bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimation of prognosis after hepatectomy for hepatocellular carcinoma
Authors: Lee W-C , Jeng L-B , Chen M-F
Published: March 2003

Background

The preferred means of treatment for hepatocellular carcinoma is surgical resection. However, the tumour recurrence rate is high. Accurate estimation of the risk of tumour recurrence after hepatectomy may facilitate the administration of adjuvant therapy after hepatectomy to patients with a high likelihood of tumour recurrence.

Methods

The clinical and pathological profiles of 176 patients undergoing hepatectomy for hepatocellular carcinoma from March 1992 to August 1998 were reviewed. The Kaplan–Meier method and log rank test were used to analyse univariate prognostic factors. The Cox proportional hazard model was used for multivariate analysis. Disease-free and overall cumulative survival rates were estimated with respect to the number of prognostic factors.

Results

Independent factors associated with a lower disease-free survival included the presence of venous infiltration, presence of daughter tumours, absence of tumour encapsulation and tumour size exceeding 5 cm. Factors decreasing the overall survival rate included the presence of venous infiltration, absence of tumour encapsulation and surgical resection margin less than 1 cm. The 1-year disease-free survival rate decreased from 77·5(s.e. 5·6) to 14·0(8·5) per cent when the number of risk factors present increased from zero to three. The 5-year survival rate decreased from 60·2(11·7) per cent to zero when the number of risk factors increased from zero to three.

Conclusion

The deterioration of disease-free or overall survival of patients with hepatocellular carcinoma after hepatectomy correlates with increasing number of risk factors. The number of risk factors can be employed to accurately estimate disease-free and overall survival.
Postoperative body-weight loss and survival after curative resection for gastric cancer
Authors: Yu W , Seo B Y , Chung H Y
Published: March 2003

Background

Body-weight loss has been reported as a poor prognostic factor for some malignancies. The purpose of this study was to evaluate the prognostic value of postoperative body-weight loss in patients with gastric cancer.

Methods

In 564 patients who underwent curative resection for gastric cancer, usual body-weight, body-weight at the time of resection and that 6 and 12 months after resection were recorded prospectively.

Results

The 5-year survival rate of patients who lost more than 5 per cent of their 6-month postoperative weight by 12 months after resection was 63 per cent while that of patients who maintained 95 per cent or more of their 6-month postoperative weight was 84 per cent (P < 0·001). Multivariate analysis revealed that serosal invasion, nodal metastasis, body-weight loss during the second 6-month interval after resection and extent of gastric resection were independent prognostic indicators.

Conclusion

When a patient loses body-weight during the second 6-month interval after curative resection for gastric cancer, recurrent disease should be suspected.
Randomized clinical trial of long-term outcome after resection of hepatitis C virus-related hepatocellular carcinoma by postoperative interferon therapy
Authors: Kubo S , Nishiguchi S , Hirohashi K , Tanaka H , Shuto T , Kinoshita H
Published: March 2003

Background

Interferon therapy seems to decrease the incidence of recurrence after resection of hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Effects of postoperative interferon therapy on the survival rate after resection of such HCC are still unclear.

Methods

A prospective randomized clinical trial of postoperative interferon therapy was performed. Thirty men were allocated randomly after liver resection to an interferon-α group (15 patients) or a control group. Patients in the interferon group received interferon-α 6 MIU intramuscularly every day for 2 weeks, then three times a week for 14 weeks and finally twice a week for 88 weeks.

Results

The response to interferon was complete in two patients, there was a biochemical response in six patients and no response in seven patients. Interferon administration was not completed in three patients because of adverse events. Liver function did not change or worsened after operation in the control group, and did not change or improved in the interferon group. The cumulative survival rate was higher in the interferon group than in the control group (P = 0·041).

Conclusion

Postoperative interferon therapy seems to improve the outcome after resection of HCV-related HCC.
Results of laparoscopic Nissen fundoplication at 2–8 years after surgery
Authors: Booth M I , Jones L , Stratford J , Dehn T C B
Published: March 2003

Background

Although the long-term results of open fundoplication for gastro-oesophageal reflux disease are well documented, there have been few reports of the long-term results of laparoscopic fundoplication.

Methods

Between January 1993 and July 1999, 179 consecutive patients underwent laparoscopic floppy Nissen fundoplication. Of these, 175 were available for long-term follow-up. Structured symptom questionnaires were completed by 140 patients (80 per cent) at 2–5 years (n = 92) or 5–8 years (n = 48) after operation.

Results

Patient satisfaction with surgery was 91 per cent at a median follow-up of 48 (range 24–99) months. Visick scores of I or II were recorded by 84 per cent. Ninety per cent of patients remained free from significant reflux symptoms. Side-effects were common (22 per cent) but rarely affected patient satisfaction. Of the 19 patients (14 per cent) taking regular antireflux medication, eight used it for non-reflux symptoms and 12 had normal postoperative pH tests.

