bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulfide dehydrogenase is identical with the SoxB protein of the thiosulfate‐oxidizing enzyme system of Paracoccus denitrificans GB17
Authors: Schneider Angela , Friedrich Cornelius
Published: July 2002

Thiosulfate induced cells of Paracoccus denitrificans GB17 oxidize thiosulfate and sulfide to sulfate. A mutant carrying a Tn5‐mob insertion in the soxB gene is unable to oxidize thiosulfate or sulfide suggesting a linkage of both activities. To test this assumption we have separated the components of the thiosulfate‐oxidizing enzyme system of the wild‐type by ion exchange chromatography. The SoxB protein coeluted with a highly active sulfide dehydrogenase. Analysis by polyacrylamide gel electrophoresis revealed one major protein of M
r 32k. Thus, the SoxB protein appeared to be identical with sulfide dehydrogenase.
The crystal structure of human CskSH3: structural diversity near the RT‐Src and n‐Src loop
Authors: Borchert T.V. , Mathieu M. , Zeelen J.Ph. , Courtneidge S.A. , Wierenga R.K.
Published: July 2002

SH3 domains are modules occurring in diverse proteins, ranging from cytoskeletal proteins to signaling proteins, such as tyrosine kinases. The crystal structure of the SH3 domain of Csk (c‐Src specific tyrosine kinase) has been refined at a resolution of 2.5 Å, with an R‐factor of 22.4%. The structure is very similar to the FynSHS crystal structure. When comparing CskSHS and FynSH3 it is seen that the structural and charge differences of the RT‐Src loop and the n‐Src loop, near the conserved Trp47, correlate with different binding properties of these SH3 domains. The structure comparison suggests that those glycines and acid residues which are very well conserved in the SH3 sequences are important for the stability of the SH3 fold.
A two‐domain model for the R domain of the cystic fibrosis transmembrane conductance regulator based on sequence similarities
Authors: Dulhanty Ann M. , Riordan John R.
Published: July 2002

CFTR belongs to a group of proteins sharing the structural motif of six transmembrane helices and a nucleotide binding domain. Unique to CFTR is the R domain, a charged cytoplasmic domain. Comparison of R domain sequences from ten species revealed that the N‐terminal third is highly conserved, while the C‐terminal two‐thirds is poorly conserved. The R domain shows no strong sequence similarity to known proteins; however, 14 viral pol proteins show limited similarity to fragments of the R domain. Analysis revealed a relationship between the N‐ and C‐terminal fragments of the R domain and two discontinuous fragments of the pol protein. These observations support a two‐domain model for the R domain.
Fcγ receptor II stimulated formation of inositol phosphates in human platelets is blocked by tyrosine kinase inhibitors and associated with tyrosine phosphorylation of the receptor
Authors: Blake Robert A. , Asselin Judith , Walker Trevor , Watson Steve P.
Published: July 2002

We report that activation of phospholipase C (PLC) by cross‐linking of the platelet low‐affinity Fcγ receptor II (Fcγ RII) is inhibited by two structurally distinct tyrosine kinase inhibitors, staurosporine and ST271. This contrasts with PLC activation induced by thrombin and U46619, a thromboxane mimetic, whose receptors have seven transmembrane domains characteristic of G‐protein coupled receptors. Several proteins undergo phosphorylation on tyrosine on FcγRII cross‐linking upstream of protein kinase C (PKC), Ca2+ and aggregation, including the FcγRII itself. The role of FcγRII phosphorylation in the regulation of PLC is discussed.
Binding of plant isoperoxidases to pectin in the presence of calcium
Authors: Penel Claude , Greppin Hubert
Published: July 2002

Some of the isoperoxidases present in an extract from zucchini hypocotyls ‐ one anionic and two cationic ‐ exhibited a Ca2+‐dependent pelletability which resulted from an interaction with pectins. Endogenous pectins could be replaced by polygalacturonic acid or pectin extracted from citrus, but not by highly esterifled pectin. The interaction between the isoperoxidases and the polysaccharides has been studied by centrifugation, by gel filtration, and with pectins attached in wells of a microtitration plate. These various binding tests have shown that the isoperoxidases had an affinity for the pectins in their Ca2+‐induced conformation.
Regulation of the human spermidine synthase mRNA translation by its 5′‐untranslated region
Authors: Kauppinen Leila
Published: July 2002

An increased mRNA content of spermidine synthase was found in phytohemagglutinin stimulated human peripheral lymphocytes and in cultured human myeloma (Sultan) cells stimulated to grow by change of the culture medium. The many‐fold increase in the amount of the message was accompanied by stimulation of the enzyme activity in activated lymphocytes, but not in stimulated myeloma cells. In the present study the effect of the 5′‐untranslated region of spermidine synthase mRNA on the post‐transcriptional control of its expression was studied both in vitro in rabbit reticulocyte system and in cultured mammalian cells. The results show that the GC‐rich 5′‐untranslated region of spermidine synthase mRNA has an inhibitory effect on its translation.
Site‐directed mutagenesis of Saccharomyces cerevisiae β‐tubulin: interaction between residue 167 and benzimidazole compounds
Authors: Li Jing , Katiyar Santosh K. , Edlind Thomas D.
Published: July 2002

