bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-8 is involved in cervical dilatation but not in prelabour cervical ripening
Authors: SAKAMOTO Y , MORAN P , SEARLE R F , BULMER J N , ROBSON S C
Published: September 2004

Our aim was to determine the amount and source of interleukin (IL)-8 and to study IL-8 receptor expression in the human cervix during pregnancy and after labour. Cervical biopsies were obtained from six non-pregnant women, eight women undergoing pregnancy termination, 17 women undergoing elective caesarean section and 11 women after vaginal delivery. IL-8 levels were compared in women with and without a ripe cervix, as determined by cervical Bishop score and cervicovaginal fetal fibronectin levels. Levels of IL-8 and IL-1β, a regulator of IL-8 expression, were determined by enzyme-linked immunosorbent assay (ELISA). IL-8, IL-1β and IL-8 receptor proteins were localized by immunohistochemistry. Compared with late pregnancy, IL-8 levels increased after labour and vaginal delivery (P < 0.01) but there was no correlation with cervical ripening. IL-8 was localized to stromal cells, macrophages and granulocytes. There were no significant differences in IL-1β levels between groups. IL-8 receptors were expressed primarily on granulocytes and macrophages after vaginal delivery. We conclude that IL-8 is involved in cervical dilatation but not in cervical ripening.
Hepatic interleuklin 15 (IL-15) expression: implications for local NK/NKT cell homeostasis and development
Authors: GOLDEN-MASON L , KELLY A M , DOHERTY D G , TRAYNOR O , MCENTEE G , KELLY J , HEGARTY J E , O'FARRELLY C
Published: September 2004

Interleukin 15 (IL-15) is critical for the development of human and murine natural killer (NK) cells and hepatic-derived NK T cells (NKT) in mice, and for the homeostatic maintenance of NK/NKT and CD8+ memory T cells. The lymphocyte repertoire of an adult human liver includes significant populations of NK and NKT-like cells, which may arise locally from hepatic haematopoietic stem cells (HSCs). We investigated hepatic IL-15 levels and the expression of IL-2/IL-15-receptor β-chain (IL-2/IL-15Rβ ; CD122) on mature hepatic lymphocytes and HSCs. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect secreted/intracellular IL-15 transcripts. IL-15 protein was localized using immunohistochemistry; levels were measured by enzyme-linked immunosorbent assay IL-2/IL-15Rβ expression by flow-cytometry. Normal hepatic IL-15 protein was detected at 0.43 ng/100 mg total protein (n = 11, range 0.10 ng−0.9 ng). There was a significant increase in HCV-infected tissue (1.78 ng, P < 0.005, n = 11, range 0.18–2.43 ng). The staining pattern suggests that infiltrating monocytes and tissue resident Kupffer cells are the main producers. IL-15 protein was detected in supernatants from cultured liver biopsy specimens in the absence of stimulation (mean 175.8 pg/100 mg wet tissue, n = 3), which increased significantly upon stimulation (P < 0.05, mean 231.21 pg). On average, 61% of hepatic HSCs expressed IL-2/IL-15Rβ suggesting a local lymphopoietic role. Eighty per cent of NK and 45.8% of CD56+ T cells expressed IL-2/IL-15Rβ, suggesting involvement in local CD56+ cell activation and expansion. Constitutive expression of IL-15 protein and IL-2/IL-15Rβ on hepatic lymphocytes suggests a key role in the generation and maintenance of the unique hepatic lymphoid repertoire. The significant increase observed in HCV-infected liver suggests a role for IL-15 in host antiviral responses in the liver.
Autoimmunity gene expression portrait: specific signature that intersects or differentiates between multiple sclerosis and systemic lupus erythematosus
Authors: MANDEL M , GUREVICH M , PAUZNER R , KAMINSKI N , ACHIRON A
Published: September 2004

Autoimmune diseases are either tissue-specific like multiple sclerosis (MS) or multisystemic like systemic lupus erythematosus (SLE), although clinically both exhibit common features. To gain insight into the properties of the genes involved in each disease we have investigated the gene expression signature of peripheral blood mononuclear cells (PBMC) in MS and SLE in comparison to healthy subjects. Total RNA was purified, hybridized to Genechip array and analysed in 36 subjects (13 relapsing-remitting MS patients, five SLE patients and 18 age-matched healthy subjects that served as controls). Additional blood samples from 15 relapsing-remitting MS patients, 8 SLE patients and 10 healthy subjects were used for confirmation of microarray gene expression findings by ELISA and RT-PCR. MS and SLE patients demonstrated a common gene expression autoimmune signature of 541 genes which differentiated them from healthy subjects. The autoimmune signature included genes that encode proteins involved in apoptosis, cell cycle, inflammation and regulation of matrix metalloproteinase pathways. Specifically, decreased TIMP1 gene expression in the autoimmunity signature suggests increased MMP activity in target tissues as a result of the lack of feedback mechanism. An additional different disease specific signature identified the gene expression pattern for MS (1031 genes), mainly associated with over-expression of adhesion molecules and down-expression of heat shock proteins; the SLE specific signature (1146 genes) mainly involved DNA damage/repair pathways that result in production of nuclear autoantibodies.

