bluuebunny/crossref_metadata_2025 huggingface dataset:
2‐Oxoglutarate increases the binding affinity of the NtcA (nitrogen control) transcription factor for the Synechococcus glnA promoter
Authors: Vázquez-Bermúdez Marı́a Félix , Herrero Antonia , Flores Enrique
Published: October 2002

The cyanobacterial NtcA global nitrogen regulator belongs to the catabolite activator protein (CAP) family and activates transcription of nitrogen assimilation genes in response to nitrogen step‐down. The binding affinity of NtcA towards a DNA fragment carrying the promoter of the glnA gene from Synechococcus sp. PCC 7942, analyzed in vitro by band‐shift assay, was increased five‐fold by 2‐oxoglutarate in the presence of Mg2+ ions. The 2‐oxoglutarate effect peaked at about 0.6 mM, a rather physiological concentration for this compound under nitrogen‐limiting conditions, and could be partially reproduced by 3‐oxoglutarate but not by oxaloacetate or glutamate. These results suggest 2‐oxoglutarate as a signal of the C to N balance of the cells to regulate NtcA activity and provide a new example of regulation in the versatile CAP family of proteins.
Involvement of the N‐terminus of Bax in its intracellular localization and function
Authors: Cartron P.-F. , Moreau C. , Oliver L. , Mayat E. , Meflah K. , Vallette F.M.
Published: October 2002

We have identified, using site‐directed mutagenesis, a proline located at position 13 of Baxα (Bax) as crucial for the maintenance of its cytosolic conformation. The substitution of this proline by a valine results in a strong binding of Bax to mitochondria and to conformational changes monitored by a decreased sensitivity of Bax to mild proteolysis and the enhancement of its oligomerization state. Deletion of the C‐terminus of Bax does not modify its intracellular localization. On the other hand, the pro‐apoptotic activity of Bax is enhanced by a deletion of the C‐terminus in the absence of the N‐terminus but is decreased in its presence. These results suggest that both extremities functionally interact to control the activity but not the subcellular localization of Bax.
Activity and inhibition of plasmepsin IV, a new aspartic proteinase from the malaria parasite, Plasmodium falciparum
Authors: Wyatt David M , Berry Colin
Published: October 2002

A new aspartic proteinase from the human malaria parasite Plasmodium falciparum is able to hydrolyse human haemoglobin at a site known to be the essential primary cleavage site in the haemoglobin degradation pathway. Thus, plasmepsin IV may play a crucial role in this critical process which yields nutrients for parasite growth. Furthermore, synthetic inhibitors known to inhibit parasite growth in red cells in culture are able to inhibit the activity of this enzyme in vitro. As a result, plasmepsin IV appears to be a potential target for the development of new antiparasitic drugs.
Restoration of UV sensitivity in UV‐resistant HeLa cells by antisense‐mediated depletion of damaged DNA‐binding protein 2 (DDB2)
Authors: Sun Nian-Kang , Kamarajan Pachiyappan , Huang Haimei , Chao Chuck C.-K
Published: October 2002

Damaged DNA‐binding activity comprises two major protein components, DDB1 and DDB2, which are implicated in the repair of ultraviolet (UV) radiation‐induced DNA damage. The possible role of DDB2 as a determinant of cellular sensitivity to UV was investigated. The abundance of DDB2 in UV‐resistant HeLa cell lines was increased compared with that in the parental UV‐sensitive cells. Stable transfection of the resistant cells with DDB2 antisense cDNA resulted in marked depletion of DDB2 protein and restored cellular sensitivity to UV‐induced apoptosis. Whereas the extent of UV‐induced activation of apoptosis executioners, including DNA fragmentation factor, and caspase‐3 were reduced in the UV‐resistant cells compared with those apparent in the sensitive cells, depletion of DDB2 from the resistant cells restored the normal activation patterns for these proteins. In contrast, overexpressing DDB2 in DDB2‐depleted cells with recombinant adenovirus, which carries ddb2 cDNA, markedly inhibited the extent of UV‐induced activation of DNA fragmentation factor, and caspase‐3. Interestingly, a mutated form of DDB2, which is defective in interacting with DDB1 and binding to UV‐damaged DNA, also markedly inhibited the activation of apoptosis executioners. These results indicate that DDB2 is a modulator of UV‐induced apoptosis, and that UV resistance can be overcome by inhibition of DDB2. The findings also suggest that modulation of UV‐induced apoptosis by DDB2 may be independent of DNA repair.
The characterization of the human Siah‐1 promoter1
Authors: Maeda Ayaka , Yoshida Tatsushi , Kusuzaki Katsuyuki , Sakai Toshiyuki
Published: October 2002

Siah‐1, the human homologue of Drosophila seven in absentia, is related to apoptosis and tumor suppression. Although it was reported that the expression of Siah‐1 is induced by p53 and p21/WAF1, little is known about the transcriptional regulation of the Siah‐1 gene. To investigate the transcriptional regulation, we isolated and sequenced the genomic fragment of the Siah‐1 promoter region. The Siah‐1 promoter has no typical TATA box or CCAAT box. Transient transfection assays using reporter plasmids in which the promoter region of the Siah‐1 gene was deleted or mutated showed that one Sp1 site was responsible for the basal promoter activity. In Northern blotting analysis, the expression of the Siah‐1 gene was upregulated by p53, but activation of the reporter plasmid by the p53 co‐transfection assay was not shown, suggesting that a p53 responsive element does not exist in the promoter region we examined in this study but might be present in another region.
Distinct regions of C‐terminus of the high affinity neurotensin receptor mediate the functional coupling with pertussis toxin sensitive and insensitive G‐proteins
Authors: Najimi Mustapha , Gailly Philippe , Maloteaux Jean-Marie , Hermans Emmanuel
Published: October 2002