Benzimidazoles are widely used as anthelmintic agents and systemic fungicides. In susceptible organisms, benzimidazoles bind to β‐tubulin and block microtubule polymerization. To further characterize this interaction, site‐directed mutagenesis followed by gene replacement was used to change Saccharomyces cerevisiae β‐tubulin residue Phe‐167 to Tyr. Consistent with previous studies, this mutation resulted in at least 3–4‐fold decreased sensitivity to the benzimidazole derivatives carbendazim and nocodazole. The Tyr‐167 mutant was cold sensitive, implying a direct effect on benzimidazole binding rather than a nonspecific increase in microtubule stability. Surprisingly, the mutant had 8‐fold increased sensitivity to the derivative benomyl, which is structurally identical to carbendazim except at position 1. This suggests that residue 167 interacts with benzimidazoles in the vicinity of the 1‐position.
Purification and biochemical characterization of the ATH1 gene product, vacuolar acid trehalase, from Saccharomyces cerevisiae
Authors: Alizadeh Parvaneh , Klionsky Daniel J.
Published: July 2002

The disaccharide trehalose plays a critical role in yeast cell survival during conditions of environmental stress. The vacuole of the yeast Saccharomyces cerevisiae contains an enzyme, acid trehalose (ATH), that is capable of degrading trehalose. Recently, a gene required for ATH activity, ATH1, was cloned and sequenced [Destruelle et al., (1995) Yeast 11, 1015–1025]. The relationship between ATH1 and ATH, however, was not determined. We have purified ATH and shown that it is the ATH1 gene product; peptide sequences from the purified protein correspond to the deduced amino acid sequence of Ath1p. In addition, antiserum to Ath1p specifically recognizes purified ATH.
Effect of gestational age on levels of serum alkaline phosphatase isoenzymes in healthy pregnant women
Authors: Okesina A.B. , Donaldson D. , Lascelles P.T. , Morris P.
Published: July 2002

Objectives: To confirm that the bone isoenzyme of alkaline phosphatase (ALP) contributes significantly to the increased ALP in normal pregnancy, and to determine the gestational age at which the increase occurs. Methods: Cross‐sectional determinations of serum total, bone and placental ALP were carried out in 67 normal pregnant women. The results were compared with those of 18 normal age‐matched non‐pregnant controls using Student's t‐test. Results: At both 31–32 weeks and 38 weeks of gestation, serum activities of bone ALP were increased significantly (P < 0.05 and P < 0.001, respectively) when compared with controls. The percentage contribution of bone ALP to the total was higher at 31–32 weeks and lower at 38 weeks when compared with the contribution from placental ALP. Conclusion: Knowledge of time of elevation of serum bone ALP activity in normal pregnancy will be helpful in the interpretation of elevated serum total ALP activity during pregnancy.
Mutational analysis of 4‐coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate‐forming enzymes
Authors: Stuible Hans-Peter , Büttner Daniela , Ehlting Jürgen , Hahlbrock Klaus , Kombrink Erich
Published: July 2002

4‐Coumarate:coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for a large variety of important plant secondary products, such as lignin, flavonoids, or phytoalexins. To identify amino acids important for 4CL activity, eight mutations were introduced into Arabidopsis thaliana At4CL2. Determination of specific activities and K
m values for ATP and caffeate of the heterologously expressed and purified proteins identified four distinct classes of mutants: enzymes with little or no catalytic activity; enzymes with greatly reduced activity but wild‐type K
m values; enzymes with drastically altered K
m values; and enzymes with almost wild‐type properties. The latter class includes replacement of a cysteine residue which is strictly conserved in 4CLs and had previously been assumed to be directly involved in catalysis. These results substantiate the close relationship between 4CL and other adenylate‐forming enzymes such as luciferases, peptide synthetases, and fatty acyl‐CoA synthetases.
A GATA binding site is involved in the regulation of 15‐lipoxygenase‐1 expression in human colorectal carcinoma cell line, Caco‐2
Authors: Kamitani Hideki , Kameda Hideto , Kelavkar Uddhav P. , Eling Thomas E.
Published: July 2002

The data presented implicate a GATA binding site in the transcriptional regulation of 15‐lipoxygenase‐1 (15‐LO‐1) gene expression in human colorectal carcinoma Caco‐2 cells. High expression of GATA‐6 mRNA and protein was observed, while GATA‐4 mRNA was expressed at a very low level in Caco‐2 cells. The expression of GATA‐6 was down‐regulated, while 15‐LO‐1 expression was dramatically up‐regulated after treatment with sodium butyrate (NaBT). A study using an electrophoretic mobility shift assay indicated that a GATA binding site of the 15‐LO‐1 promoter region binds to GATA proteins present in both undifferentiated and, to a lesser extent, NaBT‐treated (differentiated) Caco‐2 cells. Moreover, that DNA binding shift band was disrupted after the addition of GATA‐6 antibody in a supershift assay in the absence of NaBT, suggesting that GATA‐6 is bound to the GATA binding site of the 15‐LO‐1 promoter in undifferentiated cells. In contrast, the addition of GATA‐6 antibody did not affect the DNA binding ability in NaBT‐induced differentiated cells. On the other hand, mutation of the GATA site of the 15‐LO‐1 promoter decreased the transactivation of the 15‐LO‐1 promoter as measured by luciferase activity in both FBS and NaBT cultured cells, indicating an unknown GATA binding protein to up‐regulate 15‐LO‐1 expression. These implicate the GATA site at −240 of the proximal region of the 15‐LO‐1 promoter in the basic transcription of 15‐LO‐1 gene expression in Caco‐2 cells, with GATA‐6 acting to repress 15‐LO‐1 expression.
Analysis of post‐translational modification of mycobacterial proteins using a cassette expression system
Authors: Herrmann Jean Louis , Delahay Robin , Gallagher Alex , Robertson Brian , Young Douglas
Published: July 2002

