bluuebunny/crossref_metadata_2025 huggingface dataset:
Regeneration of protochlorophyllide in green and greening leaves of plants with varying proportions of protochlorophyllide forms in darkness
Authors: Amirjani Mohammad R , Sundqvist Christer
Published: June 2004

During illumination of dark‐grown plants protochlorophyllide (Pchlide) is continuously transformed to chlorophyllide (Chlide). Different dark‐grown plants, maize (Zea mays cv. Sundance), wheat (Triticum aestivum cv. Kosack), pea (Pisum sativum cv. Kelwedon wonder), the lip1 mutant of pea, and the aurea mutant of tomato (Solanum lycopersicum), have various ratios of spectral Pchlide forms in darkness. When the plants were illuminated and then returned to darkness Pchlide re‐accumulated. The proportions of different Pchlide forms within the pool of re‐accumulated Pchlide were followed by low temperature fluorescence emission and excitation spectra in green and greening leaves. After 1 h of illumination the spectral characteristics of regenerated Pchlide forms mirrored those of Pchlide in dark‐grown plants and were thus species dependent. After a prolonged illumination period (24 h) as well as in fully green leaves energy transfer to chlorophyll (Chl) masked the presence of long‐wavelength Pchlide in the fluorescence emission spectra. However, excitation spectra showed Pchlide absorption around 650 nm and its flash‐induced disappearance confirmed its nature of phototransformable Pchlide. In fact the excitation spectra showed that the proportions of different Pchlide forms in green leaves highly resembled the proportions of Pchlide forms in dark‐grown leaves and were specific for the plant variety. Thus Chl formation in both dark‐grown and light‐grown leaves can occur in a similar way through the main photoactive long‐wavelength form of Pchlide.
Position and cell type‐dependent microtubule reorientation characterizes the early response of the Arabidopsis root epidermis to ethylene
Authors: Le Jie , Vandenbussche Filip , Van Der Straeten Dominique , Verbelen Jean‐Pierre
Published: June 2004

The involvement of cortical microtubules in the control of plant cell expansion was studied in the Arabidopsis root epidermis. In the zone of fast elongation microtubules were transverse to the root axis in all epidermal cells. However when cells entered the differentiation zone cell type‐specific microtubule reorientation took place. In the trichoblasts that were then approximately 130 µm long and formed the root hair bulge, the microtubules switched to a random distribution. In the adjoining atrichoblasts microtubules adopted a slightly oblique orientation. In more proximal parts of the differentiation zone atrichoblast microtubules were found in a more oblique and finally in a longitudinal orientation. Upon exposure to ethylene or 1‐aminocyclopropane‐1‐carboxylic acid (ACC – the precursor of ethylene) at a saturating dose, cell elongation abruptly stopped. From then on trichoblast cells reached only a length of about 35 µm, and developed root hairs. Cortical microtubules changed orientation within 10 min. In trichoblasts they adopted the typical random orientation, in atrichoblasts however, they took up a longitudinal orientation. Microtubule reorientation was complete within 60 min. The possible role of microtubules in the control of cell elongation is discussed.
Sorbitol dehydrogenase expression and activity during apple fruit set and early development
Authors: Nosarszewski Marta , Clements Ann M. , Downie A. Bruce , Archbold Douglas D.
Published: June 2004

Apple (Malus domestica Borkh.) fruit set and early development is very sensitive to carbohydrate availability, but details on carbohydrate metabolism during this phase are limited. The objective of this work was to determine if sorbitol dehydrogenase (SDH, EC 1.1.1.14), the primary enzyme responsible for metabolism of the major phloem‐transported carbohydrate sorbitol, is present and active during apple fruit set and early development. Nine different genes encoding SDH were determined from analysis of cDNA‐ and genomic‐clones obtained from an apple cDNA library or PCR using genomic DNA and degenerate oligonucleotide primers, respectively. The presence of multiple genes was confirmed from Southern analysis. Comparing three apple cultivars with different maturation dates, Northern analysis showed that SDH transcript was present during the first 5 weeks of fruit development after bloom. Using antibody developed against purified recombinant SDH protein from a fragment of apple cDNA expressed in Escherichia coli, SDH was detected in whole fruit extracts at approximately 37 kDa during each of the 5 weeks after fruit set with the greatest intensity at 1–3 weeks after bloom. ELISA analysis of SDH protein indicated that SDH comprised 7–8% of the total extractable protein at this time. SDH activity was highest at 2–3 weeks after bloom in each of the three cultivars. Thus, SDH activity during apple fruit set and early development may play a primary role in defining fruit sink activity. The present work is the first to show that SDH is expressed and active in apple fruit during the first 5 weeks after bloom.
Apoptotic Cell Death and Caspase 3 (CPP32) Activation Induced by Calcium Ionophore at Low Concentrations and their Prevention by Nerve Growth Factor in PC12 Cells
Authors: Takadera Tsuneo , Ohyashiki Takao
Published: June 2004

A23187 (a calcium ionophore) at low concentration (0.1 μM) induced apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by the activation of caspase‐3 (CPP32), a member of the interleukin‐1βT‐converting enzyme protease. On the other hand, A23187 at high concentration (2 μM) induced necrotic cell death not accompanied by the activation of CPP32. Nerve growth factor inhibited the cell death and CPP32 activation induced by 0.1 μM A23187, but not the cell death induced by 2 μM A23187. Acylaspartyl‐glutamyl‐valyl‐aspartyl‐aldehyde, an inhibitor of CPP32, reduced the cell death induced by 0.1 μM A23187. These results suggest that calcium‐ion‐induced apoptotic cell death was mediated by CPP32 activation in PC12 cells.
1α,25‐Dihydroxyvitamin D3 and a Variety of its Natural Metabolites Transcriptionally Repress Nuclear‐Factor‐κB‐Mediated Interleukin‐8 Gene Expression
Authors: Harant Hanna , Andrew Penelope J. , Reddy G. Satyanarayana , Foglar Elisabeth , Lindley Ivan J. D.
Published: June 2004