The functional coupling of C‐terminally truncated mutants of the high affinity rat neurotensin (NT) receptor (NTS1) was characterized in transfected Chinese hamster ovary cells. On cells expressing NTRΔ372 (truncated NTS1 lacking the entire 52 amino acid C‐terminus), NT failed to promote [35S]guanosine 5′‐[γ‐35S]triphosphate binding whereas a robust pertussis toxin (PTx) sensitive response was observed in cells expressing a partially truncated receptor (NTRΔ401 lacking the last 23 residues). Similar results were obtained when measuring the ability of NT to induce the production of arachidonic acid. Since neither deletions impaired the NT‐induced phosphoinositide hydrolysis, these results indicate that the membrane proximal region of the C‐terminus is specifically involved in the functional coupling of the receptor with PTx sensitive G‐proteins. This region was also found to be involved in the control of receptor internalization. However, PTx failed to impair internalization, indicating that these two properties are not directly related.
RNA aptamers directed against release factor 1 from Thermus thermophilus
Authors: Szkaradkiewicz Karol , Nanninga Matthias , Nesper-Brock Martina , Gerrits Michael , Erdmann Volker A , Sprinzl Mathias
Published: October 2002

An in vitro selection/amplification (SELEX) was used to generate RNA aptamers that specifically bind Thermus thermophilus release factor 1 (RF1). From 31 isolated clones, two groups of aptamers with invariable sequences 5′‐ACCU‐3′ and 5′‐GAAAGC‐3′ were isolated. Chemical and enzymatic probing of the structure indicate that in both groups the invariable sequences are located in single‐stranded regions of hairpin structures. Complex formations between RF1 and aptamers of both groups were identified by electrophoretic shift assay and chemical footprinting. Deletion of the invariable sequences did not effect the secondary structure of the aptamers but abolished their binding to RF1. RNA motifs matching the invariable sequences of the aptamers are present as consensus sequences in the peptidyl transferase center of 23S rRNAs. T. thermophilus RF1 recognizes UAG stop codons in an Escherichia coli in vitro translation system. Aptamers from both groups inhibited this RF1 activity.
Cysteine‐scanning mutagenesis study of the sixth transmembrane segment of the Na,K‐ATPase α subunit
Authors: Guennoun Saı̈da , Horisberger Jean-Daniel
Published: October 2002

The accessibility of the residues of the sixth transmembrane segment (TM) of the Bufo marinus Na,K‐ATPase α subunit was explored by cysteine scanning mutagenesis. Methanethiosulfonate reagents reached only the two most extracellular positions (T803, D804) in the native conformation of the Na,K‐pump. Palytoxin induced a conductance in all mutants, including D811C, T814C and D815C which showed no active electrogenic transport. After palytoxin treatment, four additional positions (V805, L808, D811 and M816) became accessible to the sulfhydryl reagent. We conclude that one side of the sixth TM helix forms a wall of the palytoxin‐induced channel pore and, probably, of the cation pathway from the extracellular side to one of their binding sites.
The P2X7 receptor from Xenopus laevis: formation of a large pore in Xenopus oocytes
Authors: Paukert Martin , Hidayat Susanti , Gründer Stefan
Published: October 2002

The purinergic P2X7 receptor is an ATP‐receptor channel predominantly expressed in immune cells. P2X7 has been cloned from human, rat and mouse. Here we report cloning of the Xenopus laevis P2X7 receptor (xP2X7). xP2X7 is only about 50% identical to the mammalian homologues, shows a broad tissue expression pattern, and has the electrophysiological characteristics typical of a P2X7 receptor: low agonist affinity (EC50 about 2.6 mM) and a non‐desensitizing current. Moreover, expression of xP2X7 in Xenopus oocytes is sufficient to induce the formation of a large pore, which is permeable to large cations such as NMDG+. Identification of a non‐mammalian P2X7 receptor may help to identify functionally important parts of the protein.
Construction of the ‘minimal’ SRP that interacts with the translating ribosome but not with specific membrane receptors in Escherichia coli
Authors: Avdeeva Olga N , Myasnikov Alexander G , Sergiev Petr V , Bogdanov Alexey A , Brimacombe Richard , Dontsova Olga A
Published: October 2002

Escherichia coli signal recognition particle (SRP) consists of 4.5S RNA and Ffh protein. In contrast to eukaryotes, it remains unclear whether translation arrest takes place in prokaryotic cells. To study this problem we constructed a fusion of the M domain of Ffh protein with a cleavable affinity tag. This mutant Ffh, in a complex with 4.5S RNA, can bind signal peptide at the translating ribosome but is unable to bind the membrane. This SRP–ribosome complex should accumulate in the cell if translation is arrested. To test this, the complex was purified from the cells by ultracentrifugation and affinity chromatography. The composition of the complex was analyzed and found to consist of ribosomal RNAs and proteins, the Ffh M domain and 4.5S RNA. The accumulation of this complex in the cell in significant amounts indicated that SRP‐mediated translation arrest did occur in bacterial cells.
Nucleotide specificity of an archaeal group II chaperonin from Thermococcus strain KS‐1 with reference to the ATP‐dependent protein folding cycle
Authors: Yoshida Takao , Kawaguchi Rika , Maruyama Tadashi
Published: October 2002

