bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Cloning and Expression of an Interferon‐Inducible Protein Encoded by Gene 203 from the Gene 200 Cluster
Authors: Gribaudo Giorgio , Ravaglia Stefania , Guandalini Laura , Riera Ludovica , Gariglio Marisa , Landolfo Santo
Published: June 2004

We report here the complete coding sequence of a 203 cDNA, a member of the interferon‐inducible Ifi 200 gene family. By combining reverse‐transcriptase PCR and rapid amplification of cDNA ends (RACE) techniques we have obtained a 3.8‐kb cDNA corresponding to a 203 mRNA. When used as a probe in northern analysis, its 3’ segment hybridized to a 3.8‐kb interferon‐inducible mRNA, whereas the 5’‐end additionally hybridized to a less abundant interferon‐inducible 1.8‐kb mRNA. Nucleotide sequence analysis revealed that the two mRNAs share the 5’‐untranslated region and the same open reading frame, which encodes a hydrophilic protein composed of 408 amino acids. The difference between them is due to a 3’‐untranslated region extended by alternative polyadenylation site selection. Furthermore, 203 mRNA was found to be inducible by interferon‐α in various murine cell lines. Using polyclonal antibodies raised against a segment specific for the 203 protein, we established that p203 protein levels increase on treatment with interferon‐α in murine fibroblasts and that p203 is located in the nucleus.
Solution Structure of Reduced Microsomal Rat Cytochrome b5
Authors: Banci Lucia , Bertini Ivano , Ferroni Filippo , Rosato Antonio
Published: June 2004

The solution structure of the major form of the reduced soluble fragment of rat microsomal cytochrome b5 has been solved through 1H‐NMR spectroscopy. The protein contains 98 amino acids. Proton assignment was available for residues 1–94, except 90 [Guiles, R. D., Basus, V. J., Kuntz, I. D. & Waskell, L. (1992) Biochemistry 31, 11 365–11 375] and has been confirmed. From 1722 NOEs, of which 1203 were found to be meaningful, a family of 40 energy‐minimized structures has been obtained with average backbone rmsd (for residues 5–89) of 0.078 ± 0.018 nm and average target function of 0.0045 nm2, no distance violations being larger than 0.029 nm. The structure has been compared with the X‐ray structure of the oxidized rat mitochondria! isoenzyme and with that of the highly similar bovine microsomal isoenzyme in the oxidized form. The analysis of the elements of secondary structure is instructive in terms of their stability and of their occurrence in related structures, and of the capability of NMR and X‐ray spectroscopy to observe them. Some detailed structural variations are noticed among the solved structures of the various isoenzymes and between solid and solution. The structural features in solution of the residues proposed to be involved in protein‐protein recognition are found to be largely conserved with respect to the solid state.
Methanol: Coenzyme M Methyltransferase from Methanosarcina Barkeri
Authors: Sauer Karin , Thauer Rudolf K.
Published: June 2004

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl‐coenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated methyltransferases 1 (MT1) and 2 (MT2). Transferase MT1, which is composed of a 50‐kDa subunit, MtaB, and a 27‐kDa corrinoid‐harbouring subunit, MtaC, has been shown recently to catalyze the methylation of free cob(I)alamin with methanol [Sauer, K., Harms, U. & Thauer, R. K. (1997) Eur. J. Biochem. 243, 670–677]. We report here that this reaction is catalyzed by subunit MtaB overproduced in Escherichia coli. MtaB also catalyzed the formation of methanol from methylcobalamin and H2O, the hydrolysis being associated with a free‐energy change ΔG0′ of approximately +7.0 kJ/mol. MtaB was found to contain 1 mol zinc, and its activity to be zinc dependent (pKZn2+= 9.3). The zinc dependence of the MT2 (MtaA)‐catalyzed reaction is also described (pKZn2+= 9.6).
The TATA‐Box‐Binding Protein (TBP) of Halobacterium Salinarum
Authors: Soppa Jorg , Link Thomas A.
Published: June 2004

The TATA‐box binding protein (TBP) is a basal transcription factor involved in transcription initiation in Eucarya and Archaea. Using a tbp‐specific probe, a 4.5‐kbp genomic fragment from Halobacterium salinarum was cloned and sequenced. It contained the tbp gene and the 5’‐ends of two additional open reading frames, but surprisingly, 70% of the cloned fragment (3.2 kbp) was devoid of coding capacity or similarity to database sequences.

The deduced halobacterial TBP exhibits sequence similarities to other archaeal (41–43%) as well as to eucaryal (27–38%) TBP. A comparative analysis showed that the archaeal and eucaryal TBP form two related monophylic protein families, and the archaeal TBP possess features which separate them from eucaryal TBP. Compared with the other TBP, the halobacterial TBP is unique in having a high excess of negatively charged residues.