These results provide insights into the genetic pathways underlying autoimmune diseases, and identify specific disease-associated signatures that may enable targetted disease-related specific therapies to be developed.
Allelic polymorphisms in the repeat and promoter regions of the interleukin-4 gene and malaria severity in Ghanaian children
Authors: GYAN B A , GOKA B , CVETKOVIC J T , KURTZHALS J L , ADABAYERI V , PERLMANN H , LEFVERT A-K , AKANMORI B D , TROYE-BLOMBERG M
Published: September 2004

Immunoglobulin E has been associated with severe malaria suggesting a regulatory role for interleukin (IL)-4 and/or IgE in the pathogenesis of severe malaria. We have investigated possible associations between  polymorphisms  in  the  IL-4  repeat  region  (intron  3)  and  promoter  regions  (IL-4 +33CT  and − 590CT) in Ghanaian children with severe malaria. There was a significantly higher frequency of IL-4 intron-3 B1B1 genotype in the cerebral malaria group [P < 0.0001, odds ratio (OR) = 8.7]. The genotype and allele frequencies of the IL-4 –590 and +33 polymorphisms did not differ between the four study groups. Carriers of IL-4 +33T/−590T with cerebral malaria had elevated total IgE compared to non-carriers (P = 0.03). Our data suggest that IL-4 and/or IgE play a regulatory role in the pathogenesis of severe or complicated malaria.
Macrophages are a source of extracellular adenosine deaminase-2 during inflammatory responses
Authors: CONLON B A , LAW W R
Published: September 2004

Serum activity of the adenosine deaminase (ADA) isozyme, ADA2, has been reported to be elevated during various disease states. Macrophages have been suggested as the cellular source of extracellular ADA activity because they are one of the only cell types in which intracellular ADA2 activity has been measured, but extracellular secretion has never been demonstrated. Rat primary peritoneal macrophages (PPMs) and peripheral blood monocytes (PBMs) were harvested and incubated for 18 h in RPMI supplemented with horse serum. PPM and PBM lysates were assayed for intracellular ADA activity (ammonia production). In vitro and in vivo extracellular ADA activities were measured in media and rat serum, respectively. Activity of ADA1 was confirmed by selective inhibition with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). ADA2 activity was inhibited by 2′-deoxycoformcin only, and was increased at a low pH (6.5). Activity of both ADA isozymes was found in PPMs and PBMs, and their media. In a separate group of rats, peritonitis was induced by ip insertion of 400 mg/kg caecal slurry. PPMs were harvested 24 h later and incubated for 18 h. In PPMs from rats with peritonitis both isozymes were elevated by a similar proportion. In contrast, media from these PPMs had a lower ADA1 and a higher ADA2 activity compared to PPMs from nonseptic rats. This resulted in a greater proportion of ADA2 in media. The isozyme proportions in serum from septic rats more closely resembled that of the PPM media. The response of PBM was small relative to that of PPM. These results suggest that macrophages are a significant source of extracellular ADA isozymes, the activity of which increases during an inflammatory response. Because extracellular isozymes profiles differ from cellular concentrations, the data also suggest differential release of each isozyme from PPMs.
A mycobacterial lipoarabinomannan specific monoclonal antibody and its F(ab′)2 fragment prolong survival of mice infected withMycobacterium tuberculosis
Authors: HAMASUR B , HAILE M , PAWLOWSKI A , SCHRÖDER U , KÄLLENIUS G , SVENSON S B
Published: September 2004

Lipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis. High antibody titres against LAM are often seen in active tuberculosis (TB). The role of such LAM-specific antibodies in the immune response against TB is unknown. Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab′)2 fragment directed against LAM from M. tuberculosis strain H37Rv. MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M. tuberculosis strain Harlingen as well as to M. tuberculosis H37Rv. The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5.2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM. In passive protection experiments BALB/c mice were infected intravenously with M. tuberculosis Harlingen. MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria. The antibody proved to be protective against the M. tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival.
Stimulation of toll-like receptor 2 in mononuclear cells from HIV-infected patients induces chemokine responses: possible pathogenic consequences
Authors: HEGGELUND L , DAMÅS J K , YNDESTAD A , HOLM A M , MÜLLER F , LIEN E , ESPEVIK T , AUKRUST P , FRØLAND S S
Published: September 2004

Toll-like receptor 2 (TLR2) stimulation in monocytes may contribute to enhanced inflammation and viral replication in HIV infection. In the present study we examined if TLR2 stimulation could modulate chemokine responses in peripheral blood mononuclear cells (PBMC) from HIV-infected patients and healthy controls. Our main findings were, with similar qualitative patterns in both healthy controls and HIV-infected patients: (1) TLR2 stimulation induced up-regulation of several chemokines at the mRNA level as well as increased protein levels of macrophage inflammatory protein (MIP)-1α, interleukin (IL)-8 and regulated on activation, normal T cell expressed and secreted (RANTES); (2) TLR2 stimulation induced enhanced protein expression of CCR5 (a receptor for MIP-1α and RANTES) on monocytes; (3) In vitro stimulation with RANTES induced release of MIP-1α, MCP-1, IL-8 and interferon-γ from PBMC. While increased levels of β-chemokines possibly have antiviral effects, TLR2 stimulation may also promote a chemokine-driven inflammatory loop, potentially contributing to the immunopathogenesis of HIV infection.
Effects ofChlamydia trachomatisinfection on the expression of natural killer (NK) cell ligands and susceptibility to NK cell lysis
Authors: HOOK C E , TELYATNIKOVA N , GOODALL J C , BRAUD V M , CARMICHAEL A J , WILLS M R , GASTON J S H
Published: September 2004