A recombinant expression system was developed to analyse sequence determinants involved in O‐glycosylation of proteins in mycobacteria. By expressing peptide sequences corresponding to known glycosylation sites within a chimeric lipoprotein construct, amino acids flanking modified threonine residues were found to have an important influence on glycosylation. The expression system was used to screen mycobacterial sequences selected using a neural network (NetOglyc) trained on eukaryotic O‐glycoproteins. Evidence of glycosylation was obtained for eight of 11 proteins tested. The results suggest that sites involved in O‐glycosylation of mycobacterial and eukaryotic proteins share similar structural features.
Type Iα phosphatidylinositol 4‐phosphate 5‐kinase is a putative target for increased intracellular phosphatidic acid
Authors: Jones David R. , Sanjuan Miguel A. , Mérida Isabel
Published: July 2002

Despite the fact that phosphatidic acid (PtdOH) has been implicated as a lipid second messenger for nearly a decade, its intracellular targets have remained unclear. We sought to investigate how an increase in the level of PtdOH could modulate phosphatidylinositol 4‐phosphate 5‐kinase (PIPkin), an enzyme involved in phosphatidylinositol 4,5‐bisphosphate synthesis. Transfection of porcine aortic endothelial (PAE) cells with haemagglutinin (HA)‐tagged type Iα PIPkin followed by immunofluorescence confocal microscopy revealed the enzyme to be localised to the plasma membrane. When the transfected PAE cells were stimulated with lyso‐PtdOH, increased PIPkin activity was found to be associated with HA immunoprecipitates in an in vitro assay. This PIPkin activation was found to be greatly reduced by prior treatment of the cells with 1‐butanol, thereby implicating phospholipase D (PLD) as the in vivo generator of PtdOH. In order to determine if the PtdOH‐dependent activation of type Iα PIPkin was dictated by a specific molecular composition of PtdOH, the wild type murine and porcine α isoforms of diacylglycerol kinase (DGK) were individually co‐transfected along with type Iα PIPkin. Under these conditions an increase in type Iα PIPkin lipid kinase activity was found in HA immunoprecipitates in an in vitro assay. No increases in lipid kinase activity were observed when type Iα PIPkin was co‐transfected with either the human DGKϵ isoform or a kinase‐dead mutant of the murine DGKα isoform. These results provide the first direct evidence for the unification of the production of saturated/monounsaturated PtdOH (through two different routes, PLD and DGK) and the in vivo activation of type Iα PIPkin by this lipid second messenger.
Solution structure of α‐conotoxin SI
Authors: Benie Andrew J. , Whitford David , Hargittai Balazs , Barany George , Janes Robert W.
Published: July 2002

The nuclear magnetic resonance solution structure of α‐conotoxin SI has been determined at pH 4.2. The 36 lowest energy structures show that α‐conotoxin SI exists in a single major solution conformation and is stabilized by six hydrogen bonds. Comparisons are made between the SI solution structure and the solution and crystal structures of α‐conotoxin GI. Surprisingly, a high degree of similarity between the backbone conformations of the GI crystal and the SI solution structures is seen in the region of lowest sequence homology, namely residues Gly‐8 to Ser‐12. This similarity is more surprising when considering that in SI a proline replaces the Arg‐9 found in GI. The correspondence in conformation in this region provides the definitive evidence that it is the loss of the arginine basic charge at residue 9 which determines the differences in toxicity between GI and SI, rather than any changes in conformation induced by the cyclic proline residue.
Translocation of Akt/PKB to the nucleus of osteoblast‐like MC3T3‐E1 cells exposed to proliferative growth factors
Authors: Borgatti Paola , Martelli Alberto M , Bellacosa Alfonso , Casto Riccardo , Massari Leo , Capitani Silvano , Neri Luca M
Published: July 2002

An active phosphatidylinositol 3‐kinase (PI3K) has been shown in nuclei of different cell types. The products of this enzyme, i.e. inositides phosphorylated in the D3 position of the inositol ring, may act as second messengers themselves. Nuclear PI3K translocation has been demonstrated to be related to an analogous translocation of a PtdIns(3,4,5)P3 activated PKC, the ζ isozyme. We have examined the issue of whether or not in the osteoblast‐like clonal cell line MC3T3‐E1 there may be observed an insulin‐like growth factor‐I‐ (IGF‐I) and platelet‐derived growth factor‐ (PDGF) dependent nuclear translocation of an active Akt/PKB. Western blot analysis showed a maximal nuclear translocation after 20 min of IGF‐I stimulation or after 30 min of PDGF treatment. Both growth factors increased rapidly and transiently the enzyme activity of immunoprecipitable nuclear Akt/PKB on a similar time scale and after 60 min the values were slightly higher than the basal levels. Enzyme translocation was blocked by the specific PI3K inhibitor, LY294002, as well as cell entry into S‐phase. Confocal microscopy showed an evident increase in immunostaining intensity in the nuclear interior after growth factor treatment but no changes in the subcellular distribution of Akt/PKB when a LY294002 pre‐treatment was administered to the cells. These findings strongly suggest that the intranuclear translocation of Akt/PKB is an important step in signalling pathways that mediate cell proliferation.
Use of an activation‐specific probe to show that Rap1A and Rap1B display different sensitivities to activation by forskolin in Rat1 cells
Authors: McPhee Ian , Houslay Miles D , Yarwood Stephen J
Published: July 2002