Conclusion

Laparoscopic floppy Nissen fundoplication is an effective and durable treatment for gastro-oesophageal reflux disease. Longer-term follow-up of patients operated on beyond the learning curve can be expected to show further improvements in surgical outcome.
E2F1‐mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 gene
Authors: Koziczak Magdalena , Müller Heiko , Helin Kristian , Nagamine Yoshikuni
Published: March 2003

Gene expression of the plasminogen activation system is cell‐cycle dependent. Previously, we showed that ectopic expression of E2F1 repressed the plasminogen activator inhibitor type 1 (PAI‐1) promoter in a manner dependent on the presence of DNA‐binding and transactivation domains of E2F1 but independent of binding to pocket‐binding proteins, suggesting a novel mechanism for E2F‐mediated negative gene regulation [Koziczak, M., Krek, W. & Nagamine, Y. (2000) Mol. Cell. Biol.20, 2014–2022]. However, it remains to be seen whether endogenous E2F can exert a similar effect. We report here that down‐regulation of PAI‐1 gene expression correlates with an increase in endogenous E2F activity. When cells were treated with a cdk2/4‐specific inhibitor, which maintains E2F in an inactive state, the decline of serum‐induced PAI‐1 mRNA levels was suppressed. In mutant U2OS cells expressing a temperature‐sensitive retinoblastoma protein (pRB), a shift to a permissive temperature induced PAI‐1 mRNA expression. In U2OS cells stably expressing an E2F1‐estrogen receptor chimeric protein that could be activated by tamoxifen, PAI‐1 gene transcription was markedly reduced by tamoxifen even in the presence of cycloheximide. These results all indicate that endogenous E2F can directly repress the PAI‐1 gene. DNase I hypersensitive‐site analysis of the PAI‐1 promoter suggested the involvement of conformation changes in chromatin structure of the PAI‐1 promoter. 5′ deletion analysis of the PAI‐1 promoter showed that multiple sites were responsible for the E2F negative regulation, some of which were promoter dependent. Interestingly, one of these sites is a p53‐binding element.
Processing of natural and recombinant CXCR3‐targeting chemokines and implications for biological activity
Authors: Hensbergen Paul J. , van der Raaij‐Helmer Elisabeth M. H. , Dijkman Remco , van der Schors Roel C. , Werner‐Felmayer Gabriele , Boorsma Dick M. , Scheper Rik J. , Willemze Rein , Tensen Cornelis P.
Published: March 2003

Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post‐translational modifications have been shown to affect the effectiveness of chemokines.

Although in several studies, natural CXCR3‐targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3‐targeting chemokines expressed by interferon‐γ‐stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays.

We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C‐terminally truncated CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to an N‐terminally truncated human CXCL11, lacking the first two amino acids FP, indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3‐expressing T‐cells.

It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.
Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I
Authors: Szabò Ildikò , Rigoni Fernanda , Bianchetti Maria , Carbonera Donatella , Pierantoni Francesca , Seraglia Roberta , Segalla Anna , Giacometti Giorgio M.
Published: March 2003

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47–RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47–RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47–RC in studying primary reactions of oxygenic photosynthesis.
The role of the TolC family in protein transport and multidrug efflux.
Authors: Sharff Andrew , Fanutti Cristina , Shi Jiye , Calladine Chris , Luisi Ben
Published: March 2003

Gram‐negative bacteria are enveloped by a system of two membranes, and they use specialized multicomponent, energy‐driven pumps to transport molecules directly across this double‐layered partition from the cell interior to the extra‐cellular environment. One component of these pumps is embedded in the outer‐membrane, and the paradigm for its structure and function is the TolC protein from Escherichia coli. A common component of a wide variety of efflux pumps, TolC and its homologues are involved in the export of chemically diverse molecules ranging from large protein toxins, such as α‐hemolysin, to small toxic compounds, such as antibiotics. TolC family members thus play important roles in conferring pathogenic bacteria with both virulence and multidrug resistance. These pumps assemble reversibly in a transient process that brings together TolC or its homologue, an inner‐membrane‐associated periplasmic component, an integral inner‐membrane translocase and the substrate itself. TolC can associate in this fashion with a variety of different partners to participate in the transport of diverse substrates. We review here the structure and function of TolC and the other components of the efflux/transport pump.
B″‐associated factor(s) involved in RNA polymerase III preinitiation complex formation and start‐site selection
Authors: Andrau Jean‐Christophe , Werner Michel
Published: March 2003

The TFIIIB transcription factor is the central component of the RNA polymerase III transcriptional machinery. In yeast, this factor is composed of three essential polypeptides TBP, TFIIIB70 and TFIIIB90, that are sufficient as recombinant proteins, together with TFIIIC, to promote accurate transcription in vitro. Here we show that a partially purified fraction, named B″, that contains the TFIIIB90 subunit, displays properties distinct from recombinant TFIIIB90. This fraction contains at least a component that interacts with DNA•TFIIIC complexes, either alone or in combination with TFIIIB90, and increases the resistance of the complexes to heparin treatment. In addition, primer extension and single round transcriptions experiment reveal a different start‐site selection pattern directed by B″ or rTFIIIB90. In mixing experiments, we show that an activity in B″, distinct from TFIIIB90, can promote transcription initiation at the +1 site without affecting the rate of preinitiation complex formation. Our data suggest the existence of at least one new component that participates in preinitiation complex formation and influences start‐site selection by RNA polymerase III.
Mutations at position 125 in transmembrane helix III of rhodopsin affect the structure and signalling of the receptor
Authors: Andrés Anna , Kosoy Ana , Garriga Pere , Manyosa Joan
Published: March 2003