A histidine‐tagged version of the halobacterial TBP was produced in Escherichia coli in a denatured conformation and purified by means of Ni‐chelating chromatography. CD spectroscopy was used to monitor TBP secondary structure and the conditions necessary for folding it into a native conformation. In the absence of denaturating agents, the folded as well as the unfolded state were found to be stable over a wide range of salt concentrations. Properly folded TBP was shown to bind to a halobacterial TATA‐box‐containing DNA fragment, indicating that the fusion protein can be used to characterize DNA recognition by the halobacterial TBP.
Identification and Analysis of a Static Culture‐Specific Cell Wall Protein, Tir1p/Srp1p in Saccharomyces Cerevisiae
Authors: Kitagaki Hiroshi , Shimoi Hitoshi , Itoh Kiyoshi
Published: June 2004

A 100‐kDa protein was found to be a major cell wall protein in Saccharomyces cerevisiae cells cultured without shaking, but was not present in cells cultured with shaking. The amino acid sequence of this protein was identical to the sequence of Tir1p/Srp1p. T1R1/SRP1 has previously been identified as a gene induced by glucose, cold shock or anaerobiosis and was believed to be a cell membrane protein but not a cell wall protein. However, we found that β‐1,3‐glucanase solubilized Tir1p/Srp1p from the cell wall and the purified Tir1p/Srp1p reacted with antiserum to β‐1,6‐glucan and contained glucose. These results suggest that Tir1p/Srp1p is a major structural cell wall protein in the static‐cultured yeast cells and is bound to the cell wall through β‐1,6‐glucan. T1R1/SRP1 mRNA was transcribed only in the static culture and its transcription was regulated by the ROX1 repressor.
The Role of Tyrosine Phosphorylation in Proliferation and Maturation of Erythroid Progenitor Cells
Authors: Klingmüller Ursula
Published: June 2004

Red blood cells arise continuously from pluripotent stem cells which mature and become functionally specialized upon commitment to the erythroid lineage. In mammals, the key regulator of this process is the hormone erythropoietin (EPO). Hormone binding to the cognate receptor, the erythropoietin receptor (EPO‐R), causes receptor homodimerization and transiently triggers tyrosine phosphorylation within target cells. Although the EPO‐R lacks intrinsic enzymatic activity it couples, presumably sequentially, to the protein tyrosine kinase receptor c‐KIT and the cytosolic protein tyrosine kinase JAK2. Signaling through the EPO‐R is promoted by tyrosine phosphorylation of the cytosolic domain and the recruitment of secondary signaling molecules such as the lipid kinase inositolphospholipid 3‐kinase (phosphatidylinositol 3‐kinase) and protein tyrosine phosphatase SHP‐2 to the activated receptor. Complex formation of the activated EPO‐R with the protein tyrosine phosphatase SHP‐1 terminates signaling. In primary fetal liver cells redundant signals emanating from phosphotyrosine residues in the EPO‐R support formation of erythroid colonies in vitro. However, since the last tyrosine residue in the cytosolic domain of the EPO‐R, Y479, uniquely supports in the absence of other tyrosine residues an almost normal level of colony‐forming unit‐erythroid (CFU‐E) colony formation, Y479 represents one of the key residues required in vivo for erythroid proliferation and differentiation. The signal emanating from Y479 involves sequential EPO‐induced recruitment of phosphoinositol lipid 3‐kinase to the EPO‐R and activation of mitogen‐activated‐protein(MAP)kinase activity. The MAP‐kinase signaling cascade could serve as an intracellular switch integrating signals mediated by several phosphotyrosine residues in the cytosolic domain of the EPO‐R and provide a possible explanation for partial redundancy in signaling.
A Molecular Dynamics Study in Explicit Water of the Reduced and Oxidized forms of Yeast Iso‐1‐cytochrome c
Authors: Banci Lucia , Gori‐Savellini Giovanni , Turano Paola
Published: June 2004

Molecular dynamics calculations have been performed over long trajectories with the inclusion of explicit solvent molecules on the reduced and the oxidized states of yeast iso‐1‐cytochrome c. The resulting structures have been analyzed and compared both in terms of structural properties and dynamical behavior. The structure of the buried water molecules around the heme has been also analyzed for the two oxidation states and compared with the experimental observations on the X‐ray and the solution NMR structures. From the overall analysis we learn that, as also observed experimentally through NMR, no significant differences are present between the structures of the two oxidation states beside the arrangement of a few side chains. Also the internal mobility is similar for the two oxidation states, even if interesting differences are observed for some residues, as for Tyr67, a residue present at the heme site. The location and the mobility of the ordered water molecules, observed in solution by NMR, are completely reproduced in the molecular dynamics simulations, which have been able to predict the different displacements of the catalytically relevant water molecule WAT166, similar to those observed in solution for the two oxidation states, at variance with that observed in the starting crystallographic structures. The relevance of these findings with respect to the prediction of structural and dynamical properties is discussed.
Protein Phosphatase β, a Putative Type‐2A Protein Phosphatase from the Human Malaria Parasite Plasmodium Falciparum
Authors: Li Ji‐Liang , Baker David A.
Published: June 2004

