bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of smoothelin in avian smooth muscle and identification of a vascular‐specific isoform
Authors: Wehrens X.H.T. , Mies B. , Gimona M. , Ramaekers F.C.S. , Van Eys G.J.J.M. , Small J.V.
Published: July 2002

Smoothelin is a smooth muscle‐specific protein of minor abundance first identified via a monoclonal antibody obtained using an avian gizzard extract as antigen. Dual labelling of ultrathin sections with antibodies to smoothelin together with antibodies to other smooth muscle proteins showed that smoothelin was co‐distributed with filamin and desmin in the cytoskeleton domain of the smooth muscle cell. From the finding that smoothelin, unlike desmin, was readily extracted by Triton X‐100 as well as under conditions that solubilized myosin, β‐actin and filamin, we conclude that smoothelin is most likely associated with the actin cytoskeleton. Western blot analysis of gizzard smooth muscle tissue revealed an immunoreactive protein band with an apparent molecular weight of 59 kDa that separated into 3–4 isolated variants, while avian vascular muscle showed a polypeptide band of 95 kDa. These results point to the presence of specific isoforms in visceral and vascular smooth muscles. The 59 kDa isoform was shown to be distinct from the 60 kDa filamin‐binding protein, described by Maekawa and Sakai (FEBS Lett. 221, 68–72, 1987). As compared to other smooth muscle markers, such as calponin and SM22, smoothelin appeared very late during differentiation in the chick gizzard, on about the 18th embryonic day.
Inhibition of the M1→M2 (Mclosed→Mopen) transition in the D96N mutant photocycle and its relation to the corresponding transition in wild‐type bacteriorhodopsin
Authors: Radionov Alexey N , Kaulen Andrey D
Published: July 2002

Glutaraldehyde, lutetium ions and glycerol inhibit the blue shift of the difference spectra maximum of the M intermediate in the D96N mutant. The M formed has a spectrum indistinguishable from the M intermediate in wild‐type bacteriorhodopsin. It has been concluded that the Mopen form previously described by us is identical to the M2 and Mn intermediates postulated by Zimanyi et al. (Photochem. Photobiol. (1992) 56, 1049–1055) and Sasaki et al. (J. Biol. Chem. (1992) 267, 20782–20786), respectively. It is supposed that its formation is accompanied by the appearance of the cytoplasmic proton half‐channel. Mopen in the wild‐type protein is present in a very low amount due to the shift of the Mclosed↔Mopen equilibrium towards the Mclosed. The inhibitors used do not prevent the multiphase pattern of the M formation in either mutant or wild‐type proteins.
The transcription of NAM7/UPF1 is enhanced in the absence of Cyp1p/Hap1p concomitant with the appearance of an ISF1‐NAM7 cotranscript in Saccharomyces cerevisiae
Authors: Altamura Nicola , de Pinto Brigida , Castaldo Rosa , Verdiére Jacqueline
Published: July 2002

The two adjacent nuclear genes ISF1 and NAM7 cooperatively participate in mitochondrial functions. It is well known that Cyp1p(Hap1p) activates a number of genes involved in these same functions. We show in this paper that Cyp1p influences the transcriptional regulation of NAM7. In addition, a significant amount of ISF1–NAM7 cotranscript is observed in a cyp1 mutant context. An extensive analysis of the intergenic region which separates the two genes revealed 5′ starts of the NAM7 transcripts, additional to those previously mapped. These new 5′ starts overlap the 3′ ends of ISF1. We propose that NAM7 is under the control of a negative Cyp1p‐dependent regulator and that its absence favours a transcriptional read‐through which results in the ISF1–NAM7 cotranscript we have identified.
A single mutation in the M‐subunit of Rhodospirillum rubrum confers herbicide resistance
Authors: Sopp Gabriele , Rutherford William A , Oettmeier Walter
Published: July 2002

Cells of the photosynthetic bacterium Rhodospirillum rubrum were rendered resistant against the inhibitor 2‐(1‐phenyl)ethylamino‐3‐propionylamino‐4‐cyano‐thiazole (PPCTH). Electron transport in reaction centers prepared from one of the mutants (M6) was neither inhibited by PPCTH and other NH‐thiazoles nor terbutryn. These inhibitors are known to bind at the QB site of the L‐subunit. Compared to the wild type, chromatophores from M6 exhibited strongly altered QB
−Fe2+ and QA
−Fe2+ EPR signals. Inhibitor resistance is due to a mutation in the bacterial reaction center M‐subunit, where Glu234 is exchanged against Lys. This is the first example of an inhibitor resistance in the QB site caused by a mutation in the M‐subunit.
Bacterial expression and purification of biologically active mouse c‐Fos proteins by selective codon optimization
Authors: Deng Tiliang
Published: July 2002

A simple strategy using selective codon optimization was devised to express mouse c‐Fos protein in high levels in E. coli. Ten arginine codons located in the basic region were optimized to achieve high levels of protein expression. The c‐Fos protein was purified to near homogeneity and was demonstrated to be biologically active by assaying its several biological activities.
Inhibition of Galβ1,4GlcNAc α2,6 sialyltransferase expression by antisense‐oligodeoxynucleotides
Authors: Kemmner Wolfgang , Hohaus Kathrin , Schlag Peter M
Published: July 2002

Treatment of human colorectal carcinoma cells HT29 with specific antisense oligodeoxynucleotides led to a decreased Galβ1,4GlcNAc α2,6 sialyltransferase activity on the level of protein expression as well as on the mRNA level. Antisense treatment did not effect cell viability or cell growth. Oligodeoxynucleotides which were complementary to the region upstream of the initiation codon were particularly effective in inhibition of enzyme expression. No such inhibition was found by treatment of cells with oligodeoxynucleotides complementary to the region downstream of the initiation codon or by treatment of cells with scrambled controls or sense oligodeoxynucleotides.
Effect of excess cadmium ion on the metal binding site of cabbage histidinol dehydrogenase studied by 113Cd‐NMR spectroscopy
Authors: Kanaori Kenji , Ohta Daisaku , Nosaka Atsuko Y.
Published: July 2002