Mutation of L125R in trasmembrane helix III of rhodopsin, associated with the retinal degenerative disease retinitis pigmentosa, was previously shown to cause structural misfolding of the mutant protein. Also, conservative mutations at this position were found to cause partial misfolding of the mutant receptors. We report here on a series of mutations at position 125 to further investigate the role of Leu125 in the correct folding and function of rhodopsin. In particular, the effect of the size of the substituted amino‐acid side chain in the functionality of the receptor, measured as the ability of the mutant rhodopsins to activate the G protein transducin, has been analysed. The following mutations have been studied: L125G, L125N, L125I, L125H, L125P, L125T, L125D, L125E, L125Y and L125W. Most of the mutant proteins, expressed in COS‐1 cells, showed reduced 11‐cis‐retinal binding, red‐shifts in the wavelength of the visible absorbance maximum, and increased reactivity towards hydroxylamine in the dark. Thermal stability in the dark was reduced, particularly for L125P, L125Y and L125W mutants. The ability of the mutant rhodopsins to activate the G protein transducin was significantly reduced in a size dependent manner, especially in the case of the bulkier L125Y and L125W substitutions, suggesting a steric effect of the substituted amino acid. On the basis of the present and previous results, Leu125 in transmembrane helix III of rhodopsin, in the vicinity of the β‐ionone ring of 11‐cis‐retinal, is proposed to be an important residue in maintaining the correct structure of the chromophore binding pocket. Thus, bulky substitutions at this position may affect the structure and signallling of the receptor by altering the optimal conformation of the retinal binding pocket, rather than by direct interaction with the chromophore, as seen from the recent crystallographic structure of rhodopsin.
Female‐specific expression of a hexamerin gene in larvae of an autogenous mosquito
Authors: Zakharkin Stanislav O. , Headley Violetta V. , Kumar Nagothu K. , Buck Norman A. , Wheeler Diana E. , Beneš Helen
Published: March 2003

Fourth‐instar larvae of the autogenous mosquito, Aedes atropalpus, synthesize three hexamerins or hexameric storage proteins which are distinguished by different methionine and aromatic amino‐acid contents. One protein, Hexamerin‐1.2 (AatHex‐1.2) is only found in female larvae and pupae. In order to investigate the molecular basis for this sex‐specific accumulation, we have cloned and sequenced the cDNA encoding AatHex‐1.2 and isolated and sequenced over 1 kb of the 5′ flanking region of the AatHex‐1.2 gene. The AatHex‐1.2 transcript encodes a 81.6‐kDa hexamerin subunit which contains 19.8% phenylalanine, tyrosine and tryptophan and 8.6% methionine residues. The single‐copy AatHex‐1.2 gene consists of three exons and two small introns located at its 5′ end. A 2.3‐kb AatHex‐1.2 mRNA accumulates only in female larvae and pupae and is expressed at very low levels in adult female mosquitoes. The temporal expression profile of this transcript is typical of other mosquito hexamerin genes, with rapid disappearance of the mRNA shortly after pupation. Hence this is the first observation of exclusively female‐specific gene activity during preadult development of an insect. In the 5′ flanking region of the AatHex‐1.2 gene, we identified putative binding sites for transcription factors, such as GATA, C/EBP and Doublesex, typically involved in fat body‐ and female‐specific gene activity in Diptera. These findings suggest that mechanisms for sex‐specific transcription in the fat body may be well conserved between flies and mosquitoes.
Identification of a critical lysine residue at the active site in glyceraldehyde‐3‐phosphate dehydrogenase of Ehrlich ascites carcinoma cell
Authors: Ghosh Swapna , Mukherjee Kasturi , Ray Manju , Ray Subhankar
Published: March 2003

The involvement of the lysine residue present at the active site of Ehrlich ascites carcinoma (EAC) cell glyceraldehyde‐3‐phosphate dehydrogenase (Gra3PDH) was investigated by using the lysine specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal phosphate (PP). Both TNBS and PP inactivated EAC cell Gra3PDH with pseudo‐first‐order kinetics with the rate dependent on modifier concentration. Kinetic analysis, including a Tsou plot, indicated that both TNBS and PP apparently react with one lysine residue per enzyme molecule. Two of the substrates, d‐glyceraldehyde‐3‐phosphate and NAD, and also NADH, the product and competitive inhibitor, almost completely protected the enzyme from inactivation by TNBS. A comparative study of Gra3PDH of EAC cell and rabbit muscle indicates that the nature of active site of the enzyme is significantly different in these two cells. A double inhibition study using 5,5′‐dithiobis(2‐nitrobenzoic acid) and TNBS and subsequent reactivation of only the rabbit muscle enzyme by dithiothreitol suggested that a cysteine residue of this enzyme possibly reacts with TNBS. These studies on the other hand, confirm that an essential lysine residue is involved in the catalytic activity of the EAC cell enzyme. This difference in the nature of the active site of EAC cell Gra3PDH that may be related to the high glycolysis of malignant cells has been discussed.
Identification of molecular determinants required for interaction of ubiquitin‐conjugating enzymes and RING finger proteins
Authors: Martinez‐Noel Gustavo , Müller Ursula , Harbers Klaus
Published: March 2003