Natural killer (NK) cells are an important component of the immediate immune response to infections, including infection by intracellular bacteria. We have investigated recognition of Chlamydia trachomatis (CT) by NK cells and show that these cells are activated to produce interferon (IFN)-γ when peripheral blood mononuclear cells (PBMC) are stimulated with CT organisms. Furthermore, infection of epithelial cell lines with CT renders them susceptible to lysis by human NK cells. Susceptibility was observed 18–24 h following infection and required protein synthesis by the infecting chlamydiae, but not by the host cell; heat or UV inactivated chlamydiae did not induce susceptibility to NK cell lysis. CT infection was also shown to decrease the expression of classical and non-classical major histocompatibility complex (MHC) molecules on infected cells, thus allowing recognition by NK cells when combined with an activating signal. A candidate activating signal is MICA/B, which was shown to be expressed constitutively on epithelial cells.
Serum IgE levels of tuberculosis patients in a tropical setup with high prevalence of HIV and intestinal parasitoses
Authors: KASSU A , MOHAMMAD A , FUJIMAKI Y , MOGES F , ELIAS D , MEKONNEN F , MENGISTU G , YAMATO M , WONDMIKUN Y , OTA F
Published: September 2004

Tuberculosis remains a major health problem worldwide in the era of HIV/AIDS. Co-infection with intestinal parasites has been suggested to worsen the outcome of infection by polarizing the immune response towards Th2. This study investigated serum IgE levels of 241 tuberculosis patients and compared the IgE profiles in the tuberculosis patients either with or without intestinal helminthic infection and/or HIV infection. The serum levels of IgE in tuberculosis patients before initiation of antimycobacterial chemotherapy were found to be 1722 ± 1290 IU/ml (Mean ± SD) in HIV seronegatives and 2366 ± 1849 IU/ml in HIV seropositives. Further, the IgE level was significantly higher in patients coinfected with intestinal helminthes and HIV compared to those infected with helminthes or without coinfection (P < 0.05). Anti-tuberculosis chemotherapy significantly reduced serum IgE levels in HIV seronegative tuberculosis patients (P < 0.05). These findings might indicate an active role of therapy in shifting the immune response towards Th1 which is crucial for prognosis in tuberculosis patients.
In vitrocellular immune responses to complex and newly defined recombinant antigens ofMycobacterium tuberculosis
Authors: AL-ATTIYAH R , MUSTAFA A S , ABAL A T , EL-SHAMY A S M , DALEMANS W , SKEIKY Y A W
Published: September 2004

The immunological diagnosis and development of new antituberculosis vaccines require the characterization of Mycobacterium tuberculosis antigens inducing cell-mediated immune responses. In this study, we have tested peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients (n = 43) and Bacille Calmette–Guérin (BCG)-vaccinated healthy subjects (n = 24) for in vitro cellular immune responses, as indicated by antigen-induced proliferation and interferon (IFN)-γ secretion, in response to a panel of complex (culture filtrate and cell wall preparations) and single recombinant antigens (Mtb8.4, Mtb9.8, Mtb9.9, Mtb32A, Mtb39A, Mtb40, Mtb41 and Ag85B) of M. tuberculosis. The results of cellular responses showed that the majority (ranging from 70 to 98%) of TB patients and healthy donors responded to the complex antigens in antigen-induced proliferation and IFN-γ secretion assays. However, when PBMC from the same groups of patients and healthy donors were tested with the recombinant antigens, TB patients showed strong recognition (>50% responders) of Mtb9.8 and Mtb39A in proliferation assays (median SI = 6.2 and 6.4, respectively) and of Mtb9.8, Mtb39A, Mtb40 and Ag85B in IFN-γ assays (median delta IFN-γ= 15.5, 10.8, 7.8 and 8.1 U/ml, respectively). BCG-vaccinated healthy donors showed weak (<30% responders) to moderate (31–50% responders) responses to all of the recombinant antigens in both assays. When PBMC of a subset of TB patients (n = 11) were tested for secretion of protective Th1 cytokines [IFN-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-12] and the immunosuppressive cytokine IL-10, the complex CF and CW antigens as well as the recombinant Mtb9.8, Mtb9.9, Mtb40 and Ag85B induced the secretion of both types of cytokines. On the other hand, Mtb41 induced only IL-10, while Mtb8.4, Mtb32Aand Mtb39A induced the secretion of one or more of Th1 cytokines, but not IL-10. In conclusion, the recombinant antigens inducing the secretion of Th1 cytokines could be useful as subunit vaccine candidates against TB.
Injection of poly(β‐l‐malate) into the plasmodium of Physarum polycephalum shortens the cell cycle and increases the growth rate
Authors: Karl Michael , Anderson Roger , Holler Eggehard
Published: September 2004

Poly(β‐l‐malate) (PMLA) has been reported as an unconventional, physiologically important biopolymer in plasmodia of myxomycetes, and has been proposed to function in the storage and transport of nuclear proteins by mimicking the phospho(deoxy)ribose backbone of nucleic acids. It is distributed in the cytoplasm and especially in the nuclei of these giant, multinucleate cells. We report here for the first time an increase in growth rate and a shortening of the cell cycle after the injection of purified PMLA. By comparing two strains of Physarum polycephalum that differed in their production levels of PMLA, it was found that growth activation and cell cycle shortening correlated with the relative increases of PMLA levels in the cytoplasm or the nuclei. Growth rates of a low PMLA producer strain (LU897 × LU898) were increased by 40–50% while those of a high producer strain (M3CVIII) were increased by only 0–17% in comparison with controls. In both strains, shortening of the cell cycle occurred to a similar extent (7.2–9.5%), and this was associated with similar increases in nuclear PMLA levels. The effects showed saturation dependences with regard to the amount of injected PMLA. A steep rise of intracellular PMLA shortly after injection was followed by the appearance of histone H1 in the cytoplasm. The increase in growth rate, the shortening of the cell cycle duration and the appearance of H1 in the cytoplasm suggest that PMLA competes with nucleic acids in binding to proteins that control translation and/or transcription. Thus, PMLA could play an important role in the coordination of molecular pathways that are responsible for the synchronous functioning of the multinucleate plasmodium.
HIP/PAP, a C‐type lectin overexpressed in hepatocellular carcinoma, binds the RIIα regulatory subunit of cAMP‐dependent protein kinase and alters the cAMP‐dependent protein kinase signalling
Authors: Demaugre France , Philippe Yannick , Sar Sokavuth , Pileire Bernard , Christa Laurence , Lasserre Chantal , Brechot Christian
Published: September 2004

