bluuebunny/crossref_metadata_2025 huggingface dataset:
A Putative Digestive Cysteine Proteinase from Drosophila melanogaster is Predominantly Expressed in the Embryonic and Larval Midgut
Authors: Matsumoto Ichiro , Watanabe Hirohito , Abe Keiko , Arai Soichi , Emori Yasufumi
Published: March 2005

Plant seeds have biodefense systems for protection against insects. One of these systems may be based on the occurrence of phytocystatins, cysteine proteinase inhibitors of plant origin, that probably inhibit insect cysteine proteinases involved in digestive functions. To elucidate a molecular mechanism for this biodefense phenomenon, we isolated a gene encoding a putative digestive cysteine proteinase from Drosophila melanogaster, a suitable model species. The cloned genomic DNA fragment contained a sequence encoding a cysteine proteinase. The mature enzyme from the encoded protein, termed Drosophila cysteine proteinase‐1, consisted of 218 amino acid residues. Drosophila cysteine proteinase‐1 showed 67% similarity in its amino acid sequence to a lobster cysteine proteinase‐3 contained in the digestive juice. This enzyme also showed significant similarities to cysteine proteinases of animal origin such as cathepsins H and L, and to proteinases of plant origin such as rice oryzains α and β. In situ hybridization studies for the embryo showed that the mRNA for Drosophila cysteine proteinase‐1 was predominantly expressed in the midgut. Larval alimentary organs, such as the salivary gland and the midgut including the gastric caeca, also expressed the mRNA at significant levels. These observations, suggesting that Drosophila cysteine proteinase‐1 is a digestive cysteine proteinase which can be used as a model target of phytocystatins, will hopefully lead to new strategies for the regulation of pest insects.
Integral Cytochrome‐c Oxidase
Authors: Soulimane Tewfik , Buse Gerhard
Published: March 2005

A new rapid procedure for the preparation of monodispersed highly active cytochrome‐c oxidase from bovine heart is described. The crucial step is the separation of cytochrome‐c oxidase from cytochrome‐c reductase by selective solubilization in the non‐ionic detergents Triton X‐100 or lauryl β‐d‐maltoside. The enzyme is purified by subsequent anion‐exchange chromatography. The preparation is finished within two days yielding approximately 60% of the oxidase present in mitochondria. The enzyme has a heme a/protein ratio of 9.7 × 0.5 nmol/mg, approximately equal to the theoretical value of 9.77 nmol/mg based on a molecular mass of 204.696 kDa for the protein monomer. SDS/PAGE of the preparation reveals the presence of the well‐known thirteen protein components. Quantitative Edman degradation of the enzyme exclusively releases the known ten N‐terminal residues; three of the thirteen protein components are blocked at the N‐terminus. The preparation is highly active with maximal turnover numbers of approximately 600 s−1, identical to the maximal activity found in the mitochondrial membrane under these conditions. No g= 12 signal and no adventitious copper signal are observed in the EPR spectrum. The enzyme exhibits a fast monophasic reaction with cyanide.

Determination of the metal contents of the enzyme indicates the stoichiometric presence of three copper ions besides two iron, one magnesium and one zinc ion in relation to the 94 sulfur atoms of the protein monomer. Gel‐filtration experiments show a monodispersed dimeric association to form a complex of approximately 500 kDa. The phosphorus content, 44 ± 6.8 atoms/dimer, results from 59% cardiolipin, 23% phosphatidylethanolamine and 18% phosphatidylcholine, indicating a stable lipid shell, different from other previously described preparations.

Crystals have been obtained from these preparations and are investigated for their suitability for X‐ray work.
Studies on the Regulation and Localization of 5‐Lipoxygenase in Human B‐Lymphocytes
Authors: Jakobsson Per‐Johan , Shaskin Pavel , Larsson Pontus , Feltenmark Stina , Odlander Björn , Aguilar‐Santelises Miguel , Jondal Mikael , Biberfeld Peter , Claesson Hans‐Erik
Published: March 2005

Stimulated B‐lymphocytes, isolated from patients with chronic lymphocytic leukemia of B‐cell type (B‐CLL cells) or from human tonsils, produced similar amounts of leukotriene (LT) B4 and 5–hydroxy‐eicosatetraenoic acid (5‐HETE) as polymorphonuclear granulocytes. Unlike intact granulocytes or monocytes, human B‐lymphocytes require calcium ionophore, exogenous arachidonic acid and an oxidative environment in order to produce 5‐lipoxygenase products. Several thiol‐reactive compounds such as N‐ethylmaleimide, methyl methanethiosulfonate, azodicarboxylic acid bisfdimethylamide] (diamide) as well as hydrogen peroxide were all found to stimulate cellular leukotriene biosynthesis. Reverse transcriptase (RT)‐PCR analysis demonstrated the expression of 5‐lipoxygenase, 5‐lipoxygenase‐activating protein (FLAP) and LTA4 hydrolase mRNA in B‐CLL cells. Western blot analysis demonstrated a band corresponding to the molecular size of FLAP in the B‐CLL cell membrane. Furthermore, MK886, the FLAP‐binding cellular leukotriene biosynthesis inhibitor, reduced both LTB4 and 5‐HETE formation. Immunocy‐tochemistry showed that 5‐lipoxygenase was mainly localized in the nuclei of non‐activated B‐CLL cells, tonsillar B‐lymphocytes and monoclonal B‐cells. In contrast, neither human peripheral T‐lymphocytes nor Jurkat cells were stained. These results suggest that 5‐lipoxygenase and its products function in the nucleus of B‐lymphocytes.
Expression of Central and Peripheral Cannabinoid Receptors in Human Immune Tissues and Leukocyte Subpopulations
Authors: Galiègue Sylvaine , Mary Sophie , Marchand Jean , Dussossoy Danielle , Carrière Dominique , Carayon Pierre , Bouaboula Monsif , Shire David , LE Fur Gérard , Casellas Pierre
Published: March 2005