Recent results from several laboratories suggest that the interaction of E2 ubiquitin‐conjugating enzymes with the RING finger domain has a central role in mediating the transfer of ubiquitin to proteins. Here we present a mutational analysis of the interaction between the E2 enzyme UbcM4/UbcH7 and three different RING finger proteins, termed UIPs, which, like Parkin, contain a RING1‐IBR‐RING2 motif. The results show that the E2 enzyme binds to the RING1 domain but not to the other cysteine/histidine‐rich domains of the RING1‐IBR‐RING2 motif. Three regions within the UbcM4 molecule are involved in this interaction: the H1 α helix, loop L1, connecting the third and fourth strand of the β sheet, and loop L2, located between the fourth β strand and the second α helix. Loop L2 plays an important role in determining the specificity of interaction. The effects of L2 mutations on UbcM4/UIP interaction are different for each UIP, indicating that RING finger domains can vary considerably in their structural requirements for binding to E2 enzymes. The result that single amino‐acid changes can regulate binding of E2 enzymes to different RING finger proteins suggests a novel approach to experimentally manipulate proteolytic pathways mediated by RING finger proteins.
Development of new assays and improved procedures for the purification of recombinant human chymase
Authors: Ferry Gilles , Gillet Ludovic , Bruneau Vincent , Banales Jesus M. , Beauverger Philippe , Cogé Francis , Galizzi Jean‐Pierre , Scalbert Elizabeth , Okamoto Takehiro , Urata Hidenori , Boutin Jean A.
Published: March 2003

Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin‐angiotensin system. This enzyme is also involved in other physio‐pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C‐activated, newly raised anti‐chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96‐well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC‐based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium‐throughput screening (up to 10 000 points per day), about 800 µg·L−1 of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.
Tumour‐related enzyme alterations in the clear cell type of human renal cell carcinoma identified by two‐dimensional gel electrophoresis
Authors: Balabanov Stefan , Zimmermann Uwe , Protzel Chris , Scharf Christian , Klebingat Klaus‐Jürgen , Walther Reinhard
Published: March 2003

To identify tumour‐related enzyme alterations we have used 2D‐gels to analyse the proteome from dissected malignant and benign kidney areas from patients with clear‐cell‐type renal carcinoma. The expression of 12 proteins was diminished in tumour. Four proteins were characterized by mass spectrometry and were identified as enoyl‐CoA hydratase, α‐glycerol‐3‐phosphate dehydrogenase, aldehyde dehydrogenase 1 and aminoacylase‐I.
Inhibition of glycosyl‐phosphatidylinositol biosynthesis inPlasmodium falciparumby C‐2 substituted mannose analogues
Authors: Santos de Macedo Cristiana , Gerold Peter , Jung Nicole , Azzouz Nahid , Kimmel Jürgen , Schwarz Ralph T.
Published: March 2003

Mannose analogues (2‐deoxy‐d‐glucose, 2‐deoxy‐2‐fluoro‐d‐glucose and 2‐amino‐2‐deoxy‐d‐mannose) have been used to study glycosylphosphatidylinositol (GPtdIns) biosynthesis and GPtdIns protein anchoring in protozoal and mammalian systems. The effects of these analogues on GPtdIns biosynthesis and GPtdIns‐protein anchoring of the human malaria parasitePlasmodium falciparumwere evaluated in this study. At lower concentrations of 2‐deoxy‐d‐glucose and 2‐deoxy‐2‐fluoro‐dglucose (0.2 and 0.1 mm, respectively), GPtdIns biosynthesis is inhibited without significant effects on total protein biosynthesis. At higher concentrations of 2‐deoxy‐d‐glucose and 2‐deoxy‐2‐fluoro‐d‐glucose (1.5 and 0.8 mm, respectively), the incorporation of [3H]glucosamine into glycolipids was inhibited by 90%, and the attachment of GPtdIns anchor to merozoite surface protein‐1 (MSP‐1) was prevented. However, at these concentrations, both sugar analogues inhibit MSP‐1 synthesis and total protein biosynthesis. In contrast to 2‐deoxy‐2‐fluoro‐d‐glucose and 2‐amino‐2‐deoxy‐d‐mannose (mannosamine), the formation of new glycolipids was observed only in the presence of tritiated or nonradiolabelled 2‐deoxy‐d‐glucose. Mannosamine inhibits GPtdIns biosynthesis at a concentration of 5 mm, but neither an accumulation of aberrant intermediates nor significant inhibition of total protein biosynthesis was observed in the presence of this analogue. Furthermore, the [3H]mannosamine‐labelled glycolipid spectrum resembled the one described for [3H]glucosamine labelling. Total hydrolysis of mannosamine labelled glycolipids showed that half of the tritiated mannosamine incorporated into glycolipids was converted to glucosamine. This high rate of conversion led us to suggest that no actual inhibition from GPtdIns biosynthesis is achieved with the treatment with mannosamine, which is different to what has been observed for mammalian cells and other parasitic protozoa.
The tree squirrel HP‐25 gene is a pseudogene
Authors: Kojima Miho , Shiba Tadayoshi , Kondo Noriaki , Takamatsu Nobuhiko
Published: March 2003

