 bp of the 5′‐end was amplified from the cDNA clone and expressed in Escherichia coli. The truncated cDNA coded for a soluble protein of about 12 kDa as shown by SDS/PAGE and N‐terminal sequencing. The enriched recombinant protein exhibited redox absorbance spectra characteristic of plant microsomal cytochrome b5
Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na+/K+‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin
Authors: Antolovic Roberto , Linder Dietmar , Hahnen Josef , Schoner Wilhelm 
Published: March 2005

Na+/K+‐ATPase from pig kidney is inactivated by protein‐reactive N‐hydroxysuccinimidyl derivatives of digoxigenin. Like digoxigenin, its protein‐reactive derivatives N‐hydroxysuccinimidyl digoxigenin‐3‐methylcarbonyl‐ɛ‐aminocaproate (HDMA), 3‐amino‐3‐deoxydigoxigenin hemisuccinimide succinimidyl ester (ADHS), 3‐iodoacetylamino‐3‐deoxydigoxigenin (IAD) and digoxigenin‐3‐O‐succinyl‐[2‐(N‐maleimido)]ethylamide (DSME) inhibited the sodium pump in the presence of Na+, Mg2+ and ATP. At 37°C, half‐maximal inhibition of Na+/K+‐ATPase was seen by HDMA at 0.47 μM, by ADHS at 5.8 μM, by IAD at 8 μM and by DSME at 94 μM. Thus, all compounds bind to the cardiac steroid receptor site of Na+/K+‐ATPase. Affinity labeling of the α subunit by ‘front door’ or ‘back door’ phosphorylation was only seen with HDMA or ADHS in the range 0.1 μM. Excess of ouabain protected against affinity labeling. All the other protein‐reactive derivatives of digoxigenin labeled the enzyme independent of the formation of a phosphointermediate at much higher concentrations. This labeling was not suppressed by an excess of ouabain.Tryptic hydrolysis of the HDMA‐modified Na+/K+‐ATPase gave peptides of the apparent molecular masses 20, 12.5 and 11.2 kDa. The 11.2‐kDa and 12.5‐kDa peptides started amino‐terminally with Asp68, and the 20‐kDa peptide with Asp24. Thus, the HDMA‐labeled peptides originate from the cardioactive steroid‐binding site formed by the first and second transmembrane helix. N‐Hydroxysuccinimidyl esters such as HDMA are normally thought to modify lysine and arginine residues covalently. Since such residues do not exist in the putative cardiac glycoside‐binding site, the possibility of a thioester formation of the digoxigenin derivatives HDMA and ADHS with Cys104 in the H1 transmembrane domain was tested. In fact, hydroxylaminolysis led to the release of the covalently bound HDMA, and the formation of a free sulfhydryl group. This could be labeled by [2‐14C]ICH2COOH. We therefore propose, consistent with a recent conclusion from a site‐directed mutagenesis experiment [Canessa, C. M., Horisberger, J.‐D., Louvard, D. & Rossier, B. C. (1992) EMBO J. 11, 1681–1687], that a cysteine residue (probably Cys104) participates in the structure and function of the cardiac glycoside binding.
Kinetic Properties and Ligand Binding of the Eleven‐subunit Cytochrome‐c Oxidase from Saccharomyces cerevisiae Isolated with a Novel Large‐Scale Purification Method
Authors: Geier Birgitta M. , Schägger Hermann , Ortwein Claus , Link Thomas A. , Hagen Wilfred R. , Brandt Ulrich , Von Jagow Gebhard 
Published: March 2005