The enzymatic reaction of histidinol dehydrogenase (HDH) was stimulated by about maximally 75% on the addition of Cd2+ ion to the reaction mixture. 113Cd‐substituted HDH in the presence of excess Cd2+ has been studied by 113Cd‐NMR. 113Cd2+ less than 1 equiv. per subunit preferentially binds to the catalytic metal binding site of the apoenzyme. Further addition of the metal ions causes the structural change of the enzyme including the catalytic metal binding site. HDH takes at least three discernible states, which may correspond to the more or less active forms of the enzyme induced by metal ions.
Comparative stability studies on the iron and manganese forms of the cambialistic superoxide dismutase from Propionibacterium shermanii
Authors: Meier Beate , Parak Fritz , Desideri Alessandro , Rotilio Giuseppe
Published: July 2002

The superoxide dismutase of Propionibacterium shermanii shows similar activity with iron and manganese bound at the active site of the protein. On the other hand, the iron form, in comparison to the manganese form, exhibits higher stability towards thermal‐ and pH‐dependent inactivation. Upon inactivation the metal ions are released from the active site. Thus, in comparison to the manganese form, a higher stability of the iron–protein complex might be the triggering reason for this behavior.
Identification of the minimum segment in which the threonine246 residue is a potential phosphorylated site by protein kinase A for the LukS‐specific function of staphylococcal leukocidin
Authors: Nariya Hirofumi , Nishiyama Akihito , Kamio Yoshiyuki
Published: July 2002

Staphylococcal leukocidin and γ‐hemolysin consist of LukF and LukS for leukocidin and LukF and Hlg2 for γ‐hemolysin. In this report, we identify the minimum segment responsible for the LukS‐specific function of leukocidin. After chemical analysis and homology study of the amino acid sequence of the C‐terminal region between LukS and Hlg2, we found a unique 5‐residue sequence I242K243R244S245T246 in LukS in which the 4‐residue KRST is identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. To elucidate whether the 5‐residue segment is essential for the LukS function, we created plasmids containing a series of mutant genes corresponding to the 5‐residue sequence and expressed them in Escherichia coli. The mutant proteins were purified and assayed for their leukocytolytic activity with LukF. The mutant MLS‐TS, in which the T246 in the 5‐residue sequence was replaced by S, showed leukocidin activity 10 times higher than that of the intact LukS. However, neither mutant MLS‐TY nor MLS‐TA, in which T246 was replaced by Y or A, respectively, showed leukocidin activity. The 5‐residue segment was found to be deleted in Hlg2. The mutant of Hlg2, in which the 5‐residue segment was inserted at the position that the segment is deleted, showed leukocidin activity. The boiled LukS, MLS‐TS, and MHS‐Z were strongly phosphorylated with [γ‐32P]ATP in the presence of protein kinase A in a cell‐free system. Thus, we conclude that the 5‐residue segment I242K243R244S245T246 is the pivotal segment of LukS responsible for the LukS function of staphylococcal leukocidin.
Class 2 aldehyde dehydrogenase. Characterization of the hamster enzyme, sensitive to daidzin and conserved within the family of multiple forms
Authors: Hjelmqvist Lars , Lundgren Robert , Norin Annika , Jörnvall Hans , Vallee Bert , Klyosov Anatole , Keung Wing Ming
Published: July 2002

Mitochondrial (class 2) hamster aldehyde dehydrogenase has been purified and characterized. Its primary structure has been determined and correlated with the tertiary structure recently established for this class from another species. The protein is found to represent a constant class within a complex family of multiple forms. Variable segments that occur in different species correlate with non‐functional segments, in the same manner as in the case of the constant class of alcohol dehydrogenases (class III type) of another protein family, but distinct from the pattern of the corresponding variable enzymes. Hence, in both these protein families, overall variability and segment architectures behave similarly, with at least one `constant' form in each case, class III in the case of alcohol dehydrogenases, and at least class 2 in the case of aldehyde dehydrogenases.
A crystallographic approach to DNA bending: prediction of nucleosome formation by DNA triple repeats and other repetitive sequences
Authors: Subirana Juan A
Published: July 2002

DNA bending is due to two main factors: the inherent curvature of the sequence and its flexibility. Most methods of analysis do not allow a differentiation between these two factors. In this paper I show that the flexibility of DNA sequences can be estimated from the standard deviation of roll values determined by X‐ray crystallography for each base step. As an application of this approach, the nucleosome formation ability of triple repeat sequences has been determined and shown to be in agreement with the experimental results. Local variations in twist do not appear to have any influence on nucleosome formation.
A series of vectors to construct lacZ fusions for the study of gene expression in Schizosaccharomyces pombe
Authors: Lafuente Marı́a J , Petit Thomas , Gancedo Carlos
Published: July 2002

We have constructed a series of plasmids to facilitate the fusion of promoters with or without coding regions of genes of Schizosaccharomyces pombe to the lacZ gene of Escherichia coli. These vectors carry a multiple cloning region in which fission yeast DNA may be inserted in three different reading frames with respect to the coding region of lacZ. The plasmids were constructed with the ura4+
or the his3+
marker of S. pombe. Functionality of the plasmids was tested measuring in parallel the expression of fructose 1,6‐bisphosphatase and β‐galactosidase under the control of the fbp1+
promoter in different conditions.
Selective induction of CD8+ T cell functions by single substituted analogs of an antigenic peptide: distinct signals for IL‐10 production
Authors: Kohyama Masako , Kakehi Masahiro , Totsuka Mamoru , Hachimura Satoshi , Hisatsune Tatsuhiro , Kaminogawa Shuichi
Published: July 2002