HIP/PAP is a C‐type lectin overexpressed in hepatocellular carcinoma (HCC). Pleiotropic biological activities have been ascribed to this protein, but little is known about the function of HIP/PAP in the liver. In this study, therefore, we searched for proteins interacting with HIP/PAP by screening a HCC cDNA expression library. We have identified the RIIα regulatory subunit of cAMP‐dependent protein kinase (PKA) as a partner of HIP/PAP. HIP/PAP and RIIα were coimmunoprecipitated in HIP/PAP expressing cells. The biological relevance of the interaction between these proteins was established by demonstrating, using fractionation methods, that they are located in a same subcellular compartment. Indeed, though HIP/PAP is a protein secreted via the Golgi apparatus we showed that a fraction of HIP/PAP escaped the secretory apparatus and was recovered in the cytosol. Basal PKA activity was increased in HIP/PAP expressing cells, suggesting that HIP/PAP may alter PKA signalling. Indeed, we showed, using a thymidine kinase‐luciferase reporter plasmid in which a cAMP responsive element was inserted upstream of the thymidine kinase promoter, that luciferase activity was enhanced in HIP/PAP expressing cells. Thus our findings suggest a novel mechanism for the biological activity of the HIP/PAP lectin.
The tumor suppressor HIC1 (hypermethylated in cancer 1) is O‐GlcNAc glycosylated
Authors: Lefebvre Tony , Pinte Sébastien , Guérardel Cateline , Deltour Sophie , Martin‐Soudant Nathalie , Slomianny Marie‐Christine , Michalski Jean‐Claude , Leprince Dominique
Published: September 2004

HIC1 (hypermethylated in cancer 1) is a transcriptional repressor containing five Krüppel‐like C2H2 zinc fingers and an N‐terminal dimerization and autonomous repression domain called BTB/POZ. Here, we demonstrate that full‐length HIC1 proteins are modified both in vivo and in vitro with O‐linked N‐acetylglucosamine (O‐GlcNAc). This is a highly dynamic glycosylation found within the cytosolic and the nuclear compartments of eukaryotes. Analysis of [3H]Gal‐labeled tryptic peptides indicates that HIC1 has three major sites for O‐GlcNAc glycosylation. Using C‐terminal deletion mutants, we have shown that O‐GlcNAc modification of HIC1 proteins occurred preferentially in the DNA‐binding domain. Nonglycosylated and glycosylated forms of full‐length HIC1 proteins separated by wheat germ agglutinin affinity purification, displayed the same specific DNA‐binding activity in electrophoretic mobility shift assays proving that the O‐GlcNAc modification is not directly implicated in the specific DNA recognition of HIC1. Intriguingly, N‐terminal truncated forms corresponding to BTB‐POZ‐deleted proteins exhibited a strikingly differential activity, as the glycosylated truncated forms are unable to bind DNA whereas the unglycosylated ones do. Electrophoretic mobility shift assays performed with separated pools of glycosylated and unglycosylated forms of a construct exhibiting only the DNA‐binding domain and the C‐terminal tail of HIC1 (residues 399–714) and supershift experiments with wheat germ agglutinin or RL‐2, an antibody raised against O‐GlcNAc residues, fully corroborated these results. Interestingly, these truncated proteins are O‐GlcNAc modified in their C‐terminal tail (residues 670–711) and not in the DNA‐binding domain, as for the full‐length proteins. Thus, the O‐GlcNAc modification of HIC1 does not affect its specific DNA‐binding activity and is highly sensitive to conformational effects, notably its dimerization through the BTB/POZ domain.
Solution structure of IsTX
Authors: Yamaji Nahoko , Dai Li , Sugase Kenji , Andriantsiferana Marta , Nakajima Terumi , Iwashita Takashi
Published: September 2004

The novel sex‐specific potassium channel inhibitor IsTX, a 41‐residue peptide, was isolated from the venom of male Opisthacanthus madagascariensis. Two‐dimensional NMR techniques revealed that the structure of IsTX contains a cysteine‐stabilized α/β‐fold. IsTX is classified, based on its sequential and structural similarity, in the scorpion short toxin family α‐KTx6. The α‐KTx6 family contains a single α‐helix and two β‐strands connected by four disulfide bridges and binds to voltage‐gated K+ channels and apamin‐sensitive Ca2+‐activated K+ channels. The three‐dimensional structure of IsTX is similar to that of Heterometrus spinifer toxin (HsTX1). HsTX1 blocks the Kv1.3 channel at picomolar concentrations, whereas IsTX has much lower affinities (10 000‐fold). To investigate the structure–activity relationship, the geometry of sidechains and electrostatic surface potential maps were compared with HsTX1. As a result of the comparison of the primary structures, Lys27 of IsTX was conserved at the same position in HsTX1. The analogous Lys23 of HsTX1, the most critical residue for binding to potassium channels, binds to the channel pore. However, IsTX has fewer basic residues to interact with acidic channel surfaces than HsTX1. MALDI‐TOF MS analysis clearly indicated that IsTX was found in male scorpion venom, but not in female. This is the first report that scorpion venom contains sex‐specific compounds.
Investigations of the supercoil‐selective DNA binding of wild type p53 suggest a novel mechanism for controlling p53 function
Authors: Fojta Miroslav , Pivonkova Hana , Brazdova Marie , Nemcova Katerina , Palecek Jan , Vojtesek Borivoj
Published: September 2004