Two proteins with seven transmembrane‐spanning domains typical of guanosine‐nucleotide‐binding‐protein‐coupled receptors have been identified as cannabinoid receptors; the central cannabinoid receptor, CB1, and the peripheral cannabinoid receptor, CB2, initially described in rat brain and spleen, respectively. Here, we report the distribution patterns for both CB1 and CB2 transcripts in human immune cells and in several human tissues, as analysed using a highly sensitive and quantitative PCR‐based method. CB1 was mainly expressed in the central nervous system and, to a lower extent, in several peripheral tissues such as adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils. In contrast, the CB2 gene, which is not expressed in the brain, was particularly abundant in immune tissues, with an expression level 10–100‐fold higher than that of CB1. Although CB2 mRNA was also detected in some other peripheral tissues, its level remained very low. In spleen and tonsils, the CB2 mRNA content was equivalent to that of CB1 mRNA in the central nervous system. Among the main human blood cell subpopulations, the distribution pattern of the CB2 mRNA displayed important variations. The rank order of CB2 mRNA levels in these cells was B‐cells > natural killer cells ≫ monocytes > polymorphonuclear neutrophil cells > T8 cells > T4 cells. The same rank order was also established in human cell lines belonging to the myeloid, monocytic and lymphoid lineages. The prevailing expression of the CB2 gene in immune tissues was confirmed by Northern‐blot analysis. In addition, the expression of the CB2 protein was demonstrated by an immunohistological analysis performed on tonsil sections using specific anti‐(human CB2) IgG; this experiment showed that CB2 expression was restricted to B‐lymphocyte‐enriched areas of the mantle of secondary lymphoid follicles. These results suggest that (a) CB1 and CB2 can be considered as tissue‐selective antigens of the central nervous system and immune system, respectively, and (b) cannabinoids may exert specific receptor‐mediated actions on the immune system through the CB2 receptor.
Rice Aspartic Proteinase, Oryzasin, Expressed During Seed Ripening and Germination, has a Gene Organization Distinct from Those of Animal and Microbial Aspartic Proteinases
Authors: Asakura Tomiko , Watanabe Hirohito , Abe Keiko , Arai Soichi
Published: March 2005

The gene organization and nucleotide sequence of an aspartic proteinase (AP) of plant origin were first disclosed by cDNA and genomic DNA cloning of a rice AP (oryzasin). The deduced amino acid sequence of oryzasin 1 was significantly similar to those of other APs (34–85%), with highest similarity (85%) to barley AP (HvAP). Oryzasin 1, as well as HvAP, is distinct from animal and microbial APs in that the plant APs contain a unique 104‐amino‐acid insertion in the C‐terminal region. The oryzasin 1 gene spans approximately 6.6 kbp and is composed of 14 exons and 13 introns. The exon‐intron organization of the oryzasin 1 gene is totally different from those of genes for animal and microbial APs such as human cathepsin D, rat renin, bovine chymosin, aspergillopepsin A of Aspergillus awamori, proteinase A of Saccharomyces cerevisiae and rhizopuspepsin of Rhizopus niveus, despite the fact that oryzasin 1 shows overall sequence similarity to these APs.
Identification of the Electrophilic Substrate‐Binding Site of Glutathione S‐Transferase P by Photoaffinity Labeling
Authors: Nishihira Jun , Sakai Masaharu , Nishi Shinzo , Hatanaka Yasumaru
Published: March 2005

We determined the electrophilic substrate‐binding site of rat glutathione S‐transferase P (GST‐P) by photoaffinity labeling using the photosensitive compound S‐[2‐(2‐fluoro‐4‐nitrophenoxy)ethyl]glutathione. This photosensitive glutathione analogue inhibited the catalytic activity in a competitive manner against both glutathione and 1‐chloro‐2,4‐dinitrobenzene, a putative electrophilic substrate. The enzyme kinetics indicated that the photoactivatable glutathione analogue was specifically bound at the active site, which consisted of glutathione‐binding (G‐site) and the electrophilic substrate‐binding (H‐site) regions. The procedure involved the following steps: S‐[2‐(2‐fluoro‐4‐nitrophenoxy)ethyl]glutathione was photochemically reacted with a purified recombinant GST‐P expressed in Escherichia coli using ultraviolet irradiation for 30 min on ice. After the reaction, only the GST‐P complexed with the glutathione analogue was prepared with glutathione‐immobilized agarose. The GST‐P covalently bound with the analogue was digested with lysyl endopeptidase (Achromobacter protease I), and the peptides were separated by high‐performance liquid chromatography. Only a single major peak with appreciable absorbance at 340 nm was observed by peptide mapping. The peptide was collected and analyzed using an automated peptide sequencer (ABI 477A). Amino acid sequence analysis showed that this peptide consisted of seven amino acid residues corresponding to the sequence at positions 122–128 of GST‐P (Ala‐Leu‐Pro‐Gly‐Xaa‐Leu‐Lys). No appreciable phenylthiohydantoin‐amino acid was detected at the fifth cycle, which indicated that His126 was chemically labeled with the photosensitive glutathione analogue. It was concluded that His126 was one of the amino acid residues forming the electrophilic substrate‐binding site of GST‐P.
Thermal Sensitivity in NIH 3T3 Fibroblasts Transformed by the v‐Fos Oncogene
Authors: Fabre‐Jonca Nathalie , Gonin Sandrine , Diaz‐Latoud Chantal , Rouault Jean‐Pierre , Arrigo AndréPatrick
Published: March 2005