The archaeal chaperonin‐mediated folding of green fluorescent protein (GFP) was examined in the presence of various nucleotides. The recombinant α‐ and β‐subunit homo‐oligomers and natural chaperonin oligomer from Thermococcus strain KS‐1 exhibited folding activity with not only ATP but also with CTP, GTP, or UTP. The ADP‐bound form of both recombinant and natural chaperonin had the ability to capture non‐native GFP, but could not refold it in the presence of CTP, GTP or UTP until ATP was supplied. The archaeal chaperonin thus utilized ATP, but could not use other nucleoside triphosphates in the cytoplasm where ADP was present.
High free energy of lipid/protein interaction in biological membranes
Authors: Sandermann Heinrich
Published: October 2002

The free energy of lipid/protein interaction in biological membranes is still unknown although extensive partitioning and modelling studies have revealed many partial energetic increments. Multiple site binding kinetics are now applied to four well‐studied functional membrane proteins, and mean free energy values (±S.D.) of −4.23±0.49 kcal/mol for single lipid binding sites and of −89.7±35.4 kcal/mol for complete lipid substitution are obtained. These high free energy values point to an important bioenergetic role of lipid/protein interaction in membrane functions.
Dephosphorylation of the two regulatory components of myosin phosphatase, MBS and CPI17
Authors: Takizawa Norio , Niiro Naohisa , Ikebe Mitsuo
Published: October 2002

Dephosphorylation of the two key regulatory factors of myosin light chain phosphatase (MLCP), CPI17 and MBS (myosin binding subunit) of MLCP was studied. While Thr38 phosphorylated CPI17 is quite susceptible to protein phosphatases, phosphorylated MBS was highly resistant to dephosphorylation. Type 2A, 2B and 2C protein phosphatases (PP2A, PP2B and PP2C), but not type 1 (PP1), dephosphorylated CPI17. The majority of the CPI17 phosphatase activity in smooth muscle was attributed to PP2A and PP2C. Phospholipids inhibited dephosphorylation of MBS and arachidonic acid (AA) inhibited PP2A activity against both MBS and CPI17, raising the possibility that AA favors the preservation of active MLCP. Consistently, while the phosphorylation of CPI17 was promptly decreased when the agonist was removed, the phosphorylation of MBS was unchanged in intact smooth muscle fiber. The results suggest that MBS phosphorylation mediated regulation of MLCP is not suitable for regulating rapid change in myosin phosphorylation. On the other hand, phosphorylated CPI17 is readily dephosphorylated thus likely to play a role in regulating fast phosphorylation–dephosphorylation cycle in cells.
TheSambucus nigratype‐2 ribosome‐inactivating protein SNA‐I′ exhibits in planta antiviral activity in transgenic tobacco
Authors: Chen Ying , Peumans Willy J , Van Damme Els J.M
Published: October 2002

Transgenic tobacco (Samsun NN) plants transformed with a cDNA clone encoding SNA‐I′ fromSambucus nigrasynthesize, and correctly process and assemble, a fully active type‐2 ribosome‐inactivating protein. Expression of SNA‐I′ under the control of the 35S cauliflower mosaic virus promoter enhances the plant's resistance against infection with tobacco mosaic virus. In contrast to type‐1 ribosome‐inactivating proteins, the expression of SNA‐I′ does not affect the growth and fertility of the transgenic plants and is not accompanied by an increased expression of pathogenesis‐related proteins indicating that its antiviral activity most probably differs from that of pokeweed antiviral protein.
Differentiation of single‐cell organisms according to elongation stages crucial for gene expression efficacy
Authors: Likhoshvai Vitali A. , Matushkin Yuri G.
Published: October 2002

We analyzed the interrelation between the efficiency of a gene expression and the nucleotide composition of all protein‐coding sequences in 38 unicellular organisms whose complete genomic sequences are known. These organisms comprise 37 prokaryotic (29 eubacteria and eight archaebacteria) and one eukaryotic (yeast) species. We demonstrated that frequency analysis of gene codon composition fails to reflect adequately the gene expression efficiency of all these organisms. We constructed a measure, the elongation efficiency index, that considers simultaneously the information on codon frequencies and the degree of mRNA local self‐complementarity. This measure recognizes the ribosome‐coding genes as highly expressed in all the unicellular organisms studied. According to our analysis, these species fall into five groups differentiated by the process that makes the key contribution to the elongation rate.
Regulation of the heme A biosynthetic pathway in Saccharomyces cerevisiae
Authors: Barros Mario H , Tzagoloff Alexander
Published: October 2002

Biosynthesis of heme A, a prosthetic group of cytochrome oxidase (COX), involves an initial farnesylation of heme B. The heme O product formed in this reaction is modified by hydroxylation of the methyl group at carbon C‐8 of the porphyrin ring. This reaction was proposed to be catalyzed by Cox15p, ferredoxin, and ferredoxin reductase. Oxidation of the alcohol to the corresponding aldehyde yields heme A. In the present study we have assayed heme A and heme O in yeast COX mutants. The steady state concentrations of the two hemes in the different strains studied indicate that hydroxylation of heme O, catalyzed by Cox15p, is regulated either by a subunit or assembly intermediate of COX. The heme profiles of the mutants also suggest positive regulation of heme B farnesylation by the hydroxylated intermediate formed at the subsequent step or by Cox15p itself.
Action spectrum of psbA gene transcription is similar to that of photoinhibition in Synechocystis sp. PCC 6803
Authors: Tyystjärvi Taina , Tuominen Ilona , Herranen Mirkka , Aro Eva-Mari , Tyystjärvi Esa
Published: October 2002