Rap1A and Rap1B are small GTPases of the Ras superfamily whose activation can be measured using a probe that interacts specifically with the GTP‐bound forms of Rap1A and Rap1B. Using this procedure we demonstrate that the cyclic AMP‐elevating agent forskolin activates both Rap1A and Rap1B in Rat1 cells. Whilst the protein kinase A inhibitor H89 ablated the ability of forskolin to cause cAMP response element binding protein (CREB) phosphorylation in Rat1 cells, it did not affect the ability of forskolin to activate either Rap1A and Rap1B. Forskolin differentially activated Rap1A and Rap1B isoforms in a time‐ and dose‐dependent manner. The cAMP‐specific type 4 family phosphodiesterase inhibitor rolipram potentiated the rate of activation of both Rap1A and Rap1B by forskolin challenge of Rat1 cells. Challenge of Rat1 cells with rolipram alone was able to elicit the phosphorylation of CREB but not activation of either Rap1A or Rap1B.
Thyroglobulin type‐I‐like domains in invariant chain fusion proteins mediate resistance to cathepsin L digestion
Authors: Hitzel Christoph , Kanzler Holger , König Angelika , Kummer Markus P. , Brix Klaudia , Herzog Volker , Koch Norbert
Published: July 2002

The MHCII associated invariant chain isoform Ii41 shows homology to a repeat in thyroglobulin (TgR). We show that the Ii31 isoform, which lacks the TgR‐like domain, is sensitive to cathepsin L treatment whereas Ii41 displays substantial resistance. The TgR‐like sequence of Ii41 was exchanged for thyroglobulin type‐IA and ‐IB repeats, that contain six or four cysteine residues. Resistance to cathepsin L digestion was maintained upon substitution of the Ii41 TgR for homologous sequences from TgR type‐IA. Mutation of a conserved cysteine in the TgR domain of an Ii fusion protein strongly reduced resistance to cathepsin L digestion.
Sulfocyanin and subunit II, two copper proteins with novel features, provide new insight into the archaeal SoxM oxidase supercomplex
Authors: Komorowski Lars , Schäfer Günter
Published: July 2002

The isolation of a fully functional SoxM terminal oxidase supercomplex from the archaeon Sulfolobus acidocaldarius has failed thus far and several of its constituents have only been predicted genetically, such as the small Cu protein sulfocyanin and the subunit II bearing a CuA center. Here we report the recombinant expression of sulfocyanin and prove its transcription in Sulfolobus as well as its presence in the enriched complex. It reveals a redox potential of +300 mV and spectroscopic features that are characteristic of type I copper centers. It is highly thermostable and firmly attached to the complex by one putative transmembrane anchor. Surprisingly, subunit II is completely missing from the isolated complex and behaves as an easily dissociable constituent which is a unique case within the terminal oxidase family. Its loss into the soluble phase upon cell disruption can be considered the reason for the inactivity of the isolated membrane complex.
Phosphorylation of neurofibromin by cAMP‐dependent protein kinase is regulated via a cellular association ofNG,NG‐dimethylarginine dimethylaminohydrolase
Authors: Tokuo Hiroshi , Yunoue Shunji , Feng Liping , Kimoto Masumi , Tsuji Hideaki , Ono Tomomichi , Saya Hideyuki , Araki Norie
Published: July 2002

The neurofibromatosis type 1 (NF1) tumor suppressor (neurofibromin) is thought to play crucial roles in cellular Ras‐ and cAMP‐dependent kinase (PKA)‐associated signals. In this study, we identified a cellular neurofibromin‐associating protein,NG,NG‐dimethylarginine dimethylaminohydrolase (DDAH) that is known as a cellular NO/NOS regulator. The interaction of DDAH was mainly directed to the C‐terminal domain (CTD) and to the cysteine/serine‐rich domain (CSRD) of neurofibromin, coinciding with the regions containing specific PKA phosphorylation sites. DDAH increased PKA phosphorylation of native neurofibromin in a dose‐dependent manner, especially affecting the phosphorylation of CSRD. These findings suggest that the PKA accessibility of neurofibromin was regulated via DDAH interaction, and this regulation may modulate the cellular function of neurofibromin that is implicated in NF1‐related pathogenesis.
Mitochondrial deenergization underlies neuronal calcium overload following a prolonged glutamate challenge
Authors: Khodorov B. , Pinelis V. , Vergun O. , Storozhevykh T. , Vinskaya N.
Published: July 2002