The effect of v‐fos transformation on the cellular response to heat shock has been investigated. NIH 3T3 fibroblasts were transfected with the FBR p75gag‐fos gene fusion under the control of the long terminal repeat (LTR) promoter of Finkel‐Biskin‐Reilly (FBR) murine sarcoma virus and with the gene encoding hygromycin resistance. Several hygromycin‐resistant clone isolates, that expressed various levels of p75gag‐ros oncoprotein, were analyzed as they displayed properties of transformed cells, such as altered morphology, shorter doubling time, serum‐independent growth and foci formation in soft agar. The thermal response of these clones was compared to that of the control cells expressing the hygromycin‐resistance gene only. Here, we report that the v‐fos‐transformed clones displayed an enhanced thermosensitivity which resulted in a reduced tolerance to thermal stress. Heat‐treated v‐fos‐transformed cells displayed a decreased expression and accumulation of the major stress proteins Hsp68 (68‐kDa heat‐shock protein) and Hsp25 which probably resulted of a reduced accumulation of the corresponding mRNAs. This effect was particularly intense at the level of Hsp25. These alterations in cell survival and stress‐protein expression appeared correlated to the level of p75gag‐fos. At least for Hsp68, the transcription of this gene was not found altered by v‐fos expression suggesting that this oncogene increases the turn‐over of Hsp68 mRNA. After the heat‐shock treatment, v‐fos transformation also reduced the time period during which the constitutively expressed stress protein Hsc70 redistributes inside the nucleus. Since Hsp68 and Hsp25 are molecular chaperones that in vivo protect cells against the deleterious effects of heat shock, it is conceivable that their reduced accumulation and altered cellular distribution following heat shock may contribute, at least in part, to the thermosensitivity of v‐fos‐transformed NIH 3T3 fibroblasts.
Bacterial Expression, Characterization and DNA Binding Studies on Drosophila Melanogaster c‐Myb DNA‐Binding Protein
Authors: Madan Anup , Radha Plachikkat K. , Hosur Ramakrishna V. , Padhy Lakshmi C.
Published: March 2005

The Drosophila Myb homologue retains an evolutionarily conserved typical sequence of three imperfect tandem tryptophan repeat units (R1‐R2–R3) of 51–53 amino acids towards its N‐terminus as its presumptive DNA binding domain. Using PCR amplification and the TV expression vector pET 11d, we have overproduced this tryptophan repeat domain of Drosophila Myb in Escherichia coli and the protein has been purified. Circular dichroic measurements indicate that the protein has a high helical component (58.6%) in its overall structure. The protein is found to recognize the same cognate target sequence TAACGG, as recognized by the vertebrate proteins. The DNA binding properties of the protein have been investigated in detail by fluorescence spectroscopy taking advantage of the large number of tryptophan residues present in the protein. The fluorescence of the native Drosophila R123 was quenched when synthetic duplex DNA oligomers were added to the protein. The oligomers containing specific Myb target sites quenched the protein fluorescence to a greater extent than the non‐specific DNA. Binding constants of the protein to the targets were also length dependent for smaller oligomers. Experiments with the collisional quencher acrylamide and cysteine modification reagent indicated that the specific and nonspecific target sequences interact with the protein differently. In the former case both the buried and the exposed tryptophan residues were affected by DNA binding whereas in the latter only the solvent‐exposed residues were involved.
Regulation of Rat Liver Apolipoprotein A‐I, Apolipoprotein A‐II and Acyl‐Coenzyme A Oxidase Gene Expression by Fibrates and Dietary Fatty Acids
Authors: Berthou Laurence , Saladin Régis , Yaqoob Parveen , Branellec Didier , Calder Philip , Fruchart Jean‐Charles , Denèfle Patrice , Auwerx Johan , Staels Bart
Published: March 2005

The regulation by fibrates and dietary fatty acids of the hepatic gene expression of apolipoproteins (apo) A‐I and A‐II, the major protein constituents of high‐density lipoproteins, as well as of acyl‐CoA oxidase, the rate‐limiting enzyme of the peroxisomal β‐oxidation pathway, was studied in vivo in the rat and in vitro in primary cultures of rat hepatocytes. In primary hepatocytes, different fibrates decreased apo A‐I and increased acyl‐CoA oxidase mRNA levels, whereas apo A‐II mRNA only decreased in level after treatment with fenofibric acid, but not after bezafibrate, gemfibrozil or Wy‐14643 treatment. Treatment with fenofibric acid counteracted the increase in apo A‐I mRNA levels observed after dexamethasone or all‐trans retinoic acid treatment, whereas simultaneous addition of fenofibric acid together with all‐trans retinoic acid or dexamethasone resulted in a superinduction of acyl‐CoA oxidase mRNA. Addition of the n‐3 polyunsaturated fatty acids (PUFAs), docosanohexaenoic acid and eicosanopentaenoic acid, or the fatty acid derivative α‐bromopalmitate, decreased apo A‐I and increased acyl‐CoA oxidase mRNA in a dose‐dependent and time‐dependent manner, whereas apo A‐II mRNA did not change significantly. Nuclear run‐on experiments demonstrated that fenofibric acid and α‐bromopalmitate decreased apo A‐I and increased acyl‐CoA oxidase gene expression at the transcriptional level. When rats were fed isocaloric diets enriched in saturated fat (hydrogenated coconut oil), n‐6 PUFAs (safflower oil) or n‐3 PUFAs (fish oil), a significant decrease in liver apo A‐I and apo A‐II mRNA levels was only observed after fish oil feeding. Compared to feeding low fat, liver acyl‐CoA oxidase mRNA increased after fat feeding, but this effect was most pronounced (twofold) in rats fed fish oil. Results from these studies indicate that fish oil feeding reduces rat liver apo A‐I and apo A‐II gene expression, similar to results obtained after feeding fenofibrate. Fibrates and n‐3 fatty acids (and the fatty acid derivative, α‐bromopalmitate) down‐regulate apo A‐I and induce acyl‐CoA oxidase gene expression through a direct transcriptional action on the hepatocyte. In contrast, only fenofibric acid, but not the other fibrates or fatty acids tested, decrease apo A‐II gene expression in vitro.
Cyclic Voltammetry and 1H‐NMR of Rhodopseudomonas Palustris Cytochrome c2 pH‐Dependent Conformational States
Authors: Battistuzzi Gianantonio , Borsari Marco , Ferretti Silvia , Sola Marco , Soliani Enrico
Published: March 2005