The photosystem II (PSII) reaction center protein D1 undergoes rapid light‐dependent turnover, which is caused by photoinhibition. To identify the photoreceptor(s) involved in the light‐dependent expression of the psbA gene encoding the D1 protein, we determined the action spectra of psbA transcription, PSII activity, photosynthesis and photoinhibition in Synechocystis sp. PCC 6803. In accordance with its phycobilisome antenna, PSII showed the highest activity in the spectral region from yellow to red and only low activity in the ultraviolet‐A (UV‐A) to green region. Photoinhibition, in turn, was fastest in UV‐A to violet light and a minor peak was found in the orange region. The action spectrum of psbA transcription resembled closely that of photoinhibition, suggesting that photoinhibition creates a signal for up‐regulation of the psbA gene.
Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types
Authors: Selth Luke A. , Randles John W. , Rezaian M.Ali
Published: October 2002

We have previously shown that the soil‐borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication‐competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low‐level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using β‐glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.
Crystal structure of human nicotinamide mononucleotide adenylyltransferase in complex with NMN
Authors: Werner Erik , Ziegler Mathias , Lerner Felicitas , Schweiger Manfred , Heinemann Udo
Published: October 2002

The final step in the biosynthesis of nicotinamide‐adenine dinucleotide, a major coenzyme in cellular redox reactions and involved in intracellular signaling, is catalyzed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT). The X‐ray structure of human NMNAT in complex with nicotinamide mononucleotide was solved by the single‐wavelength anomalous dispersion method at a resolution of 2.9 Å. Human NMNAT is a symmetric hexamer whose subunit is formed by a large six‐stranded parallel β‐sheet with helices on both sides. Human NMNAT displays a different oligomerization compared to the archaeal enzyme. The protein–nicotinamide mononucleotide interaction pattern provides insight into ligand binding in the human enzyme.
Trans‐translation mediated by Bacillus subtilis tmRNA
Authors: Ito Ken-ichi , Tadaki Toshimasa , Lee SungGa , Takada Kazuma , Muto Akira , Himeno Hyouta
Published: October 2002

Trans‐translation, in which a ribosome switches between translation of an mRNA and a tmRNA, produces a chimera polypeptide of an N‐terminal truncated polypeptide and a C‐terminal tag‐peptide encoded by tmRNA. One of the tmRNA binding proteins, a ribosomal protein S1, has not been found in a group of Gram‐positive bacteria. In this study, the trans‐translation reaction with tmRNA from Bacillus subtilis belonging to this group was examined. When a truncated gene lacking a termination codon was expressed in B. subtilis, a 15‐amino acid tag‐peptide derived from tmRNA was identified in the C‐termini of the trans‐translation products. An identical tag‐peptide was also found at the C‐termini of the products from a truncated gene, when it was coexpressed with B. subtilis tmRNA in Escherichia coli. B. subtilis tmRNA was functional, although much less efficiently, in the in vitro poly(U)‐dependent tag‐peptide synthesis system of E. coli. A comparison of two bacterial tmRNAs suggests that the rule for determining the tag‐initiation point on tmRNA may be the same in Gram‐positive and Gram‐negative bacteria.
A chimera of a gelatinase inhibitor peptide with streptavidin as a bifunctional tumor targeting reagent
Authors: Farlow Samuel J. , Wang Ruo Jie , Pandori Mark W. , Sano Takeshi
Published: October 2002

A chimeric protein, consisting of streptavidin fused to a cyclic decapeptide with potent inhibitory activity for matrix metalloproteinases (MMP), has been produced in Escherichia coli and purified. The purified chimera formed a tetramer and showed full biotin‐binding ability. The chimera was also capable of both binding to MMP‐2 and inhibiting its activity. Thus, both the streptavidin moiety and the decapeptide of the chimera are fully functional. This bifunctional nature of the chimera should facilitate the application of the decapeptide since the streptavidin moiety can be used as a specific conjugation site for almost any materials upon biotinylation.
NF‐κB activation upon interaction of HIV‐1 envelope glycoproteins with cell surface CD4 involves IκB kinases
Authors: Bossis Guillaume , Salinas Sara , Cartier Christine , Devaux Christian , Briant Laurence
Published: October 2002

Human immunodeficiency virus type‐1 envelope glycoprotein (gp120
env
) binding to cell surface CD4 receptor triggers a broad range of intracellular effects leading to T cell activation and cell cycle entry. Among these effects we and others previously reported on the nuclear translocation of the nuclear factor‐κB (NF‐κB) transcription factor. The present work further investigates the signal transduction pathways involved in gp120
env
‐induced NF‐κB activation. We demonstrate that gp120
env
–CD4 interaction stimulates the hyperphosphorylation of IκB‐α inhibitory protein. Conversely, overexpression of a dominant‐negative IκB‐α transgene mutated at S32 and S36 residues, abolishes gp120
env
‐induced NF‐κB activation. IκB kinases (IKKs) activity was found to be selectively enhanced following CD4 engagement with gp120
env
and to mediate the phosphorylation of IκB‐α while co‐transfection experiments using dominant‐negative forms of IKKs inhibited gp120
env
‐induced NF‐κB activation. Taken together, these results confirm that IKKs complex play a key role in gp120
env
‐induced NF‐κB activation.
A novel positive tetracycline‐dependent transactivator (rtTA) variant with reduced background activity and enhanced activation potential
Authors: Kämper Martin R , Gohla Gabi , Schlüter Gregor
Published: October 2002