The gene for the hibernation‐specific protein HP‐25 is expressed in the liver in hibernating species of the squirrel family (chipmunk and ground squirrel), but not in a nonhibernating species (tree squirrel). To investigate why the HP‐25 gene is not expressed in the tree squirrel, we isolated the tree squirrel HP‐25 gene and compared its gene structure and promoter activity with that of the chipmunk. The tree squirrel HP‐25 gene is composed of three exons, and the gene structures are conserved between the tree squirrel and chipmunk. However, the tree squirrel HP‐25 gene has an insertional mutation of 13 nucleotides in exon 2 that disrupts the ORF. In the chipmunk HP‐25 gene, the 80‐bp 5′ flanking sequence is sufficient for the liver‐specific promoter activity, and HNF‐4, which binds to the sequence from nucleotides −67 to −51, is involved in its transcriptional regulation. In contrast, the corresponding tree squirrel 5′ flanking sequence had almost no promoter activity in HepG2 cells, and HNF‐4 did not bind to the corresponding region of the tree squirrel HP‐25 gene. Furthermore, a tree squirrel‐type G to A mutation at −57 in the chipmunk HP‐25 gene promoter context abolished its binding to and transactivation by HNF‐4. Thus, the point mutation in the HNF‐4‐binding site is likely to be involved in the lack of HP‐25 gene expression in the tree squirrel.
The sialate‐pyruvate lyase from pig kidney
Authors: Traving Christina , Bruse Petra , Wächter Andrea , Schauer Roland
Published: March 2003

The first complete primary structure of a mammalian sialate‐pyruvate lyase, namely of the enzyme from porcine kidney, was elucidated by a combination of different PCR techniques followed by sequencing of the resulting fragments. The primers used were either deduced from four porcine lyase peptides or from an alignment of human and mouse expressed sequence tags (ESTs), which were found to be homologous to already known microbial lyase sequences, and cDNA alone or after ligation with a plasmid vector served as a template.

The lyase primary structure consists of 319 amino acids with a calculated protein molecular mass of ≈ 35 kDa, which fits well to the value determined for the native enzyme. The porcine lyase sequence made it possible to assemble several ESTs from mouse and man in order to obtain the complete putative lyase genes. The three mammalian sequences reveal a high degree of homology both on the nucleotide (83% of the nucleotides are identical between all three sequences) and on the amino‐acid level (72% of the amino acids are identical between all three sequences), and thus form a tightly related group. In contrast, the identity between the lyase primary structures from pig kidney and the microbial enzyme from Clostridium perfringens is much less pronounced (25%). Thirty‐one amino acids were found to be absolutely conserved in all lyase sequences. Among them are two amino acids (lysine 173 and tyrosine 143 in the porcine lyase) that are most important for the catalytic reaction. After expression cloning, recombinant enzyme activity was expressed in Escherichia coli BL21(DE3)pLysS, which confirms the identity of the cloned sequence and verifies one of the putative human and murine sequences. After SDS/PAGE of a cell extract of the expression clone, a band of 35kDa was stained on the gel.
Expression of glucose transporter‐2, glucokinase and mitochondrial glycerolphosphate dehydrogenase in pancreatic islets during rat ontogenesis
Authors: García‐Flores Marta , Zueco José Antonio , Arenas Joaquín , Blázquez Enrique
Published: March 2003

To gain better insight into the insulin secretory activity of fetal β cells in response to glucose, the expression of glucose transporter 2 (GLUT‐2), glucokinase and mitochondrial glycerol phosphate dehydrogenase (mGDH) were studied. Expression of GLUT‐2 mRNA and protein in pancreatic islets and liver was significantly lower in fetal and suckling rats than in adult rats. The glucokinase content of fetal islets was significantly higher than of suckling and adult rats, and in liver the enzyme appeared for the first time on about day 20 of extrauterine life. The highest content of hexokinase I was found in fetal islets, after which it decreased progressively to the adult values. Glucokinase mRNA was abundantly expressed in the islets of all the experimental groups, whereas in liver it was only present in adults and 20‐day‐old suckling rats. In fetal islets, GLUT‐2 and glucokinase protein and their mRNA increased as a function of increasing glucose concentration, whereas reduced mitochondrial citrate synthase, succinate dehydrogenase and cytochrome c oxidase activities and mGDH expression were observed. These findings, together with those reported by others, may help to explain the decreased insulin secretory activity of fetal β cells in response to glucose.
Interferon‐alpha inhibits Stat5 DNA‐binding in IL‐2 stimulated primary T‐lymphocytes
Authors: Erickson Sven , Matikainen Sampsa , Thyrell Lena , Sangfelt Olle , Julkunen Ilkka , Einhorn Stefan , Grandér Dan
Published: March 2003