The pH‐induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c2 from Rhodopseudomonas palustris were monitored by electrochemical and spectroscopic measurements. In the pH range 1.5–11, the E° values (and/or the peak potentials) determined by cyclic voltammetry, the electronic spectra and the hyperfine‐shifted 1H‐NMR resonances of the protein are sensitive to a number of acid/base equilibria. In particular, four equilibria have been determined for the oxidized protein with pKa values of 2.5, 5.5, 6.6 and 9.0. The lowest pK, most probably involves disruption of both axial heme iron bonds and protein unfolding. The subsequent pKa is associated with a low‐pH oxidation of the protein by dioxygen, which is accompanied by a conformational change. The equilibrium with an apparent pKa of 6.6 modulates the E° values without determining any detectable spectral change and most likely involves the acid/base equilibrium of an histidine residue in close vicinity of the heme (possibly His53). Finally, the alkaline ionization is due to the replacement of the methionine axially bound to the heme iron with a stronger (most probably N‐donor) ligand. The reduced alkaline form is unstable and spontaneously converts to the neutral reduced form with a kinetic constant of 0.98 s−1 at pH 9.2.
Characterisation of Fast, Slow and Cardiac Muscle Tropomyosins from Salmonid Fish
Authors: Heeley David H. , Bieger Thorben , Waddleton Deena M. , Hong Caroline , Jackman Donna M. , Mcgowan Colin , Davidson William S. , Beavis Ronald C.
Published: March 2005

Tropomyosin (TM) has been isolated from the cardiac muscle, and fast and slow trunk (myotomal) muscles of the mature salmonid fish Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri). When examined electrophoretically, isoforms of TM were detected which were specific, and exclusive, to each type of muscle. Cardiac and fast muscles contained single and distinct isoforms, while slow muscle contained two distinct isoforms, closely related in terms of apparent Mr and pI. There was no detectable difference between the same TM type from either salmon or trout. On a variety of gel systems, the cardiac and slow isoforms migrated in close proximity to each other and to rabbit α‐TM. The fast isoform comigrated with rabbit β‐TM. In developing salmon fry, a more acidic (unphosphorylated) variant of TM was present in addition to, and of similar Mr to, the fast adult isoform. This TM declined in steady‐state level during maturation and was virtually undetected in adult muscle. All of the isolated TMs contained little or no covalently bound phosphate and were blocked at the N‐terminus. The amino acids released by carboxypeptidase A, when ordered to give maximal similarity to other muscle TMs, were consistent with the following sequences: fast (LDNALNDMTSI) and cardiac (LDHALNDMTSL). The C‐terminal region of the slow TM contained His but was heterogeneous. In viscosity measurements, performed as a function of increasing protein concentration, at low ionic strength (t= 5°C, pH 7.00), fast TM exhibited the highest relative viscosity values. Lower and equivalent levels of polymerisation occurred with the cardiac and slow TMs. Polymerisation of all three isoforms was temperature‐dependent, with cardiac TM being least sensitive and fast TM being most sensitive. Determination of the complete coding sequence of adult fast TM confirmed the findings of the carboxypeptidase analysis, but the remainder of the sequence more closely resembled α‐type TMs than β‐type TMs. Overall, salmon fast TM contains 20 (mostly conservative) substitutions compared to rabbit striated muscle α‐TM and 40 (mostly conservative) substitutions compared to rabbit striated muscle β‐TM. This demonstrates that electrophoretic mobility is not, in all instances, a suitable method to assess the isomorphic nature of striated muscle TMs.
Immunochemical Probing of the Functional Role of the 238–246 and 567–574 Sequences of Myosin Heavy Chain
Authors: Blotnick Edna , Miller Carl , Groschel‐Stewart Ute , Muhlrad Andras
Published: March 2005

Polyclonal site‐directed peptide antibodies were raised against the 567–574 and 238–246 sequences of the rabbit skeletal muscle myosin heavy chain. These sequences, which are located in the subfragment 1 (S1) segment of myosin, have been implicated by former studies in actin and nucleotide binding of the molecule and in the communication between the two binding sites. The antibodies obtained from rabbit sera were found to be conformation‐sensitive since they specifically reacted with S1 in solid‐phase binding assay but not in Western blot. The binding of both antibodies to S1 was strongly inhibited by actin. The antibody against the 567–574 sequence, Ab567–574, moderately decreased the binding of S1 to actin filaments in rigor but not in the weakly–attached state, while Ab238–246 did not influence the binding of S1 to actin under either conditions. Both antibodies inhibited the actin activation of the MgATPase of S1 but did not affect MgATPase without actin or the Ca‐ and K(EDTA)‐activated ATPase activities of S1. The sliding velocity of actin filaments in the in vitro motility assays were also reduced in the presence of the antibodies. Ab567–574 had especially strong inhibitory effect on the movement of actin filaments. The results indicate that the binding of antibodies may induce conformational changes, which propagate in the S1 structure, perturb the coupling between the binding sites and impair the motor function of myosin.
Inhibition of Bovine Cytochrome P‐45011β by 18‐Unsaturated Progesterone Derivatives
Authors: Delorme Cécile , Piffeteau Annie , Viger Antoinette , Marquet Andrée
Published: March 2005