The tetracycline‐controlled transcription system has become one of the most potent systems for experimental manipulations of transcription levels in vivo. Here we report on rtTA variants, which were generated by combining the existing positively regulated Tet repressor domains of rtTA and rtTA‐M2 with a modified and multimerized minimal transactivation domain from VP16 (L‐domain). A transactivator with multimerized L‐domains shows drastically reduced background activity and enhanced transcriptional activation on different tetracycline‐responsive promoters. The new rtTA variants require higher doses of doxycycline and display a more linear dose–response curve than the original rtTA or rtTA‐M2 proteins.
Zinc is required for structural stability of the C‐terminus of archaeal translation initiation factor aIF2β
Authors: Gutiérrez Pablo , Coillet-Matillon Stéphane , Arrowsmith Cheryl , Gehring Kalle
Published: October 2002

aIF2β is an archaeal homolog of eukaryotic translation factor eIF2β necessary for translation initiation and involved in recognition of the initiation codon. In the present study, we demonstrate for the first time zinc binding to the C2–C2 zinc finger at the C‐terminus of aIF2β. Nuclear magnetic resonance backbone assignments were also determined and the secondary structural elements identified.
Two distinct classes of FixJ binding sites defined by in vitro selection
Authors: Ferrières Lionel , Kahn Daniel
Published: October 2002

The phosphorylated FixJ transcriptional activator is key to nitrogen fixation in Sinorhizobium meliloti, switching both the nifA and fixK genes on. Previously no consensus picture emerged concerning the nature of FixJ binding sites. Here we used in vitro DNA selection in order to systematically characterise FixJ binding sequences. This led to the definition of two classes of sites. Class I sites share the CTAAGTAGTTTCCC sequence found in the fixK promoter, whereas class II sites are defined by a GTAMGTAG consensus octamer. Our results indicate that FixJ∼P binds DNA following two distinct binding modes.
Endothelial Rho signaling is required for monocyte transendothelial migration
Authors: Strey Anke , Janning Annette , Barth Holger , Gerke Volker
Published: October 2002

Bacterial toxins affecting Rho activity in microvascular endothelial cells were employed to elucidate whether endothelial Rho participates in regulating the migration of monocytes across monolayers of cultured endothelial cells. Inactivation of Rho by the Clostridium C3 exoenzyme resulted in an increased adhesion of peripheral blood monocytes to the endothelium and a decreased rate of transendothelial monocyte migration. Cytotoxic necrotizing factor 1‐mediated activation of endothelial Rho also reduced the rate of monocyte transmigration, but did not affect monocyte–endothelium adhesion. Thus, efficient leukocyte extravasation requires Rho signaling not only within the migrating leukocytes but also within the endothelial lining of the vessel wall.
Molecular cloning and characterization of the human p19INK4d gene promoter
Authors: Matsuzaki Youichirou , Miyazawa Kazuhiro , Yokota Tomoya , Hitomi Toshiaki , Yamagishi Hisakazu , Sakai Toshiyuki
Published: October 2002

p19INK4d, a member of the INK4 family of cyclin‐dependent kinase (CDK) inhibitors, negatively regulates the cyclin D–CDK4/6 complexes, which promote G1/S transition by phosphorylating the retinoblastoma tumor‐suppressor gene product. To investigate the mechanism of transcriptional regulation of the p19INK4d gene, we characterized the 5′‐flanking region of the human p19INK4d gene. The cap‐site hunting method revealed that the transcription starts at −16 nucleotide (nt) upstream of the initiation codon. The 5′‐flanking region of the human p19INK4d gene was ligated to a luciferase reporter gene and possessed functional promoter activity. Luciferase assay with a series of truncated 5′‐flanking regions indicated that the region from −81 to −2 nt could drive the transcription of the p19INK4d gene. Several Sp1 and activating protein 2 binding sites are located within the region from −81 to −2 nt. Mutation of the second Sp1 binding site from −33 to −25 nt decreased the promoter activity. Collectively, it was demonstrated that the human p19INK4d gene is under the control of TATA‐less promoter and the Sp1 binding site is involved in the transcription.
Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F‐actin studied by 1H NMR spectroscopy
Authors: Kany Harry , Wolf Jones , Kalbitzer Hans Robert
Published: October 2002

Mg‐F‐actin occurs in two conformational states, I and M, where the N‐terminal amino acids are either immobile or highly mobile. In the rigor or ADP complex of rabbit myosin S1 with Mg‐F‐actin the N‐terminal acetyl group of actin stays in its highly mobile state. The same is true for the complexes with the myosin motor domain from Dictyostelium discoideum. This excludes a direct strong interaction of the N‐terminal amino acids with myosin in the rigor state as suggested. An interaction of the N‐terminus of F‐actin with myosin is also not promoted by occupying its low‐affinity binding site(s) with divalent ions. The N‐terminal high‐mobility region may be part of a structural system which has evolved for releasing inadequate stress applied to the actin filaments
Interplay between ascorbic acid and lipophilic antioxidant defences in chloroplasts of water‐stressed Arabidopsis plants
Authors: Munné-Bosch Sergi , Alegre Leonor
Published: October 2002

The effects of low ascorbic acid (Asc) on lipophilic antioxidant defences and lipid peroxidation in chloroplasts were evaluated in the vtc‐1 mutant of Arabidopsis thaliana, which had an Asc deficiency in chloroplasts of ca. 60%. Although low Asc did not cause oxidative stress in optimal growth conditions, it increased malondialdehyde levels in chloroplasts by ca. 60%, and reduced α‐tocopherol and β‐carotene by ca. 85% and 40%, respectively, in water‐stressed (WS) mutants. These results are indicative of the interplay between Asc and lipophilic antioxidants in chloroplasts of WS plants in vivo, and show that Asc contributes to the protection of thylakoid membrane lipids from oxidation in stressed plants.
A mutation in the β interaction domain of the Ca2+ channel α1C subunit reduces the affinity of the (+)‐[3H]isradipine binding site
Authors: Hitzl Monika , Striessnig Jörg , Neuhuber Birgit , Flucher Bernhard E.
Published: October 2002