A novel, large‐scale method for the purification of cytochrome‐c oxidase from the yeast Saccharomyces cerevisiae is described. The isolation procedure gave highly pure and active enzyme at high yields. The purified enzyme exhibited a heme a/protein ratio of 9.1 nmol/mg and revealed twelve protein bands after Tricine/SDS/PAGE. N‐terminal sequencing showed that eleven of the corresponding proteins were identical to those recently described by Taanman and Capaldi [Taanman, J.‐W. & Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481–2248]. 15 of the N‐terminal residues of the 12th band were identical to subunit VIII indicating that this band represents a dimer of subunit VIII (Mr 5364). We conclude that subunit XII postulated by Taanman and Capaldi is the subunit VIII dimer and that cytochrome‐c oxidase contains eleven rather than twelve subunits.We obtained the complete sequence of subunit VIa by Edman degradation. The protein contains more than 25% of charged amino acids and hydropathy analysis predicts one membrane‐spanning helix.The purified enzyme had a turnover number of 1500 s−1 and the ionic‐strength dependence of the Km value for cytochrome‐c was similar to that described for other preparations of cytochrome‐c oxidase. This was also true for the cyanide‐binding characteristics of the preparation. When the enzyme was isolated in the presence of chloride, more than 90% of the preparation showed fast cyanide‐binding kinetics and was resistant to formate incubation, indicating that chloride was bound to the binuclear center. When the enzyme was isolated in the absence of chloride, approximately 70% of the preparation was in the fast form. This high content of fast enzyme was also reflected in the characteristics of optical and EPR spectra for cytochrome‐c oxidase purified with our method.
Evolution of Transthyretin in Marsupials
Authors: Duan Wei , Richardson Samantha J. , Babon Jeffrey J. , Heyes Rebecca J. , Southwell Bridget R. , Harms Paul J. , Wettenhall Richard E. H. , Dziegielewska Katarzyna M. , Selwood Lynne , Bradley Adrian J. , Brack Charlotte M. , Schreiber Gerhard 
Published: March 2005

The evolution of the expression and the structure of the gene for transthyretin, a thyroxine‐binding plasma protein formerly called prealbumin, was studied in three marsupial species: the South American polyprotodont Monodelphis domestica, the Australian polyprotodont Sminthopsis macroura and the Australian diprotodont Petaurus breviceps. The transthyretin gene was found to be expressed in the choroid plexus of all three species. In liver it was expressed in P. breviceps and in M. domestica, but not in S. macroura. This, together with previous studies [Richardson, S. J., Bradley, A. J., Duan, W., Wettenhall, R. E. H., Harms, P. J., Babon, J. J., Southwell, B. R., Nicol, S., Donnellan, S. C. & Schreiber, G. (1994) Am. J. Physiol. 266, R1359–R1370], suggests the independent evolution of transthyretin synthesis in the liver of the American Polyprotodonta and the Australian Diprotodonta.The results obtained from cloning and sequencing of the cDNA for transthyretin from the three species suggested that, in the evolution of the structure of transthyretin in vertebrates, marsupial transthyretin structures are intermediate between bird/reptile and eutherian transthyretin structures. In marsupials, as in birds and reptiles, a hydrophobic tripeptide beginning with valine and ending with histidine was found in transthyretin at a position which has been identified in eutherians as the border between exon 1 and intron 1. In humans, rats and mice, the nine nucleotides, coding for this tripeptide in marsupials/reptiles/birds, are found at the 5′ end of intron 1. They are no longer present in mature transthyretin mRNA. This results in a change in character of the N‐termini of the subunits of transthyretin from hydrophobic to hydrophilic. This change might affect the accessibility of the thyroxine‐binding site in the central channel of transthyretin, since, at least in humans, the N‐termini of the subunits of transthyretin are located in the vicinity of the channel entrance [Hamilton, J. A., Steinrauf, L. K., Braden, B. C., Liepnieks, J., Benson, M. D., Holmgren, G., Sandgren, O. & Steen, L. (1993) J. Biol. Chem. 268, 2416–24241.
Novel Forms of B‐Domain‐Deleted Recombinant Factor VIII Molecules
Authors: Lind Peter , Larsson Kerstin , Spira Jack , Sydow‐Báckman Mona , Almstedt Annelie , Gray Eva , Sandberg Helena 
Published: March 2005

