bluuebunny/crossref_metadata_2025 huggingface dataset:
Ionizing radiation can overcome resistance to TRAIL in TRAIL‐resistant cancer cells
Authors: Kim Mi-Ra , Lee Jeong-Yim , Park Moon-Taek , Chun Yong-Jin , Jang Young-Joo , Kang Chang-Mo , Kim Hye Sun , Cho Chul-Koo , Lee Yun-Sil , Jeong Hee-Young , Lee Su-Jae
Published: October 2002

Although the majority of cancer cells are killed by TRAIL (tumor necrosis factor‐related apoptosis‐inducing ligand treatment), certain types show resistance to it. Ionizing radiation also induces cell death in cancer cells and may share common intracellular pathways with TRAIL leading to apoptosis. In the present study, we examined whether ionizing radiation could overcome TRAIL resistance in the variant Jurkat clones. We first selected TRAIL‐resistant or ‐sensitive Jurkat clones and examined cross‐responsiveness of the clones between TRAIL and radiation. Treatment with γ‐radiation induced significant apoptosis in all the clones, indicating that there seemed to be no cross‐resistance between TRAIL and radiation. Combined treatment of radiation with TRAIL synergistically enhanced killing of TRAIL‐resistant cells, compared to TRAIL or radiation alone. Apoptosis induced by combined treatment of TRAIL and radiation in TRAIL‐resistant cells was associated with cleavage of caspase‐8 and the proapoptotic Bid protein, resulting in the activation of caspase‐9 and caspase‐3. No changes in the expressions of TRAIL receptors (DR4 and DR5) and Bcl‐2 or Bax were found after treatment. The caspase inhibitor z‐VAD‐fmk completely counteracted the synergistic cell killing induced by combined treatment of TRAIL and γ‐radiation. These results demonstrated that ionizing radiation in combination with TRAIL could overcome resistance to TRAIL in TRAIL‐resistant cells through TRAIL receptor‐independent synergistic activation of the cascades of the caspase‐8 pathway, suggesting a potential clinical application of combination treatment of TRAIL and ionizing radiation to TRAIL‐resistant cancer cells.
Age‐related loss of skeletal muscle function and the inability to express the autocrine form of insulin‐like growth factor‐1 (MGF) in response to mechanical overload
Authors: Owino Vivian , Yang Shi Yu , Goldspink Geoffrey
Published: October 2002

The response of insulin‐like growth factor‐1 (IGF‐1) signalling and the capacity of skeletal muscle to adapt to mechanical overload was studied using synergistic muscle ablation. Overload of the plantaris and soleus resulted in marked hypertrophy and activation of satellite cells (as indicated by MyoD expression), particularly in young rats. Two muscle IGF‐1 splice variants were measured and found to be differentially regulated at the RNA level. The significant changes associated with the inability of the older muscles to respond to mechanical overload included the considerably lower expression of the local splice variant mechano growth factor, and the failure to up‐regulate IGF‐1 receptor and MyoD mRNA.
Evidence for cell surface association between CXCR4 and ganglioside GM3 after gp120 binding in SupT1 lymphoblastoid cells
Authors: Sorice Maurizio , Garofalo Tina , Misasi Roberta , Longo Agostina , Mattei Vincenzo , Sale Patrizio , Dolo Vincenza , Gradini Roberto , Pavan Antonio
Published: October 2002

CXCR4 (fusin) is a chemokine receptor which is involved as a coreceptor in gp120 binding to the cell surface. In this study we provide evidence that binding of gp120 triggers CXCR4 recruitment to glycosphingolipid‐enriched microdomains. Scanning confocal microscopy showed a nearly complete localization of CXCR4 within GM3‐enriched plasma membrane domains of SupT1 cells and coimmunoprecipitation experiments revealed that CXCR4 was immunoprecipitated by IgG anti‐GM3 after gp120 pretreatment. These findings reveal that gp120 binding induces a strict association between CXCR4 and ganglioside GM3, supporting the view that GM3 and CXCR4 are components of a functional multimolecular complex critical for HIV‐1 entry.
Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function
Authors: Ibrahim Hisham R , Matsuzaki Tetsuji , Aoki Takayoshi
Published: October 2002

A catalytically inactive mutant of hen egg white lysozyme was constructed by site‐directed mutagenesis to elucidate the role of enzymatic activity on its antimicrobial activity against Gram‐positive bacteria. The catalytic residue aspartic acid at position 52 of lysozyme was substituted with serine (D52S‐Lz) and the mutant cDNA was inserted into a yeast expression vector, pYES‐2. Western blot analysis indicated that the mutation did not affect secretion of the D52S‐Lz lysozyme into the medium of the expressing Saccharomyces cerevisiae, INVSC1. In addition, circular dichroism and fluorescence spectral analysis revealed no change in the structure of D52S‐Lz compared to that of wild‐type (Wt‐Lz) lysozyme. The mutation (D52S) abolished the catalytic activity of lysozyme. Antimicrobial tests against Staphylococcus aureus and Bacillus subtilis revealed that the catalytically inactive D52S‐Lz was as bactericidal as the Wt‐Lz lysozyme. Heat treatment leading to enzyme inactivation had no effect on the bactericidal activity of either wild‐type or the mutant D52S‐Lz lysozyme. The binding affinity of D52S‐Lz to the isolated peptidoglycan of S. aureus was unaffected. Our results provide the first demonstration of direct genetic evidence that the antimicrobial activity of lysozyme is operationally independent of its muramidase activity, and strongly suggest the antimicrobial action of lysozyme is due to structural factors.
Localization of neutral ceramidase in caveolin‐enriched light membranes of murine endothelial cells
Authors: Romiti Elena , Meacci Elisabetta , Tanzi Giuditta , Becciolini Laura , Mitsutake Susumu , Farnararo Marta , Ito Makoto , Bruni Paola
Published: October 2002