The molecular mechanisms of how α1 and β subunits of voltage‐gated Ca2+ channels interact with one another are still controversial. Here we show that despite a mutation in the β interaction domain that has previously been shown to disrupt binding, α1CY467S and β1a‐myc still formed immunoprecipitable complexes when coexpressed in tsA201 cells. However, the α1CY467S–β1a‐myc complexes had a decreased affinity to (+)‐[3H]isradipine. This indicates that the β interaction domain in the I–II loop of the α1 subunit is not merely an anchor required for the functional interaction of the two Ca2+ channel subunits but is itself part of the effector pathway for β‐induced channel modulation.
Nitration of PPARγ inhibits ligand‐dependent translocation into the nucleus in a macrophage‐like cell line, RAW 264
Authors: Shibuya Atsuhito , Wada Koichiro , Nakajima Atsushi , Saeki Makio , Katayama Kazufumi , Mayumi Tadanori , Kadowaki Takashi , Niwa Hitoshi , Kamisaki Yoshinori
Published: October 2002

Nitration of tyrosine residues in proteins has been observed in many inflammatory tissues of arthritis, ulcerative colitis, septic shock and ischemia‐reperfusion injury. Although several studies have been carried out, it is still unclear what type of protein is nitrated and whether tyrosine nitration interferes with protein function. Peroxisome proliferator‐activated receptor gamma (PPARγ) is a nuclear receptor whose activation is linked to several physiological pathways including regulation of insulin sensitivity and control of inflammation. PPARγ possesses several tyrosine residues, which might be potential targets for nitration by peroxynitrite during inflammatory responses. Here we have investigated whether PPARγ is nitrated in macrophage‐like RAW 264 cells and the effect of nitration on the translocation of PPARγ into the nucleus. Western blot analysis showed that tumor necrosis factor‐α, lipopolysaccharide or peroxynitrite treatment significantly increases the nitration of PPARγ. Cell fractionation analysis and immunofluorescence coupled with confocal laser microscopy revealed that nitration of PPARγ inhibits its ligand‐dependent translocation from the cytosol into the nucleus. Together, these results indicate that nitration of PPARγ during inflammation may be involved in a reduction in the control of inflammatory responses and also in the development of resistance to PPARγ ligand‐based therapies against inflammation.
Ribosomal protein S1 from Thermus thermophilus: its detection, identification and overproduction1
Authors: Shiryaev Vyacheslav M. , Selivanova Olga M. , Hartsch Thomas , Nazimov Igor V. , Spirin Alexander S.
Published: October 2002

Ribosomal protein S1 has been identified in Thermus thermophilus ribosomes. The gene of ribosomal protein S1 from Thermus thermophilus has been cloned and overexpressed in Escherichia coli. A procedure for purification of the protein has been developed.
Perinatal outcome of induced and spontaneous pregnancies of primiparous women aged 35 or over
Authors: Kozinszky Z , Orvos H , Katona M , Zoboki T , Pál A , Kovács L
Published: October 2002

Objectives: To compare the neonatal and maternal morbidity data associated with induced or naturally conceived pregnancies of primiparous women aged 35 years and older. Methods: We recruited primiparous women aged 35 years and older, who delivered between January 1995 and December 2000. The outcomes of the induced (n=62) and naturally conceived (n=132) pregnancies were compared. The Fisher exact test was used for univariate analysis in order to compare the delivery and pregnancy characteristics in the two groups. Results: Cesarean section featured with a 0.76 times lower prevalence among the induced pregnant women, than among the spontaneous ones, but the difference was not significant statistically. The induced pregnancies were not associated with a significantly higher rate of perinatal complications. Conclusions: Induced pregnancy does not involve a higher risk of maternal complications. The incidence of premature newborns and intrauterine growth retardation was high in both subgroups, but without a statistically significant difference.
Effects of levonorgestrel implant upon TSH and LH levels in male infants during lactation
Authors: Bassol S , Nava‐Hernandez M.P , Hernandez‐Morales C , Trujillo‐Macias A.M , Lopez‐Lozano M.R , Recio R
Published: October 2002

Objectives: The objective of the study is to evaluate the effects of levonorgestrel transferred through breast milk on thyroid stimulating hormone (TSH) and luteinizing hormone (LH) levels in full breast‐fed infants. Methods: Forty healthy postpartum women and their male newborns were recruited for the study. Women were randomly allocated to two study groups: Group 1, IUD users and group 2, Norplant® users. Blood and milk samples were collected on the day of IUD or Norplant® insertion and 3 and 6 months later for TSH and levonorgestrel measurements. Results: The results disclosed a significant decrease in TSH levels, and a negative relationship between LNG levels and TSH concentration in breast feeding infants at 3 months after implant insertion. The lowest TSH levels were observed at 6 months in the women from group 2. Conclusions: The overall data indicate that the LNG levels transferred to fully breast‐fed infants through breast milk from Norplant® users significantly modified their TSH levels.
Specific enhancement of acylase I and acylpeptide hydrolase activities by the corresponding N‐acetylated substrates in primary rat hepatocyte cultures
Authors: Perrier Josette , Giardina Thierry , Durand Anne , Puigserver Antoine
Published: October 2002