The purpose of our work was to study the relationship between glutamate (GLU)‐induced mitochondrial depolarization and deterioration of neuronal Ca2+ homeostasis following a prolonged GLU challenge. The experiments were performed on cultured rat cerebellar granule cells using the fluorescent probes, rhodamine 123 and fura‐2. All the cells, in which 100 μM GLU ( 10 μM glycine, 0 Mg2+) induced only relatively slight mitochondrial depolarization (1.1‐1.3‐fold increase in rhodamine 123 fluorescence), retained their ability to recover [Ca2+]i following a prolonged GLU challenge. In contrast, the cells in which GLU treatment induced pronounced mitochondrial depolarization (2–4‐fold increase in rhodamine 123 fluorescence), exhibited a high Ca2+ plateau in the post‐glutamate period. Application of 3–5 mM NaCN or 0.25–1 μM FCCP during this Ca2+ plateau phase usually failed to produce a further noticeable increase in [Ca2+]1. Regression analysis revealed a good correlation (r
2 = 0.88 ± 0.03, n = 19) between the increase in the percentage of rhodamine 123 fluorescence and the postglutamate [Ca2+]i Collectively, the results obtained led us to conclude that the GLU‐induced neuronal Ca2+ overload was due to the collapse of the mitochondrial potential and subsequent ATP depletion.
Non‐random usage of ‘degenerate’ codons is related to protein three‐dimensional structure
Authors: Adzhubei Alexei A. , Adzhubeib Ivan A. , Krasheninnikov Igor A. , Neidle Stephen
Published: July 2002

We report an analysis of a novel sequence‐structure database of mammalian proteins incorporating nucleotide sequences of the exon regions of their genes together with protein sequence and structural information. We find that synonymous codon families (i.e. coding the same residue) have non‐random codon distribution frequencies between protein secondary structure types. Their structural preferences are related to the third, ‘silent’ nucleotide position in a codon. We also find that some synonymous codons show very different or even opposite structural preferences at the N‐ or C‐termini of structure fragments, relative to those observed for their amino acid residues.
Effect of substance P on cytokine production by human astrocytic cells and blood mononuclear cells: characterization of novel tachykinin receptor antagonists
Authors: Derocq Jean-Marie , Ségui Michel , Blazy Chantal , Emonds-Alt Xavier , Le Fur Gérard , Brelière Jean-Claude , Casellas Pierre
Published: July 2002

Substance P (SP) has been reported to induce inflammatory cytokine production in human neuroglial cells and peripheral lymphoid cells as well. In order to evaluate the potency of novel non‐peptide antagonists of the tachykinin receptors as inhibitors of SP‐induced cytokines, we used the astrocytoma cell line U373MG and blood mononuclear cells as models of central and peripheral SP‐target cells, respectively. In the first part of this study, we showed that SR 140333, an NK1 tachykinin receptor antagonist, was able to inhibit strongly the SP‐induced production of interleukin (IL)‐6 and IL‐8 in the astrocytoma cell line. The antagonistic activity of SR 140333 toward SP‐induced cytokine production was specific and could not be attributed to a general anti‐cytokine effect, since cytokine release induced by another inflammatory protein such as IL‐1β was not blocked by this compound. In addition, NK2 and NK3 agonist neuropeptides were at least 1000‐fold less effective than SP, while SR 48968 and SR 142801 which are selective NK2 and NK3 receptor antagonists, respectively, displayed a 2.5–3 orders of magnitude lower inhibitory potency than SR 140333. All these data indicated that SR 140333 blocked SP‐induced cytokine production in U373MG astrocytic cells via a specific NK1 receptor‐mediated process. Since SP has also been described to trigger peripheral blood mononuclear cells (PBMNC) or monocytes to release inflammatory cytokines, we attempted, in the second part of this study, to evaluate the potential antagonistic effect of our compounds on these cells. Experiments on human PBMNC from different donors were carried out to determine first their pattern of cytokine production upon SP stimulation. Surprisingly, we noticed that SP at concentrations ranging from 0.1 to 1000 nM was unable to stimulate the release of any inflammatory cytokine tested. This raises the question of the specificity of the reported in vitro effects of SP on cytokine production by human peripheral immune cells.
Isolation and characterization of a cDNA encoding a Translin‐like protein, TRAX
Authors: Aoki Katsunori , Ishida Reiko , Kasai Masataka
Published: July 2002

Translin is a DNA binding protein which specifically binds to consensus sequences at breakpoint junctions of chromosomal translocations in many cases of lymphoid malignancies. To investigate its functional significance at such recombination hotspots, we examined whether Translin interacts with other proteins using a yeast two‐hybrid system and identified an associated 33 kd protein partner, TRAX, with extensive amino acid homology. The TRAX protein was established to contain bipartite nuclear targeting sequences in its N‐terminal region, suggesting a possible role in the selective nuclear transport of Translin protein lacking any nuclear targeting motifs.
Identification of a novel angiotensin‐I‐converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine β‐lactoglobulin
Authors: Mullally Margaret M , Meisel Hans , FitzGerald Richard J
Published: July 2002