The tumor suppressor protein, p53, selectively binds to supercoiled (sc) DNA lacking the specific p53 consensus binding sequence (p53CON). Using p53 deletion mutants, we have previously shown that the p53 C‐terminal DNA‐binding site (CTDBS) is critical for this binding. Here we studied supercoil‐selective binding of bacterially expressed full‐length p53 using modulation of activity of the p53 DNA‐binding domains by oxidation of cysteine residues (to preclude binding within the p53 core domain) and/or by antibodies mapping to epitopes at the protein C‐terminus (to block binding within the CTDBS). In the absence of antibody, reduced p53 preferentially bound scDNA lacking p53CON in the presence of 3 kb linear plasmid DNAs or 20 mer oligonucleotides, both containing and lacking the p53CON. Blocking the CTDBS with antibody caused reduced p53 to bind equally to sc and linear or relaxed circular DNA lacking p53CON, but with a high preference for the p53CON. The same immune complex of oxidized p53 failed to bind DNA, while oxidized p53 in the absence of antibody restored selective scDNA binding. Antibodies mapping outside the CTDBS did not prevent p53 supercoil‐selective (SCS) binding. These data indicate that the CTDBS is primarily responsible for p53 SCS binding. In the absence of the SCS binding, p53 binds sc or linear (relaxed) DNA via the p53 core domain and exhibits strong sequence‐specific binding. Our results support a hypothesis that alterations to DNA topology may be a component of the complex cellular regulatory mechanisms that control the switch between latent and active p53 following cellular stress.
What makes biochemical networks tick?
Authors: Goldstein Boris N. , Ermakov Gennady , Centelles Josep J. , Westerhoff Hans V. , Cascante Marta
Published: September 2004

In view of the increasing number of reported concentration oscillations in living cells, methods are needed that can identify the causes of these oscillations. These causes always derive from the influences that concentrations have on reaction rates. The influences reach over many molecular reaction steps and are defined by the detailed molecular topology of the network. So‐called ‘autoinfluence paths’, which quantify the influence of one molecular species upon itself through a particular path through the network, can have positive or negative values. The former bring a tendency towards instability. In this molecular context a new graphical approach is presented that enables the classification of network topologies into oscillophoretic and nonoscillophoretic, i.e. into ones that can and ones that cannot induce concentration oscillations. The network topologies are formulated in terms of a set of uni‐molecular and bi‐molecular reactions, organized into branched cycles of directed reactions, and presented as graphs. Subgraphs of the network topologies are then classified as negative ones (which can) and positive ones (which cannot) give rise to oscillations. A subgraph is oscillophoretic (negative) when it contains more positive than negative autoinfluence paths. Whether the former generates oscillations depends on the values of the other subgraphs, which again depend on the kinetic parameters. An example shows how this can be established. By following the rules of our new approach, various oscillatory kinetic models can be constructed and analyzed, starting from the classified simplest topologies and then working towards desirable complications. Realistic biochemical examples are analyzed with the new method, illustrating two new main classes of oscillophore topologies.
Specific interaction between the classical swine fever virus NS5B protein and the viral genome
Authors: Xiao Ming , Gao Jufang , Wang Wei , Wang Yujing , Chen Jun , Chen Jiakuan , Li Bo
Published: September 2004

The NS5B protein of the classical swine fever virus (CSFV) is the RNA‐dependent RNA polymerase of the virus and is able to catalyze the viral genome replication. The 3′ untranslated region is most likely involved in regulation of the Pestivirus genome replication. However, little is known about the interaction between the CSFV NS5B protein and the viral genome. We used different RNA templates derived from the plus‐strand viral genome, or the minus‐strand viral genome and the CSFV NS5B protein obtained from the Escherichia coli expression system to address this problem. We first showed that the viral NS5B protein formed a complex with the plus‐strand genome through the genomic 3′ UTR and that the NS5B protein was also able to bind the minus‐strand 3′ UTR. Moreover, it was found that viral NS5B protein bound the minus‐strand 3′ UTR more efficiently than the plus‐strand 3′ UTR. Further, we observed that the plus‐strand 3′ UTR with deletion of CCCGG or 21 continuous nucleotides at its 3′ terminal had no binding activity and also lost the activity for initiation of minus‐strand RNA synthesis, which similarly occurred in the minus‐strand 3′ UTR with CATATGCTC or the 21 nucleotide fragment deleted from the 3′ terminal. Therefore, it is indicated that the 3′ CCCGG sequence of the plus‐strand 3′ UTR, and the 3′ CATATGCTC fragment of the minus‐strand are essential to in vitro synthesis of the minus‐strand RNA and the plus‐strand RNA, respectively. The same conclusion is also appropriate for the 3′ 21 nucleotide terminal site of both the 3′ UTRs.
Use of hydrostatic pressure to produce ‘native’ monomers of yeast enolase
Authors: Kornblatt M. Judith , Lange Reinhard , Balny Claude
Published: September 2004