Recombinant molecules similar to the smallest active plasma‐derived factor VIII molecule, a complex of an 80‐kDa and a 90‐kDa polypeptide chain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80+90‐kDa complex. Three types of single‐chain cDNAs encoding B‐domain‐deleted derivatives factor VIII were designed, taking account of sites at Arg740 and Glut 649, assumed to be important for processing factor VIII.In the type 1 constructs, either Arg747, Arg752, or Arg776 in the N‐terminal region of factor VIII B domain was fused to the N‐terminus (Glu1649) of the 80‐kDa subunit. In the type 2 construct r‐VIII SQ, Ser743 was fused to Gln1638, creating a link of 14 amino acids between the C‐terminus (Arg740) of the 90‐kDa chain and N‐terminus of the 80‐kDa chain, whereas in type 2 r‐VIII RH, Arg747 was fused to His1646. In the type 3 constructs, the B‐domain was completely removed or replaced with 1–4 Arg residues.After expression in Chinese hamster ovary cells, the type 1 derivatives and the type 3 derivatives with 0–2 Arg residues inserted were found to be only partially processed and contained a large amount of the 170‐kDa primary translation product. In contrast, most of the type 2 derivatives r‐VIII SQ and r‐VIII RH and the type 3 derivatives r‐VIII R4 and r‐VIII R5 containing three or four extra Arg residues preceding the N‐terminus of the 80‐kDa chain were processed into the desired 80+90‐kDa chain complexes. The feature common to the most efficiently processed factor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane‐bound subtilisin‐like protease furin, which is expressed in most types of cells; that is, basic amino acid residues at positions –1 and –4 relative to the cleavage site at Glu1649. Biochemical studies of r‐VIII SQ and r‐VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N‐ and C‐termini of the 80‐kDa and 90‐kDa chains corresponding to those found in plasma‐derived factor VIII.
The Detection of an Unidentified Type of Adenovirus in the Stools of Calves with Weak Calf Syndrome by Use of a Commercial Kit Designed for the Detection of Human Adenoviruses
Authors: Brenner J. , Elad D. , Bernstein M. , Dagoni I. , Pálfi V. , Yadin H. 
Published: March 2005

SummaryAn outbreak of polyarthritis in newborn calves in a large collective dairy herd was characterized by intra‐articular blood‐tinged synoviae, blood tainted faeces and massive subcorneal haemorrhages. Faecal samples from eight clinical newborn cases, 10 from unrelated dairy farms and 10 faecal samples from healthy calves were examined by the Rida® Quick rotavirus/adenovirus‐combi test . A specific adenovirus antigen precipitin‐line was seen in the reaction in all the faecal samples from the diseased calves (n = 8), while all the others (n = 20) were negative. In addition, the same positive reaction was noted when one aqueous humor and two synovial samples were tested with this kit. Several other enteropathogens were found sporadically, but no conclusive significance could be attributed to their presence. Bovine viral diarrhoea and infectious bovine rhinothracheitis viruses as well as Chlamydia spp. and Mycoplasma spp. were not involved in this episode.
Shortening of the Bovine Tongue According to Regulation (EC) 999/2001 is not Complying with the Current Legal Definition of Specified Risk Material – a Macroscopical and Histological Preliminary Study
Authors: Kühne M. , Klein G. , Gasse H. 
Published: March 2005

SummaryThe full elimination of all specified risk material (SRM) in food of animal origin is crucial for consumer protection and is of high priority in inner EU trade. Among other tissues, the tonsils of cattle are considered as SRM. The aim of this study was to evaluate whether the ‘cut at the back of the tongue just before the tongue bones’ required by EC regulation is sufficient to remove tonsils and lymphatic tissue completely. Eight skulls from cattle were collected for the simulation of a vertical cut according to the EC regulation and the detection of the target at the back of the tongue. Further, specimens of the lingual mucosa were cut out from two tongues and examined microscopically. The most caudal of these specimens was from the macroscopically visible part of the lingual tonsil. The most rostral specimen contained the most caudal Papilla vallata. Simulation of the obligatory ventro‐dorsal cut yielded hits at varying locations on the dorsal surface of the tongue, sometimes including tissue of the lingual tonsil. Histological examination of the lingual mucosa gave clear evidence that lymphatic tissue resembling the tissue of a tonsil in terms of its histological organization and infiltration of the mucosal epithelium could even be found in areas with no macroscopically visible lingual tonsils.
Effect of dietary docosahexaenoic acid on the endothelium‐dependent vasorelaxation in diabetic rats
Authors: Goirand Françoise , Ovide‐Bordeaux Stéphanie , Renaud Jean‐François , Grynberg Alain , Lacour Bernard 
Published: March 2005