The CD8+ T cell clone 5F1 produces interleukin 10 (IL‐10) and interferon γ (IFN‐γ) in response to stimulation with a peptide corresponding to region 142–149 of bovine αs1‐casein (p142–149). Ninety analog peptides derived from p142–149 with single amino acid substitutions of putative T cell receptor contact residues were prepared to examine whether production of IL‐10 and IFN‐γ by 5F1 can be altered by stimulation with these peptides. We found that some peptides triggered only IL‐10 production whereas others induced production of IFN‐γ alone or both of these cytokines. Peptides inducing IFN‐γ production triggered both cytotoxicity and a proliferative response, whereas peptides inducing production of IL‐10 but not IFN‐γ triggered neither of these responses. Our results clearly demonstrate that the signaling pathway required for IL‐10 production in CD8+ T cells differs from that required for IFN‐γ production. The distinct cellular signals for IL‐10 production appear to be independent of those for cytotoxicity and the proliferative response of CD8+ T cells.
EcoRII endonuclease has two identical DNA‐binding sites and cleaves one of two co‐ordinated recognition sites in one catalytic event
Authors: Petrauskene O.V. , Babkina O.V. , Tashlitsky V.N. , Kazankov G.M. , Gromova E.S.
Published: July 2002

EcoRII is a typical restriction enzyme that cleaves DNA using a two‐site mechanism. EcoRII endonuclease is unable to cleave DNA which contains a small number of EcoRII recognition sites but the enzyme activity can be stimulated in the presence of DNA with a high frequency of EcoRII sites. To investigate the mechanism of activation, the kinetics of stimulated EcoRII cleavage has been studied. A 14 bp substrate activated the cleavage of the 71 bp substrate, containing one EcoRII recognition site (trans‐activation) by a competitive mechanism: the activator increased substrate binding but not catalysis. The activation increased if the substrate concentration decreased and if the activator had a lower affinity for the enzyme than the substrate. The introduction of the second recognition site into the 71 bp duplex also enabled cleavage of this substrate (cis‐activation). Pyrophosphate bonds were incorporated into one of two recognition sites to switch off the cleavage of the phosphodiester bonds. Analysis of cleavage products of these modified substrates showed that EcoRII cuts one of two coordinated recognition sites in one catalytic event.
A deficiency in Syk enhances ceramide‐induced apoptosis in DT40 lymphoma B cells
Authors: Qin Suofu , Ding Junyi , Kurosaki Tomohiro , Yamamura Hirohei
Published: July 2002

Syk deficiency significantly enhanced ceramide‐induced apoptosis. Ectopic expression of wild‐type or kinase‐inactive Syk rendered Syk‐negative cells resistant to ceramide‐induced apoptosis. Furthermore, ceramide could not activate Syk, indicating that Syk protected DT40 cells from ceramide‐induced apoptosis, via a mechanism independent of its activity. In addition, a deficiency in Lyn also resulted in the cells becoming susceptible to ceramide‐induced apoptosis. However, no difference of Ara‐C‐induced apoptosis between wild‐type and mutant cells was observed. c‐Jun N‐terminal kinases appeared not to be important in mediating the enhanced apoptosis, as they were still activated in mutant cells following ceramide treatment.
Covalent complex of phenylalanyl‐tRNA synthetase with 4‐thiouridine‐substituted tRNAPhe gene transcript retains aminoacylation activity1
Authors: Moor Nina A , Favre Alain , Lavrik Olga I
Published: July 2002

s4U‐containing transcripts of tRNAPhe gene have been prepared by complete substitution of 16 U residues or by random incorporation of s4U residues followed by affinity electrophoresis isolation of s4U‐monosubstituted tRNA transcripts. Both analogs have been cross‐linked to Thermus thermophilus phenylalanyl‐tRNA synthetase (PheRS) and the specificity of the cross‐linking has been demonstrated. Functional activity of the covalent complex of PheRS with the s4U‐monosubstituted transcript has been shown by aminoacylation of 60% of the enzyme‐cross‐linked tRNA. This is the first instance in which biological activity of aminoacyl‐tRNA synthetase and cross‐linked tRNA in a specific complex has been revealed.
FK506 (tacrolimus) inhibits extravasation of lymphoid cells by abrogating VLA‐4/VCAM‐1 mediated transendothelial migration
Authors: Tsuzuki Shinobu , Toyama-Sorimachi Noriko , Kitamura Fujiko , Tobita Yoshimi , Miyasaka Masayuki
Published: July 2002

Extravasation is a critical process for the physiological lymphocyte traffic as well as the hematogenous spread of malignant hemopoietic cells. Here we report that abrogation of calcineurin activity leads to in vitro transendothelial migration and in vivo infiltration of human lymphoma Nalm‐6 cells, which are associated with the abrogation of the VLA‐4/VCAM‐1 mediated pathway. Rapamycin, which can antagonize FK506 but not CsA to inhibit calcineurin, abrogates FK‐506 mediated but not CsA mediated inhibition of in vitro transendothelial migration. FK506 may exert its potent immunosuppressive action partly by inhibiting VLA‐4/VCAM‐1 mediated transendothelial migration or insinuation of lymphoid cells to tissues.
Neurotensin type 1 receptor‐mediated activation of krox24, c‐fos and Elk‐1: preventing effect of the neurotensin antagonists SR 48692 and SR 142948
Authors: Portier Marielle , Combes Thérèse , Gully Danièle , Maffrand Jean-Pierre , Casellas Pierre
Published: July 2002