It has previously been shown that IFN‐α is a potent inhibitor of IL‐2 induced proliferation in primary T‐lymphocytes, by selectively abrogating the downstream effects of IL‐2 on the core cell cycle machinery regulating the G1/S transition. Theoretically this could be mediated through cross‐talk between the signalling cascades activated by these cytokines, as several signalling components are known to be shared. IL‐2 activates multiple signalling pathways that are important for T‐cell proliferation and differentiation. In the present study, the effects of IFN‐α on IL‐2 signal transduction was investigated. The IFN‐α induced inhibition of IL‐2 induced proliferation in activated T‐lymphocytes, was associated with a suppressed Jak3 protein expression as well as an inhibited prolonged Stat5 DNA binding, and a partially reduced expression of the Stat5 inducible gene IL‐2Rα. Our results provide a possible molecular link between the prominent antiproliferative effects of IFN‐α on IL‐2 induced T‐cell proliferation and the signal transduction pathways emerging from the IL‐2 receptor.
Expression of the uncoupling protein 1 from the aP2 gene promoter stimulates mitochondrial biogenesis in unilocular adipocytes in vivo
Authors: Rossmeisl Martin , Barbatelli Giorgio , Flachs Pavel , Brauner Petr , Zingaretti Maria Cristina , Marelli Mariella , Janovská Petra , Horáková Milada , Syrový Ivo , Cinti Saverio , Kopecký Jan
Published: March 2003

Mitochondrial uncoupling protein 1 (UCP1) is a specific marker of multilocular brown adipocytes. Ectopic UCP1 in white fat of aP2‐Ucp1 mice mitigates development of obesity by both, increasing energy expenditure and decreasing in situ lipogenesis. In order to further analyse consequences of respiratory uncoupling in white fat, the effects of the ectopic UCP1 on the morphology of adipocytes and biogenesis of mitochondria in these cells were studied. In subcutaneous white fat of both aP2‐Ucp1 and young control (5‐week‐old) mice, numerous multilocular adipocytes were found, while they were absent in adult (7‐ to 9‐month‐old) animals. Only unilocular cells were present in epididymal fat of both genotypes. In both fat depots of aP2‐Ucp1 mice, the levels of the UCP1 transcript and UCP1 antigen declined during ageing, and they were higher in subcutaneous than in epididymal fat. Under no circumstances could ectopic UCP1 induce the conversion of unilocular into multilocular adipocytes. Presence of ectopic UCP1 in unilocular adipocytes was associated with the elevation of the transcripts for UCP2 and for subunit IV of mitochondrial cytochrome oxidase (COX IV), and increased content of mitochondrial cytochromes. Electron microscopy indicated changes of mitochondrial morphology and increased mitochondrial content due to ectopic UCP1 in unilocular adipocytes. In 3T3‐L1 adipocytes, 2,4‐dinitrophenol increased the levels of the transcripts for both COX IV and for nuclear respiratory factor‐1. Our results indicate that respiratory uncoupling in unilocular adipocytes of white fat is capable of both inducing mitochondrial biogenesis and reducing development of obesity.
Coincidence of immune‐mediated diseases driven by Th1 and Th 2 subsets suggests a common aetiology. A population‐based study using computerized General Practice data
Authors: Simpson C. R. , Anderson W. J. A. , Helms P. J. , Taylor M. W. , Watson L. , Prescott G. J. , Godden D. J. , Barker R. N.
Published: March 2003

Background The recent rise in the prevalence of immune‐mediated diseases has been attributed to environmental factors such as a lack of microbial challenge, or dietary change, that deviate the overall balance between mutually antagonistic subsets of T helper (Th) cells.

Objective An alternative proposal is that recent environmental changes have resulted in an immune system that is more likely to produce both Th1 and Th 2 responses against benign antigens. The prediction of this hypothesis, that Th1 and Th 2‐mediated diseases are not mutually exclusive, and may be positively associated, is tested here in a whole population.

Methods Data from General Practices participating in the Scottish Continuous Morbidity Recording (CMR) project were used to determine the coincidence of the major Th 2‐mediated atopic diseases; asthma, eczema and allergic rhinitis, with the Th1‐mediated autoimmune conditions; type I diabetes, rheumatoid arthritis and psoriasis. We also identified the prescription rates of inhaled therapy for asthma in patients with Th1‐mediated disease.

Results There was a significant increase in the risk of presenting with a Th1‐mediated autoimmune condition in patients with a history of allergic disease (standardized prevalence ratio (95% confidence interval) 1.28 (1.18–1.37)). Likewise, the standardized prevalence ratios of presenting with either eczema (1.67 (1.48–1.87)) or allergic rhinitis (1.22 (1.02–1.44)) were significantly increased in subjects with a history of Th1‐mediated disease. There was a particularly strong association between current psoriasis and current eczema (standardized prevalence ratio of psoriasis in subjects with eczema 2.88, 95% confidence interval (CI) 2.38–3.45). There was also a significant increase in prescriptions for inhaled asthma therapy in patients with Th1 disease.