Regulation of interleukin‐8 (IL‐8) gene transcription occurs mainly through the sequences ‐94 to ‐71 of the 5′‐flanking region of the IL‐8 gene, involving the transcription factors nuclear factor for interleukin‐6 (NF‐IL‐6) and nuclear factor κB (NF‐κB). The human melanoma cell line A3 was derived from G‐361 cells by stable transfection with an IL‐8 promoter–luciferase construct containing these sequences. 1α,25‐Dihydroxyvitamin D3 (calcitriol) repressed IL‐8 promoter activity induced by tumor necrosis factor‐α (TNF‐α) by 50%, compared to 30% inhibition using dexamethasone, an effect consistent with its effect on TNF‐α‐induced IL‐8 release and IL‐8 mRNA levels. A variety of vitamin D metabolites caused the same repressive effect on IL‐8 promoter activation as calcitriol. However, only those metabolites which were able to transactivate a classical vitamin D response element had the ability to repress IL‐8 promoter activation, suggesting that this repression is mediated via vitamin D receptor (VDR). Furthermore, overexpression of VDR in the parental G‐361 cell line enhanced the repressive effect of calcitriol on activation of the IL‐8 promoter by either TNF‐α stimulation or overexpression of the NF‐κB subunit p65. Electrophoretic mobility shift assays using nuclear extracts from A3 cells showed that calcitriol decreased the abundance of nuclear factors bound to the NF‐κB binding site of the IL‐8 promoter and this reduced binding of NF‐κB proteins presumably contributes to its inhibitory action.
Genomic Organization, cDNA Sequence, Bacterial Expression, and Purification of Human Seryl‐tRNA Synthase
Authors: Vincent Christine , Tarbouriech Nicolas , Härtlein Michael
Published: June 2004

In this paper, we report the cDNA sequence and deduced primary sequence for human cytosolic seryl‐tRNA synthetase, and its expression in Escherichia coli. Two human brain cDNA clones of different origin, containing overlapping fragments coding for human seryl‐tRNA synthetase were sequenced: HFBDN14 (fetal brain clone); and IB48 (infant brain clone). For both clones the 5′ region of the cDNA was missing. This 5′ region was obtained via PCR methods using a human brain 5′ RACE‐Ready cDNA library. The complete cDNA sequence allowed us to define primers to isolate and characterize the intron/exon structure of the serS gene, consisting of 10 introns and 11 exons. The introns' sizes range from 283 bp to more than 3000 bp and the size of the exons from 71 bp to 222 bp. The availability of the gene structure of the human enzyme could help to clarify some aspects of the molecular evolution of class‐II aminoacyl‐tRNA synthetases. The human seryl‐tRNA synthetase has been expressed in E. coli, purified (95% pure as determined by SDS/PAGE) and kinetic parameters have been measured for its substrate tRNA. The human seryl‐tRNA synthetase sequence (514 amino acid residues) shows significant sequence identity with seryl‐tRNA synthetases from E. coli (25%), Saccharomyces cerevisiae (40%), Arabidopsis thaliana (41%) and Caenorhabditis elegans (60%). The partial sequences from published mammalian seryl‐tRNA synthetases are very similar to the human enzyme (94% and 92% identity for mouse and Chinese hamster seryl‐tRNA synthetase, respectively). Human seryl‐tRNA synthetase, similar to several other class‐I and class‐II human aminoacyl‐tRNA synthetases, is clearly related to its bacterial counter‐parts, independent of an additional C‐terminal domain and a N‐terminal insertion identified in the human enzyme. In functional studies, the enzyme aminoacylates calf liver tRNA and prokaryotic E. coli tRNA.
Homologous Regions of the α Subunit of Eukaryotic Translational Initiation Factor 2 (eIF2α) and the Vaccinia Virus K3L Gene Product Interact with the Same Domain within the dsRNA‐Activated Protein Kinase (PKR)
Authors: Sharp Tyson V. , Witzel J. Eric , Jagus Rosemary
Published: June 2004

The vaccinia virus K3L gene product, pK3, binds to the dsRNA‐activated protein kanase, PKR, reducing its ability to interact with and phosphorylate eIF2α. On the basis of this characteristic and the homology of pK3 to the N‐terminus of eIF2α, several laboratories have utilized pK3 to investigate the molecular determinants that specify substrate recognition by PKR. The data presented here demonstrate that the natural substrate, eIF2α, also binds to PKR in vitro and interacts with the same or an overlapping domain within PKR. A truncated form of eIF2α, representing the N‐terminal 123 amino acids and containing the regions of homology to pK3, retains the ability to bind PKR. pK3, eIF2α, and the truncated form of eIF2α all bind to the C‐terminus of PKR containing the catalytic domain, but not to the regulatory N‐terminus. Variants of pK3 and eIF2α, des‐(75–78)‐K3L (PK3GYID), and des‐(80–83)‐eIF2α (eIF2αGYID), from which the conserved amino acids GYID have been deleted, exhibit a decreased ability to interact with PKR. Similarly, the in vitro binding of pK3, eIF2α, and the truncated form of eIF2α to PKR can be competed with purified pK3 but not with pK3GYID. In addition, the deletion of GYID from eIF2α significantly reduces its ability to be phosphorylated by PKR, demonstrating that PKR recognizes its substrate, at least in part through interaction with sequences remote from the phosphorylation site. In summary, we have shown that the region within PKR that interacts with the pseudosubstrate, pK3, is the same region that interacts with the authentic substrate, eIF2α. In addition, we have shown that the N‐terminal 123 amino acids of eIF2α contains structural elements necessary for recognition by PKR. The results pinpoint the GYID motif, shared between pK3 and eIF2α and distant from the phosphorylation site, as being important for the interaction of eIF2α with PKR, as well as its phosphorylation.
Identification of the Common Antigenic Determinant Shared by Streptococcus pneumoniae Serotypes 35A and 20 Capsular Polysaccharides — Structural Analysis of the Streptococcus pneumoniae Serotype 35A Capsular Polysaccharide
Authors: Beynon Linda M. , Richards James C. , Perry Malcolm B.
Published: June 2004