Stimulation of neurotensin (NT) type 1 receptors (NT1‐R) in transfected CHO cells is followed by the activation of mitogen‐activated protein kinases and the expression of the early response gene krox24. By making point mutations and internal deletions in the krox24 promoter, we show that proximal serum responsive elements (SRE) are involved in transcriptional activation by NT. In addition, we show that the related early response gene c‐fos and the Ets protein Elk‐1 are also induced by NT. The involvement of NT1‐R in NT‐mediated activation of krox24, c‐fos and Elk‐1 was demonstrated by the preventing effect of the specific antagonists SR 48692 and SR 142948. Finally, we show that the activation of krox24 and Elk‐1 on the one hand, and that of c‐fos on the other hand, result from independent transduction pathways since the former are pertussis toxin‐sensitive whereas the latter is insensitive to pertussis toxin.
Expression of the SOX10 gene during human development
Authors: Bondurand Nadege , Kobetz Alexandra , Pingault Veronique , Lemort Nicole , Encha-Razavi Ferechte , Couly Gerard , Goerich Derk E , Wegner Michael , Abitbol Marc , Goossens Michel
Published: July 2002

SOX10, a new member of the SOX gene family, is a transcription factor defective in the Dom (Dominant megacolon) mouse and in the human Shah‐Waardenburg syndrome. To help unravel its physiological role during human development, we studied SOX10 gene expression in embryonic, fetal, and adult human tissues by Northern blot and in situ hybridization. As in mice, the human SOX10 gene was essentially expressed in the neural crest derivatives that contribute to the formation of the peripheral nervous system, and in the adult central nervous system. Nevertheless, it was more widely expressed in humans than in rodents. The spatial and temporal pattern of SOX10 expression supports an important function in neural crest development.
Expression of pICln in Escherichia coli gives a strong tolerance to hypotonic stress
Authors: Tao Guo-Zhong , Kobayashi Akira , Itoh Hideaki , Tashima Yohtalou
Published: July 2002

We amplified the coding region DNA sequence from a rat renal pICln cDNA by PCR and expressed the protein in Escherichia coli cells. The cells were exposed to hypotonic conditions followed by spreading them onto LB plates for subsequent colony survival assay. The present study demonstrated that the cells expressing pICln exhibit a strong resistance to hypotonic stress. Moreover, the resistance was specifically inhibited by extracellular ATP and some anion channel inhibitors. These findings indicate that the expression of pICln directly confers tolerance to hypotonic stress, and pICln is concluded to be an important molecule for cell‐volume regulation.
Positive feedback between ethanolamine‐specific phospholipid base exchange and cytochrome P450 activities in rat liver microsomes. The effect of clofibric acid
Authors: Lenart Jacek , Komańska Izabela , Pikuła Sławomir , Jasińska Renata
Published: July 2002

The results of the present investigation relate the effects of the nutritional state and administration of clofibric acid (CLA), a hypolipidaemic drug and peroxisomal proliferator, on phosphatidylethanolamine (PE) synthesis in rat liver and fatty acid metabolism. Fasting and CLA treatment of animals causes an increase in the amount of PE in endoplasmic reticulum (ER) membranes and mitochondria, as well as in the PE/phosphatidylcholine (PC) ratio. Moreover, the activity of the ethanolamine‐specific phospholipid base exchange (PLBE) enzyme in liver ER membranes of fasted animals was enhanced by 75% in comparison to that of animals fed ad libitum. The effect of CLA treatment was additive to that of starvation; PE synthesis tested in vitro via the Ca2+‐sensitive PLBE reaction increased 3‐fold in comparison to rats fed ad libitum. This is confirmed by an increased V
max for the reaction, but the affinity of the enzyme for ethanolamine was not significantly changed. These effects were accompanied by an enhanced expression of cytochrome P450 CYP4A1 isoform and elevated activity of the enzyme upon CLA administration. The stimulatory effect of CLA administration on the efficiency of the ethanolamine‐specific PLBE reaction can be explained by elimination of lauric acid, a known inhibitor of de novo PE synthesis, during the course of ω‐hydroxylation catalysed by CYP4A1, and by increased expression of the PLBE enzyme. The products of ω‐hydroxylation of lauric acid, which are then converted by dehydrogenase to 1,12‐dodecanedioic acid, did not significantly affect the in vitro synthesis of PE.
Phosphorylation of the translational regulator, PHAS‐I, by protein kinase CK2
Authors: Fadden Patrick , Haystead Timothy A.J , Lawrence John C
Published: July 2002

The primary site in PHAS‐I for phosphorylation by protein kinase CK2 in vitro was identified as Ser111. A relatively small amount of phosphorylation of Ser99 was also detected, and mutating Ser99 to Ala in PHAS‐I slightly decreased phosphorylation by CK2 in vitro. In contrast, mutating Ser111 to Ala almost abolished phosphorylation, confirming Ser111 as the preferred site for CK2. Phosphorylation of Ser111 did not decrease binding of PHAS‐I to eIF4E, and results of peptide mapping experiments with PHAS‐I immunoprecipitated from 32P‐labeled adipocytes indicated that Ser111 was not phosphorylated in cells. These results support the conclusion that CK2 is not involved in the control of PHAS‐I.
α‐Adrenergic inhibition of proliferation in HepG2 cells stably transfected with the α1B‐adrenergic receptor through a p42MAP kinase/p21Cip1/WAF1‐dependent pathway
Authors: Auer Kelly L , Spector Mark S , Tombes Robert M , Seth Prem , Fisher Paul B , Gao Bin , Dent Paul , Kunos George
Published: July 2002