Sphingomyelinase (SMase) and ceramidase (CDase) activities participate in sphingomyelin (SM) metabolism and have a role in the signal transduction of a variety of ligands. In this study evidence is presented that caveolin‐enriched light membranes (CELMs) of murine endothelial cells, characterized by high SM, ceramide (Cer) and cholesterol content, bear acid and neutral SMase as well as neutral CDase activities. Localization of neutral CDase in CELMs was confirmed by Western analysis. Notably, cell treatment with cyclodextrin, which depleted cell cholesterol, did not affect acid or neutral SMase activities but significantly enhanced neutral CDase activity in CELMs, indicating a negative role for cholesterol in CDase regulation. These findings suggest that neutral CDase is implicated, together with SMase activities, in the control of caveolar Cer content that may be critical for caveola dynamics.
Mechanisms mediating the antiproliferative effects of nitric oxide in cultured human airway smooth muscle cells
Authors: Hamad Ahmed M , Knox Alan J
Published: October 2002

We have characterised the mechanisms involved in the antiproliferative effect of NO in human airway smooth muscle cells (HASMC). S‐Nitroso‐N‐acetyl penicillamine, a nitric oxide donor, inhibited proliferation in both G1 and S phases of the cell cycle. Additionally, experiments with 8‐bromo‐cGMP, haemoglobin, a NO scavenger and zaprinast, a cGMP‐specific phosphodiesterase inhibitor, showed that both effects were NO‐mediated. The G1 phase inhibition was cGMP‐dependent whereas the S phase inhibition was due to a cGMP‐independent inhibition of ribonucleotide reductase. These results demonstrate that NO inhibits HASMC proliferation by cGMP‐dependent and ‐independent mechanisms acting at distinct points in the cell cycle.
Signaling pathways utilized by tumor necrosis factor receptor 1 in adipocytes to suppress differentiation
Authors: Xu Haiyan , Hotamisligil Gökhan S.
Published: October 2002

Tumor necrosis factor‐α (TNFα) has profound effects on cultured adipocytes, one of which is the inhibition of terminal differentiation. Previous studies in TNF receptor (TNFR)‐deficient preadipocytes have demonstrated that the anti‐adipogenic effect of both secreted and transmembrane TNFα is mediated solely by TNFR1. In this study, we performed a structure–function analysis of the intracellular domains of TNFR1 and investigated the signaling pathway(s) involved in TNFR1‐mediated inhibition of adipocyte differentiation. Our results show that repression of adipogenesis required the juxtamembrane and death domains and was independent of the pathways involving nuclear factor κB and neutral sphingomyelinase.
A plant steroid, diosgenin, induces apoptosis, cell cycle arrest and COX activity in osteosarcoma cells
Authors: Moalic Sandra , Liagre Bertrand , Corbière Cécile , Bianchi Arnaud , Dauça Michel , Bordji Karim , Beneytout Jean L.
Published: October 2002

Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid into prostanoids which are involved in apoptosis and inflammation. Two distinct COXs have been identified: COX‐1 which is constitutively expressed and COX‐2 which is induced by different products such as tumor promoters or growth factors. Previously, we demonstrated that a plant steroid, diosgenin, was a new megakaryocytic differentiation inducer of human erythroleukemia cells. In our study, we investigated the effect of diosgenin on the proliferation rate, cell cycle distribution and apoptosis in the human osteosarcoma 1547 cell line. The effects of this compound were also tested on COX expression and COX activities. Diosgenin treatment caused an inhibition of 1547 cell growth with a cycle arrest in G1 phase and apoptosis induction. Moreover, we found a correlation between p53, p21 mRNA expression and nuclear factor‐κB activation and we observed a time‐dependent increase in PGE2 synthesis after diosgenin treatment.
In vitro transactivation ofBacillus subtilisRNase P RNA
Authors: Kim Hadong , Poelling Richard R , Leeper Thomas C , Meyer Melissa A , Schmidt Francis J
Published: October 2002

Deletion of the ‘signature’ PL5.1 stem‐loop structure of a Type II RNase P RNA diminished its catalytic activity. Addition of PL5.1 intransincreased catalytic efficiency (kcat/KM) rather thankcat. Transactivation was due to the binding of a single PL5.1 species per ribozyme with an apparentKdnear 600 nM. The results are consistent with the role of PL5.1 being to position the substrate near the active site of the ribozyme, and with the hypothesis that ribozymes can evolve by accretion of preformed smaller structures.
Structural basis for the emission of violet bioluminescence from a W92F obelin mutant
Authors: Deng Lu , Vysotski Eugene S. , Liu Zhi-Jie , Markova Svetlana V. , Malikova Natalia P. , Lee John , Rose John , Wang Bi-Cheng
Published: October 2002

Mutation of the Trp92 that is known to lie within the active site of the photoprotein obelin fromObelia longissima, results in a shift of the bioluminescence color from blue (λmax=485 nm) to violet. The corrected spectrum shows a new band withλmax=410 nm now contributing equally to the one at longer wavelength. The crystal structure of this W92F obelin determined at 1.72 Å resolution shows that there is no significant change in the dimensions of the active site between WT obelin (recombinant Ca2+‐regulated photoprotein fromObelia longissima) and the mutant. It is proposed that the bioluminescence spectral shift results from removal of a hydrogen bond from the indole of W92 nearby a hydroxyl belonging to the 6‐phenyl substituent of the substrate coelenterazine. Propagation of this change through a conjugated bond system in the excited state of the product coelenteramide affects the coupling of the N1‐position and the hydrogen‐bonded Y138.
Abnormal IgG galactosylation and arthritis in MRL‐Faslpr or MRL‐FasLgld mice are under the control of the MRL genetic background
Authors: Kuroda Yasuhiro , Nakata Munehiro , Nose Masato , Kojima Naoya , Mizuochi Tsuguo
Published: October 2002