The specific polysaccharide antigen of Streptococcus pneumoniae serotype 35A was shown, by a combination of one‐ and two‐dimensional NMR methods and chemical analyses, to be a high‐molecular‐mass polymer composed of d‐galactose, d‐glucose, mannitol, and phosphate (3:1:1:1). The pentasaccharide repeating unit is polymerized through phosphate diester linkages to give the structure,
image

O‐Acetyl substituents are present at postions 5 and 6 of the 3)‐β‐d‐Galf residue and at position 2 of the 6)‐β‐d‐Galf residue. The capsular polysaccharides of S. pneumoniae serotypes 20 and 35A both contain the disaccharide unit →3)‐β‐d‐Galf‐(1→3)‐β‐d‐Glcp‐(1→ which is the probable structural determinant responsible for the serological cross reactivity of the two polysaccharides.
Development and Evaluation of Peptide‐Based Prolyl Oligopeptidase Inhibitors — Introduction of N‐Benzyloxycarbonyl‐Prolyl‐3‐Fluoropyrrolidine as a Lead in Inhibitor Design
Authors: Goossens Filip , Vanhoof Greet , De Meester Ingrid , Augustyns Koen , Borloo Marjan , Tourwe Dirk , Haemers Achiel , Scharpé Simon
Published: June 2004

The current study has been undertaken to develop new and biocompatible inhibitors for prolyl oligopeptidase, a highly specific endopeptidase, proposed to be involved, through its affinity for neuropeptides and kinins, in the processes of learning and memory and in the control of blood pressure. For in vitro evaluation of the inhibitors, human platelet prolyl oligopeptidase was purified to homogeneity and characterized. Northern blot analysis showed that mRNA coding for prolyl oligopeptidase was present in all tissues examined and only one transcript of 3.1 kb was detected. In addition to the human platelet enzyme, we also purified rat brain prolyl oligopeptidase, which proved to have the same characteristics as the human enzyme. In a series of tested peptides, bradykinin was found to be the best substrate. Based on this information, peptides bearing pseudopeptide bonds were generated and evaluated as inhibitors. The experiments clearly demonstrated that changes to the scissile peptide bond significantly decrease the affinity of prolyl oligopeptidase for the peptide derivatives. In our series of synthetic N‐terminal blocked dipeptides, N‐benzyloxycarbonyl‐prolyl‐3‐fluoropyrrolidine was the most potent compound. Inhibition was reversible, but the inhibitor was bound tightly. Calculation of its Ki according to Henderson [Henderson, J. P. (1972) Biochem. J. 127, 321–333] yielded a value of 0.8 nM. This compound was not cytotoxic in a cell culture system and inhibited the purified prolyl oligopeptidase from rat as well as from human origin. In vivo evaluation in male Whistar rats showed no acute toxicity. 5 h after administration, the most profound decrease in prolyl oligopeptidase activity was found in the thymus, brain, and testis. This study demonstrates that N‐benzyloxycarbonyl‐prolyl‐3‐fluoropyrrolidine is a potent inhibitor and a promising compound suitable to investigate the physiologic function of the enzyme in vitro and In vivo.
Human and Murine Serine‐Palmitoyl‐CoA Transferase
Authors: Weiss Bertram , Stoffel Wilhelm
Published: June 2004

Serine palmitoyltransferase (SPT, EC 2.3.1.50) is the key enzyme in sphingolipid biosynthesis. It catalyzes the pyridoxal‐5’‐phosphate‐dependent condensation of L‐serine and palmitoyl‐CoA to 3‐oxo‐sphinganine. Human expressed‐sequence‐tag (EST) clones are similar to the two yeast genes for synthesis of long‐chain bases, LCB1 and LCB2, which are believed to encode two subunits of SPT [Buede, R., Pinto, W. J., Lester, R. L. & Dickson, R. C. (1991) J. Bacteriol. 173, 4325–5332; Nagiec, M. M., Baltisberger, J. A., Wells, G. B., Lester, R. L. & Dickson, R. C. (1994) Proc. Natl Acad. Sci. USA 91, 7899–7902]. We have cloned and characterized two complete human and murine cDNA sequences named hLCB1 & mLCB1 and hLCB2 & mLCB2, respectively, similar to the yeast LCB1 and LCB2 genes.

Human embryonic kidney cells (HEK 293) transfected with murine sequences of LCB1 (mLCB1) and LCB2 (mLCB2) independently and in coexpression showed an overexpression of the transcripts on the mRNA and protein level. The enzymatic activity of cells expressing mLCB2 alone or coexpressed with mLCB1 was three times higher than the activity of untransfected HEK cells. mLCB1 expression was not required for the synthesis of 3‐oxo‐sphinganine in mammalian cells. Transcription/translation in vitro yielded mLCB1 (53 kDa) and mLCB2 (63 kDa). The two proteins do not contain a signal peptide nor are they glycosylated. The endogenous and overexpressed SPT activity were both sensitive to common SPT inhibitors. Labeling studies with [1‐14C]palmitic acid indicated that cell lines transfected with mLCB2 preferentially use the excess sphingoid bases for glucocerebroside and galactocerebroside synthesis. Our results provide conclusive genetic and biochemical evidence that the human and murine LCB2 genes described here encode serine palmitoyltransferase. Further studies will be required to unravel the function of the LCB1 gene in mammalian cells.
Platination of A GG Site on Single‐Stranded and Double‐Stranded forms of A 14‐Base Oligonucleotide with Diaqua Cisplatin followed by NMR and HPLC
Authors: Reeder Franziska , Guo Zijian , Murdoch Piedad del Socorro , Corazza Alessandra , Hambley Trevor W. , Berners‐Price Susan J. , Chottard Jean‐Claude , Sadler Peter J.
Published: June 2004