Activation of α1B adrenergic receptors (α1BAR) promotes DNA synthesis in primary cultures of hepatocytes, yet expression of α1BAR in hepatocytes rapidly declines during proliferative events. HepG2 human hepatoma cells, which do not express α1BAR, were stably transfected with a rat α1BAR cDNA (TFG2 cells), in order to study the effects of maintained α1BAR expression on hepatoma cell proliferation. TFG2 cells had a decreased rate of growth compared to mock transfected HepG2 cells as revealed by a decrease in [3H]thymidine incorporation into DNA. Stimulation of α1BAR with phenylephrine caused a further large reduction in TFG2 cell growth, whereas no effect on growth was observed in mock transfected cells. Reduced cell growth correlated with increased percentages of cells found in G0/G1 and G2/M phases of the cell cycle. In TFG2 cells, phenylephrine increased p42MAP kinase activity by 1.5‐ to 2.0‐fold for up to 24 h and increased expression of the cyclin dependent kinase inhibitor protein p21Cip1/WAF1. Treatment of TFG2 cells with the specific MEK1 inhibitor PD98059, or infection with a −/− MEK1 recombinant adenovirus permitted phenylephrine to increase rather than decrease [3H]thymidine incorporation. In addition, inhibition of MAP kinase signaling by PD98059 or MEK1 −/− blunted the ability of phenylephrine to increase p21Cip1/WAF1 expression. In agreement with a role for increased p21Cip1/WAF1 expression in causing growth arrest, infection of TFG2 cells with a recombinant adenovirus to express antisense p21Cip1/WAF1 mRNA blocked the ability of phenylephrine to increase p21Cip1/WAF1 expression and to inhibit DNA synthesis. Antisense p21Cip1/WAF1 permitted phenylephrine to stimulate DNA synthesis in TFG2 cells, and abrogated growth arrest. These results suggest that transformed hepatocytes may turn off the expression of α1BARs in order to prevent the activation of a growth inhibitory pathway. Activation of this inhibitory pathway via α1BAR appears to be p42MAP kinase and p21Cip1/WAF1 dependent.
Up‐regulation of the expression of major histocompatibility complex class I antigens by plasmid DNA transfection in non‐hematopoietic cells
Authors: Park Joo-Hung , Chang Sun-Hee , Kim Min-Chul , Shin Sung-Hye , Youn Hyun-Joo , Kim Jin-Kyoo , Jang Yong-Suk , Kim Chul-Woo
Published: July 2002

The effect of DNA on the surface expression of major histocompatibility (MHC) class I antigens was examined in non‐hematopoietic tumor cell lines. Transfection with plasmid DNA via liposome or electroporation significantly increased the surface expression of MHC class I molecules in a transient manner. Northern blot analysis showed that levels of MHC class I mRNA were increased by DNA transfection, probably via transcriptional activation. In contrast, the expression of the MHC class II and β‐actin genes was not affected, suggesting that the up‐regulation of MHC class I expression by plasmid DNA works in a gene‐specific manner.
Hydroxamate derivatives of substrate‐analogous peptides containing aminomalonic acid are potent inhibitors of matrix metalloproteinases1
Authors: Krumme Dirk , Wenzel Herbert , Tschesche Harald
Published: July 2002

Novel peptides containing the sequence ‐Pro‐Leu‐Ama(NHOH)‐ were synthesized and characterized by spectroscopic techniques. Their inhibitory properties towards the activated form of native human gelatinase B (MMP‐9) and the catalytic domain of neutrophil collagenase (cdMMP‐8) were determined. The most effective inhibitor synthesized exhibits K
i values of 2×10−6 M (cdMMP‐8) and 5×10−9 M (MMP‐9) thus attaining interesting discrimination between the tested metalloproteinases. A most important feature of this type of inhibitor is its peptide nature making the compounds similar to natural substrates. In spite of the peptide character of the inhibitors synthesized, the P1‐P1′‐peptide bond shows a high resistance to cleavage by the proteinases.
Histone H4 acetylation during interleukin‐2 stimulation of mouse T cells
Authors: Taplick Jan , Kurtev Vladislav , Lagger Gerda , Seiser Christian
Published: July 2002

Proliferation and cell cycle progression of eukaryotic cells are closely linked to changes in chromatin structure and gene expression. By reversible histone acetylation the cell is able to modulate chromatin condensation and accessibility of specific regions within the chromatin. Here, we examined histone H4 acetylation patterns during growth induction of the murine interleukin‐2 dependent T cell line B6.1. In order to detect acetylation on each of the four potential target residues we produced a set of antibodies recognizing specifically acetylated lysine 5, 8, 12 and 16 in the N‐terminal tail of histone H4. Acetylation was generally low in resting T cells, but increased after stimulation with a specific kinetics for each lysine. Lysine 16 was acetylated during the G1 phase and deacetylated during S phase. H4 acetylation on lysine 5, 8 and 12, in contrast, was induced before cells started to replicate, and persisted until cells entered mitosis. Treatment of resting B6.1 cells with the specific deacetylase inhibitor trichostatin A (TSA) led to H4 hyperacetylation at all four lysine residues indicating that the histone modification can occur in the absence of replication. After release from TSA treatment normal H4 acetylation levels were reestablished by extremely rapid deacetylation of lysines 5, 8, 12 and 16. The deacetylation step was 60–100 times faster than TSA induced acetylation and equally efficient in resting and exponentially growing T cells. Our results indicate the presence of cell cycle regulated lysine specific acetylating and deacetylating activities in mouse T cells.
His84 rather than His35 is the active site histidine in the corrinoid protein MtrA of the energy conserving methyltransferase complex from Methanobacterium thermoautotrophicum
Authors: Sauer Karin , Thauer Rudolf K
Published: July 2002

The energy conserving corrinoid containing MtrA‐H complex from Methanobacterium thermoautotrophicum is composed of eight different subunits of which MtrA harbors the corrinoid prosthetic group, the corrinoid being bound in the base‐off/His‐on configuration. Based on sequence comparisons it was recently proposed that His35 of MtrA is the active site histidine. We report here that His84 rather than His35 is the axial ligand to the cobamide in MtrA.
Linker histones: paradigm lost but questions remain
Authors: Belikov Sergey , Karpov Vadim
Published: July 2002