The last step of aldosterone biosynthesis, an 11β‐hydroxylation followed by two 18‐hydroxylations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P‐45011β (deoxycorticosterone (DOC) → corticosterone → 18‐hydroxycorticosterone → aldosterone). The 11β‐ and 18‐hydroxylase activities were studied separately with a reconstituted enzymic system, using 11‐deoxy[14C]corticosterone and [3H]corticosterone, respectively, as substrates.

The inhibition of 11β‐hydroxylase activity by corticosterone was competitive (Ki= 60 μM) showing that transformation of both substrates occurs at the same site. Double‐label/double‐substrate experiments, using an equimolar mixture of 11‐deoxy[14C]corticosterone and [3H]corticosterone, suggested that 18‐hydroxycorticosterone is directly formed from 11‐deoxycorticosterone without the intermediate corticosterone leaving the enzyme.

Inhibitions by 18‐vinylprogesterone and 18‐ethynylprogesterone, potent inhibitors of aldosterone biosynthesis [Viger, A., Coustal, S., Pérard, S., Piffeteau, A. & Marquet, A. (1989) J. Steroid Biochem. 33, 119–124], were characterized for both activities (11β‐ and 18‐hydroxylase). The value of reversible Ki for the 18‐hydroxylation (Ki= 5 μM for 18‐vinylprogesterone and 30 μM for 18‐ethynylprogesterone) is lower than that for the 11β‐hydroxylation (30 μM and 100–150 μM, respectively); the former inhibitor is stronger than the latter for both steps.

The binding of substrates and inhibitors to the active site was also examined by difference absorption spectroscopy. 18‐Vinylprogesterone gave rise to a type I spectrum with a Ks value of 35 μM close to that of progesterone, while 18‐ethynylprogesterone showed a reverse type I spectrum with a much higher Ks value (140 μM). Based on these results, a hypothetical model, involving a conformational change of the enzyme for the second step, is proposed.
Occurrence of Isospora suis in Germany, Switzerland and Austria
Authors: Mundt H.‐C. , Cohnen A. , Daugschies A. , Joachim A. , Prosl H. , Schmäschke R. , Westphal B.
Published: March 2005

Nationwide surveys for the occurrence of Isospora suis were carried out in Germany, Austria and Switzerland including a questionnaire regarding herd size, health status and management practices and a coccidiosis sampling kit for pooled faecal samples from litters of suckling piglets. A total of 184 veterinary practices participated in the survey and returned 1745 samples (331 kits) from 324 farms in the north (n = 98), south (n = 84), centre/east (n = 42) and west (n = 10) of Germany, Austria (n = 61) and Switzerland (n = 29) with larger farms in north and centre/east (average number of sows: 270 and 500) and smaller ones in the south (95), Austria (60) and Switzerland (43). Larger farms tended to have better hygienic standards (slatted floors, disinfection of the farrowing units). The majority of the participating farms (93.5%) reported problems with diarrhoea in piglets at 2–3 weeks of age, significantly associated (P < 0.001) with uneven weaning weights (94.9%). Toltrazuril (5%; Baycox®) was used only rarely; however, in these farms unevenness of weaning weights was less frequently observed (P = 0.011). A 76.2% of the farms were positive for I. suis (samples contained mostly low or moderate oocyst numbers), especially in the south (P < 0.001). Oocysts were more frequently found in samples from farms with reported diarrhoea (P = 0.011), uneven weight gain (P = 0.019) or in herds of small size (P < 0.001). Disinfection, floor type or treatment with toltrazuril did not affect the frequency of observation of oocysts.
Marked structural and functional heterogeneity in CXCR4: Separation of HIV‐1 and SDF‐1α responses
Authors: Sloane Andrew J , Raso Vic , Dimitrov Dimiter S , Xiao Xiaodong , Deo Shivashni , Muljadi Nick , Restuccia David , Turville Stuart , Kearney Christine , Broder Christopher C , Zoellner Hans , Cunningham Anthony L , Bendall Linda , Lynch Garry W
Published: March 2005

CXCR4, the chemotactic cell receptor for SDF‐1α, is essential for immune trafficking and HIV infection. CXCR4 is remarkably heterogeneous and the purpose of this study was to better identify the isoforms expressed by cells and compare their structure and function. We found that cells express either a predominant isoform or multiple isoforms. These were best resolved on SDS‐PAGE using sucrose‐gradient‐fractionated, triton‐insoluble, membrane extracts. We hypothesized that glycosyl modification may underpin some of this heterogeneity and that cell isoform(s) differences may underscore CXCR4's multiple cell functions. A comparison of wild‐type (WT) and dual N‐linked glycosylation site, N11A/N176A, mutant CXCR4 expressed in 3T3 and HEK‐293 cells served to implicate variabilities in glycosylation and oligomerization in almost half of the isoforms. Immunoprecipitation of CXCR4 revealed monomer and dimer non‐glycosylated forms of 34 kDa and 68 kDa from the N11A/N176A mutant, compared with glycosylated 40 kDa and 47 kDa and 73 kDa and 80 kDa forms from WT. The functional specificity of isoform action was also implicated because, despite CEMT4 cells expressing high levels of CXCR4 and 11 different isoforms, a single 83 kDa form was found to bind gp120 for HIV‐1 IIIB infection. Furthermore, comparative studies found that in contrast to SDF‐1α‐responsive Nalm‐6 cells that expressed similar levels of a single isoform, CEMT4 cells did not show a Ca++ flux or a chemotactic response to SDF‐1α. Thus, CXCR4 can differ both structurally and functionally between cells, with HIV‐1 infection and chemotaxis apparently mediated by different isoforms. This separation of structure and function has implications for understanding HIV‐1 entry and SDF‐1α responses and may indicate therapeutic possibilities.
Cooperation between CD4 T helper cells is required for the generation of alloantigen‐specific, IFN‐γ‐producing human CD4 T cells
Authors: Strutt Tara , Bretscher Peter
Published: March 2005