The angiotensin‐I‐converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine β‐lactoglobulin (β‐lg) was investigated. Intact β‐lg essentially did not inhibit ACE while the tryptic digest gave an 84.3% inhibition of ACE. Peptide material eluting between 20 and 25% acetonitrile during C18 solid‐phase extraction of the β‐lg tryptic digest inhibited ACE by 93.6%. This solid‐phase extraction fraction was shown by mass spectroscopy to contain β‐lg f(142–148). This peptide had an ACE IC50 value of 42.6 μmol/l. The peptide was resistant to further digestion with pepsin and was hydrolysed to a very low extent with chymotrypsin. The contribution of specific amino acid residues within the peptide to ACE inhibitory activity and the potential application of this peptide as a nutraceutical is discussed.
Synaptotagmin restores kinetic properties of a syntaxin‐associated N‐type voltage sensitive calcium channel
Authors: Wiser Ofer , Tobi Dror , Trus Michael , Atlas Daphne
Published: July 2002

© 1997 Federation of European Biochemical Societies.
The interaction of synaptic vesicle‐associated membrane protein/synaptobrevin with botulinum neurotoxins D and F
Authors: Pellizzari Rossella , Mason Silvia , Shone Clifford C. , Montecucco Cesare
Published: July 2002

Botulinum neurotoxins type D and F are zinc‐endopeptidases with a unique specificity for VAMP/synaptobrevin, an essential component of the exocytosis apparatus. VAMP contains two copies of a nine residue motif, termed V1 and V2, which are determinants of the interaction with tetanus and botulinum B and G neurotoxins. Here, we show that V1 plays a major role in VAMP recognition by botulinum neurotoxins D and F and that V2 is also involved in F binding. Site‐directed mutagenesis of V1 and V2 indicates that different residues are the determinants of the VAMP interaction with the two endopeptidases. The study of the VAMP‐neurotoxins interaction suggest a pairing of the V1 and V2 segments.
An antagonist of ATP‐regulated potassium channels, the guanidine derivative U‐37883A, stimulates the synthesis of phosphatidylserine in rat liver endoplasmic reticulum membranes
Authors: Makowski Piotr , Szewczyk Adam , Jasińska Renata , Pikuła Sławomir
Published: July 2002

The guanidine derivative U‐37883A has been found to stimulate in vitro synthesis of phosphatidylserine in endoplasmic reticulum membranes, catalyzed exclusively by a serine‐specific base exchange enzyme. The stimulation of the enzyme activity by the drug was concentration‐dependent, with EC50 of 54 μM, while the biologically inactive analog of U‐37883A, U‐42069, was without effect. The stimulation caused by U‐37883A was enhanced under the conditions when active transport of Ca2+ into the lumen of microsomal vesicles was induced, whereas it was inhibited by a calcium ionophore, A23187, and by a specific inhibitor of Ca2+‐ATPase, thapsigargin. On the other hand, a potassium ionophore, valinomycin, had no effect on phosphatidylserine synthesis. U‐37883A did not affect the K
m of the base exchange enzyme for serine, but greatly reduced the EC50 value of the enzyme for calcium. Furthermore, Ca2+ uptake by endoplasmic reticulum vesicles has been found to increase in the presence of U‐37883A. These observations suggest that U‐37883A enhances phosphatidylserine synthesis indirectly by acting on calcium transport, thus affecting calcium concentration within the lumen of endoplasmic reticulum membranes. Alternatively, the effect of the drug could be propagated via the mechanism by which phospholipid flip‐flop movement, known to regulate the serine‐specific base exchange reaction, is modulated.
Advanced glycation endproducts stimulate the MAP‐kinase pathway in tubulus cell line LLC‐PK1
Authors: Simm Andreas , Münch Gerald , Seif Farshid , Schenk Oliver , Heidland August , Richter Hedwig , Vamvakas Spiridou , Schinzel Reinhard
Published: July 2002

Advanced glycation endproducts (AGEs) are suggested to play an important role in diabetic nephropathy. They induce specific cellular responses such as the release of cytokines in different cell lines. The effect of AGEs on signal transduction pathways was investigated in the renal tubulus cell line LLC‐PK1. Using a serine‐phosphate‐specific antibody AGE‐induced cellular responses associated with phosphorylation/dephosphorylation events were demonstrated. In particular, the p42MAP kinase and its downstream target, the AP‐1 complex, are shown to be activated by AGE‐BSA but not by BSA. In contrast, only partial phosphorylation is observed for the p70S6‐kinase. Thus, AGEs appear to induce specific signal transduction pathways.
Tissue‐specific expression of the human laminin α5‐chain, and mapping of the gene to human chromosome 20q13.2‐13.3 and to distal mouse chromosome 2 near the locus for the ragged (Ra) mutation
Authors: Durkin Marian E. , Loechel Frosty , Mattei Marie-Geneviève , Gilpin Brent J. , Albrechtsen Reidar , Wewer Ulla M.
Published: July 2002

To investigate the function of the laminin α5‐chain, previously identified in mice, cDNA clones encoding the 953‐amino‐acid carboxy terminal G‐domain of the human laminin α5‐chain were characterized. Northern blot analysis showed that the laminin α5‐chain is expressed in human placenta, heart, lung, skeletal muscle, kidney, and pancreas. The human laminin α5‐chain gene (LAMA5) was assigned to chromosome 20q13.2‐q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra) mutation.
cDNA cloning of three cecropin‐like antimicrobial peptides (Styelins) from the tunicate, Styela clava
Authors: Zhao Chengquan , Liaw Lilian , Lee In Hee , Lehrer Robert I
Published: July 2002