The specific effects of N‐acetyl‐L‐methionine on acylase I activity and of both N‐acetyl‐L‐alanyl‐L‐alanine and N‐acetyl‐L‐methionyl‐L‐alanine on N‐acylpeptide hydrolase activity were investigated in primary rat hepatocyte cultures. Each of the above two substrates is known to be much more rapidly hydrolyzed than other derivatives of the same type under optimum enzyme assay conditions. After a two‐day incubation of the substrates in the presence of primary rat hepatocyte cultures, the N‐acetylaminoacid was found to specifically induce an increase in the acylase I activity, whereas the two N‐acetylated peptides increased the acylpeptide hydrolase activity in the soluble 100000 × g fraction from the culture medium. No change in any of the enzyme activities could be detected during the same period of time when the medium was not supplemented with N‐acetylated substrates. In addition, the acylase I activity showed a dose dependent response when the N‐acetyl‐L‐methionine concentration increased from 10 fold to 50 fold. It is therefore suggested that the efficient hydrolysis of each type of substrate that occured in the 48 h hepatocyte cell cultures was due to the increase observed in the overall activity of the corresponding enzymes. The ratio of acylpeptide hydrolase to that of acylase I increased considerably when the medium was supplemented with N‐acetylpeptides, and decreased in the presence of the N‐acetylaminoacid.
Work team performance over time: three case studies of South African manufacturers
Authors: Grütter Anton W , Field Joy M , Faull Norman H.B
Published: October 2002

In this paper, we report on three case studies of South African manufacturing firms that made significant efforts to implement shopfloor improvement teams. Following Meredith’s [J. Operat. Manage. 16 (1998) 441] suggestion to use case studies as a basis for theory formulation, insights from the cases were used to extend existing theory by generating hypotheses pertaining to the timing and sustainability of performance gains following the implementation of performance improvement teams, focusing, in particular on ongoing performance improvement teams. Because of the richness of the case study data, we delve deeper than other studies into the actions of teams and management to better understand how and why successful performance improvement teams evolve as they do. Stated in temporal order, our main hypotheses are: work team engagement is positively associated with early implementation credibility‐building activities; both outcome‐orientation and substantive participation are positively associated with a rapid rate of performance gains; and team institutionalization is positively associated with sustaining performance gains. The findings of the case studies and the associated hypotheses are summarized in a time‐phased framework for work team implementation. Limitations and directions for further research are discussed.
Medial collateral ligament autografts have increased creep response for at least two years and early immobilization makes this worse
Authors: Thornton G. M. , Boorman R. S. , Shrive N. G. , Frank C. B.
Published: October 2002

Recent evidence has shown that 10–40% of knee joints reconstructed with soft‐tissue autografts have a recurrence of abnormal joint laxity over time. One possible explanation is the “stretching out” (or unrecovered creep) of the graft tissue. To test in vitro creep and creep recovery of fresh anatomic ligament autografts in an extra‐articular environment, 16 rabbits underwent an orthotopic medial collateral ligament (MCL) autograft procedure to one hindlimb. Three subgroups of animals had either unrestricted cage activity for 1 year (n = 5) or 2 years (n = 5) or pin‐immobilization for the first 6 weeks followed by cage activity for the remainder of 1 year (n = 6). Following laxity measurements, to test their creep response, isolated MCL grafts were cyclically and then statically creep tested in vitro at 4.1 MPa, allowed to recover at zero load for 20 min, and finally elongated to failure. Due to differences in cross‐sectional area between the grafts and normal MCLs, two normal control groups were tested: stress‐matched tested at 4.1 MPa (16.2 N; n = 7) and force‐matched tested at 29.1 N (7.1 MPa; n = 6). Ligament grafts had normal laxity but significantly increased creep and decreased creep recovery compared to normal MCLs after 1 and 2 years of healing (p < 0.0004). Graft failure stress was also significantly less than normal (p < 0.0001). Immobilized grafts had significantly greater creep compared to non‐immobilized grafts at 1 year of healing (p < 0.05). These results support previous observations concerning material inferiority of fresh anatomic rabbit MCL autografts, but add the concept that such grafts also have increased potential to creep with either slower or incomplete recovery when subjected to low stresses in vitro. Joint and ligament laxities in situ were normal in this model, however, suggesting either that in vivo MCL graft stresses are lower than those used here in vitro or that these tissues have other mechanisms by which they can recover their functional length in vivo. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Collagen expression and biomechanical response to human recombinant transforming growth factor beta (rhTGF‐β2) in the healing rabbit MCL
Authors: Spindler K. P. , Dawson J. M. , Stahlman G. C. , Davidson J. M. , Nanney L. B.
Published: October 2002

We investigated biomechanical and collagen expression in a healing bilateral rabbit medial collateral ligament (MCL) model to human recombinant transforming growth factor beta (rhTGF‐β2) at three and six weeks. Each rabbit had rhTGF‐β2 in a bioabsorbable pellet administered in one side, with the contralateral side serving as control (no rhTGF‐β2). All MCL healed with rhTGF‐β2 producing a profoundly increased scar mass at three weeks which decreased in size toward control at six weeks. In‐situ hybridization demonstrated collagen expression (type I and III) no different than control at three weeks, but by six weeks elevated expression of type I was seen. Biomechanical analysis at three weeks showed no effect of rhTGF‐β2 on structural properties. However, at six weeks rhTGF‐β2 significantly inhibited both the maximum load (p < 0.05) and energy absorbed (p < 0.05) with no change in stiffness. Despite increased type I collagen expression and profound increase in early scar mass, rhTGF‐β2 did not improve the structural properties. Whether the dose or mode of delivery is responsible for decline in structural properties cannot be determined in this design. We hypothesize investigations of healing ligaments to cytokines should have biologic and biomechanical properties correlated in the same study at a minimum of two time points. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Epidural injection of cyclooxygenase‐2 inhibitor attenuates pain‐related behavior following application of nucleus pulposus to the nerve root in the rat
Authors: Kawakami Mamoru , Matsumoto Takuji , Hashizume Hiroshi , Kuribayashi Koichi , Tamaki Tetsuya
Published: October 2002