MRL mice bearing the lpr (Fas) or gld (Fas ligand) mutation, MRL‐Faslpr
or MRL‐FasLgld
, respectively, develop arthritis similar to rheumatoid arthritis, but C3H and C57BL/6 mice bearing such mutations do not. In MRL‐Faslpr
mice, agalactosylated oligosaccharides in serum IgG increase significantly in comparison to MRL‐+/+ mice without arthritis. In this study, an increased level of agalactosylation in IgG, as compared to MRL‐+/+, was found in both MRL‐Faslpr
and MRL‐FasLgld
mice. In contrast, the incidence of IgG without galactose was comparable among C3H‐Faslpr
, C3H‐FasLgld
, and C3H‐+/+ mice as well as between C57BL/6‐Faslpr
and C57BL/6‐+/+ mice. These results suggest that the increase in agalactosylated IgG and the development of arthritis in MRL‐Faslpr
and MRL‐FasLgld
mice are controlled by the MRL genetic background.
Induction of caspase‐11 by inflammatory stimuli in rat astrocytes: lipopolysaccharide induction through p38 mitogen‐activated protein kinase pathway
Authors: Hur Jinyoung , Kim Sun Yeou , Kim Hocheol , Cha Sang-hoon , Lee Myung-Shik , Suk Kyoungho
Published: October 2002

Caspase‐11 plays a crucial role in both inflammation and apoptosis. Caspase‐11 not only activates caspase‐1, that is required for the maturation of proinflammatory cytokines such as interleukin (IL)‐1 and IL‐18, but also activates caspase‐3, leading to cellular apoptosis under pathological conditions. Here, we cloned the rat homolog of caspase‐11, and investigated its inducibility by inflammatory stimuli and signal transduction pathways involved. Deduced amino acid sequence of rat caspase‐11 showed 88.7% similarity to mouse caspase‐11, and in vitro translation of rat caspase‐11 cDNA yielded approximately a 43 kDa polypeptide, which was in agreement with predicted protein size generated from full‐length rat caspase‐11 cDNA. The expression of caspase‐11 was strongly induced at both mRNA and protein levels by inflammatory stimuli such as lipopolysaccharide (LPS), interferon‐γ, and tumor necrosis factor‐α in C6 rat glial cells as well as primary astrocytes. LPS induced activation of both p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinase (ERK) in C6 cells. However, SB203580 (specific inhibitor of p38 kinase), but not PD98059 (specific inhibitor of ERK kinase), inhibited LPS induction of caspase‐11, indicating that induction of caspase‐11 by LPS in astrocytes was mediated through the p38 MAPK pathway. Inflammatory induction of caspase‐11 in astrocytes may play an important role in both inflammatory responses involving these cells and auto‐regulatory apoptosis of activated astrocytes in inflammatory sites.
Identification of rabaptin‐5, rabex‐5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER
Authors: Valsdottir Rebekka , Hashimoto Hitoshi , Ashman Keith , Koda Toshiaki , Storrie Brian , Nilsson Tommy
Published: October 2002

The role of rab33b, a Golgi‐specific rab protein, was investigated. Microinjection of rab33b mutants stabilised in the GTP‐specific state resulted in a marked inhibition of anterograde transport within the Golgi and in the recycling of glycosyltransferases from the Golgi to the ER, respectively. A GST‐rab33b fusion protein stabilised in its GTP form was found to interact by Western blotting or mass spectroscopy with Golgi protein GM130 and rabaptin‐5 and rabex‐5, two rab effector molecules thought to function exclusively in the endocytic pathway. A similar binding was seen to rab1 but not to rab6, both Golgi rabs. In contrast, rab5 was as expected, shown to bind rabaptin‐5 and rabex‐5 as well as the endosomal effector protein EEA1 but not GM130. No binding of EEA1 was seen to any of the Golgi rabs.
Reversible inhibition of cathepsin L‐like proteases by 4‐mer pseudopeptides
Authors: Lecaille Fabien , Cotton Joël , McKerrow James H , Ferrer-Di Martino Michèle , Boll-Bataillé Emmanuelle , Gauthier Francis , Lalmanach Gilles
Published: October 2002

A library of 121 pseudopeptides was designed to develop reversible inhibitors of trypanosomal enzymes (cruzain from Trypanosoma cruzi and congopain from Trypanosoma congolense). The peptides share the framework: Cha‐X1‐X2‐Pro (Cha=cyclohexyl‐alanine, X1 and X2 were phenylalanyl analogs), based on a previous report [Lecaille, F., Authié, E., Moreau, T., Serveau, C., Gauthier, F. and Lalmanach, G. (2001) Eur. J. Biochem. 268, 2733–2741]. Five peptides containing a nitro‐substituted aromatic residue (Tyr/Phe) and one a 4‐chloro‐phenylalanine at the X1 position, and 3‐(2‐naphthyl)‐alanine, homocyclohexylalanine or 3‐nitro‐tyrosine (3‐NO2‐Tyr) at the X2 position, were selected. They inhibited congopain more effectively than cruzain, except Cha‐4‐NO2‐Phe‐3‐NO2‐Tyr‐Pro which bound the two parasitic enzymes similarly. Among this series, Cha‐3‐NO2‐Tyr‐HoCha‐Pro and Cha‐4‐NO2‐Phe‐3‐NO2‐Tyr‐Pro are the most selective for congopain relative to host cathepsins. No hydrolysis occurred upon prolonged incubation time with purified enzymes. In addition introduction of non‐proteogenic residues in the peptidyl backbone greatly enhanced resistance to proteolysis by mammalian sera.
An in vitro DNA virus for in vitro protein evolution
Authors: Tabuchi Ichiro , Soramoto Sayaka , Nemoto Naoto , Husimi Yuzuru
Published: October 2002

In vitro virus is a molecular construct for in vitro protein evolution, which requires some mechanism to link phenotype to genotype. The first in vitro virus was realized by bonding a nascent protein with its coding mRNA via puromycin in in vitro translation. We report a new construct of in vitro DNA virus. The virion was a covalent cDNA–protein fusion, and virion formation did not require any modification of mRNA. Due to intactness of mRNA, this type of in vitro DNA virus will take the next step toward in vitro autonomous evolution, just like in vivo viral evolution in a cellstat.
The solution structure of the viral binding domain of Tva, the cellular receptor for subgroup A avian leukosis and sarcoma virus1
Authors: Tonelli Marco , Peters Reuben J. , James Thomas L. , Agard David A.
Published: October 2002