Linker histones (LH) represent a diverse family of proteins that bind to nucleosomes and bring them together to form a 30‐nm chromatin fiber. Although the structure of the globular domain of linker histones H1 and H5 has been solved, the details of its interaction with the nucleosome are not understood in full. Recent data on the location of LH in nucleosome are discussed here.
Chimeric Gαq mutants harboring the last five carboxy‐terminal residues of Gαi2 or Gαo are resistant to pertussis toxin‐catalyzed ADP‐ribosylation
Authors: Joshi Sushma A. , Fan Kenneth P.K. , Ho Vanessa W.S. , Wong Yung H.
Published: July 2002

Three widely‐used Gαq chimeras harboring the last five residues of Gαi, Gαo and Gαz (qi5, qo5 and qz5) were examined for their ability to serve as substrates for pertussis toxin (PTX)‐catalyzed ADP‐ribosylation. In COS‐7 cells coexpressing one of the three opioid receptors (μ, δ, and κ) and a Gαq chimera, agonist‐induced stimulation of phosphoinositide‐specific phospholipase C (PI‐PLC) was largely insensitive to PTX treatment. Only the qi5‐mediated stimulation of PI‐PLC by κ‐opioids was partially inhibited by PTX. In βγ‐release assays, PTX treatment did not affect the ability of opioid receptors to activate these chimeras. [32P]ADP‐ribosylation labeled Gαi/o but not qi5 or qo5, although the expression of these chimeras was confirmed by immunodetection. Thus, Gαq chimeras with a Gαi/o‐like tail are insensitive to PTX treatment.
Palmitoylation of the rat μ opioid receptor
Authors: Chen Chongguang , Shahabi Vafa , Xu Wei , Liu-Chen Lee-Yuan
Published: July 2002

We examined whether the μ opioid receptor was palmitoylated and attempted to determine sites of palmitoylation. Following metabolic labeling with [3H]palmitic acid and immunoaffinity purification of the μ opioid receptor, SDS‐PAGE and fluorography revealed a broad labeled band with M
r of ∼80 kDa in CHO cells stably expressing the rat μ receptor, but not in CHO cells transfected with the vector alone, indicating that the μ receptor is palmitoylated. Activation of the receptor with morphine did not affect the extent of palmitoylation. Hydroxylamine or dithiothreitol treatment removed most of the radioactivity, demonstrating that [3H]palmitic acid is incorporated into Cys residue(s) via thioester bond(s). Surprisingly, mutations of the only two Cys residues in the C‐terminal domain did not reduce [3H]palmitic acid incorporation significantly. Thus, unlike many G‐protein coupled receptors, the palmitoylation site(s) of the rat μ opioid receptor do(es) not reside in the C‐terminal domain.
Recombinant bovine lactoperoxidase as a tool to study the heme environment in mammalian peroxidases
Authors: Watanabe Shikiko , Varsalona Franca , Yoo Yung-Choon , Guillaume Jean-Paul , Bollen Alex , Shimazaki Keiichi , Moguilevsky Nicole
Published: July 2002

The cDNA encoding bovine lactoperoxidase (LPO) has been expressed in CHO cells. The recombinant LPO was secreted as an enzymatically active single chain molecule presenting two immunoreactive forms of 88 kDa and 82 kDa, differing by their glycosylation. rLPO exhibited the characteristic absorbance spectrum with a Soret peak at 413 nm. Engineering of rLPO into a myeloperoxidase (MPO)‐like molecule was attempted by substituting Gln‐376 by Met, a residue known to achieve covalent binding with the heme in MPO. However, the resulting bovine LPO mutant failed to acquire the peculiar absorbance spectrum and the chlorinating activity of MPO, underlining the complex nature of interactions in the heme vicinity.
Effect of magnetic resonance imaging on human respiratory burst of neutrophils
Authors: Heine Joern , Scheinichen Dirk , Jaeger Karsten , Herzog Thomas , Sümpelmann Robert , Leuwer Martin
Published: July 2002

It is known that low intensity magnetic fields increase superoxide anion production during the respiratory burst of rat peritoneal neutrophils in vitro. We investigated whether the high intensity magnetic fields (1.5 T) during magnetic resonance imaging can influence the human neutrophil function under in vivo conditions. Blood samples were obtained from 12 patients immediately before and after magnetic resonance imaging (mean time 27.6(±11.4 min)). The induced respiratory burst was investigated by the intracellular oxidative transformation of dihydrorhodamine 123 to the fluorescent dye rhodamine 123 via flow cytometry. The respiratory burst was induced either with phorbol 12‐myristate 13‐acetate, Escherichia coli, N‐formyl‐methionyl‐leucylphenylalanine or priming with tumor necrosis factor followed by FMLP stimulation. There was no significant difference between the respiratory burst before and after magnetic resonance imaging, irrespective of the stimulating agent. Short time exposure to a high intensity magnetic field during magnetic resonance imaging seems not to influence the production of radical species in living neutrophils.
Identification of the catalytic triad in the haloalkane dehalogenase from Sphingomonas paucimobilis UT26
Authors: Hynková Kamila , Nagata Yuji , Takagi Masamichi , Damborský Jiřı́
Published: July 2002

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the γ‐hexachlorocyclohexane degradation. This enzyme hydrolyses a broad range of halogenated aliphatic compounds via an alkyl‐enzyme intermediate. LinB is believed to belong to the family of α/β‐hydrolases which employ a catalytic triad, i.e. nucleophile‐histidine‐acid, during the catalytic reaction. The position of the catalytic triad within the sequence of LinB was probed by a site‐directed mutagenesis. The catalytic triad residues of the haloalkane dehalogenase LinB are proposed to be D108, H272 and E132. The topological location of the catalytic acid (E132) is after the β‐strand six which corresponds to the location of catalytic acid in the pancreatic lipase, but not in the haloalkane dehalogenase of Xanthobacter autotrophicus GJ10 which contains the catalytic acid after the β‐strand seven.
Reconstitution of an electrogenic auxin transport activity mediated by Arabidopsis thaliana plasma membrane proteins
Authors: Szponarski Wojciech , Guibal Olivier , Espuna Meriam , Doumas Patrick , Rossignol Michel , Gibrat Rémy
Published: July 2002