Cyclooxygenase‐2 (COX‐2), the inducible isoform of COX, has been identified as the key enzyme to regulate prostaglandin E2 synthesis in inflammatory conditions. Although it has been reported that COX‐2 is present in herniated disc samples obtained from patients, little is known concerning the relationships between COX‐2 and painful radiculopathy. The purpose of this study was to evaluate whether epidural injection of COX‐2 inhibitor abolishes hyperalgesia induced by nucleus pulposus, which is a pain‐related behavior in the rat. Rats, in which nucleus pulposus was relocated on the nerve root, exhibited evidence of mechanical hyperalgesia. Epidural injection of COX‐2 inhibitor resulted in decrease in mechanical hyperalgesia 1 h, 3 and 7 days after the epidural injection of COX‐2 inhibitor (0.1 mg/kg SC‐236 dissolved in the vehicle). There were no significant differences in sensitivity to thermal noxious stimuli after either application of the nucleus pulposus or epidural injections. These results suggest that prostaglandins and thromboxane, which are produced by COX‐2 in inflammatory cells, appear to be related to the inflammatory process produced by application of nucleus pulposus to the nerve root. It is possible that COX‐2 plays a significant role in painful radiculopathy following herniated nucleus pulposus. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Wrist kinetics after scapholunate dissociation: the effect of scapholunate interosseous ligament injury and persistent scapholunate gaps
Authors: Tang Jin Bo , Ryu Jaiyoung , Omokawa Shohei , Wearden Stanley
Published: October 2002

The purpose of this study was to investigate the effects of cutting of the scapholunate interosseous ligament (SLIL) and persistent widening of the scapholunate (SL) joint on changes in moment arms of the principal wrist motor tendons. In seven fresh frozen cadaveric upper extremities, excursions of the extensor carpi radialis longus (ECRL) and brevis (ECRB), extensor carpi ulnaris (ECU), flexor carpi radialis (FCR), and flexor carpi ulnaris (FCU) were recorded simultaneously with wrist joint angulation during wrist flexion–extension and radioulnar deviation. Tendon excursions were measured in intact wrists, then in the wrists with complete SLIL sectioning and in those with moderate or severe persistent SL joint widening. The data were converted to moment arms of the tendons. The results showed that moment arms of the ECRL and ECRB tendons after SLIL sectioning were, respectively, 110 ± 6% and 105 ± 3% of those in the intact wrist. In the wrists with moderate or severe SL joint widening, moment arms of the flexors significantly increased (P < 0.01 and P < 0.001, respectively). During radioulnar deviation, moment arms of the ECRL, ECRB, ECU, and FCU tendons decreased after SLIL sectioning and the SL joint widening. However, moment arms of the FCR tendon significantly increased 122 ± 23% after the SLIL section, 133 ± 28% after the moderate SL joint widening, and 138 ± 24% after the severe SL joint widening compared with those of the intact wrists. This study demonstrated that integrity of the SLIL and appropriate SL joint space are important for mechanics of wrist motor tendons. Loss of integrity of the SLIL and persistent SL joint widening increase mechanical effects of the radial side wrist motor tendons, which may contribute to the pathomechanics of scaphoid malrotation, scapholunate advanced collapse, and early osteoarthritis in the radioscaphoid joint interface seen in SL dissociation. The results also suggest that reduction of the displaced SL joint is imperative to the recovery of wrist kinetics after SL dissociation. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Endogenous norepinephrine regulates blood flow to the intact rat tibia
Authors: Feitelson J. B. A. , Kulenovic E. , Beck D. J. , Harris P. D. , Passmore J. C. , Malkani A. L. , Fleming J. T.
Published: October 2002

The goal of our study was to determine if endogenous norepinephrine (NE) has a role in the regulation of basal blood flow to intact bone. The experimental plan was to measure bone blood flow before and after pharmacological blockade of alpha‐adrenergic receptors. A significant increase in blood flow after receptor blockade would suggest that endogenous norepinephrine exerts a tonic constrictor effect on the vessels supplying blood to the bone. Mature, male rats were anesthetized with Inactin. Arterial blood pressure and left tibia blood flow (laser Doppler flowmetry) were measured. A cannula was inserted into the right iliac artery and advanced to the aortic bifurcation to deliver drugs into the left hindlimb circulation, including the left tibia vasculature. Bolus injection of norepinephrine caused a dose‐dependent decrease in bone blood flow (30–40%). Blockade of alpha‐adrenergic receptors with phentolamine or phenoxybenzamine attenuated by more than 50% the norepinephrine‐induced decrease in bone blood flow. In separate rats that had not received exogenous norepinephrine, injection of phentolamine alone decreased bone vascular resistance by 34 ± 3%. Similarly, phenoxybenzamine decreased resistance by 25 ± 4%. These results are consistent with the conclusion that alpha‐adrenergic receptors mediate a significant constriction of blood vessels which participate in the partial control of basal blood flow to the intact rat tibia. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.