The cellular receptor for subgroup A avian leukosis and sarcoma virus (ALSV‐A) is Tva, which contains a motif related to repeats in the low density lipoprotein receptor (LDLR) ligand binding repeat (LBr) and which is necessary for viral entry. As observed with LBr repeats of LDLR, the 47 residue LBr domain of Tva (sTva47) requires calcium during oxidative folding to form the correct disulfide bonds, and calcium enhances the structure of correctly oxidized sTva47, as well as its ability to bind the viral envelope protein (Env). However, solution nuclear magnetic resonance studies indicate that, even in the presence of excess calcium, sTva47 exists in an ensemble of conformations. Nonetheless, as reported here, the structure of the predominant sTva47 solution conformer closely resembles that of other LBr repeats, with identical S–S binding topology and octahedral calcium coordination. The location of W48 and other critical residues on the surface suggests a region of the molecule necessary for Env binding and to mediate post‐binding events important for ALSV‐A cell entry.
The Arabidopsis thaliana ethylene‐responsive element binding protein (AtEBP) can function as a dominant suppressor of Bax‐induced cell death of yeast
Authors: Pan Ling , Kawai Maki , Yu Li-Hua , Kim Kyung-Min , Hirata Aiko , Umeda Masaaki , Uchimiya Hirofumi
Published: October 2002

We identified genes based on screening of an Arabidopsis cDNA library for functional suppressors of mouse Bax‐induced cell death of yeast cells. Interestingly, the cDNA encoding AtEBP, known as Arabidopsis thaliana ethylene‐responsive element binding protein, was isolated numerous times in the functional screen (82% of all suppressors). Full‐length AtEBP and its localization to the nucleus were essential for the suppression of Bax‐induced cell death. Morphological abnormality of intracellular network that is a hallmark of Bax‐induced cell death was attenuated by expression of AtEBP.
Antigen‐independent selection of stable intracellular single‐chain antibodies
Authors: Auf der Maur Adrian , Escher Dominik , Barberis Alcide
Published: October 2002

The intracellular expression of single‐chain Fv antibody fragments (scFv) in eukaryotic cells has an enormous potential in functional genomics and therapeutics [Marasco (1997) Gene Ther. 4, 11–15; Richardson and Marasco (1995) Trends Biotechnol. 13, 306–310]. However, the application of these so‐called intrabodies is currently limited by their unpredictable behavior under the reducing conditions encountered inside eukaryotic cells, which can affect their stability and solubility properties [Wörn et al. (2000) J. Biol. Chem. 275, 2795–2803; Biocca et al. (1995) Bio/Technology 13, 1110–1115]. We present a novel system that enables selection of stable and soluble intrabody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single‐chain antibodies fused to a selectable marker that can control gene expression and cell growth. Our results show that the activity of a selectable marker fused to well characterized scFvs [Wörn et al. (2000) J. Biol. Chem. 275, 2795–2803] correlates with the solubility and stability of the scFv moieties. This method provides a unique tool to identify stable and soluble scFv frameworks, which subsequently serve as acceptor backbones to construct intrabody complementarity determining region libraries by randomization of hypervariable loops.
Non‐steroidal anti‐inflammatory drugs inhibit matrix metalloproteinase‐2 expression via repression of transcription in lung cancer cells
Authors: Pan Mei-Ren , Chuang Lea-Yea , Hung Wen-Chun
Published: October 2002

Recent studies show that up‐regulation of cyclooxygenase‐2 (COX‐2) in human cancer cells induces activation of matrix metalloproteinases (MMPs) and increase of metastatic potential. In this study, we investigate the effect of a COX‐2 selective inhibitor, NS398, on the expression and enzymatic activity of MMPs in human lung cancer cells. We found that NS398 inhibited MMP‐2, not MMP‐9, mRNA expression. NS398 also reduced the amount of MMP‐2, not MMP‐9, released into the medium. Additionally, this COX‐2 inhibitor attenuated the degrading activity of MMP‐2 as demonstrated by gelatin zymography. Investigation of cellular MMP‐2 by Western blotting indicated that synthesis and processing of MMP‐2 was significantly suppressed by NS398. We performed promoter activity assay to address whether NS398 might affect MMP‐2 gene transcription. Our results indicated that NS398 directly inhibited MMP‐2 promoter activity. However, the inhibitory effect of NS398 is not fully dependent on inhibition of COX‐2 because a high concentration of NS398 was needed to suppress MMP‐2 expression and addition of prostaglandin E2 only partially reversed the action of NS398. Moreover, a non‐selective COX inhibitor indomethacin also suppressed the expression of MMP‐2. Taken together, these results indicate that non‐steroidal anti‐inflammatory drugs suppress MMP‐2 expression via repression of transcription and support the notion that COX inhibitors may be useful in inhibition and/or prevention of metastasis.
Overexpression of hepatocyte growth factor/scatter factor promotes vascularization and granulation tissue formation in vivo
Authors: Toyoda Mitsuo , Takayama Hisashi , Horiguchi Norio , Otsuka Toshiyuki , Fukusato Toshio , Merlino Glenn , Takagi Hitoshi , Mori Masatomo
Published: October 2002

The effect of hepatocyte growth factor/scatter factor (HGF/SF) during wound healing in the skin was investigated, using HGF/SF‐overexpressing transgenic mouse model. Histological analysis of HGF/SF transgenic mouse excisional wound sites revealed increased granulation tissue with marked vascularization. Northern blot analysis demonstrated that, relative to control, vascular endothelial growth factor (VEGF) expression in transgenic skin was significantly higher at baseline and was robustly up‐regulated during wound healing. Elevated levels of VEGF protein were detected immunohistochemically, predominantly in endothelial cells and fibroblasts within the granulation tissue of HGF/SF transgenic skin. Serum levels of VEGF were also elevated in HGF/SF transgenic mice. Thus, results from our study suggest that HGF/SF has a significant effect on vascularization and granulation tissue formation during wound healing in vivo, involving with induction of VEGF.
Selenium spares ascorbate and α‐tocopherol in cultured liver cell lines under oxidant stress
Authors: Li Xia , Hill Kristina E , Burk Raymond F , May James M
Published: October 2002