Protein phosphatases play a critical role in the regulation of the eukaryotic cell cycle and signal transduction. A putative protein serine/threonine phosphatase gene has been isolated from the human malaria parasite Plasmodium falciparum. The gene has an unusual intron that contains four repeats of 32 nucleotides and displays a high degree of size polymorphism among different strains of P. falciparum. The open reading frame reconstituted by removal of the intron encodes a protein of 466 amino acids with a predicted molecular mass of approximately 53.7 kDa. The encoded protein, termed protein phosphatase β (PP‐β), is composed of two distinct domains. The C‐terminal domain comprises 315 amino acids and exhibits a striking similarity to the catalytic subunits of the type‐2A protein phosphatases. Database searches revealed that the catalytic domain has the highest similarity to Schiwsaccharomyces pombe Ppal (58% identity and 73% similarity). However, it contains a hydrophilic insert consisting of five amino acids. The N‐terminal domain comprises 151 amino acid residues and exhibits several striking features, including high levels of charged amino acids and asparagine, and multiple consensus phosphorylation sites for a number of protein kinases. An overall structural comparison of PP‐β with other members of the protein phosphatase 2A group revealed that PP‐β is more closely related to Saccharomyces cerevisiae PPH22. Southern blots of genomic DNA digests and chromosomal separations showed that PP‐β is a single‐copy gene and is located on chromosome 9. A 2800‐nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage (gametocytes). The results indicate that PP‐β may be involved in sexual stage development.
Purification and Characterization of Transcription Factor IIIA from Higher Plants
Authors: Wyszko Eliza , Barciszewska Mirostawa
Published: June 2004

Transcription factor IIIA (TF IIIA) binds and specifically activates transcription of eukaryotic 5S rRNA genes. It also forms a 7S ribonucleoprotein complex with mature 5S rRNA. Here, we describe the purification and properties of pTF IIIA from higher plants. The purified protein from tulip (Tulipa whittalii) has a molecular mass of about 40 kDa and also binds 5S rRNA and 5S rRNA genes. pTF IIIA also facilitates the transcription of a 5S rRNA gene in a HeLa cell extract.
The Na+‐Translocating NADH:Ubiquinone Oxidoreductase from Vibrio alginolyticus
Authors: Steuber Julia , Krebs Walter , Dimroth Peter
Published: June 2004

The FAD prosthetic group of the Na+‐motive NADH: ubiquinone oxidoreductase (Na+‐NQR) from Vibrio alginolyticus was investigated by ultraviolet–visible and fluorescence spectroscopy. The reduction of Na+‐NQR by excess NADH in the presence of 6–13 μM O2 resulted in the formation of the blue flavosemiquinone radical. If the concentration of dioxygen was further reduced to 0.1 μM O2, neither the reduction of Na+‐NQR by NADH nor its reoxidation with ubiquinone‐1 (Q‐l) yielded a stable flavosemiquinone in equilibrium with reductant or oxidant, respectively, but the fully reduced (FlredH2) or oxidized flavin (Flox) prevailed. During reoxidation of FlredH2 with Q‐l, the intermediate formation of an absorbance band around 800 nm was observed, which was tentatively assigned as the FlredH−‐NAD+ charge‐transfer complex. Complete reoxidation of FlredH2 in Na+‐NQR was achieved by a fivefold excess of Q‐1 over NADH. These results indicated that only a small fraction of FAD was in the flavosemiquinone redox state during turnover to mediate the electron transfer between the hydride donor, NADH, and the one‐electron acceptor [2Fe‐2S]. The titration of Na+‐NQR with Ag+, a specific inhibitor, was followed by the fluorescence emission spectra of FAD (Flox). The addition of Ag+ resulted in a marked increase of the flavin fluorescence (16% at 200 nM Ag+), with half‐maximal saturation at approximately 50 nM Ag+, indicating dissociation of FAD from the enzyme. The increase in fluorescence intensity correlated with the loss of enzyme activity. Gel filtration of the Ag+ treated Na+‐NQR confirmed that FAD had been displaced from the holo‐enzyme.
Differential Splicing of Pre‐Messenger RNA Produces Multiple Forms of Mature Caprine αs1‐Casein
Authors: Ferrant Pasquale , Added Francesco , Malorni Antonio , Chianese Lina , Leroux Christine , Martin Patrice
Published: June 2004

The identity of multiple forms of caprine αs1‐casein in variants A, B, and C has been determined by structural characterisation using mass spectrometry, automated Edman degradation and peptide mapping. Mature goat αs1‐casein exists as a mixture of at least four molecular species which differ in peptide chain length. The main component corresponds to the 199‐residues form already described. The other three, in lesser amounts, were shorter forms of αs1‐casein and differed for the deleted peptides 141–148, as shown previously for ovine αs1‐casein, peptide 110–117, or Gln78. Analysis of αs1‐casein mRNA from milk somatic cells demonstrated that these forms originated from skipping events at the level of exon 13 (codifying for peptide 110–117) and 16 (codifying for peptide 141–148) and from the presence of a cryptic splice site within exon 11 (whose first CAG triplet encodes Gln78) during primary transcript processing. The finding of these splicing abnormalities in the three common variants A, B, and C suggests that this is a general feature of αs1‐casein in goat.

A further source of heterogeneity of caprine αs1‐casein was identified in the discrete phosphorylation of seryl residues. Eight serine residues (at positions 44, 46, 64 to 68 and 75) are fully phosphorylated (except in variant A because of the replacement Glu77→Gln which prevents phosphorylation of Ser75). Conversely, Ser115 and Ser41 are phosphorylated only to about 50% and 20%, respectively. Ser12, although located in a consensus triplet, is never phosphorylated, similarly to the ovine αs1‐casein variants.