We have used MLR to assess the cell interactions that enable human PBL stimulated with irradiated, allogeneic PBL to give rise to allospecific, IFN‐γ‐producing CD4 T cells. We were able to generate alloantigen‐specific, IFN‐γ‐producing T cells from peripheral blood. The responder and stimulator cell numbers required for the optimal generation of such cells, enumerated using an enzyme‐linked immunospot assay, were separately determined for various combinations of responders and stimulators. The number of antigen‐specific, IFN‐γ‐producing cells generated per 106 responder cells, seeded at the initiation of culture, is critically dependent on the density of the responder cells, with minimal generation of IFN‐γ‐producing T cells at low responder densities. These and further observations with fractionated responding PBL show that CD4 T‐cell/CD4 T‐cell interactions, which are completely independent of CD8 T cells, are necessary for the in vitro generation of alloantigen‐specific, IFN‐γ‐producing CD4 T cells.
Increased peripheral blood T‐cell apoptosis and decreased Bcl‐2 in chronic obstructive pulmonary disease
Authors: Hodge Sandra , Hodge Greg , Holmes Mark , Reynolds Paul N
Published: March 2005

Chronic obstructive pulmonary disease (COPD) is an inflammatory airway disease, usually associated with cigarette smoking. Stimulated peripheral blood T cells from patients with COPD have an increased propensity to undergo apoptosis. The mitochondrial apoptotic pathway is regulated by pro‐apoptotic proteins (including p53 and Bax) as well as anti‐apoptotic proteins (e.g. Bcl‐2) and cytokines (IL‐2, IL‐4 and IL‐7). We hypothesized that alterations in expression of these apoptosis‐related proteins, cytokines and cytokine receptors may be important in determining the susceptibility of T cells to undergoing apoptosis in COPD. We further hypothesized that inhaled corticosteroids (GCS) contribute to the increased rates of T‐cell apoptosis observed in COPD. The process of apoptosis (assessed by Annexin V and ssDNA staining), as well as Bcl‐2, Bax, p53, IL‐2, IL‐4 and receptors IL‐7R, IL‐4R and IL‐2Rγ were investigated in PHA‐stimulated peripheral blood‐derived T cells, using flow cytometry. Fifteen patients with COPD receiving inhaled GCS (four of who received additional prednisolone), eight patients with COPD receiving symptom control medication, and 16 control subjects were studied. T cells (CD4+ and CD8+) from GCS‐treated COPD patients showed an increased propensity to undergo apoptosis, associated with significantly decreased Bcl‐2 and IL‐7 receptor expression. No significant differences were observed for the COPD patients who were receiving symptom control medication. These findings may suggest a negative peripheral effect of inhaled GCS on the immune system in COPD, although the clinical significance of these effects remains uncertain.
Development of hydrogel patch for controlled release of alpha‐hydroxy acid contained in tamarind fruit pulp extract
Authors: Viyoch J. , Sudedmark T. , Srema W. , Suwongkrua W.
Published: March 2005

Synopsis

The aim of this study was to develop hydrogel patch using crosslinked chitosan–starch as polymeric matrix for controlling the release of the natural alpha‐hydroxy acid (AHA) contained in the extract of tamarind's fruit pulp. The chitosan (MW 100 000) was blended with corn, tapioca or rice starch in various ratios and then crosslinked with glutaraldehyde. The physical characteristics, mechanical resistance, bio‐adhesion property and surface morphology of the prepared hydrogel patches with and without the extract were investigated. The release patterns of the hydrogel patches containing the extract were investigated by measuring the amount of tartaric acid, a major AHA present in the tamarind's fruit pulp extract, accumulated in the receptor medium of the vertical diffusion cell at various time intervals over a period of 6 h. The results indicated that the formulations of chitosan : corn starch 4.5 : 0.5 with glutaraldehyde 0.02% w/w (C4.5C0.5G0.02) or 0.04% w/w (C4.5C0.5G0.04), chitosan : tapioca starch 4.5 : 0.5 with glutaraldehyde 0.04% w/w (C4.5T0.5G0.04) or 0.05% w/w (C4.5T0.5G0.05), and chitosan : rice starch 4.5 : 0.5 with glutaraldehyde 0.04% w/w (C4.5R0.5G0.04) and chitosan : rice starch 4.0 : 1.0 with glutaraldehyde 0.03% w/w (C4.0R1.0G0.03) provided the flexible and elastic patches with good bio‐adhesive property. The tensile strength values ranged from 5 to15 N mm−2 and the elasticity ranged from 30 to 60%. The addition of the extract in these formulations significantly increased the tensile strength values of the obtained patches. The patch of C4.0R1.0G0.03 formulation containing the extract showed relatively highest porosity, corresponding to its highest amount (12.02 ± 0.33 mg) and rate (0.452 ± 0.012 mg mm−2 min−1/2) of tartaric acid released. The amounts of tartaric acid released from the developed hydrogel patches were proportional to a square root of time (Higuchi's model), particularly the release from C4.0R1.0G0.03 (R2, 0.9978 ± 0.0020) and C4.5R0.5G0.04 (R2, 0.9961 ± 0.0024) patches.
The Relative Contribution of Trait and Social Influences to the Links Among Perceived Social Support, Affect, and Self‐Esteem
Authors: Lakey Brian , Scoboria Alan
Published: March 2005