SUMMARY1. The aim of the present study was to investigate the responses to acetylcholine (ACh; 3 nmol/L−30 µmol/L) and sodium nitroprusside (SNP; 3 nmol/L−30 µmol/L) of precontracted aortic rings from diabetic rats supplemented with docosahexaenoic acid (DHA).2. Diabetes was induced by streptozotocin (STZ; 55 mg/kg). Diabetic and sham rats were fed, over a period of 8 weeks, either control diet or a DHA‐supplemented diet. Aortic endothelial fatty acid composition was analysed by gas chromatography. The involvement of endothelial‐derived nitric oxide (NO) and cyclo‐oxygenase (COX) metabolites in response to ACh was assessed using the NO synthase inhibitor NG‐nitro‐l‐arginine methyl ester (100 µmol/L) and the COX inhibitor indomethacin (1 µmol/L), respectively.3. The DHA‐supplemented diet induced a small increase in n‐3 polyunsaturated fatty acids (PUFA; P < 0.001) owing to the incorporation of DHA in the endothelial cells of sham animals (1.6 ± 0.2% in the DHA group compared with traces in the control group; P < 0.001) and diabetic animals (1.3 ± 0.2% in the DHA group compared with traces in control group; P < 0.001), without a decrease in n‐6 PUFA, despite a small decrease in arachidonic acid content (P < 0.05). Diabetes did not modify the incorporation of DHA in endothelial cells, but did significantly increase the arachidonic acid content (0.6 ± 0.0 vs 0.4 ± 0.1% in control group in the STZ and sham groups, respectively; P < 0.001). Acetylcholine‐induced relaxation was significantly reduced in STZ groups compared with the sham groups (P < 0.001) and the DHA‐supplemented diet did not modify these effects. In contrast, neither the DHA‐supplemented diet nor diabetes affected the aortic relaxation induced by SNP. NG‐Nitro‐l‐arginine methyl ester strongly inhibited the relaxant effects of ACh in the sham groups (P < 0.001) and abolished ACh‐induced relaxation in the STZ groups (P < 0.001). The diet did not modify these effects. In the presence of indomethacin, the relaxation induced by ACh was decreased in the sham groups (P < 0.01), but not in the STZ groups. The DHA‐supplemented diet did not have any effect on these responses.4. In conclusion, these results suggest that, in the present study, the endothelial dysfunction occurring in the rat model of STZ‐induced diabetes is associated with modifications of both the synthesis of COX derivatives and NO metabolism and is not affected by dietary supplementation with DHA.
The characterization of the semi‐solid W/O/W emulsions with low concentrations of the primary polymeric emulsifier
Authors: Vasiljevic D. , Vuleta G. , Primorac M. 
Published: March 2005

SynopsisSemi‐solid multiple W/O/W emulsions with low concentrations (0.8, 1.6 and 2.4% w/w) of lipophilic polymeric primary emulsifier PEG‐30‐dipolyhydroxystearate (PDHS) have been formulated. Both emulsions, primary and multiple, were prepared with high content of inner phase (Φ1 = Φ2 = 0.8). All the formulations differ only in the lipophilic emulsifier concentration. Evaluating several parameters such as macroscopic and microscopic aspect, droplet size, accelerated stability under centrifugation and flow and oscillatory rheological behaviour, assessed the multiple systems. It is possible to formulate the semi‐solid W/O/W multiple emulsions with low concentrations of PDHS as the primary emulsifier. It appeared that the highest long‐term stable multiple emulsion with the lowest droplet size, the highest apparent viscosity and highest elastic characteristic, was the sample with the highest concentration (2.4% w/w) of the primary emulsifier.
Methotrexate for psoriasis: current European practice. A postal survey
Authors: Boffa MJ 
Published: March 2005

ABSTRACTThe aim of this survey was to determine current practice throughout Europe regarding use and monitoring of methotrexate therapy for psoriasis. A structured questionnaire with questions on methotrexate prescribing and monitoring was mailed to 150 dermatologists in 32 European countries in June 2002. Dermatologists’ names were chosen at random from the 2001 European Academy of Dermatology and Venereology membership directory. A reply was received from 69 dermatologists of whom 59 prescribed methotrexate regularly. In those patients receiving systemic treatment for psoriasis, methotrexate was the most widely used drug (4.4 patients of every 10) followed by acitretin (3.2) and cyclosporin (1.6). Myelosuppression was the commonest reported fatal side‐effect of methotrexate (eight of a total of 10 cases). None of the respondents routinely requested a baseline liver biopsy before starting methotrexate treatment and of every 10 of their patients on long‐term methotrexate it was estimated that less than two had had a liver biopsy some time during treatment. Serum measurement of the amino‐terminal peptide of type III procollagen (PIIINP) was used routinely to detect liver fibrosis by 12 (20%) of the 59 respondents who regularly prescribed methotrexate. This survey demonstrates that, despite the advent of new therapies, methotrexate retains a central role in the treatment of severe psoriasis in Europe. The responses demonstrated variation in, among others, the methotrexate dose regimen utilized and the use of folate supplementation, confirming the need for randomized controlled studies to address these issues.
Eosinophilic cellulitis of papulonodular presentation (Wells’ syndrome)
Authors: Ghislain P‐D , Van Eeckhout P 
Published: March 2005