Detailed studies of the kinetics of platination of the single‐stranded 14‐base DNA oligonucleotide d(ATACATGGTACATA) and the corresponding duplex by cis‐[Pt(NH3)2(H2O)2]2+ show that HPLC and NMR are complementary methods which provide similar results. The 5'‐G and 3'‐G monofunctional intermediates were trapped, separated and characterized by NMR (via 15NH3 labeling) and enzymatic digestion followed by mass spectrometry. The kinetic data are compared with those for the corresponding reactions of cis‐[PtCl2(NH3)2] (cisplatin) and its monohydrolysed analogue. For both single and double strands of the oligonucleotide, the aqua complex shows little selectivity for the 5'‐G or the 3'‐G in the initial platination step, whereas the chloro‐complex preferentially platinates the 3'‐G. The base on the 3' side of the GG sequence appears to play an important role in controlling this selectivity; replacement of T by C increases the selectivity of duplex platination by the diaqua complex by a factor of about 6, and the selectivity of chelation of the 3'‐G monofunctional adduct by a factor of about 3. In general the reactivity of the 5'‐G in a GG sequence appears to be enhanced in a duplex compared with a single‐strand. For both the aqua‐monoadduct and chloro‐monoadduct, cis‐[Pt(NH3)2(NG)(H2O or Cl)], the 5'‐G monoadduct is much longer lived (t½≈ 4 h at 288 K for aqua, 80 h at 298 K for chloro) than the 3'‐G monoadduct (t½≤ 45 min at 288 K for aqua, 6 h at 298 K for chloro). Inspection of molecular mechanics models of the end states of various monofunctional adducts provided insight into H‐bonding and destacking interactions in these adducts and the sequence selectivity observed in their formation. Such adducts may play an important role in the mechanism of action of platinum anticancer drugs.
Dynamics of Ubiquitin Conjugation during Heat‐Shock Response Revealed by using a Monoclonal Antibody Specific to Multi‐Ubiquitin Chains
Authors: Fujimuro Masahiro , Sawada Hitoshi , Yokosawa Hideyoshi
Published: June 2004

Levels of intracellular multi‐ubiquitinated proteins in heat‐shocked HeLa cells were investigated using a monoclonal antibody specific to multi‐ubiquitin chains. After heat‐shock treatment at 42–44°C for 30 min, the level of multi‐ubiquitinated proteins increased within the first 2h at 37°C and returned to the initial level within the following 2h. The accumulation of multi‐ubiquitin conjugates was elevated by increasing the temperature, while the opposite was the case for the level of ubiquitinated histone H2A. Immunocytochemical analysis revealed that the amount of ubiquitin conjugates rapidly increased in the cytosol and concomitantly decreased in the nucleus under heat‐shock conditions. The heat‐shock treatment elicited little apparent change in the activity of the 26S proteasome, but it did induce a gradual increase in activity of the ubiquitinating enzyme system. These results strongly suggest that the level of cytoplasmic multi‐ubiquitinated proteins and that of nuclear ubiquitinated histone H2A increases and decreases, respectively, in response to heat shock and that the heat‐shock‐induced accumulation of multi‐ubiquitinated proteins is caused by activation of the ubiquitinating enzyme system rather than inactivation of the 26S proteasome.
The Ca2+‐Mobilizing Potency of α‐Thrombin and Thrombin‐Receptor‐Activating Peptide on Human Platelets
Authors: Heemskerk Johan W. M. , Feijge Marion A. H. , Henneman Lidewij , Rosing Jan , Hemker H. Coenraad
Published: June 2004

In single platelets and in suspensions of platelets, α‐thrombin evokes dose‐dependent, transient increases in cytosolic Ca2+ concentration, [Ca2+]ij, which are more prolonged than the [Ca2+]; transients evoked by other platelet agonists such as the thrombin‐receptor‐activating hexapeptide SFLLRN, thromboxane A2 analog U46619, and ADP As a quantity taking into account both the magnitude and length of the Ca2+ response, we defined the Ca2+‐mobilizing potency (CMP) of an agonist as the integrated rise in [Ca2+]i during the time of the Ca2+ signal. It was observed that: (a) the CMP increased with the agonist concentration in a saturating way, its maximal value being about four‐times higher with α‐thrombin than with SFLLRN; (b) the high CMP of α‐thrombin was for only a small part due to endogenous production of ADP or thromboxane, and was mainly a consequence of prolonged influx of external Ca2+; (c) the CMP declined when a‐thrombin was inactivated during the course of the Ca2+ signal; (d) CMP values increased with the agonist concentration upon sequential addition of increasing amounts of α‐thrombin or SFLLRN; (e) when α‐thrombin was gradually added to the platelets or formed by an in situ reconstituted prothrombinase system (with factor Xa, factor Va, and prothrombin), integrated Ca2+ responses were a function of the product of the α‐thrombin concentration and the time of its presence. However, in these cases, the final CMP values were independent of the rate of α‐thrombin addition or formation. We conclude that α‐thrombin‐induced Ca2+ signals in platelets rely largely upon Ca2+ influx, are not, or only slightly, subjected to homologous desensitization, and reflect the enzymatic capacity of α‐thrombin to cleave protease‐activated receptors. Thus, the high and prolonged Ca2+ signal induced by α‐thrombin is due to continuous receptor cleavage without desensitizing effects of previously cleaved receptors.
The Role of Ferredoxin‐NADP+ Reductase in the Concerted Cell Defense Against Oxidative Damage
Authors: Krapp Adriana R. , Tognetti Vanesa B. , Carrillo Néstor , Acevedo Alberto
Published: June 2004