Plasma membrane proteins from Arabidopsis thaliana leaves were reconstituted into proteoliposomes and a K+ diffusion potential was generated. The resulting ionic fluxes, determined in the presence of the plant hormone auxin (indole‐3 acetic acid), showed an additional electrogenic and saturable component, with a K
M of 6 μM. This flux was neither detected in liposomes in the presence of indole‐3 acetic acid, nor in proteoliposomes in the presence of an inactive auxin analog and was completely inhibited by 3 μM naphtylphthalamic acid, a specific inhibitor of the auxin efflux carrier. The efficiency of the reconstituted carrier and the mechanism of its regulation by naphtylphthalamic acid are discussed.
Mapping of the MYCL2 processed gene to Xq22‐23 and identification of an additional L MYC‐related sequence in Xq27.2
Authors: Redolfi E , Pizzuti A , Di Bacco A , Susani L , Labella T , Affer M , Montagna C , Reinbold R , Mumm S , Vezzoni P , Zucchi I
Published: July 2002

We report here the identification of a human genomic sequence from the q27.2 region of the X chromosome which shows a high homology to the L‐MYC proto‐oncogene. This sequence is not the MYCL2 homology, previously mapped to the long arm of the X chromosome at q22‐qter by Morton et al., as we located the MYCL2‐processed gene in Xq22‐23, using a panel containing a combination of hybrid DNA carrying different portions of the human X chromosome. Based on computer analysis, the MYC‐like sequence (MYCL3) is 98.2% identical to a portion of exon 3 of the MYCL1 gene and maps to the Xq27.2 region, between the DXS312 and DXS292 loci.
Ultrastructure of the eukaryotic aminoacyl‐tRNA synthetase complex derived from two dimensional averaging and classification of negatively stained electron microscopic images
Authors: Trempe Norcum Mona
Published: July 2002

Several aminoacyl‐tRNA synthetases in higher eukaryotes are consistently isolated as a multi‐enzyme complex for which little structural information is yet known. This study uses computational methods for analysis of electron microscopic images of the particle. A data set of almost 2000 negatively stained images was processed through reference‐free alignment and multivariate statistical analysis. Interpretable structural information was evident in five eigenvectors. Hierarchical ascendant classification extracted clusters corresponding to distinct image orientations. The class averages are consistent with rotations around and orthogonal to a central particle axis and provide particle measurements: approximately 25 nm in height, 30 nm at the widest point and 23 nm thick. The results also provide objective evidence in support of the working structural model and demonstrate the feasibility of obtaining the three dimensional structure of the multisynthetase complex by single particle reconstruction methods.
The inositol (1,4,5)‐trisphosphate 3‐kinase of Xenopus oocyte is activated by CaMKII and involved in the regulation of InsP3‐mediated Ca2+ release
Authors: Hague Frédéric , Matifat Fabrice , Brûlé Gérard , Collin Thibault
Published: July 2002

The effect of Ca2+ on inositol (1,4,5)‐trisphosphate 3‐kinase (3‐kinase) activity was measured on Xenopus oocyte cytosolic extracts. The Ca2+‐evoked elevation in 3‐kinase activity appeared to be mediated by calmodulin (CaM) and the calmodulin‐dependent protein kinase II (CaMKII). The results observed in vitro were totally retrieved in intact oocytes and tend to demonstrate the involvement of a CaMKII‐mediated phosphorylation in the regulation of 3‐kinase activity. Finally, electrophysiological recordings of InsP3‐elicited chloride current transients in the presence of CaM/CaMKII inhibitors allowed to postulate an involvement of 3‐kinase activity in the regulation of InsP3‐mediated Ca2+ release.
Mutation of the N‐terminal proline 9 of BLMA from Streptomyces verticillus abolishes the binding affinity for bleomycin
Authors: Kumagai Takanori , Hibino Ryu , Kawano Yoshiaki , Sugiyama Masanori
Published: July 2002

A gene, blmA, from bleomycin (Bm)‐producing Streptomyces verticillus, encodes a Bm‐binding protein, designated BLMA. The expression of BLMA conferred resistance to Bm in the Escherichia coli host, whereas a mutant protein, designated Pro‐9/Leu, with the N‐terminal proline 9 residue in BLMA replaced by leucine, did not. We created a fusion protein between the maltose‐binding protein (MBP) and a mutant protein Pro‐9/Leu/Leu with Met‐94 in Pro‐9/Leu replaced by leucine. Pro‐9/Leu/Leu from the fusion protein, obtained by digestion with CNBr digestion, did not inhibit DNA‐cleaving and antibacterial activities of Bm. Native‐polyacrylamide gel electrophoresis (PAGE) and gel filtration column chromatographic analysis showed that the molecular size of Pro‐9/Leu/Leu is roughly half of that of BLMA, suggesting that the mutant protein cannot form dimeric structure. Furthermore, Far‐UV circular dichroism (CD) spectrum of Pro‐9/Leu/Leu was quite different from that of BLMA and similar to the spectra obtained from unordered proteins [Venyaminov, S.Y. and Vassilenko, K.S. (1994) Anal. Biochem. 222, 176–184], suggesting that the secondary structure of Pro‐9/Leu/Leu is disrupted. These results indicate that the mutation abolishes not only dimer formation but also the secondary structure of BLMA, which results in the loss of its function as a Bm‐resistance determinant.
Characterization of CFTR expression in a human pulmonary mucoepidermoid carcinoma cell line, NCI‐H292 cells
Authors: Nagayama Shin-ichi , Kai Hirofumi , Okiyoneda Tsukasa , Horikawa Sachiko , Miyata Takeshi
Published: July 2002