The effects of hydrostatic pressure on yeast enolase have been studied in the presence of 1 mm Mn2+. When compared with apo‐enolase, and Mg‐enolase, the Mn‐enzyme differs from the others in three ways. Exposure to hydrostatic pressure does not inactivate the enzyme. If the experiments are performed in the presence of 1 mm Mg2+, or with apo‐enzyme, the enzyme is inactivated [Kornblatt, M.J., Lange R., Balny C. (1998) Eur. J. Biochem251, 775–780]. The UV spectra of the high pressure forms of the Mg2+‐ and apo‐forms of enolase are identical and distinct from the spectrum of the form obtained in the presence of 1 mm Mn2+; this suggests that Mn2+ remains bound to the high pressure form of enolase. With Mn‐enolase, the various spectral changes do not occur in the same pressure range, indicating that multiple processes are occurring. Pressure experiments were performed as a function of [Mn2+] and [protein]. One of the changes in the UV spectra shows a dependence on protein concentration, indicating that enolase is dissociating into monomers. The small changes in the UV spectrum and the retention of activity lead to a model in which enolase, in the presence of high concentrations of Mn2+, dissociates into native monomers; upon release of pressure, the enzyme is fully active. Although further spectral changes occur at higher pressures, there is no inactivation as long as Mn2+ remains bound. We propose that the relatively small and polar nature of the subunit interface of yeast enolase, including the presence of several salt bridges, is responsible for the ability of hydrostatic pressure to dissociate this enzyme into monomers with a native‐like structure.
Synergistic activation of signalling to extracellular signal‐regulated kinases 1 and 2 by epidermal growth factor and 4β‐phorbol 12‐myristate 13‐acetate
Authors: Hornberg Jorrit J. , Tijssen Marloes R. , Lankelma Jan
Published: September 2004

Signal transduction pathways are often embedded in complex networks, which result from interactions between pathways and feedback circuitry. In order to understand such networks, qualitative information on which interactions take place and quantitative data on their strength become essential. Here, we have investigated how the multiple interactions between the mitogen‐activated protein kinase cascade and protein kinase C (PKC) affect the time profile of extracellular signal‐regulated kinase (ERK) phosphorylation upon epidermal growth factor (EGF) stimulation in normal rat kidney fibroblasts. This profile is a major determinant for the cellular response that is evoked. We found that EGF stimulation leads to a biphasic ERK‐PP pattern, consisting of an initial peak and a relaxation to a low quasi‐steady state‐phase. Costimulation with the EGF and PKC activator, 4β‐phorbol 12‐myristate 13‐acetate (PMA) resulted in a similar pattern, but the ERK‐PP concentration in the quasi‐steady state‐phase was synergistically higher than after stimulation with either EGF or PMA only. This resulted in prolonged signalling to ERK. PMA increased the EGF concentration sufficient to obtain half‐maximum ERK phosphorylation. These data suggest that PKC amplifies EGF‐induced signalling to ERK, without increasing its sensitivity to low EGF concentrations. Furthermore, PKC inhibition did not affect the ERK‐PP time profile upon EGF stimulation and a cellular phospholipase A2 (cPLA2) inhibitor did not decrease the synergistic effect of EGF and PMA. This indicates that the positive feedback loop from ERK to Raf via cPLA2 and PKC does not contribute significantly to signalling from EGF to ERK in normal rat kidney cells. Taken together, we provide a quantitative description of which reported interactions in this network affect the time profile of ERK phosphorylation.
Critical roles of Asp40 at the haem proximal side of haem‐regulated phosphodiesterase from Escherichia coli in redox potential, auto‐oxidation and catalytic control
Authors: Watanabe Miki , Kurokawa Hirofumi , Yoshimura‐Suzuki Tokiko , Sagami Ikuko , Shimizu Toru
Published: September 2004

In haem‐regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS), haem is bound to the PAS domain, and the redox state of the haem iron regulates catalysis by the PDE domain. We generated mutants of Asp40, which forms a hydrogen bond with His77 (a proximal haem axial ligand) via two water molecules, and a salt bridge with Arg85 at the protein surface. The redox potential of haem was markedly increased from 67 mV vs. the standard hydrogen electrode in the wild‐type enzyme to 95 mV and 114 mV in the Ala and Asn mutants, respectively. Additionally, the auto‐oxidation rate of Ec DOS PAS was significantly increased from 0.0053 to 0.051 and 0.033 min−1, respectively. Interestingly, the catalytic activities of the Asp40 mutants were abolished completely. Thus, Asp40 appears to play a critical role in the electronic structure of the haem iron and redox‐dependent catalytic control of the PDE domain. In this report, we discuss the mechanism of catalytic control of Ec DOS, based on the physico‐chemical characteristics of the Asp40 mutants.
Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis
Authors: Canaan Stéphane , Maurin Damien , Chahinian Henri , Pouilly Bénédicte , Durousseau Cécile , Frassinetti Frédéric , Scappuccini‐Calvo Loréna , Cambillau Christian , Bourne Yves
Published: September 2004