Abstract
Although perceived support is influenced by both the personality traits of support recipients as well as various social factors, it is unknown to what extent these two types of influences account for perceived support's link to mental health. We investigated these relations using multivariate generalizability analyses. In three samples, both the trait and social influence components of perceived support were related to favorable affect and to self‐esteem. The magnitude of the correlations between perceived support and mental health was similar for both the trait and social influence components. Similar findings were obtained for social conflict, although the links between conflict and mental health varied somewhat depending upon the level of analysis. These findings suggest that social support theories and interventions should include both trait and social mechanisms to explain and modify perceived support and mental health.
Costs of Self‐Handicapping
Authors: Zuckerman Miron , Tsai Fen‐Fang
Published: March 2005

Abstract
Four studies examined the relation of trait self‐handicapping with health‐related measures. Study 1 showed that, over time, self‐handicapping and maladjustment reinforce each other. Study 2 showed that self‐handicappers reported a loss in competence satisfaction which, in turn, mediated the relation of self‐handicapping with negative mood. Study 3 found that, over time, self‐handicappers report an increase in substance use. Study 4 showed that self‐handicappers reported a loss in intrinsic motivation for their jobs. It was suggested that people with unstable (or contingent) self‐esteem use self‐handicapping to bolster a fragile self‐concept.
Hope Against the Cold: Individual Differences in Trait Hope and Acute Pain Tolerance on the Cold Pressor Task
Authors: Snyder C. R. , Berg Carla , Woodward Julia T. , Gum Amber , Rand Kevin L. , Wrobleski Kristin K. , Brown Jill , Hackman Ashley
Published: March 2005

AbstractHope theory (seeSnyder, 1994) is presented as a useful framework for understanding reactions to pain. In Study 1, persons scoring higher on the trait Hope Scale (Snyder, Harris et al., 1991) kept their hands in the freezing water (of a cold pressor task) for significantly longer. In Study 2, the higher‐hope males, and not females, as measured by both trait and state hope (Snyder, Sympson et al., 1996), recognized the onset of the pain threshold significantly later. Moreover, in Study 2, results showed that individual differences measures of optimism, self‐efficacy, depression, and positive and negative affects did not relate to the pain onset and tolerance variables. The implications of hope as related to the pain process and related research are discussed.
Assessing Settlement of High‐Rise Structures by 3D Simulations
Authors: Katzenbach R. , Schmitt A. , Turek J.
Published: March 2005

Abstract:  A number of new high‐rise buildings in the Frankfurt region are currently in their planning stage. Most of these high‐rise structures will be erected on combined piled‐raft foundations (CPRFs). Due to the complex interaction between piles, raft, and subsoil the difficult design of these foundations will be carried out by three‐dimensional finite‐element (FE) simulations. For the 121 m high City‐Tower, which is currently under construction, the design procedure based on a three‐dimensional FE simulation of the CPRF is described. The design process for the new 228 m high office tower Max, which will be located in the financial district of Frankfurt, in the direct vicinity of already existing high‐rise buildings, has just started. To improve and verify the input parameters for the constitutive modeling and to allow for a cost optimized foundation design, a numerical back‐analysis of the 110 m high Eurotheum, located close to the building site of Max, has been performed. For this building comprehensive measurements were carried out starting in the construction stage and lasting up to the present day.
Pityriasis versicolor alba
Authors: Thoma W , Krämer H‐J , Mayser P
Published: March 2005

Pityriasis versicolor alba is a hypopigmented or depigmented variant of pityriasis versicolor characterized by maculous, partly pityriasiform, scaly depigmented lesions occurring particularly in seborrhoeic areas. Long‐persisting hypopigmentation after healing of the pityriasis versicolor was first described by Gudden in 1853. Hypopigmentation and depigmentation were later differentiated as an independent variant of the disease. In 1848, Eichstedt recognized the pathogen‐related character of pityriasis versicolor in its hyperpigmented form. Today it is generally accepted that the disease is caused by yeasts of the genus Malassezia, of which nine species are differentiated. It is controversial whether a single species is responsible for the disease. The pathogenesis of depigmentation has not been established. A screening effect by the scale layer as well as toxic effects on pigment synthesis by fungal metabolites have been discussed. With regard to the second mechanism, the newly discovered tryptophan‐derived metabolites of M. furfur might be significant. Evidence‐based data concerning the therapy of pityriasis versicolor alba do not exist. According to current recommendations, pityriasis versicolor should be rapidly treated with antimycotics, followed by ultraviolet therapy to induce maturation of existent melanosomes and accelerate repigmentation. However, depigmented lesions are difficult to improve by ultraviolet therapy.
Management of erosive lichen planus with topical tacrolimus and recurrence secondary to metoprolol
Authors: Meyer S , Burgdorff T , Szeimies RM , Vogt T , Landthaler M , Karrer S
Published: March 2005