Conclusion It is concluded that Th1‐ and Th 2‐mediated diseases are significantly associated in a large General Practice population. This finding supports the proposal that autoimmune and atopic diseases share risk factors that increase the propensity of the immune system to generate both Th1‐ and Th 2‐mediated inappropriate responses to non‐pathological antigens.
A comparison of once daily fexofenadine versus the combination of montelukast plus loratadine on domiciliary nasal peak flow and symptoms in seasonal allergic rhinitis
Authors: Wilson A. M. , Orr L. C. , Coutie W. J. R. , Sims E. J. , Lipworth B. J.
Published: March 2003

Background The combination of montelukast (ML) and loratadine (LT) has previously been shown to be superior to either drug alone in managing seasonal allergic rhinitis (SAR), whilst fexofenadine (FEX) has been shown to be better than LT as monotherapy.

Objectives We wished to compare ML + LT vs. FEX alone for effects on daily measurements (am/pm) of peak inspiratory flow (PIF) and symptoms.

Methods Thirty‐seven patients with SAR (skin prick positive to grass pollen) were randomised into a single‐blind, double‐dummy placebo (PL)‐controlled cross‐over study during the grass pollen season, comparing 2 weeks of once daily treatment with (a) 120 mg FEX or (b) 10 mg ML + 10 mg LT. There was a 7–10 day placebo run‐in and washout prior to each randomised treatment. The average of am/pm PIF (the primary outcome variable) was analysed. Patients recorded their symptom scores (from 0 to 3) twice daily, for nasal blockage, discharge, itching and sneezing with; total eye symptoms, ocular cromoglycate use, and daily activity. The total nasal symptom score was calculated as a composite (out of 24).

Results There were no significant differences between baselines after the run‐in and washout placebos for any variables. There were significant (P < 0.05, Bonferroni) improvements in all symptoms and PIF compared to pooled placebo with both treatments for all end‐points, but no differences between the two treatment regimes (as means and within‐treatment 95% confidence intervals): PIF: PL 102 (98–107), FEX 111 (107–116), ML + LT 113 (109–118); total nasal symptoms: PL 7.4 (6.7–2.0), FEX 5.0 (4.3–5.7), ML + LT 4.0 (3.3–4.7).

Conclusions Once daily FEX as monotherapy was equally effective as the combination of once daily ML + LT in improving nasal peak flow and controlling symptoms in SAR. Further studies are indicated to assess whether ML confers additional benefits to FEX in SAR.
Joint laxity in the index finger and thumb and its relationship to pencil grasps used by children
Authors: Summers Janet
Published: March 2003

The pencil grasps of 55 Australian 7‐year‐old children were analysed to determine if joint laxity in the distal interphalangeal joint of the index finger and the interphalangeal joint and metacarpophalangeal joint of the thumb influenced the development of grasps that deviate from the dynamic tripod grasp. Laxity was determined using torque range of motion, and descriptive analysis of the pencil grasp was undertaken during three writing tasks. Laxity occurred in 71% of the children in at least one joint. Four pencil grasps were identified based on the position of the thumb and the number of digits on the pencil shaft. Trends indicated that laxity, especially in the interphalangeal joint of the thumb, is an influence on the pencil grasp used; however, these trends were not statistically significant.
A survey of the apes in the Dzanga‐Ndoki National Park, Central African Republic: a comparison between the census and survey methods of estimating the gorilla (Gorilla gorilla gorilla) and chimpanzee (Pan troglodytes) nest group density
Authors: Blom A. , Almaši A. , Heitkönig I. M. A. , Kpanou J.‐B. , Prins H. H. T.
Published: March 2003

A survey of apes was carried out between October 1996 and May 1997 in the Dzanga sector of the Dzanga‐Ndoki National Park, Central African Republic (CAR), to estimate gorilla (Gorilla gorilla gorilla) and chimpanzee (Pan troglodytes) densities. The density estimates were based on nest counts. The strip transect census and the line transect survey method (Standing Crop Nest Count) were used to estimate the gorilla nest group density. The strip transect has been most commonly used to date. It assumes that all nest groups within the width of the strip are detected, but as this assumption is easily violated in the dense tropical rain forest, the line transect survey was also used. In this method, only the nest groups on the transect line itself should be detected. This method proved to be an adequate and easy technique for estimating animal densities in dense vegetation. The gorilla density of 1.6 individuals km−2 (line transect survey method) found for the Dzanga sector is one of the highest densities ever reported in the literature for the Western lowland gorilla. The density estimate for chimpanzees was 0.16 individuals km−2 (census method). The results of this study confirm the importance of the Dzanga‐Ndoki National Park for primate conservation.
Bushbuck (Tragelaphus scriptus Pallas) habitat in Mole National Park, northern Ghana
Authors: Dankwa‐Wiredu Bernice , Euler David L.
Published: March 2003

This study examined the habitat and activity patterns of bushbucks (Tragelaphus scriptus Pallas), in Mole National Park, Ghana. Three bushbucks, one with a radio transmitter attached to its ear, served as focal‐animals for the study. The animals were followed and data on their habitat, food and activity patterns were recorded.