The Mycobacterium tuberculosis genome contains an unusually high number of proteins involved in the metabolism of lipids belonging to the Lip family, including various nonlipolytic and lipolytic hydrolases. Driven by a structural genomic approach, we have biochemically characterized the Rv1399c gene product, LipH, previously annotated as a putative lipase. Rv1399c was overexpressed in E. coli as inclusion bodies and refolded. Rv1399c efficiently hydrolyzes soluble triacylglycerols and vinyl esters. It is inactive against emulsified substrate and its catalytic activity is strongly inhibited by the diethyl paranitrophenyl phosphate (E600). These kinetic behaviors unambiguously classify Rv1399c as a nonlipolytic rather than a lipolytic hydrolase. Sequence alignment reveals that this enzyme belongs to the α/β hydrolase fold family and shares 30–40% amino acid sequence identity with members of the hormone‐sensitive lipase subfamily. A model of Rv1399c derived from homologous three‐dimensional structures reveals a canonical catalytic triad (Ser162, His290 and Asp260) located at the bottom of a solvent accessible pocket lined by neutral or charged residues. Based on this model, kinetic data of the Arg213Ala mutant partially explain the role of the guanidinium moiety, located close to His290, to confer an unusual low pH shift of the catalytic histidine in the wild type enzyme. Overall, these data identify Rv1399c as a new nonlipolytic hydrolase from M. tuberculosis and we thus propose to reannotate its gene product as NLH‐H.
Testosterone 1β‐hydroxylation by human cytochrome P450 3A4
Authors: Krauser Joel A. , Voehler Markus , Tseng Li‐Hong , Schefer Alexandre B. , Godejohann Markus , Guengerich F. Peter
Published: September 2004

Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6β‐hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6β‐ and 2β‐hydroxy products, was identified as 1β‐hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC‐solid phase extraction‐cryoprobe NMR/time‐of‐flight mass spectrometry system, with an estimated total of ≈ 6 µg of this product. Mass spectrometry established the formula as C19H29O3 (MH+ 305.2080). The 1‐position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the β‐stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1α‐H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes.
Priapism secondary to leukemia: Effective management with prompt leukapheresis
Authors: PONNIAH ALLAN , BROWN CHRISTIAN T , TAYLOR PETER
Published: September 2004

Abstract Priapism is a urological emergency; without prompt treatment the corpora cavernosa undergoes necrosis and fibrosis that may result in erectile dysfunction. Treatment initially involves conservative measures, such as corporeal aspiration and irrigation with saline or dilute phenylephrine. If this fails, embolization or surgical shunting is usually required. Hyperviscosity resulting from leukemia is a rare cause of priapism. We report a case of a 19‐year‐old man with an 18‐hour history of priapism secondary to undiagnosed chronic myeloid leukaemia. We discuss the method of leukapheresis (mechanical white cell depletion) to reduce viscosity and achieve detumescence after failed aspiration.
Squamous cell carcinoma in the renal pelvis of a horseshoe kidney
Authors: MIZUSAWA HIROYA , KOMIYAMA ITSUKI , UENO YOKO , MAEJIMA TOSHITAKA , KATO HARUAKI
Published: September 2004

Abstract We report a rare case of squamous cell carcinoma in the renal pelvis of a horseshoe kidney. An 80‐year‐old woman was referred to the National Nagano Hospital for the examination of occult blood in her urine. Microscopic hematuria was found, but pyuria was not seen. Computed tomography and magnetic resonance imaging showed a mass in the left renal pelvis of the horseshoe kidney. No renal stone or hydronephrosis was found. Cytopathological examination in the voided urine specimen was positive. Left nephroureterectomy with the splitting of the isthmus of the horseshoe kidney was performed without renal pedicle clamping using a microwave tissue coagulator. No bleeding was encountered after separating the isthmus. A final pathological diagnosis of squamous cell carcinoma with a tumor thrombus was made. Lymph node metastasis had developed and rapidly progressed and the patient died of disseminated malignancy 4 months after the operation.

We reviewed 24 cases of renal pelvic tumor in horseshoe kidneys previously reported in Japan. Seven cases (30%) included components of squamous cell carcinoma. The incidence is higher than that of renal pelvic tumors in the general population.
Increased expression of lung resistance‐related protein in lower grade urothelial carcinoma of the renal pelvis and ureter
Authors: KONG CHUI‐ZE , ZENG YU , WU XIU‐XIAN , LI JIA‐QING , ZHU YU‐YAN , CHEN YAN
Published: September 2004

Abstract Background: Lung resistance‐related protein (LRP), like multidrug resistance gene 1 (MDR1) and multidrug resistance‐associated proteins (MRP), has been associated with intrinsic therapeutic resistance in various malignancies. To date, there has been no study on the expression of LRP in urothelial carcinomas of the renal pelvis and ureter. We investigated the protein and mRNA expression levels of LRP, MDR1 and MRP1 in this malignancy and the clinical significance of their expression was evaluated.

Methods: Forty urothelial carcinomas of the renal pelvis and ureter and 31 normal upper urothelial samples were examined by immunohistochemistry and reverse transcription polymerase chain reaction to determine the protein and mRNA levels of the multidrug resistance‐related genes, respectively.

Results: The positive staining rates and mRNA levels of LRP were the highest among these multidrug resistance‐related genes in both normal urothelium and carcinoma examinations. In contrast to the up‐regulated expression of MDR1, the expression of LRP tended to be down‐regulated in carcinomas. Moreover, the expression of LRP inversely correlated with tumor grades, but this correlation was not found for the other two genes. However, there was no correlation among the expression of the three genes observed.