These results confirm that there are stabilised mechanisms of simultaneous synthesis of αs1‐casein at different length and of post‐translational modification in both caprine and ovine species.
Effect ofEscherichia coliHeat‐labile Enterotoxin on the Myoelectric Activity of the Duodenum in Weaned Pigs
Authors: Yao G. , Woliński J. , Zabielski R.
Published: June 2004

The objective of the present study was to elucidate the effect of subclinical doses ofEscherichia coliheat‐labile enterotoxin (LT) on the antro‐duodenal myoelectric activities of weaned pigs. Twelve weaned pigs were surgically implanted with three pairs of electrodes on the antrum 3 cm before the pylorus, 5 and 20 cm after the pylorus on the duodenum, respectively. An infusion cannula was inserted into the duodenum between duodenal electrodes. Using a wireless telemetry recording system, an electromyography (EMG) tracing lasting at least 24 h was recorded as the control, then another 24‐h EMG recording was performed with a bolus intraduodenal infusion of LT (0.1 and 0.5 μg/kg b.w.). After a 1‐ to 2‐day break, a 5‐fold higher dose of LT was administered using the same protocol. In the antrum, LT administration barely modified the EMG signal. However, in the duodenum it prolonged the duration of phase II and the migrating myoelectric complex (MMC) cycle when compared with the control. The number of duodenal MMC cycles was also significantly diminished. Moreover, the migrating velocity of phase III was increased. The migrating action potential complex (MAPC) was present both without and with LT, but occurred more frequently following LT administration. In conclusion, LT caused a dose‐dependent, lagged alteration in the duodenal MMC in weaned pigs, involving a reduction of the MMC number by lengthening phase II, increased phase III migration velocity, and increased MAPC frequency. The disturbances did not, however, result in diarrhoea and may reflect the induction of a local protection mechanism of the gut to expel unwanted foreign content from the lumen of the upper gut.
Tissue Doppler Imaging and Echo‐Doppler Findings Associated with a Mitral Valve Stenosis with an Immobile Posterior Valve Leaflet in a Bull Terrier
Authors: Tidholm A. , Nicolle A. P. , Carlos C. , Gouni V. , Caruso J. L. , Pouchelon J. L. , Chetboul V.
Published: June 2004

A mitral valve stenosis was diagnosed in a 2‐year‐old female Bull Terrier by use of two‐dimensional (2‐D) and M‐mode echocardiography, colour‐flow imaging and spectral Doppler examinations. Tissue Doppler Imaging was also performed to assess the segmental radial myocardial motion. The mitral valve stenosis was characterized by a decreased mitral orifice area/left ventricle area ratio (0.14), an increased early diastolic flow velocity (E wave = 1.9 m/s), a prolonged pressure half‐time (106 ms) and a decreased E‐F slope (4.5 cm/s) on pulsed‐wave Doppler examination. This mitral stenosis was associated with an immobile posterior leaflet, as seen on 2‐D and M‐mode echocardiography. Immobility of the posterior mitral leaflet is considered to be a rare finding in humans and, to our knowledge, has not been precisely documented in dogs with mitral valve stenosis.
Metabolic Profiles and Bile Acid Extraction Rate in the Liver of Cows with Fasting‐induced Hepatic Lipidosis
Authors: Mohamed T. , Oikawa S. , Iwasaki Y. , Mizunuma Y. , Takehana K. , Endoh D. , Kurosawa T. , Sato H.
Published: June 2004

This study was designed to monitor lipid profile in the portal and hepatic blood of cows with fasting‐induced hepatic lipidosis, and to compare the results with those in the jugular blood. The work was also carried out to investigate bile acid (BA) in these vessels, and further to investigate BA extraction rate in the liver. Five cows were equipped with catheters in the portal, hepatic and jugular veins (day 0), fasted for 4 days (day 1–day 4) and then refed (day 5–day 11). Before morning feeding, blood was sampled before, during and after fasting from the catheterized vessels. In the portal blood, the concentration of non‐esterified fatty acids (NEFA) showed a progressive increase and at day 5 there was an approximate twofold rise. Increased NEFA concentrations were also found similarly in the other two veins. At day 5,β‐hydroxybutyrate (BHBA) in the portal, hepatic and jugular blood rose to 197, 190 and 186% of the pre‐fasting value, respectively. However, the concentrations of NEFA and BHBA in the three veins gradually returned to pre‐fasting concentration during the refeeding period. Compared with the pre‐fasting value at day 0, the content of liver triglyceride (TG) increased significantly at day 5 (P < 0.01). In the liver, the hepatic extraction rate of BA dropped from 3.1 times pre‐fasting to 2.2 times during fasting. There were no significant differences in the concentrations of glucose, TG, total cholesterol, cholesterol esters, free cholesterol and phospholipids. The results of the current study show that metabolic alterations occur in the portal, hepatic and jugular veins during induction of hepatic lipidosis in cows, and mostly metabolites, with exception of BA concentration, run parallel. The decreased BA extraction rate in the liver of fasted cows was considered to reflect hepatic cell impairment caused by TG accumulation. Hopefully, the findings, at least in part, contribute to the explanation of the pathophysiology of hepatic lipidosis in dairy cows.
Comparison of Plasma/Serum Urea and Creatinine Concentrations in the Dog: A 5‐Year Retrospective Study in a Commercial Veterinary Clinical Pathology Laboratory
Authors: Médaille C. , Trumel C. , Concordet D. , Vergez F. , Braun J. P.
Published: June 2004