We cloned precursors of three new antimicrobial peptides, Styelins C, D and E, from a pharyngeal cDNA library of a tunicate, Styela clava. Preprostyelins resembled dipteran preprocecropins, while the mature domain of Styelin C resembled Cecropin P1, an antimicrobial peptide purified from the porcine intestine. Beginning with the last 6 residues of their signal sequences, Styelin C and Cecropin 1 from Drosophila virilis had 8/11 identical amino acids (72.7%). Moreover, 4 of the last 6 residues of their mature peptide domains were also identical. Styelins were shorter, by 8 residues, than dipteran cecropins and preprostyelins contained a conserved, polyanionic C‐terminal extension that was absent in preprocecropins. Delineation of cecropin‐like antimicrobial peptides in a protochordate supports the antiquity of this family as effectors of innate immunity in animals and it increases the likelihood that additional cecropin‐like peptides will be found among other evolutionary descendants of protochordates — vertebrates.
Oxa1p mediates the export of the N‐ and C‐termini of pCoxII from the mitochondrial matrix to the intermembrane space
Authors: Hell Kai , Herrmann Johannes , Pratje Elke , Neupert Walter , Stuart Rosemary A.
Published: July 2002

Oxa1p is a mitochondrial protein reported to be involved in the assembly of the cytochrome oxidase complex. In the absence of a functional Oxa1p, subunit II of the cytochrome oxidase accumulates as its precursor form (pCoxII). Using mitochondria isolated from a yeast strain bearing a temperature sensitive mutation in the Oxa1p, pet ts1402, we have analyzed the function of the Oxa1p protein. We demonstrate that the accumulation of pCoxII in the pet ts1402 mitochondria does not reflect a compromised Imp1p activity in this mutant. Furthermore, measurement of the membrane potential has shown it to be sufficient to support the export of CoxII from the matrix. Rather, we found that newly synthesized pCoxII accumulates in the matrix of the pet ts1402 mitochondria, because export across the inner membrane is inhibited in the pet ts1402 mitochondria. In conclusion, Oxa1p mediates the export of the N‐ and C‐termini of the mitochondrially encoded subunit II of cytochrome oxidase from the matrix to the intermembrane space.
Isolation by differential display and characterization of a tobacco auxin‐responsive cDNA Nt‐gh3, related to GH3
Authors: Roux Camille , Perrot-Rechenmann Catherine
Published: July 2002

By use of differential display, we have isolated a new early auxin‐responsive cDNA in Nicotiana tabacum. Nt‐gh3 had 70% identity with the unique GH3 sequence isolated in soybean by Hagen et al. [Planta 162 (1984) 147–153] and is thus the first reported cDNA related to this gene until now. Nt‐gh3 mRNA accumulates within a short time after auxin treatment, responds to very low concentrations of NAA (as little as 10−9 M) and specifically to active auxins. Nt‐gh3 mRNA is demonstrated to be one of the most relevant early molecular markers of primary auxin response.
Transcription of unr (pstream of ‐as) down‐modulates N‐ras expression in vivo
Authors: Boussadia Oréda , Amiot Franck , Cases Sylvaine , Triqueneaux Gérard , Jacquemin-Sablon Hélène , Dautry François
Published: July 2002

The ras proteins (Harvey, Kirsten and N‐ras) are key regulators of signal transduction and a perturbation of their GDP/GTP cycle is frequently observed in tumors. In mammals, N‐ras constitutes with unr (pstream of ‐as) a tightly linked tandem of ubiquitously expressed genes. Although unr and N‐ras appear to be involved in distinct functions, this unusual genetic organization could be important for the regulation of N‐ras expression. Specifically, transcription of unr could negatively regulate that of N‐ras by transcriptional interference. To investigate this possibility, we have deleted the unr promoter by homologous recombination in murine embryonic stem cells. Analysis of tissues of heterozygous mice revealed an increase in N‐ras mRNA accumulation ranging between 20 and 65%, in agreement with the suppression of a transcriptional interference.
Involvement of the YIGSR sequence of laminin in protein tyrosine phosphorylation
Authors: Bushkin-Harav I , Littauer U.Z
Published: July 2002

We have examined the mechanism of signaling by the 67 kDa YIGSR binding protein of laminin and its properties in neuroblastoma cells. Ligand displacement analysis showed that the interaction with the C(YIGSR)3‐NH2 peptide amide is of intermediate affinity (1.5×10−7 M). Cross‐linking experiments with sulfo‐MBS detected an additional protein with a molecular mass of 116 kDa that binds the YIGSR sequence. Incubation of neuroblastoma cells with C(YIGSR)3‐NH2 peptide amide or antibody directed against the 67 kDa laminin binding protein induces tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa and another heterogeneous protein group of 32 kDa.
Inhibition of matrix metalloproteinase 2 maturation and HT1080 invasiveness by a synthetic furin inhibitor
Authors: Maquoi Erik , Noël Agnès , Frankenne Francis , Angliker Herbert , Murphy Gillian , Foidart Jean-Michel
Published: July 2002