Ferredoxin‐NADP+ reductases (FNR) participate in cellular defense against oxidative damage. Escherichia coli mutants deficient in FNR are abnormally sensitive to methyl viologen and hydrogen peroxide. Tolerance to these oxidants was regained by expression of plant FNR, superoxide dismutase, or catalase genes in the mutant cells. FNR contribution to the concerted defense against viologen toxicity under redox‐cycling conditions was similar to that of the two major E. coli superoxide dismutases together, as judged by the phenotypes displayed by relevant mutant strains. However, FNR expression in sodA sodB strains failed to increase their tolerance to viologens, indicating that the FNR target is not the superoxide radical. Sensitivity of FNR‐deficient cells to oxidants is related to extensive DNA damage. Incubation of the mutant bacteria with iron chelators or hydroxyl radical scavengers provided significant protection against viologens or peroxide, suggesting that oxidative injury in FNR‐deficient cells was mediated by intracellular iron through the formation of hydroxyl radicals in situ. The NADP(H)‐dependent activities of the reductase were necessary and sufficient for detoxification, without participation of either ferredoxin or flavodoxin in the process. Possible mechanisms by which FNR may exert its protective role are discussed.
Structure of the Rat Methionine Adenosyltransferase 2A Gene and its Promoter
Authors: Hiroki Tomoko , Horikawa Saburo , Tsukada Kinji
Published: June 2004

In this study, to understand the regulation of methionine adenosyltransferase (MAT) gene expression, we isolated the rat MAT2A gene encoding MATa2, the catalytic subunit of non‐hepatic‐type enzyme MAT II and characterized its structural organization and 5′‐flanking region. The gene spans approximately 7 kbp and consists of nine exons interrupted by eight introns. The transcription initiation site, as demonstrated by primer extension analysis, is located 123 bp upstream of the translation start codon. Comparison of the structural organization of the rat MAT2A gene to that of the mouse MAT1 A gene encoding MATα1, the subunit of liver‐type enzymes MAT I and III, shows that the exon structure of two genes is very similar and the insertion sites of all corresponding introns are identical. A canonical TATA box and a GC box, the potential Sp1‐binding site, are found 32 bp and 70 bp upstream of the transcription initiation site, respectively. The 5′‐flanking region also contains potential recognition sites for various transcription factors including AP‐1, AP‐2 and NF‐IL6 (C/EBPβ), and a large GC‐rich domain with the characteristics of a CpG island. The 5′‐flanking sequence of the rat MAT2A gene has no significant similarity with those of the MAT1A genes. Transient transfection experiments using a luciferase reporter gene snowed that the first 820‐bp sequence of the 5′‐flanking region directed high levels of luciferase activity in cultured rat kidney fibroblast (NRK‐49F) and hepatocellular carcinoma (FAA‐HTC1) cells, but not in primary rat hepatocytes. Deletion analysis suggested that the first 343 bp of the 5′‐flanking region contained cell‐type‐specific promoter elements of this gene.
Transferrin Receptor Functions as a Signal‐Transduction Molecule for its Own Recycling Via Increases in the Internal Ca2 Concentration
Authors: Sainte‐Marie Josette , Lafont Virginie , Pécheur Eve‐Isabelle , Favero Jean , Philippot Jean R. , Bienvenüe Alain
Published: June 2004

Transferrin binding to its receptor modulates transferrin receptor (Tf‐R) recycling rates in several cells [Klausner, R. D., Van Renswoude, J., Ashwell, G., Kempf, C., Schechter, A., Dean, A. & Bridges, K. R. (1983a) J. Biol. Chem. 258, 4715–4724; Gironès, N. & Davis, R. J. (1989) Biochem. J. 264, 35–46; Sainte‐Marie, J., Vidal, M., Bette‐Bobillo, P., Philippot, J. R. & Bienvenüe, A. (1991) Eur. J. Biochem. 201, 295–302]. To delineate the mechanism of this regulation, we hypothesized that the binding of the ligand to its receptor could lead to activation of several second‐messenger pathways, which may redundantly stimulate recycling of the receptor. The effects of different regulators of Ca2 flux or concentrations were investigated on the Tf‐R–recycling pathway; these studies were carried out in two cell types. Perhexiline, a calcium antagonist, slowed receptor recycling in comparison with the control by more than 80% in L2C cells and by 60% in Jurkat cells (B and T lymphoblasts, respectively) but did not affect their internalization rate. Perhexiline thus trapped considerable amounts of Tf‐R in the internal compartment. Ca2 chelators, such as EGTA or 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N,N′‐tetraacetic acid, and a Ca2‐channel inhibitor (Ni2) decreased drastically the recycling rate of Tf‐R. Tf‐R recycling was shown to be slowed by a calmodulin antagonist. Conversely, artificial elevation of free internal Ca2 in L2C cells, using lectin, accelerated the recycling rate. These results suggest that the intracellular Ca2 concentration plays an important role in the outward flow of transferrin receptors. Consequently, we examined the role of transferrin in internal free Ca2 regulation. The addition of transferrin or anti‐(Tf‐R) Ig specifically elicited a rise in [Ca2], as demonstrated by inefficacy of apotransferrin or irrelevant antibodies. These results suggest that Ca2 is a regulator of Tf‐R recycling and that Tf‐R seems to function as a signal‐transduction molecule (perhaps in conjunction with other membrane proteins) rather than merely as an endocytic receptor.
Regulation of Expression of Ferritin H‐chain and Transferrin Receptor by Protoporphyrin IX
Authors: Coccia Eliana M. , Perrotti Edvige , Stellacci Emilia , Orsatti Roberto , del Russo Nicoletta , Marziali Giovanna , Testa Ugo , Battistini Angela
Published: June 2004