Bushbucks were found to have a low variability in their diet. The forage species were mainly herbaceous and dicotyledonous. Each individual developed its own activity pattern and followed specific paths while foraging. Bushbucks used marshes proportionally more than the open savanna and the riverine forest. At temperatures below 30°C bushbucks remained active. When the temperature exceeded 31°C, the bushbucks retreated to the bushes/thickets to rest and ruminate.
Fibromyalgia in Maori and European New Zealanders
Authors: Klemp Patrick , Williams Sheila M. , Stansfield Shelley A.
Published: March 2003

Aim: The objectives of this study were to determine: (i) the prevalence of fibromyalgia (FM) in Maori and European New Zealanders; and (ii) whether an association exists between hypermobility and FM.

Methods: The 1990 American College of Rheumatology classification criteria were used to determine the prevalence of FM (both criteria), widespread pain (WP) only, and a tender point score (TPS) ≥ 11 only, in subjects aged 12 years and older. Beighton's method was used to determine hypermobility.

Results: The prevalence of FM in Maori (1.1%) and European (1.5%) New Zealanders was similar (an overall prevalence of 1.3%); 1.7% Maori and 3.9% Europeans had WP only; and 1.7% Maori and 0.9% Europeans had a TPS of ≥ 11 only. In Maori, the prevalence of WP and a TPS ≥ 11 were the same and similar to the prevalence of FM. In Europeans, WP was 2.6‐fold more common than FM. None of the subjects with FM, WP only or a TPS ≥ 11 only was hypermobile.

Conclusion: The prevalence of FM was similar in Maori and European New Zealanders, and similar to most other population studies. In Maori, WP and a TPS ≥ 11 were equally specific for FM whereas in Europeans, WP was more sensitive but less specific for FM than a TPS ≥ 11. There was no association between hypermobility and FM.
The challenges associated with the biologic repair of cartilage defects
Authors: Ghosh P.
Published: March 2003

As observed by John Hunter as long ago as 1743, superficial lesions of articular cartilage (AC) do not undergo spontaneous healing and can lead to osteoarthritis in later years. While AC defects which penetrate the subchondral plate may fill with a blood clot and show early signs of healing, the regenerated hyaline cartilage progressively undergoes degeneration and transformation to fibrocartilage and eventually fails. In 1994, Brittberg and colleagues (NEJM 1994; 331: 889–94) reported favourable clinical outcomes in young patients when autologous chondrocytes were transplanted into small AC defects and covered by periosteum. However, subsequent studies in experimental animals failed to demonstrate filling of the defects by normal AC which was well integrated with the edges and calcified cartilage the defect. It was clear to many researchers at the time that any attempts to regenerate or repair cartilage defects would need to take into account a multiplicity of factors including, at the very least (a) the types of cells placed into the defect and whether they would differentiate or maintain the chondrocytes phenotype, (b) the composition and structure of the matrix or scaffold used to support them, and (c) the availability of growth factors or other external factors which could influence their rate of growth and/or gene expression.

In the last 10 years, there has been an exponential growth in reports describing various combinations of (a)–(c) which purport to achieve AC repair to a variable extent. To date, however, none can be said to be really satisfactory, although mesenchymal stem cells (MSC) in combination with collagen‐ or hyaluronan‐based support matrices have shown promising results. Moreover, the incorporation of growth factors such as BMP‐2 or BMP‐7 into such scaffolds or when genes for these proteins have been used to transfect the transplanted cells employed has improved chondrogenesis and implant success even more. More recently the complex technologies of bioengineering have been used to produce cartilage anlage of predetermined size. AC plugs made in rotating bioreactors have been press‐fitted into defects thereby providing the immediate mechanical stability necessary for cell survival and re‐differentiation in vivo. The incorporation of growth factors into these man‐made explants, together with surfacing of defects with adhesion proteins or other bonding materials before transplantation has provided additional avenues for improvement in the future.
Root and leaf production, mortality and longevity in response to soil heterogeneity
Authors: PÄRTEL M. , WILSON S. D.
Published: March 2003

Patches of fertile soil support concentrations of roots, but whether this reflects increased production or increased longevity is not known. We examined the production and longevity of roots of the grass Festuca rubra in response to soil heterogeneity. We also explored the extent to which root dynamics reflect shoot responses to heterogeneous soils.

 Root and leaf dynamics were followed in pots of heterogeneous or homogeneous soils containing the same total amount of nutrients. Digital minirhizotron images of roots and leaves were collected weekly.

 Root length was significantly greater in homogeneous than heterogeneous soils. This was caused by significantly larger root production but shorter life span. In contrast, soil heterogeneity had no effect on leaf production or longevity.

 Within heterogeneous pots, root and leaf production were strongly concordant, both being significantly greater in fertilized patches. More roots died in fertilized patches, but leaf mortality was not affected. Longevity of neither roots nor leaves was affected by the location of a fertile patch.

 Spatial variation in production of roots and shoots in response to nutrient patches was concordant. Roots and shoots, however, showed independent responses to the presence of within‐pot heterogeneity. A decrease in total root length in heterogeneous soils was a counterintuitive result of decreased production and increased longevity.