Serum/plasma urea (S/P‐urea) and creatinine (S/P‐creatinine) concentrations are routinely assayed as indirect markers of glomerular filtration rate and have been reported to be highly correlated. The aim of this study was to evaluate the relationship between the two analytes in a large number of unselected samples submitted to a commercial laboratory. In 4799 pairs of results, the correlation was high (r = 0.795) and was not affected by sex or age. The relationship between the two analytes was best represented by a second‐order polynomial equation. However, the dispersion of results was large and there was a high percentage of cases (27.5%) where S/P‐urea was increased and S/P‐creatinine normal (≤120 μmol/l for this laboratory), while there was a low percentage of cases where S/P‐creatinine (1.6%) was increased and S/P‐urea normal (≤8 mmol/l for this laboratory). The discrepancy between increases in S/P‐urea and S/P‐creatinine might not only reflect a high incidence of non‐renal factors of variation for S/P‐urea but also an effect of the size or muscle mass of the dogs on the limit of decision for S/P‐creatinine. In dogs with normal S/P‐urea, there was a significant effect of the size on the 0.975 quantile of S/P‐creatinine, ranging from 106 μmol/l in very small dogs to 133 μmol/l in large and very large dogs. This study shows that isolated increases in S/P‐urea could be misleading for the diagnosis of renal diseases and that the reference intervals of S/P‐creatinine should be re‐evaluated according to breed or muscle mass of dogs.
The role of experience in work and learning among design engineers
Authors: Collin Kaija
Published: June 2004

Taking an approach to learning in the workplace as a phenomenon situated and accumulated in the course of work activities and everyday practice, the purpose of this study was to investigate how workers (development engineers and designers) themselves perceive the role of experience in work and learning. The empirical material consists of interviews with 18 employees in two enterprises in Finland. Narrative analysis of the holistic core stories yielded four thematic categories. The categories indicate that learning from experience plays an essential part in coping and learning along with other people, in understanding holistic work processes and in creating a personal perception of one's work and the learning that takes place in its course. It is suggested that the essence of learning through experience in everyday practice challenges the employers, first, to pay attention to the maintenance of a collaborative working atmosphere, second, to arrange possibilities for the continuous analysis of tasks within working teams and networks and, third to see their experienced fellow workers as mentors in order to harness the know‐how embedded in teams and organisations to better benefit all workers.
HRD involvement in the investigative phase of a merger & acquisition
Authors: Waight Consuelo L.
Published: June 2004

This qualitative study describes the involvement of human resource development (HRD) professionals in the investigative phase of merger and acquisitions (M&As). Telephone interviews were completed with 38 HRD professionals and 17 business managers in 12 organizations that had undergone M&As  between  1996 and 1999. The results show that there  is some cognizance between business managers and HRD professionals on what HRD professionals do during an M&A. What is most significant, however is that the activities identified by both business managers and HRD professionals are all human‐capital related; this shows that business managers and some organizations are not only involved in finance related but also human capital due‐diligence during an M&A. The study results show that M&A activities are team‐oriented and HRD professionals need to be prepared to work in different projects during an M&A, especially during the investigative phase. The notion that HRD professionals will be solely working on HRD‐related activities is non‐existent during the investigative phase of an M&A.
Evaluation of cyclooxygenase‐1 and cyclooxygenase‐2 expression and the effect of cyclooxygenase inhibitors in canine prostatic carcinoma
Authors: Sorenmo K. U. , Goldschmidt M. H. , Shofer F. S. , Goldkamp C. , Ferracone J.
Published: June 2004

Prostatic carcinoma occurs primarily in older castrated male dogs and is typically a fatal disease (most dogs die within few months after the initial diagnosis). Surgery, i.e., total prostatectomy, or radiation therapy is often not pursued due to risks of complications and a high rate of distant metastasis. Cyclooxygenase‐2 (Cox‐2) expression has been documented in several malignancies, including canine prostatic carcinoma. Cox‐2 inhibition has been reported to have preventative effects on several human malignancies and has therapeutic effects on both laboratory and spontaneous tumour models. The purpose of this retrospective study was to evaluate Cox expression and the effects of Cox inhibitors on survival in dogs with prostatic carcinoma. 94.1 and 88.2% of the tumours expressed Cox‐1 and Cox‐2, respectively. Furthermore, dogs treated with Cox inhibitors (piroxicam or carprofen) lived significantly longer than untreated dogs, 6.9 versus 0.7 months (P < 0.0001), suggesting that Cox inhibitors may have an important role in canine prostate cancer therapy.
Calponin expression and myoepithelial cell differentiation in canine, feline and human mammary simple carcinomas
Authors: De Las Mulas J. Martín , Reymundo C. , De Los Monteros A. Espinosa , Millán Y. , Ordás J.
Published: June 2004