AbstractEosinophilic cellulitis (Wells’ syndrome) is a rare condition of unknown aetiopathogenesis and is characterized by erythemal plaques and a histological picture of eosinophilic infiltration of the dermis with ‘flame’ figures. Here we describe a patient with the papulonodular variant of eosinophilic cellulitis associated with an unusual hepatic dysfunction.
Intravenous infusion for the treatment of diabetic and ischaemic non‐healing pedal ulcers
Authors: Agarwal P , Agrawal PK , Sharma D , Baghel KD 
Published: March 2005

ABSTRACTDiabetic and ischaemic non‐healing pedal ulcers have a tendency for chronicity and increased chances of infection, which may threaten the viability of the foot. Systemic administration of therapeutic agents may be insufficient in these cases. We have assessed the role of retrograde venous perfusion (RVP) for the treatment of nine diabetic and 10 ischaemic non‐healing pedal ulcers. Agents used were soda bicarbonate, heparin, lignocaine, gentamicin and pentoxiphylline. Five of nine diabetic non‐healing ulcers showed complete healing and the remaining four improved. The complete recovery in the cases of diabetic ulcer occurred in 10–24 days (mean 16 days), while ischaemic ulcers took 10–14 days for complete recovery (mean 13.6 days). There was a reduction of rest pain in all 10 patients with ischaemic disease; five patients showed complete healing of ulcers, and the other five improved significantly. In two patients, pre‐gangrene changes were reversed. RVP is a useful adjunct to conservative or surgical treatment of non‐healing pedal ulcers. Its main impact was in improving ischaemia and promoting healing.
Effects and side‐effects of spironolactone therapy in women with acne
Authors: Yemisci A , Gorgulu A , Piskin S 
Published: March 2005

ABSTRACTBackground and aims  Androgen hormones play an important role in the pathogenesis of acne. Despite the demonstrated effects, spironolactone, an androgen receptor blocker, is not commonly used to treat acne. We planned an open‐labelled, prospective study to evaluate the effects and side‐effects of spironolactone therapy in women with acne.Materials and methods  Thirty‐five consecutive patients with acne were treated with spironolactone 100 mg/day, 16 days each month for 3 months. The patients were divided according to the clinical severity of the lesions as having mild, moderate and severe acne. Serum total testosterone and dehydroepiandrosterone sulfate (DHEAS) levels were measured before and after treatment. Lesion numbers and hormone levels before and after treatment were compared with one‐sampled t‐test.Results  The mean age of the patients was 21.4 ± 3.5 years. Two patients discontinued the study due to side‐effects. Five patients were lost in the follow‐up. Clinically significant improvement was noted in 24 patients (85.71%). No response was seen in four patients. All of the nonresponding patients had received previous unsuccessful therapies. Mean number of lesions and mean DHEAS levels of the 24 patients with clinical improvement decreased significantly after treatment (P < 0.01 and P < 0.05, respectively). There was no change in the mean total testosterone levels before and after treatment (P > 0.05).Conclusion  Spironolactone is a safe and effective medication for women with acne vulgaris. Although its side‐effects seem to be high, they are in the majority of cases not a reason to stop treatment.
Dithranol irritation in psoriasis treatment: a study of 68 inpatients
Authors: Kucharekova M , Lieffers L , Van De Kerkhof PCM , Van Der Valk PGM 
Published: March 2005

ABSTRACTThe irritant response of perilesional skin is a serious limitation of dithranol therapy in psoriasis. No data are available on the actual prevalence and severity of irritation during 24‐h dithranol treatment in an inpatient setting. Using a retrospective analysis of 68 patients with psoriasis visiting our inpatient department for dithranol treatment, the occurrence of dithranol irritation was studied. We found a relatively high frequency of dithranol irritation. Furthermore, most irritation occurs at the start of the therapy with relatively low concentrations.