The effect of protoporphyrin IX (hemin without iron) on the expression of transferrin receptor and ferritin was investigated in Friend leukemia cells. Cells treated with protoporphyrin IX exhibit enhanced transferrin‐receptor expression and markedly reduced ferritin synthesis. Stimulation of transferrin‐receptor expression is observed at both the mRNA and protein level. The effect on ferritin synthesis is mediated by translational inhibition of the mRNA, hich, in contrast, is transcriptionally stimulated by protoporphyrin IX treatment. The regulation of transferrin receptor and ferritin in response to iron erturbations has been studied extensively and is mediated by the binding of iron‐regulatory proteins (IRP) to the iron‐responsive elements (IRE) present in the 3′ and 5′ untranslated regions of the transferrin‐receptor and ferritin mRNA, espectively. To elucidate the molecular mechanisms underlying the effects of protoporphyrin IX on ferritin and transferrin‐receptor expression, the role of the IRE sequence was investigated both in vivo by transfection experiments, with a construct containing the coding region for the chloramphenicol acetyltransferase (CAT) reporter gene under the translational control of the ferritin IRE, and in vitro by RNA band‐shift assays. Whereas, examination of IRP binding to the IRE by in vitro assays suggests an apparent inactivation of IRP by protoporphyrin IX treatment, CAT assays indicate that proto porphyrin IX is able to induce in vivo a translational inhibition similar to that obtained by treatment with the iron chelator Desferal. This observation raises the possibility of different effects on the IRP activity exerted by porphyrin treatment in intact tissue‐culture cells and in vim. We conclude that translation of ferritin mRNA and degradation of transferrin‐receptor mRNA are inhibited in intact tissue‐culture cells by protoporphyrin IX through a mechanism similar to that exerted by iron chelation, thus involving depletion of the intracellular iron pool. These results can improve the understanding of the regulation of fenitin gene expression in some pathological conditions associated with disturbed heme synthesis.
Flavin Adenine Dinucleotide and Flavin Mononucleotide Metabolism in Rat Liver
Authors: Barile Maria , Brizio Carmen , De Virgilio Caterina , Delfine Sebastiano , Quagliariello Ernesto , Passarella Salvatore
Published: June 2004

In order to gain some insight into mitochondrial flavin biochemistry, rat liver mitochondria essentially free of lysosomal and microsomal contamination were prepared and their capability to metabolise externally added and endogenous FAD and FMN tested both spectroscopically and via HPLC.

The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD→FMN and FMN→riboflavin conversion, respectively, is shown. They differ from each other and from extramitochondrial enzymes, as judged by their pH profile and inhibitor sensitivity, and can be separated in a partial FAD pyrophosphatase purification.

Digitonin titration and subfractionation experiments show that FAD pyrophosphatase is located in the outer mitochondrial membrane and FMN phosphohydrolase in the intermembrane space. Since these enzymes can metabolise endogenous FAD and FMN, which are made available by using both Triton X‐100 and the effector oxaloacetate, a proposal is made that FAD pyrophosphatase and FMN phosphohydrolase play a major role in mitochondrial flavoprotein turnover.
Inhibitory Guanine‐nucleotide‐binding‐regulatory Protein α Subunits in Medaka (Oryzias latipes) Oocytes
Authors: Oba Yuichi , Yoshikuni Michiyasu , Tanaka Minoru , Mita Masatoshi , Nagahama Yoshitaka
Published: June 2004

We have previously shown that pertussis‐toxin‐sensitive inhibitory guanine‐nucleotide‐binding‐regulatory proteins (G proteins) are involved in the signal transduction of steroidal maturation‐inducing hormone (MIH) of rainbow trout (Oncorhynchus mykiss) oocytes, 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17α,20β‐DP) [Yoshikuni, M. & Nagahama, Y. (1994) Dev. Biol. 166, 615–622]. In this study, we obtained five different cDNA fragments of G protein α subunits from medaka (Oryzias latipes) intact ovarian follicles (three subtypes of Giα, Giαa, Giαb and Giαc; two subtypes of Gsα Gsαe, and Gαe). Using a newly developed extraction method for medaka oocyte RNA, we demonstrated that oocytes expressed both Giαa and Giαc, but not Giαb. Full‐length cDNA clones for Giαa and Giαc were then isolated from a medaka ovarian follicle cDNA library. The predicted amino acid sequences of Giαa and Giαc exhibited significant similarity with Giα1 and Giαa2 of other species, respectively. Both Giα1 and Giα2 possessed a specific Cys residue in the C‐terminal region that was the site for ADP‐ribosylation by pertussis toxin. Goo, another G protein that is ADP‐ribosylated by pertussis toxin, was not detected in oocytes, although it was expressed in brain tissue. Western blot analyses using a specific antibody against Giα1 and Giα2 subunit proteins revealed that in both medaka and rainbow trout Giα subunit protein (40 kDa) contents were abundant in plasma membranes of postvitellogenic immature oocytes, decreased in mature oocytes, and were absent in ovulated eggs. Furthermore, specific 17α,20β‐DP binding to plasma membranes was higher in postvitellogenic immature oocytes than in ovulated eggs. Taken together, these results suggest that Giαa and/or Giαc may be involved in the transduction of the signal from 17α,20β‐DP receptors during oocyte maturation of fish oocytes.
Okadaic Acid Interferes with Phorbol‐Ester‐Mediated Down‐Regulation of Protein Kinase C‐α, C‐δ and C‐ɛ
Authors: Gatti Andrea , Robinson Phillip J.
Published: June 2004