Calponin is a 34‐kDa smooth muscle‐specific protein that has been shown to be a highly sensitive marker of myoepithelial cells in canine, feline and human mammary tissue and tumours. The expression of calponin was studied in 15 canine, 32 feline and 28 human simple mammary carcinomas using a monoclonal mouse antihuman calponin antibody and the avidin–biotin peroxidase complex (ABC) immunohistochemical technique. Calponin expression was compared with the expression of cytokeratin 14, a marker of normal mammary myoepithelial cells in the three species. Four different types of calponin‐positive cells were identified: (1) Type 1: cytokeratin‐14‐positive pre‐existing myoepithelial cells forming a continuous layer with images of focal disruptions; (2) Type 2: cytokeratin‐14‐positive isolated nests of fusiform, polygonal or round cells without atypia; (3) Type 3: cytokeratin‐14‐positive atypical cells indistinguishable from non‐reactive atypical cells, which should have never been detected in haematoxylin and eosin‐stained sections and (4) Type 4: cytokeratin‐14‐negative stromal fusiform cells around the neoplastic growth or cell nests, identified as myofibroblasts. Calponin‐negative and cytokeratin‐14‐positive atypical neoplastic cells were observed in three canine, 28 feline and two human carcinomas. The latter were indicative of altered expression of high‐molecular‐weight cytokeratins in luminal epithelial‐type simple carcinomas. Our findings show that calponin is a good marker of myoepithelial cell differentiation in feline, human and, particularly, canine simple carcinomas. The high number (six out of 15) of canine tumours with type 3 cells points to the need of both introducing calponin examination in the routine diagnostic schedule and performing further studies on its prognostic significance.
Parameter Analysis of the Differential Model of Hysteresis
Authors: Ma F. , Zhang H. , Bockstedte A. , Foliente G. C. , Paevere P.
Published: June 2004

The extended Bouc-Wen differential model is one of the most widely accepted phenomenological models of hysteresis in mechanics. It is routinely used in the characterization of nonlinear damping and in system identification. In this paper, the differential model of hysteresis is carefully reexamined and two significant issues are uncovered. First, it is shown that the unspecified parameters of the model are functionally redundant. One of the parameters can be eliminated through suitable transformations in the parameter space. Second, local and global sensitivity analyses are conducted to assess the relative sensitivity of each model parameter. Through extensive Monte Carlo simulations, it is found that some parameters of the hysteretic model are rather insensitive. If the values of these insensitive parameters are fixed, a greatly simplified model is obtained.
Experimental Investigation on the Plasticity of Hexagonal Aluminum Honeycomb Under Multiaxial Loading
Authors: Mohr Dirk , Doyoyo Mulalo
Published: June 2004

A new custom-built universal biaxial testing device (UBTD) is introduced and successfully used to investigate the response of aluminum honeycomb under various combinations of large shear and compressive strains in its tubular direction. At the macroscopic level, different characteristic regimes are identified in the measured shear and normal stress-strain curves: elastic I, elastic II, nucleation, softening, and crushing. The first elastic regime shows a conventional linear elastic response, whereas the second elastic regime is nonlinear due to the generation of elastic buckles in the honeycomb microstructure. Nucleation is the point at which the cellular structure loses its load carrying capacity as a result of plastic collapse. It precedes a rapid drop of stress levels in the softening regime as pronounced plastic collapse bands emerge in the microstructure. Formation and growth of plastic folds dominate the microstructural response in the crushing phase. The mechanical features of this phase are long stress plateaus for both the corresponding shear and compressive stress-strain curves. Based on these observations, honeycomb plasticity is established by making analogies of plastic hinge lines and folding systems in the cellular microstructure with dislocations and slip line systems in a solid lattice, respectively. The initial yield surface is found to take the form of an ellipse in stress space, while the crushing behavior is described by a linear envelope along with a nonassociated flow rule based on total strain increments.
Metastability of Natural Gas Hydrates in the Presence of N2 and H2S
Authors: Lubas´ Jan , Darłak Barbara
Published: June 2004

The paper presents the results of laboratory experiments on formation and dissociation of hydrates of natural gases containing nitrogen and hydrogen sulphide. The hydrate metastability region and subcooling temperatures were measured and analyzed. The relationship between the gas composition and temperature of subcooling is investigated.
Analytical Solution of Coupled Diffusion Equations in Semi-Infinite Media
Authors: Fudym O. , Batsale J.-C. , Santander R. , Bubnovich V.
Published: June 2004

Some analytical solutions of the one dimensional transient coupled heat and mass transfer linear equations in a semi-infinite medium are proposed, based on an extension of the thermal quadrupole formalism, in both axial and radial coordinate systems. A direct relationship is proposed between the local temperature and moisture content, both at wall and inner locations. For a previous characterized medium, these relationships seem to be relevant in order to deduce the moisture content from temperature measurements. Another application would be the transport parameters estimation.
EHD-Enhanced Drying With Auxiliary Heating From Below
Authors: Wong D. S. , Lai F. C.
Published: June 2004

The present study investigates the possible drying enhancement provided by auxiliary heating to the sample in addition to the application of electric field. The electric field is produced by charging a needle electrode with direct current at a sufficiently high voltage. The auxiliary heating is provided through a thermofoil heater attached to the bottom plate of the sample container. The samples used are 3 mm and 6 mm glass beads saturated with water. Both positive and negative corona discharges have been employed. Two sets of experiments have been conducted, with and without cross-flow, at 1 kV increment from the corona threshold voltage until the occurrence of sparkover. For each case, a companion experiment was carried out simultaneously under the same ambient conditions but without the application of electric field or auxiliary heating. The result of which is used as a basis in the evaluation of drying enhancement using electric field (with or without cross-flow). The drying enhancement is evaluated as a function of the governing electric field parameters. In addition, the performance of EHD drying is compared with the conventional drying techniques.
Transient Behavior of Crossflow Heat Exchangers With Longitudinal Conduction and Axial Dispersion
Authors: Mishra Manish , Das P. K. , Sarangi Sunil
Published: June 2004