Conclusion: Lung resistance‐related protein was strongly expressed in urothelial carcinomas of the renal pelvis and ureter, particularly in well‐differentiated carcinomas.
Phyllodes tumor of the prostate: Recurrent obstructive symptom and stromal proliferative activity
Authors: SHIRAISHI KOJI , MOHRI JUN , EGUCHI SATOSHI , KAMIRYO YORIAKI , SANEFUJI HAYATO
Published: September 2004

Abstract We report the case of a 59‐year‐old man with a metachronous development of phyllodes tumor and adenocarcinoma of the prostate. He complained of urinary obstruction and transurethral resections of the prostate (TUR‐P) had been performed six times in 10 years. Microscopic examination showed cystically dilated glands consisting of bizarre cells with pleomorphic, hyperchromatic nuclei in the stroma at the sixth TUR‐P. Radical prostatectomy was performed against recurrences and adenocarcinoma was incidentally detected. Apparent up‐regulation of proliferative nuclear antigens (PCNA), but not p53, was observed in the prostatectomy specimen by Western blotting. Active proliferation of stromal cells is considered to have caused the recurrent obstructive symptom.
Torsion of the hernia sac within a hydrocele of the scrotum in a child
Authors: MATSUMOTO AKIHIKO , NAGATOMI YUTAKA , SAKAI MASATO , OSHI MASAYA
Published: September 2004

Abstract Torsion of the hernia sac is a rare disease that presents as acute scrotum in children. Including the present case, only six cases have been reported in the English literature.

We report a 10‐year‐old boy who presented with pain and swelling of his right scrotum. Ultrasonography revealed a hypoechoic region adjacent to the normal right testis. The inflammatory changes of the right scrotum deteriorated. The patient underwent surgery and a necrotic cyst was recognized within a hydrocele of the scrotum. The cyst was not connected with the testis or epididymis and was twisted at an angle of 270°. The cause of the necrotic cyst observed was anatomical and pathological torsion of the hernia sac.
Lower pole ratio: A new and accurate predictor of lower pole stone clearance after shockwave lithotripsy?
Authors: FONG YAN KIT , PEH SAM OON HUI , HO SIEW HONG , NG FOO CHEONG , QUEK PEARLLYN LENG CHOO , NG KOK KIT
Published: September 2004

Abstract Background: Lower pole spatial anatomy is an important determinant of success after extracorporeal shockwave lithotripsy. In the present study, we determine whether there is a significant relationship between lower pole ratio (infundibular length : infundibular width) on preoperative intravenous urograms and stone fragment clearances after shockwave lithotripsy.

Methods: A total of 42 patients with isolated lower pole stones were retrospectively reviewed. Anatomical factors, such as infundibular length, width and infundibulopelvic angle were measured and the lower pole ratio was calculated on pretreatment intravenous urogram. Stone fragment clearance was assessed at three months with a plain abdominal X‐ray.

Results: The overall three‐month stone‐free rate was 62%. Mean stone size ± SD was 10 ± 4.8 mm, mean infundibular length was 21.7 ± 6.9 mm, mean infundibular width was 6.1 ± 2.3 mm, mean infundibulopelvic angle was 62.1 ± 30.1 degrees and mean lower pole ratio was 4.3 ± 2.8. Stone‐free status after shockwave lithotripsy was significantly related to infundibular length and width as well as to lower pole ratio, but not to infundibulo‐pelvic angle. Infundibular length less than 30 mm, width greater than 5 mm and lower pole ratio less than 3.5 were noted to have an improved three‐month stone‐free rate (P = 0.049, 0.01 and <0.01, respectively).

Conclusion: Caliceal anatomy is an important consideration for lower pole stone clearance after shockwave lithotripsy. The present study suggests that a lower pole ratio of less than 3.5, which considers both infundibular length and width, is a promising predictor for stone‐free status.
Bilateral double testis: Evaluation by magnetic resonance imaging
Authors: DEVECI SERKAN , AYGÜN CEM , AGILDERE A. MUHTESEM , ÖZKARDES HAKAN
Published: September 2004

Abstract Bilateral double testis, a varient of polyorchidism, is a rare malformation of which six cases have been reported in the published literature. Magnetic resonance imaging was used in the presented case to confirm the diagnosis without surgical exploration.
Long‐term consequence of renal function following nephrectomy for renal cell cancer
Authors: SHIRASAKI YOSHINORI , TSUSHIMA TOMOYASU , NASU YASUTOMO , KUMON HIROMI
Published: September 2004

Abstract Aim: A retrospective analysis was performed involving patients who had undergone radical nephrectomy for renal cell cancer to determine the long‐term outcome of this surgery on renal status.

Materials and Methods: Between 1977 and 2001, 172 patients having undergone radical nephrectomy for renal cell cancer were followed for a period of more than one year at the Department of Urology Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan. The preoperative and postoperative serum creatinine values of these individuals were reviewed. Long‐term effects of radical nephrectomy on renal function and factors influencing postoperative renal dysfunction were analyzed.

Results: Six (3.5%) of the 172 individuals progressed to hemodialysis. Eleven (6.4%) patients displayed serum creatinine values of ≥1.6 mg/dL. The remaining 155 patients (90.1%) demonstrated postoperative serum creatinine values of <1.6 mg/dL. The present study suggests that aging, a high degree of proteinuria, hypertension and diabetes mellitus were the predominant risk factors for renal deterioration following nephrectomy.

Conclusions: We have primarily selected radical nephrectomy for cases exhibiting smaller renal tumors with a normal contralateral kidney. We encourage nephron‐sparing surgery where possible in patients displaying risk factors, including a high degree of proteinuria, hypertension, or diabetes mellitus.