The selenoenzyme thioredoxin reductase (TR) can recycle ascorbic acid, which in turn can recycle α‐tocopherol. Therefore, we evaluated the role of selenium in ascorbic acid recycling and in protection against oxidant‐induced loss of α‐tocopherol in cultured liver cells. Treatment of HepG2 or H4IIE cultured liver cells for 48 h with sodium selenite (0–116 nmol/l) tripled the activity of the selenoenzyme TR, measured as aurothioglucose‐sensitive dehydroascorbic acid (DHA) reduction. However, selenium did not increase the ability of H4IIE cells to take up and reduce 2 mM DHA, despite a 25% increase in ascorbate‐dependent ferricyanide reduction (which reflects cellular ascorbate recycling). Nonetheless, selenium supplements both spared ascorbate in overnight cultures of H4IIE cells, and prevented loss of cellular α‐tocopherol in response to an oxidant stress induced by either ferricyanide or diazobenzene sulfonate. Whereas TR contributes little to ascorbate recycling in H4IIE cells, selenium spares ascorbate in culture and α‐tocopherol in response to an oxidant stress.
Human macrophage inflammatory protein‐3α/CCL20/LARC/Exodus/SCYA20 is transcriptionally upregulated by tumor necrosis factor‐α via a non‐standard NF‐κB site
Authors: Harant Hanna , Eldershaw Suzy A. , Lindley Ivan J.D.
Published: October 2002

The 5′‐flanking sequences of the human macrophage inflammatory protein‐3α/CCL20 gene were cloned and transfected into G‐361 human melanoma cells in a luciferase reporter construct. Tumor necrosis factor‐α (TNF‐α) treatment stimulated luciferase expression, and promoter truncations demonstrated that TNF‐α inducibility is conferred by a region between nt −111 and −77, which contains a non‐standard nuclear factor‐κB (NF‐κB) binding site. The requirement for NF‐κB was demonstrated as follows: (i) mutations in this NF‐κB site abrogated TNF‐α responsiveness; (ii) TNF‐α activated a construct containing two copies of the CCL20 NF‐κB binding site; (iii) overexpression of NF‐κB p65 activated the CCL20 promoter; (iv) NF‐κB from nuclear extracts of TNF‐α‐stimulated cells bound specifically to this NF‐κB site.
Biochemical and structural studies of the prion protein polymorphism
Authors: Petchanikow Cyril , Saborio Gabriela P. , Anderes Laurence , Frossard Marie-Jose , Olmedo Maria I. , Soto Claudio
Published: October 2002

A hallmark event in transmissible spongiform encephalopathies is the conversion of the physiological prion protein into the disease‐associated isoform. A natural polymorphism at codon 129 of the human prion gene, resulting in either methionine or valine, has profound influence on susceptibility and phenotypic expression of the disease in humans. In this study, we investigated the local propensity of synthetic peptides, corresponding to the region of the polymorphism and containing either methionine or valine, to adopt a β‐sheet‐rich structure similar to the pathological protein. Circular dichroism studies showed that the methionine‐containing peptide has a greater propensity to adopt a β‐sheet conformation in a variety of experimental conditions. The higher β‐sheet tendency of this peptide was also associated with an increased ability to aggregate into amyloid‐like fibrils. These results suggest that methionine at position 129 of the prion protein increases its susceptibility to switch to the abnormal conformation, in comparison with the presence of valine at the same position.
Mining DNA microarray data using a novel approach based on graph theory
Authors: del Rio Gabriel , Bartley Theodore F , del-Rio Heberto , Rao Rammohan , Jin KunLin , Greenberg David A , Eshoo Mark , Bredesen Dale E
Published: October 2002

The recent demonstration that biochemical pathways from diverse organisms are arranged in scale‐free, rather than random, systems [Jeong et al., Nature 407 (2000) 651–654], emphasizes the importance of developing methods for the identification of biochemical nexuses – the nodes within biochemical pathways that serve as the major input/output hubs, and therefore represent potentially important targets for modulation. Here we describe a bioinformatics approach that identifies candidate nexuses for biochemical pathways without requiring functional gene annotation; we also provide proof‐of‐principle experiments to support this technique. This approach, called Nexxus, may lead to the identification of new signal transduction pathways and targets for drug design.
Structural and functional characterization of a monoclonal antibody specific for the preS1 region of hepatitis B virus
Authors: Pizarro Juan Carlos , Vulliez-le Normand Brigitte , Riottot Marie-Madeleine , Budkowska Agata , Bentley Graham A.
Published: October 2002

The monoclonal antibody 5a19, raised against the ay serotype of hepatitis B virus, binds to the segment of the preS1 region comprising residues 37–43, which is implicated in attachment of the virus to hepatocytes. The dissociation constant, derived from kinetic studies using surface plasmon resonance techniques, is in the low nanomolar range. The nucleotide sequence of the variable domains has been determined and the corresponding germ‐line genes have been identified. The three‐dimensional structure of the Fab fragment has been determined by X‐ray crystallography to 2.6 Å resolution.
Mass spectrometric analysis reveals a cysteine bridge between residues 2 and 61 of the auxin‐binding protein 1 from Zea mays L
Authors: Feckler Christian , Muster Gerhard , Feser Werner , Römer Axel , Palme Klaus
Published: October 2002