Transient temperature response of the crossflow heat exchangers with finite wall capacitance and both fluids unmixed is investigated numerically for step, ramp and exponential perturbations provided in hot fluid inlet temperature. Effect of two-dimensional longitudinal conduction in separating sheet and axial dispersion in fluids on the transient response has been investigated. Conductive heat transport due to presence of axial dispersion in fluids have been analyzed in detail and shown that presence of axial dispersion in both of the fluid streams neutralizes the total conductive heat transport during the energy balance. It has also been shown that the presence of axial dispersion of high order reduces the effect of longitudinal conduction.
A Combined Fourier Series–Galerkin Method for the Analysis of Functionally Graded Beams
Authors: Zhu H. , Sankar B. V.
Published: June 2004

The method of Fourier analysis is combined with the Galerkin method for solving the two-dimensional elasticity equations for a functionally graded beam subjected to transverse loads. The variation of the Young’s modulus through the thickness is given by a polynomial in the thickness coordinate and the Poisson’s ratio is assumed to be constant. The Fourier series method is used to reduce the partial differential equations to a pair of ordinary differential equations, which are solved using the Galerkin method. Results for bending stresses and transverse shear stresses in various beams show excellent agreement with available exact solutions. The method will be useful in analyzing functionally graded structures with arbitrary variation of properties.
Frictional Collapse of Granular Assemblies
Authors: Suiker Akke S. J. , Fleck Norman A.
Published: June 2004

The frictional collapse of an assembly of equisized spheres is studied by a discrete element model. The macroscopic constitutive response is determined as a function of the level of Coulomb friction between particles. It is found that the level of Coulomb friction has a strong effect upon the relative proportion of sliding and rolling between particles, and consequently upon the macroscopic strength of the granular assembly. The discrete element predictions are shown to be in good agreement with experimental results obtained from triaxial tests on an aggregate of steel spheres. It is demonstrated that the shape of the collapse surface can be adequately represented by the Lade-Duncan continuum model.
A Nondimensional Number to Classify Composite Compressive Failure
Authors: Yerramalli Chandra S. , Waas Anthony M.
Published: June 2004

A new nondimensional number (η) to predict the dominant failure mechanism of fiber reinforced composites under compression loading is presented. Results from previous experimental investigations on the failure of glass fiber reinforced and carbon fiber reinforced vinylester matrix composites, respectively, were used to motivate and develop η. Experimental results available in the open literature are used to compare the predictions of η. This number can be used as a design tool to develop new composite materials with a preferred failure mode. The exercise of developing such a number provides insight into parameters that control the compressive strength of fiber reinforced composite materials.
Measurement and Simulation of the Performance of a Lightweight Metallic Sandwich Structure With a Tetrahedral Truss Core
Authors: Rathbun H. J. , Wei Z. , He M. Y. , Zok F. W. , Evans A. G. , Sypeck D. J. , Wadley H. N. G.
Published: June 2004

Metallic sandwich panels with tetrahedral truss cores have been fabricated and their structural performance evaluated. A fabrication technique involving deformation-shaping and brazing has been used. The responses of the structure in core shear and panel bending have been measured. The results demonstrate robust behavior beyond the limit load. A finite element simulation of the core shear response duplicates the features found experimentally. When combined with the constitutive properties of the face sheet material, these shear characteristics have been shown to predict, with good fidelity, the limit load for panels in bending.
Antimalarial Quinolines and Artemisinin Inhibit Endocytosis in
Plasmodium falciparum
Authors: Hoppe Heinrich C. , van Schalkwyk Donelly A. , Wiehart Ursula I. M. , Meredith Sandra A. , Egan Joanne , Weber Brandon W.
Published: June 2004

Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake. Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole. In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in
Plasmodium falciparum
. By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite. During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers. By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40%. Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion. This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin. These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites.
In Vitro and In Vivo Activities of E5700 and ER-119884, Two Novel Orally Active Squalene Synthase Inhibitors, against
Trypanosoma cruzi
Authors: Urbina Julio A. , Concepcion Juan Luis , Caldera Aura , Payares Gilberto , Sanoja Cristina , Otomo Takeshi , Hiyoshi Hironobu
Published: June 2004