The NCI‐H292 cell, a human pulmonary mucoepidermoid carcinoma cell line, is commonly used for studying bacterial and viral infections of airway epithelial cells. Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is the main cause of fetal lung infection in cystic fibrosis patients. In this study, we examined CFTR expression in NCI‐H292 cells to determine whether NCI‐H292 cells possess sufficient, normally functioning CFTR. The results of RT‐PCR and Northern blotting analysis indicated that the CFTR gene expression level was much lower in NCI‐H292 cells than in T84 cells. However, Western blotting analysis showed that protein expression in NCI‐H292 cells was comparable to that in T84 cells. Furthermore, whole‐cell and cell‐attached patch clamp electrophysiological techniques indicated that the Cl− current induced by intracellular cAMP elevation in NCI‐H292 cells was comparable to that in T84 cells. These findings suggest that NCI‐H292 cells with a low level of CFTR gene expression possess enough functional CFTR to show a physiological response.
Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen
Authors: Mogi Tatsushi , Sato-Watanabe Mariko , Miyoshi Hideto , Orii Yutaka
Published: July 2002

To probe the functional role of a bound ubiquinone‐8 in cytochrome bo‐type ubiquinol oxidase from Escherichia coli, we examined reactions with ubiquinol‐1 and dioxygen. Stopped‐flow studies showed that anaerobic reduction of the wild‐type and the bound ubiquinone‐free (ΔUbiA) enzymes with ubiquinol‐1 immediately takes place with four kinetic phases. Replacement of the bound ubiquinone with 2,6‐dibromo‐4‐cyanophenol (PC32) suppressed the anaerobic reduction of the hemes with ubiquinol‐1 by eliminating the fast phase. Flow‐flash studies in the reaction of the fully reduced enzyme with dioxygen showed that the heme b‐to‐heme o electron transfer occurs with a rate constant of ∼1×104 s−1 in all three preparations. These results support our previous proposal that the bound ubiquinone is involved in facile oxidation of substrates in subunit II and subsequent intramolecular electron transfer to low‐spin heme b in subunit I.
Repression and activation of transcription of phosphoenolpyruvate carboxykinase gene during liver development
Authors: Cassuto Hanoch , Aran Adi , Cohen Hannah , Eisenberger Carol L , Reshef Lea
Published: July 2002

Transcriptional activation of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene at birth is critical since PEPCK appearance initiates hepatic gluconeogenesis. A delayed appearance results in hypoglycemia, while a premature appearance results in neonatal diabetes, both are incompatible with sustaining life. Experiments using transgenic mice and transfected hepatoma cells suggest that both repression and activation underlie the correct onset of hepatic PEPCK gene transcription. In transgenic mice, transgenes driven by the proximal PEPCK promoter are prematurely expressed in the fetal liver and over‐expressed in the neonatal liver, indicating that sequences upstream of the proximal promoter restrain perinatal expression. In Hepa1c1c7 cells, which mimic the fetal liver, the proximal PEPCK promoter (597 bp) exhibited a 3.5–10‐fold higher activity than longer promoters. Repression of the longer promoter (2000 bp) was diminished upon deletion of the sequence spanning positions −840 to −1116 which contains a PPAR/RXR recognition element. The intact 2000 bp PEPCK promoter could be markedly activated by co‐transfecting the transcription factor HNF‐1 together with C/EBP. It could be repressed by co‐transfection with RXRα and adding PPARα relieved this inhibition.
Differential interaction patterns in binding assays between recombinant syntaxin 1 and synaptobrevin isoforms
Authors: Pérez-Brangulı́ Francesc , Ruiz-Montasell Bonaventura , Blasi Juan
Published: July 2002

Syntaxin 1 and synaptobrevin play an essential role in synaptic vesicle exocytosis. Two isoforms for each of these proteins, syntaxin 1A and 1B and synaptobrevin 1 and 2, have been found in nerve endings. Previous morphological studies have revealed a characteristic co‐localization of syntaxin 1 and synaptobrevin isoforms in nervous and endocrine systems; however, the physiological significance of differential distribution is not known. In the present study an in vitro assay has been used to study a possible isoform specific interaction between syntaxin and synaptobrevin isoforms. The results show that although both syntaxin 1A and 1B may interact with synaptobrevin 1 and 2, this interaction is not uniform, showing different affinity patterns depending on the syntaxin 1/synaptobrevin isoform combination. The addition of SNAP‐25 increased the binding capacity of syntaxin and synaptobrevin isoforms without affecting specific interactions.
Analysis of the V genes coding for a monospecific human antibody to myosin and functional expression of single chain Fv fragments
Authors: Laroche-Traineau Jeanny , Jacobin Marie-Josée , Biard-Piechaczyk Martine , Vuillemin Luce , Chagnaud Jean-Luc , Pau Bernard , Nurden Alan T. , Clofent-Sanchez Gisèle
Published: July 2002

A monospecific human IgM monoclonal antibody (mAb), reactive with myosin from human heart, has been obtained by EBV transformation. This mAb may have a diagnostic potential in the imaging of myocardial necrosis. However, owing to the fact that the molecular mass of an IgM is 900 kDa, a poor diffusion and a slow penetration inside necrotic myocytes could reduce its capacity for scintigraphic detection. In order to alleviate these problems, we constructed the scFv by cloning the VH and VL domains into the pHOG21 vector. Analysis of the V genes proved an unmutated configuration showing that the immortalized B cell issued from the primary IgM repertoire. The expression product in Escherichia coli was a 35 kDa scFv fragment with the antigen‐binding specificity of the parental mAb.