The major auxin‐binding protein (ZmERabp1) from maize (Zea mays L.) has been structurally characterized. We determined the position of a disulfide bridge in ZmERabp1 by mass‐spectrometric analysis. We show that Cys2 and Cys61 are covalently linked and that residue Cys155 bears the free sulfhydryl group. By making use of electrospray mass spectrometry, the molecular mass of ZmERabp1 was determined to be 20 243 Da comprising a sugar moiety of 1865 Da, corresponding to a high mannose‐type glycan structure. Due to the high homology among all characterized ABPs, the information on the disulfide bonds will be important for functional analysis of recombinantly expressed ABP1.
The direct determination of protein structure by NMR without assignment
Authors: Atkinson R.Andrew , Saudek Vladimı́r
Published: October 2002

Assignment of the resonances in nuclear magnetic resonance spectra is considered a pre‐requisite for the interpretation of spectra that yield structural information. The determination of the three‐dimensional structure of a biological macromolecule may, however, be achieved directly without spectral assignment, using the same set of heteronuclear scalar and dipolar coupling experiments as normally used. A cross‐peak in any of the spectra may be interpreted as a distance between atoms, yielding a set of distances between unassigned atoms that serves to define the tertiary structure of the molecule. The principle is illustrated using the 76 amino acid protein ubiquitin.
The F286Y mutation of PrlA4 tempers the signal sequence suppressor phenotype by reducing the SecA binding affinity
Authors: de Keyzer Jeanine , van der Does Chris , Swaving Jelto , Driessen Arnold J.M.
Published: October 2002

SecYEG forms the protein‐conducting channel of the Escherichia coli translocase. It binds the peripheral ATPase SecA that drives the preprotein translocation reaction. PrlA4 is a double mutant of SecY that enables the translocation of preproteins with a defective or even missing signal sequence. The effect of the individual mutations, F286Y and I408N, was studied with SecYEG proteoliposomes. SecY(I408N) is responsible for the increased translocation of preproteins with a defective and normal signal sequence, and exhibits a stronger prl phenotype than PrlA4. This activity correlates with an elevated SecA‐translocation ATPase and SecA binding affinity. SecY(F286Y) supports only a low SecA binding affinity, preprotein translocation and SecA translocation ATPase activity. These results suggest that the second site F286Y mutation reduces the strength of the I408N mutation of PrlA4 by lowering the SecA binding affinity.
Introns in protein‐coding genes in Archaea
Authors: Watanabe Yoh-ichi , Yokobori Shin-ichi , Inaba Toshiro , Yamagishi Akihiko , Oshima Tairo , Kawarabayasi Yutaka , Kikuchi Hisasi , Kita Kiyoshi
Published: October 2002

Introns in protein‐coding genes are ubiquitous in eukaryotic cells, but pre‐mRNA splicing has yet to be reported in archaeal and its viral genomes. We present evidence of introns in genes encoding a homolog of eukaryotic Cbf5p (centromere‐binding factor 5; a subunit of a small nucleolar ribonucleoprotein) in three Archaea; Aeropyrum pernix, Sulfolobus solfataricus and Sulfolobus tokodaii. Splicing of pre‐mRNAs in vivo was demonstrated by reverse transcriptase‐mediated polymerase chain reaction. The exon–intron boundaries of these genes are predicted to be folded into a structure similar to the bulge–helix–bulge motif, suggesting that splicing of these pre‐mRNAs probably depends on the splicing system elucidated for archaeal pre‐tRNAs and rRNAs.
Kinetic properties and inhibition of Trypanosoma cruzi 3‐hydroxy‐3‐methylglutaryl CoA reductase
Authors: Hurtado-Guerrrero Ramón , Peña-Dı́az Javier , Montalvetti Andrea , Ruiz-Pérez Luis M , González-Pacanowska Dolores
Published: October 2002

A detailed kinetic analysis of the recombinant soluble enzyme 3‐hydroxy‐3‐methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower proportion compared to the anabolic reaction. We report that the catalytically active species of HMGR in solution is the tetrameric form. Fluvastatin inhibited competitively the enzyme while cerivastatin binds by a mechanism which is more accurately described by a biphasic process characteristic of a class of ‘slow, tight‐binding’ inhibitors.
WH2 domain: a small, versatile adapter for actin monomers
Authors: Paunola Eija , Mattila Pieta K. , Lappalainen Pekka
Published: October 2002

The actin cytoskeleton plays a central role in many cell biological processes. The structure and dynamics of the actin cytoskeleton are regulated by numerous actin‐binding proteins that usually contain one of the few known actin‐binding motifs. WH2 domain (ASP omology domain‐) is a ∼35 residue actin monomer‐binding motif, that is found in many different regulators of the actin cytoskeleton, including the β‐thymosins, ciboulot, WASP (iskott ldrich yndrome rotein), verprolin/WIP (ASP‐nteracting rotein), Srv2/CAP (adenylyl yclase‐ssociated rotein) and several uncharacterized proteins. The most highly conserved residues in the WH2 domain are important in β‐thymosin's interactions with actin monomers, suggesting that all WH2 domains may interact with actin monomers through similar interfaces. Our sequence database searches did not reveal any WH2 domain‐containing proteins in plants. However, we found three classes of these proteins: WASP, Srv2/CAP and verprolin/WIP in yeast and animals. This suggests that the WH2 domain is an ancient actin monomer‐binding motif that existed before the divergence of fungal and animal lineages.
Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: formation of the fluorescein is independent of the generation of reactive oxygen species
Authors: Ohashi Tomoko , Mizutani Atsushi , Murakami Akira , Kojo Shosuke , Ishii Tetsuro , Taketani Shigeru
Published: October 2002