Chagas' disease is a serious public health problem in Latin America, and no treatment is available for the prevalent chronic stage. Its causative agent,
Trypanosoma cruzi
, requires specific endogenous sterols for survival, and we have recently demonstrated that squalene synthase (SQS) is a promising target for antiparasitic chemotherapy. E5700 and ER-119884 are quinuclidine-based inhibitors of mammalian SQS that are currently in development as cholesterol- and triglyceride-lowering agents in humans. These compounds were found to be potent noncompetitive or mixed-type inhibitors of
T. cruzi
SQS with
K

i

values in the low nanomolar to subnanomolar range in the absence or presence of 20 μM inorganic pyrophosphate. The antiproliferative 50% inhibitory concentrations of the compounds against extracellular epimastigotes and intracellular amastigotes were ca. 10 nM and 0.4 to 1.6 nM, respectively, with no effects on host cells. When treated with these compounds at the MIC, all of the parasite's sterols disappeared from the parasite cells. In vivo studies indicated that E5700 was able to provide full protection against death and completely arrested the development of parasitemia when given at a concentration of 50 mg/kg of body weight/day for 30 days, while ER-119884 provided only partial protection. This is the first report of an orally active SQS inhibitor that is capable of providing complete protection against fulminant, acute Chagas' disease.
Intracellular and Plasma Pharmacokinetics of Saquinavir-Ritonavir, Administered at 1,600/100 Milligrams Once Daily in Human Immunodeficiency Virus-Infected Patients
Authors: Ford Jennifer , Boffito Marta , Wildfire Adrian , Hill Andrew , Back David , Khoo Saye , Nelson Mark , Moyle Graeme , Gazzard Brian , Pozniak Anton
Published: June 2004

Ritonavir-boosted saquinavir (SQV/r) is currently licensed as a twice-daily regimen. Reducing the pill burden with once-daily dosing may improve adherence. Intracellular concentrations of drugs must be related to the clinical efficacy of protease inhibitors. The aims of the study were to determine the cellular and plasma saquinavir and ritonavir concentrations, to determine the half-lives (t1/2s) of the drugs in each compartment, and to examine relationships between drug accumulation and lymphocyte subset P glycoprotein (P-gp) expression. Venous blood samples from 12 human immunodeficiency virus-infected patients receiving a hard-gel formulation of SQV/r (1,600/100 mg once daily) were collected at 2, 6, 12, and 24 h after dosing. Peripheral blood mononuclear cells were separated by density gradient centrifugation, and P-gp expression was measured by dual-color flow cytometry. Plasma and intracellular (cell-associated) drug concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry. The ratio of the intracellular drug area under the concentration-time curve from 0 to 24 h (AUC0-24 h) to plasma drug AUC0-24 hwas calculated to determine cellular drug accumulation. The median (range) AUC0-24 hof saquinavir in plasma was 16.2 (5.7 to 39.3) mg · h · liter−1, and that in cells was 46.3 (24.7 to 114.6) mg · h · liter−1. Corresponding ritonavir values were 7.5 (1.5 to 14.6) mg · h · liter−1and 10.4 (3.2 to 13.7) mg · h · liter−1, respectively. The median accumulation ratios of cellular AUC to plasma AUC for saquinavir and ritonavir were 3.31 (range, 1.49 to 6.69) and 1.46 (range, 0.83 to 4.15), respectively. Significant differences between the plasma and intracellular saquinavirt1/2s (4.5 h [range, 2.5 to 9.3 h] and 5.9 h [range, 4.0 to 17.7 h];P= 0.034) and between the plasma and intracellular ritonavirt1/2s (4.1 h [range, 2.6 to 8.3 h] and 6.2 h [range, 3.9 to 18.6 h];P= 0.032) were observed. No relationship was observed between the accumulation of saquinavir or ritonavir and lymphocyte subset P-gp expression. The intracellulart1/2s of saquinavir and ritonavir were longer than the plasmat1/2s, indicating that intracellular drug may be available at a time when concentrations in plasma are below the minimum effective concentration.
Diversity and Evolution of the Class A Chromosomal Beta-Lactamase Gene inKlebsiella pneumoniae
Authors: Hæggman S. , Löfdahl S. , Paauw A. , Verhoef J. , Brisse S.
Published: June 2004

We investigated the diversity of the chromosomal class A beta-lactamase gene inKlebsiella pneumoniaein order to study the evolution of the gene. A 789-bp portion was sequenced in a panel of 28 strains, representative of three phylogenetic groups, KpI, KpII, and KpIII, recently identified inK. pneumoniaeand of different chromosomal beta-lactamase variants previously identified. Three groups of sequences were found, two of them corresponding to the families SHV (pI 7.6) and LEN (pI 7.1), respectively, and one, more heterogeneous, corresponding to a new family that we named OKP (for otherK. pneumoniaebeta-lactamase). Levels of susceptibility to ampicillin, cefuroxime, cefotaxime, ceftazidime, and aztreonam and inhibition by clavulanic acid were similar in the three groups. One new SHV variant, seven new LEN variants, and four OKP variants were identified. The OKP variants formed two subgroups based on nucleotide sequences, one with pIs of 7.8 and 8.1 and the other with pIs of 6.5 and 7.0. The nucleotide sequences of the housekeeping genesgyrA, coding for subunit A of gyrase, andmdh, coding for malate dehydrogenase, were also determined. Phylogenetic analysis of the three genes studied revealed parallel evolution, with the SHV, OKP, and LEN beta-lactamase families corresponding to the phylogenetic groups KpI, KpII, and KpIII, respectively. This correspondence was fully confirmed for 34 additional strains in PCR assays specific for the three beta-lactamase families. We estimated the time since divergence of the phylogenetic groups KpI and KpIII at between 6 and 28 million years, confirming the ancient presence of the beta-lactamase gene in the genome ofK. pneumoniae.