Metoprolol, a widely prescribed β‐adrenergic receptor blocker, has occasionally been associated with a diversity of cutaneous reactions. We present a 79‐year‐old male patient with erosive lichen planus (LP) on the feet and hands who was successfully treated with topical tacrolimus. Six months after the lesions had been cured the patient received the β‐receptor blocker metoprolol for the treatment of hypertonus. Within only 2 weeks of metoprolol intake the erosive lesions on the palms and feet recurred. After discontinuation of the drug and repetitive topical treatment with tacrolimus a complete remission of the lesions could be achieved. The recurrence of erosive LP probably secondary to metoprolol and the therapeutic success of topical tacrolimus in the treatment of LP are discussed.
Livedo reticularis associated with interferon α therapy in two melanoma patients
Authors: Ruiz‐Genao DP , García‐F‐Villalta MJ , Hernández‐Núñez A , Ríos‐Buceta L , Fernández‐Herrera J , García‐Díez A
Published: March 2005

We report two patients who developed intense livedo reticularis clearly related to the administration of interferon α 2b as an adjuvant therapy for melanoma. Histological studies showed scattered perivascular infiltrates without vasculitis. Laboratory tests excluded any underlying condition. Resolution of the symptoms was observed in both patients when interferon α was withdrawn. These cases highlight the occurrence of livedo reticularis as an uncommon side‐effect of interferon α treatment.
The effects of foot disease on quality of life: results of the Achilles Project
Authors: Katsambas A , Abeck D , Haneke E , Van De Kerkhof P , Burzykowski T , Molenberghs G , Marynissen G
Published: March 2005

Background  Foot diseases have a high prevalence in the general population, but their impact on quality of life has not been assessed in large‐scale studies. The Achilles Project surveyed foot disease in patients visiting their primary care physician or dermatologist.

Methods  A total of 43 593 patients were asked about the impact of their condition on their quality of life: pain, discomfort in walking, limitations in daily activities, and embarrassment.

Results  Overall, 52.5% of patients had some aspect of their quality of life affected by their foot disease. More specifically, 30.7% of patients experienced pain, 40.3% had discomfort in walking, 19.6% had their daily activities limited, and 27.3% were embarrassed. The survey indicated a larger impact of foot disease on the quality of life of women vs. men in all categories, except for daily activities. Similarly, the elderly (≥ 65 years) were more affected by their foot disease, although they suffered no more embarrassment than other age groups. Participation in sports seemed to lower the proportion of patients who had their quality of life adversely affected. Non‐fungal foot diseases, particularly ulcer and gangrene, are more likely to cause pain, discomfort in walking and limit daily activities, than fungal diseases.

Conclusions  In general, non‐fungal foot diseases caused pain, discomfort in walking and limitations in daily activities in more patients than fungal foot diseases, but a higher proportion of patients with fungal foot diseases were embarrassed by their condition than patients with non‐fungal foot diseases. The study found that the impact of foot disease on quality of life may be greater than previously suggested. Given that effective treatments are available, routine examination of patients’ feet by dermatologists and primary care physicians may help to reduce the burden of these foot conditions.
Successful UVA1 phototherapy in a patient with scleredema adultorum
Authors: Eberlein‐König B , Vogel M , Katzer K , Hein R , Köhn F‐M , Ring J , Abeck D
Published: March 2005

Scleredema adultorum is a rare connective tissue disorder of unknown cause. Both bath‐PUVA and cream‐PUVA therapy were reported to be effective. We describe a patient with scleredema adultorum who showed a striking clinical improvement with a medium‐dose UVA1 phototherapy (single dose, 50 J/cm2; 35 treatments).
Autodestruction of penis due to carcinoma
Authors: Agarwal P , Sharma D
Published: March 2005

Four unusual cases of autodestruction of the penis are described with their aetiology, diagnosis and management. Causes for delay in diagnosis and therapy, prevention and early recognition of this condition are emphasized.
Dermatitis artefacta: unusual appearance in an older woman
Authors: Gregurek‐Novak T , Novak‐Bilić G , Vučić M
Published: March 2005

Dermatitis artefacta is a rare and difficult condition for diagnosis and treatment, with the highest incidence of onset in late adolescence to early adult life. Most patients are young women who have a personality disorder; borderline features are common and the patient's denial of psychological distress makes management and treatment difficult. Patients use a variety of means to cause the skin changes. Clinical presentation of the skin lesions does not conform to those of known dermatoses and are located on easily reached parts of the skin. We report an unusual case of a 72‐year‐old woman with symmetrical changes under the breasts and in the right inguinal region. The lesions were composed partly of haemorrhagic round lesions and partly of scars. A skin biopsy was taken and consultations with the psychiatrist, internist and the patient's family led to the diagnosis of self‐induced dermatitis. The skin lesions were covered by occlusion techniques and the lesions improved very rapidly. The patient was discharged from the hospital under psychiatric and family care.
MODULE FOUR: STANDARDS OF CARE AND CLINICAL TRIALS
Authors: SELGELID MICHAEL J.
Published: March 2005

This module examines ethical debates about the level of care that should be provided to human research participants. Particular attention is placed on the question of what should be considered an ethically acceptable control arm. You will also learn what relevant international and domestic regulatory documents say about standards of care.
MODULE SIX: SPECIAL ISSUES
Authors: SCHNEIDER BENJAMIN , SCHÜKLENK UDO
Published: March 2005

The objective of this module is to cover ground that was not covered in‐depth in any of the other modules, including: scientific misconduct, issues concerning the publication and ownership of research results (authorship guidelines – who is eligible to be considered an author, or contributor to a scientific paper etc.), special problems occurring in social science and epidemiological research, and the problems pertaining to conflicts of interest the various players in biomedical research activities could encounter.