Oxidative stress and the generation of reactive oxygen species (ROS) have been implicated in the pathogenesis of cellular damage. These events have usually been reported in terms of oxidation of a reporter molecule such as 2′,7′‐dichlorodihydrofluorescin diacetate (DCFH‐DA). Treatment of HeLa cells with hemin or metalloporphyrins resulted in a rapid oxidation of DCFH in a time‐ and dose‐dependent manner. This oxidation was inhibited by treatment of the cells with a large amount of superoxide dismutase and catalase, which is different from observations that these enzymes had no effect on the induction of heme oxygenase‐1, a stress‐induced protein, in hemin‐treated cells. To examine the possibility that the oxidation of DCFH is independent of the generation of ROS, the oxidation was measured using hemoglobin‐synthesizing erythroleukemia K562 cells. When K562 cells were treated with δ‐aminolevulinic acid, a precursor of heme, oxidation of DCFH increased depending on the heme content in cells. Then DCFH‐DA was oxidized directly with heme, hemoglobin, myoglobin and cytochrome c. These results suggest that oxidation of DCFH is not always related to the generation of ROS but may be related to heme content in cells.
Potent transforming activity of the small GTP‐binding protein Rit in NIH 3T3 cells: evidence for a role of a p38γ‐dependent signaling pathway
Authors: Sakabe Kaoru , Teramoto Hidemi , Zohar Muriel , Behbahani Babak , Miyazaki Hiroshi , Chikumi Hiroki , Gutkind J.Silvio
Published: October 2002

A novel branch of the Ras family, Rit, was recently identified. Rit exhibits a distinct C‐terminus and effector domain, and does not activate mitogen‐activated protein kinase (MAPK) but can cooperate with Raf to transform fibroblasts. Here, we found that when overexpressed, activated mutants of Rit transform NIH 3T3 cells efficiently, and stimulate p38γ but not MAPK, p38α, p38β, p38δ, or ERK5. Furthermore, we provide evidence that p38γ activation is required for the ability of Rit to stimulate gene expression and cellular transformation. These findings suggest that this unique GTPase stimulates proliferative pathways distinct from those regulated by other Ras family members.
Suppression of tumor growth by novel peptides homing to tumor‐derived new blood vessels
Authors: Asai Tomohiro , Nagatsuka Mayumi , Kuromi Koichi , Yamakawa Satoru , Kurohane Kohta , Ogino Koichi , Tanaka Michinori , Taki Takao , Oku Naoto
Published: October 2002

Novel peptides homing to angiogenic vessels were recently isolated from a phage‐displayed random pentadecapeptide library. One of the isolated peptides, ASSSYPLIHWRPWAR, significantly suppressed the migration of VEGF‐stimulated human umbilical vein endothelial cells. Dendoric ASSSYPLIHWRPWAR‐peptide suppressed the formation of new blood vessels in dorsal air sac model mice. Furthermore, ASSSYPLIHWRPWAR‐peptide and the fragment peptides containing WRP, which is revealed to be an epitope sequence, significantly suppressed the tumor growth, although 15‐mer shuffled peptide derived from ASSSYPLIHWRPWAR and pentapeptides with alanine substitution of each residue of WRP did not. Taken together, ASSSYPLIHWRPWAR‐peptide may cause tumor dormancy through inhibition of angiogenesis, and the WRP sequence may be the minimal and essential sequence for this activity.
The unfolding mechanism and the disulfide structures of denatured lysozyme
Authors: Chang Jui-Yoa , Li Li
Published: October 2002

The mechanism of denaturation and unfolding of lysozyme has been characterized here using the method of disulfide scrambling. Under denaturing conditions (urea, guanidinium hydrochloride (GdmCl), guanidinium thiocyanate (GdmSCN), or elevated temperature) and in the presence of thiol initiator, lysozyme denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled isomers. To denature 50% of the native lysozyme requires 1.1 M of GdmSCN, 2.8 M of GdmCl and 7.4 M of urea, respectively. High temperature (75°C) denatures the native lysozyme quantitatively within 20 min. Analysis by reversed‐phase high‐performance liquid chromatography reveals that urea and GdmCl denatured lysozyme comprise a single predominant disulfide isomer, designated as X‐lysozyme‐a, regardless of the concentration of the denaturant. X‐Lysozyme‐a was shown to adopt thebeads‐formstructure with its four disulfide bonds formed by four consecutive pairs of cysteines (Cys6–Cys30, Cys64–Cys76, Cys80–Cys94, Cys115–Cys127). The conspicuous absence of partially structured unfolding intermediates of lysozyme contrasts to that found in the case of α‐lactalbumin and accounts for the widely observed two‐stage mechanism of lysozyme unfolding.
Anion exchanger isoform 2 operates in parallel with Na+/H+ exchanger isoform 1 during regulatory volume decrease of human cervical cancer cells
Authors: Shen Meng-Ru , Wilkins Robert J. , Chou Cheng-Yang , Ellory J.Clive
Published: October 2002

Intracellular pH (pHi) homeostasis was investigated in human cervical cancer SiHa cells undergoing regulatory volume decrease (RVD) to determine which transport systems were involved. Using isoform‐specific primers, mRNA transcripts of Na+/H+ exchanger isoform 1 (NHE1) and isoform 3 were identified by reverse transcriptase polymerase chain reaction (RT‐PCR) and the results confirmed by Western immunoblotting. From anion exchanger isoforms 1–3 (AE1–3), only the mRNA transcript of AE2 was identified by RT‐PCR and the identity was confirmed by digestion with a specific restriction endonuclease. SiHa cells loaded with the fluorescent dye 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein and resuspended in isotonic media showed a stable pHi. In contrast, a gradual internal acidification took place following resuspension in hypotonic media. The NHE inhibitors, HOE694 (10 μM) and amiloride (1 mM), showed a similar potency in enhancing the rate and extent of the hypotonicity‐induced internal acidification. The absence of extracellular Na+ also substantially enhanced the acidification during RVD. These results suggest that internal acidification during RVD is mainly compensated by the operation of NHE1. Extracellular Cl− was critically necessary for the pHi acidification during RVD. The hypotonicity‐induced acidification was significantly attenuated by 100 μM 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid, a concentration inhibiting more than 90% AE activity. This indicates that AE2 mediates a net Cl− influx with compensating HCO3
− efflux during RVD. We conclude that AE2 operates in parallel with NHE1 to regulate pHi during RVD of human cervical cancer cells.
Transport of N‐acetyl‐D‐mannosamine and N‐acetyl‐D‐glucosamine in Escherichia coli K1: effect on capsular polysialic acid production
Authors: Revilla-Nuin Beatriz , Reglero Ángel , Martı́nez-Blanco Honorina , Bravo Ignacio G , Ferrero Miguel A , Rodrı́guez-Aparicio Leandro B
Published: October 2002