A prolonged cell exposure of all examined cell types to tumour‐promoting phorbol esters leads to a substantial inactivation and degradation of protein kinase C (PKC), a phenomenon known as downregulation. With a combination of one‐ and two‐dimensional immunoblot analyses we have previously shown the existence in PC12 cells of distinct PKC‐a forms that differentially respond to cell treatment with phorbol ester [Gatti, A. & Robinson, P. J. (1996) J. Biol. Chem. 271, 31718–31722]. Using the same experimental model, in the present study we investigated a possible relationship between PKC‐α phosphorylation and its down‐regulation. The exposure of PC 12 cells to okadaic acid, a potent inhibitor of biologically relevant protein phosphatases, was found to partially protect PKC‐α against phorbol‐ester‐mediated down‐regulation. Further, a similar protective effect of okadaic acid was observed for PKC‐α and PKC‐α, which are also expressed in PC12 cells. These results indicate that the tumour‐promoting activity of okadaic acid itself may be due to a sustained phosphorylation of PKC.
Role of Protein‐Phosphorylation Events in the Anoxia Signal‐Transduction Pathway Leading to the Inhibition of Total Protein Synthesis in Isolated Hepatocytes
Authors: Tinton Sandrine , Tran‐Nguyen Qui‐Nhi , Buc‐Calderon Pedro
Published: June 2004

Incubation of isolated hepatocytes under N2/CO2 (no O2) produced a rapid and strong inhibition of overall polypeptide biosynthesis, which was neither related to cell death nor to the appearance of specific stress proteins. Treatment of the cells with the tyrosine‐kinase inhibitor genistein or with the serine/threonine‐protein‐kinase inhibitor H7 did not modify the impairment of protein synthesis induced by oxygen deprivation, indicating that such signal‐transduction pathways are probably not involved in the anoxia‐mediated effect. Okadaic acid (100 nM) and Na3VO4 (1 mM) reduced the incorporation of [14C]Leu into proteins of hepatocytes maintained under aerobic conditions (93.3 kPa O2). The effects of oxygen deprivation and okadaic acid were additive, whereas sodium vanadate did not enhance the impairment of protein synthesis induced by anoxia. This observation suggests that a common mechanism, involving the net phosphorylation of protein tyrosine residues, that is insensitive to genistein might participate in the negative control of the translation induced by oxygen deprivation. The effect of anoxia on the synthesis of proteins was fully and rapidly reversible upon the restoration of oxygen supply, thus indicating that hepatocytes are able to sense O2. Although high concentrations of cobalt chloride partially mimic the effect of oxygen deprivation on protein biosynthesis, the nature of such an oxygen sensor remains unknown, and appears unlikely to be a part of a classic haem protein.
Peptide Fragments of DNA Topoisomerase II with Helix‐Forming and Coiled‐Coil‐Forming Properties Act as Inhibitors of the Enzyme
Authors: Frère‐Gallois Valérie , Krebs Daniel , Scala Denis , Troalen Frédéric , Fermandjian Serge
Published: June 2004

We have previously shown that a synthetic peptide (dL) consisting of amino acids 1013–1056 of human α topoisomerase II adopted an α‐helix structure and formed a stable dimer coiled‐coil in solution [Frére, V., Sourgen, F., Monnot, M., Troalen, F. & Fermandjian, S. (1995) J. Biol. Chem. 270, 17502–17507]. Here we studied two peptides, dP and dLshort, which are related to dL but which have a double substitution Leul026→Pro, Leu 1037→Pro and a deletion of the 15 C‐terminal residues, respectively. The peptides were studied for their ability to form α‐helix structures, coiled coils, and to inhibit topoisomerase II activity. In combining circular dichroism spectra with AGADIR prediction for helix structures, we demonstrated that the dLshort peptide, like its parent dL peptide, adopts an a‐helix structure and can autoassociate into coiled‐coils, while dP is completely devoid of such properties. Remarkably, only the dL and dLshort peptides act as good inhibitors of topoisomerase II in various in vitro assays. However, the dLshort peptide has a stronger helix potential and behaves as a much more potent inhibitor (5 uM versus 200 μM) compared to the dL peptide. All these data strongly suggest that the greater inhibitory effect demonstrated by the dLshort peptide is related to its higher ability to form a stable amphiphilic helix, which in turn better recognizes its homologous helical segment in topoisomerase II. Finally, we propose that the dL and the dLshort peptides could interfere with the enzymatic activity of topoisomersase II in modifying its autoassociation or translocation properties. Such peptides may serve as useful models for developing simpler and more specific inhibitors of topoisomerase II.
Characterization of a Recombinant Neisseria Meningitidesα‐2,3‐Sialyltransferase and its Acceptor Specificity
Authors: Gilbert Michel , Cunningham Anna‐Maria , Watson David C. , Martin Adèle , Richards James C. , Wakarchuk Warren W.
Published: June 2004