N‐Acetyl‐D‐mannosamine (ManNAc) and N‐acetyl‐D‐glucosamine (GlcNAc) are the essential precursors of N‐acetylneuraminic acid (NeuAc), the specific monomer of polysialic acid (PA), a bacterial pathogenic determinant. Escherichia coli K1 uses both amino sugars as carbon sources and uptake takes place through the mannose phosphotransferase system transporter, a phosphoenolpyruvate‐dependent phosphotransferase system that shows a broad range of specificity. Glucose, mannose, fructose, and glucosamine strongly inhibited the transport of these amino‐acetylated sugars and GlcNAc and ManNAc strongly affected ManNAc and GlcNAc uptake, respectively. The ManNAc and the GlcNAc phosphorylation that occurs during uptake affected NeuAc synthesis in vitro. These findings account for the low in vivo PA production observed when E. coli K1 uses ManNAc or GlcNAc as a carbon source for growth.
The involvement of reactive oxygen species (ROS) and p38 mitogen‐activated protein (MAP) kinase in TRAIL/Apo2L‐induced apoptosis
Authors: Lee Myoung-Woo , Park Soon Cheol , Yang Young Geun , Yim Soon Ock , Chae Hee Sun , Bach Jae-Hyung , Lee Hyun Jung , Kim Kyung Yong , Lee Won Bok , Kim Sung Su
Published: October 2002

To determine the apoptotic signaling pathway which tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL/Apo2L) induced, we investigated the contribution of reactive oxygen species (ROS), p38 mitogen‐activated protein (MAP) kinase and caspases in human adenocarcinoma HeLa cells. Here we show that upon TRAIL/Apo2L exposure there was pronounced ROS accumulation and activation of p38 MAP kinase, and that activation of caspases and apoptosis followed. Pretreatment with antioxidants such as glutathione or estrogen attenuated TRAIL/Apo2L‐induced apoptosis through a reduction of ROS generation and diminished p38 MAP kinase and caspase activation. The p38 MAP kinase inhibitor SB203580 prevented apoptosis through a blockage of caspase activation, although ROS generation was not attenuated. Furthermore, the pan‐caspase inhibitor Z‐Val‐Ala‐DL‐Asp‐fluoromethyl ketone fully prevented apoptosis, while neither ROS accumulation nor p38 MAP kinase activation were affected. Therefore, our results suggest that TRAIL/Apo2L‐induced apoptosis is mediated by ROS‐activated p38 MAP kinase followed by caspase activation in HeLa cells.
Serine (threonine) phosphatase(s) acting on cAMP‐dependent phosphoproteins in mammalian mitochondria
Authors: Signorile Anna , Sardanelli Anna Maria , Nuzzi Rosanna , Papa Sergio
Published: October 2002

Immunochemical and functional evidence showing the existence in the inner membrane and matrix fraction of mammalian mitochondria of serine/threonine phosphatases acting on cAMP‐dependent phosphoproteins is presented. Mg2+‐dependent Ca2+‐inhibitable PP2C phosphatase, associated to the inner membrane, dephosphorylates the 18 kDa (NDUFS4 gene) of complex I.
A novel approach to study the activity and stoichiometry simultaneously for microsomal pentoxyresorufin‐O‐dealkylase reaction
Authors: Rastogi Shipra , Das Mukul , Khanna Subhash K
Published: October 2002

A simple approach to study the activity and stoichiometry of cytochrome P‐450 IIB1‐catalyzed metabolism of pentoxyresorufin (PRF) has been investigated. It involves the continuous spectral analysis of reaction mixture containing PRF, microsomes from phenobarbital‐induced rats and NADPH. The kinetics of NADPH consumption, PRF utilization, NADP and resorufin formation was monitored at λ
max of 338, 484, 260 and 572 nm, respectively. The stoichiometry of the enzyme reaction tabulated either by specific activity or by V
max value showed that 10 molecules of NADPH were required for the conversion of one molecule of PRF to one molecule of resorufin along with 10 molecules of NADP. Further, it was observed that almost six molecules of NADPH are consumed in the incubation mixture devoid of PRF indicating the possibility of metabolism of endogenous substrates. Interestingly, the stoichiometry ratio of 1:1 for PRF and resorufin was established even in the presence of P‐450 inhibitors with a lower rate of metabolism. However, the ratio of NADPH to PRF was altered in the presence of inhibitors, suggesting that the simultaneous monitoring of the substrate, electron donor and the products could be useful in understanding the modifications of stoichiometry of electron donor and substrate/product.
Mai1p is essential for maturation of proaminopeptidase I but not for autophagy
Authors: Barth Henning , Meiling-Wesse Khuyen , Epple Ulrike D. , Thumm Michael
Published: October 2002

We here identify Mai1p, a homologue of the autophagy protein Aut10p, as a novel component essential for proaminopeptidase I (proAPI) maturation under non‐starvation conditions. In mai1Δ cells mature vacuolar proteinases are detectable and vacuolar acidification is normal. In mai1Δ cells autophagy occurs, though at a somewhat reduced level. This is indicated by proAPI maturation during starvation and accumulation of autophagic bodies during starvation with phenylmethylsulfonyl fluoride. Homozygous diploid mai1Δ cells sporulate, but with a slightly reduced frequency. Biologically active Ha‐tagged Mai1p, chromosomally expressed under its native promoter, is at least in part peripherally membrane‐associated. In indirect immunofluorescence it localizes to the vacuolar membrane or structures nearby. In some cells Ha‐tagged Mai1p appears concentrated at regions adjacent to the nucleus.