The structure and specificity of the recombinant α‐2,3‐sialyltransferase from Neisseria meningitides are reported. This enzyme showed an unusual acceptor specificity in that it could use α‐terminal and β‐terminal Gal residues as acceptors. In addition (β→4)‐linked and (β→3)‐linked terminal Gal served as acceptors. These properties distinguish the bacterial enzyme from the more widely investigated mammalian equivalents. The protein was expressed as a membrane‐associated protein in Escherichia coli at a level of 750 U/l (≈250 mg/l). The protein could be extracted with buffers containing 0.2% Triton X‐100 and purified to homogeneity using immobilized‐metal‐affinity chromatography. Electrospray‐ionization mass spectrometry of peptides obtained by cleavage with cyanogen bromide and trypsin confirmed over 95% of the deduced amino acid sequence. When used for enzymatic synthesis in coupled reactions with recombinant CMP‐Neu5Ac synthetase, the α‐2,3‐sialyltransferase could sialylate fluorescent derivatives of N‐acetyllactosamine with N‐acetylneuraminic acid, N‐propionylneuraminic acid and N‐glycoloylneuraminic acid.
PCTAIRE 2, A Cdc2‐Related Serine/Threonine Kinase, is Predominantly Expressed in Terminally Differentiated Neurons
Authors: Hirose Takashi , Tamaru Teruya , Okumura Nobuaki , Nagai Katsuya , Okada Masato
Published: June 2004

PCTAIRE are members of a subfamily of Cdc2‐related kinases that have been shown to be preferentially expressed in postmitotic cells. To examine the neural functions of PCTAIRE, rat cDNA clones encoding PCTAIRE 1, 2, and 3 were isolated, and their expression patterns in the brain were analyzed. Among the three rat PCTAIREs, only PCTAIRE 2 was found to be specifically expressed in the brain. Furthermore, its expression was transiently increased during brain development, peaking 7–15 days after birth. Within the brain, PCTAIRE 2 was concentrated in the neuronal layers of the hippocampus and olfactory bulb, which mostly consist of post‐mitotic neurons. In an immunocytochemical experiment, immunoreactivity for PCTAIRE 2 was detected in the cell bodies and extended neurites of neurons, but not in astrocytes. The PCTAIRE 2 protein was recovered in the particulate fraction and resistant to solubilization with non‐ionic detergent, suggesting that PCTAIRE 2 might be present as a component of a large protein complex. An immunoprecipitation assay revealed that the PCTAIRE 2 was associated with Ser/Thr‐phosphorylating activity for histone H1, and that its activity depended on association with a regulatory partner that can be released under high‐salt conditions. These findings suggest that PCTAIRE 2 is a Ser/Thr kinase that might play a unique role in terminally differentiated neurons.
The 2‐His‐1‐Carboxylate Facial Triad — An Emerging Structural Motif in Mononuclear Non‐Heme Iron(II) Enzymes
Authors: Hegg Eric L. , Jr Lawrence Que
Published: June 2004

A 2‐His‐1‐carboxylate facial triad is a common feature of the active sites in a number of mononuclear non‐heme iron(II) enzymes. This structural motif was established crystallographically for five different classes of enzymes and inferred from sequence similarity for two other classes. The 2‐His‐1‐carboxylate facial triad anchors the iron in the active site and at the same time maintains three additional cis‐oriented sites. These sites can be used to bind other endogenous ligands or exogenous ligands such as substrate and/or O2, giving the metal center great flexibility to use different mechanistic strategies to perform a variety of chemical transformations.
Neuronal Cell Nuclear Factor
Authors: Bauer Uta‐Maria , Schneider‐hirsch Stefan , Reinhardt Sigrid , Pauly Thorsten , Maus Arno , Wang Fan , Heiermann Reinhard , Rentrop Martin , Maelicke Alfred
Published: June 2004

We have cloned from a cDNA library of neuronal derivatives of retinoic‐acid‐induced embryonic carcinoma cells a nuclear receptor that may be involved in the control of late neurogenesis and early neuronal differentiation. The receptor which is practically identical in sequence with germ cell nuclear factor, has been designated neuronal cell nuclear factor (NCNF). NCNF is exclusively expressed in the neuronal derivatives of PCC7‐Mz1 cells, with the expression beginning within hours of exposure to retinoic acid. In the developing mouse brain, NCNF is expressed in the marginal zones of the neuroepithelium which are known to contain young postmitotic neurons. NCNF binds to the DRO sequence there by silencing transcription. Because NCNF does not recognize hormone response elements of other nuclear receptors tested and does not heterodimerize with these, it probably binds exclusively as a homodimer. NCNF may induce neuronal differentiation by repressing the activity of genes that permit cell fates other than the neuronal one.
Analysis of the Mechanism of Action of Anti‐human Interleukin‐6 and Anti‐human Interleukin‐6 Receptor‐neutralising Monoclonal Antibodies
Authors: Kalai Michael , Montero‐julian Fèlix A. , Brakenhoff Just P. J. , Fontaine Véronique , De Wit Lucas , Wollmer Axel , Brailly Hervé , Content Jean , Grötzinger Joachim
Published: June 2004

Anti‐human interleukin‐6 (human IL‐6) and anti‐human IL‐6 receptor (IL‐6R)‐neutralising monoclonal antibodies (mAbs) are among the most promising human lL‐6‐specific inhibitors and have been shown to exert short‐term beneficial effects in clinical trials. Simultaneous treatment with different anti‐human IL‐6 or anti‐human IL‐6R mAbs was recently suggested to be a potent way to inhibit the action of the cytokine in vivo. Although some of these mAbs are already used, their mechanisms of action and the location of their epitopes on the surface of human IL‐6 and human IL‐6R are still unknown. Here, we analysed the capacity of several anti‐human IL‐6 and anti‐human IL‐6R mAbs to inhibit the interaction between human IL‐6, human IL‐6R, and human glycoprotein 130 (gp130). We mapped the epitopes of several of these mAbs by studying their binding to human IL‐6 and human IL‐6R mutant proteins. Our results show that several anti‐human IL‐6 and anti‐human IL‐6R‐neutralising mAbs block the binding between human IL‐6 and human IL‐6R, whereas others block the binding to gpl 30. We provide evidence that some of the latter mAbs inhibit interaction with gp130β1, whereas others interfere with the binding to gp130β2. Our results suggest that residues included in the C′D′ loop of human IL‐6R interact with gp130β2.