The close correlation observed between matrix metalloproteinase 2 (MMP‐2) activation and metastatic progression in various tumors suggests that MMP‐2 is a `master switch' triggering tumor spread. Recently, membrane type 1 MMP (MT1‐MMP) was identified as a potential physiological activator of MMP‐2. Like all other MMPs, MT1‐MMP possesses a pro‐domain which must be removed for the enzyme to acquire its catalytic potential. The presence of a typical recognition motif (RXKR) for the furin‐like convertases at the end of its pro‐domain suggests a potential role for these proteinases in MT1‐MMP processing. In order to evaluate the implication of furin in pro‐MT1‐MMP processing, we treated HT1080 cells with a synthetic furin inhibitor and monitored their ability to activate pro‐MMP‐2 as well as their invasive potential. Our results demonstrated that the furin inhibitor decreased pro‐MT1‐MMP processing as well as pro‐MMP‐2 activation and cell invasiveness. Therefore, our data bring further evidence that furin is a key factor in the maturation of MMPs associated with the invasive and metastatic potential of tumor cells.
p53‐independent DNA repair and cell cycle arrest in embryonic stem cells
Authors: Prost Sandrine , Bellamy Christopher O.C , Clarke Alan R , Wyllie Andrew H , Harrison David J
Published: July 2002

The role of p53 in DNA repair and cell cycle checkpoint after ultraviolet irradiation was investigated in an embryonic stem cell line homozygous for a targeted deletion of p53. Results indicate that loss of p53 does not alter the capacity of ES cells to respond to DNA damage. Wild‐type and p53‐deficient cells showed similar cessation of DNA synthesis after UV damage and similar ultimate capacity to repair a transiently transfected reporter plasmid. Interestingly, in the absence of DNA damaging treatment, the transit of p53‐deficient cells through S phase was slower than wild‐type cells. We suggest that this may result from the absence of a p53‐dependent response to endogenous DNA damage: without p53 sensing endogenous damage leading to immediate repair, such damage may persist and thus delay DNA synthesis.
Efficient introduction of alkene functionality into proteins in vivo
Authors: van Hest Jan C.M , Tirrell David A
Published: July 2002

The methionine analogue 2‐amino‐5‐hexenoic acid (homoallylglycine, Hag) can be utilized by Escherichia coli in the initiation and elongation steps of protein biosynthesis. Use of an E. coli methionine auxotroph and Hag‐supplemented medium resulted in replacement of ca. 85% of the methionine residues in mouse dihydrofolate reductase expressed under control of a bacteriophage T5 promoter. N‐terminal sequencing indicated 92±5% occupancy of the initiator site by Hag. The vinyl function of Hag remains intact in the purified protein and suggests new chemistries for modification of natural and artificial proteins prepared in bacterial hosts.
Adhesion‐related glycocalyx study: quantitative approach with imaging‐spectrum in the energy filtering transmission electron microscope (EFTEM)
Authors: Soler Mireille , Desplat-Jego Sophie , Vacher Béatrice , Ponsonnet Laurence , Fraterno Marc , Bongrand Pierre , Martin Jean-Michel , Foa Colette
Published: July 2002

Large polysaccharide molecules composing the glycocalyx have been shown to prevent cell adhesion. However, this process was not observed microscopically. Terbium labeling, combined with a new quantitative imaging method based on electron energy loss spectroscopy, allowed specific glycocalyx staining with excellent contrast. Image analysis enabled us to compare glycocalyx structure in free membrane areas and contacts between monocytic cells and bound erythrocytes. Apparent glycocalyx thickness, in contact areas, was half of the sum of glycocalyx thicknesses in free areas without label density increase. Ultrastructural immunogold localization of CD43 molecules, a major component of glycocalyx, was also demonstrated to be excluded from contact areas during adhesion. Thus, both approaches strongly suggest that some glycocalyx elements must exit from contact to allow binding of adhesion molecules.
Posttranslational modifications of trichomonad tubulins; identification of multiple glutamylation sites
Authors: Schneider André , Plessmann Uwe , Felleisen Richard , Weber Klaus
Published: July 2002

The α‐ and β‐tubulins present in cytoskeletons of Tritrichomonas mobilensis are extensively glutamylated. Automated sequencing and mass spectrometry of the carboxyterminal peptides identifies 4 glutamylation sites in α‐ and 2 sites in β‐tubulin. They are marked by asterisks in the terminal sequences GDE*E*E*E*DDG (α) and EGE*E*DEEAEA (β). This is the first report that tubulin glutamylation can occur at multiple sites. Although T. mobilensis has four flagellae the tubulins lack polyglycylation. Thus glycylation is not necessary for formation or function of axonemal microtubules. α‐Tubulin is completely acetylated at lysine 40 and shows no tyrosine cycle. Peptide sequences establish two distinct β‐tubulins.
Effects of mastoparan B and its analogs on the phospholipase D activity in L1210 cells
Authors: Lee Sang Yoon , Park Nam Gyu , Choi Myung-Un
Published: July 2002

Mastoparan B (MP‐B), an amphiphilic α‐helical peptide isolated from hornet venom, and its Ala‐substituted analogs were examined for their effectiveness on phospholipase D (PLD) activity in L1210 cells. PLD activity was determined by measuring phosphatidylethanol produced from [3H]myristate‐labelled cells in the presence of ethanol. PLD activity was stimulated by MP‐B, 4MP‐B (Lys4→Ala), and 12MP‐B (Lys12→Ala), but not by 3MP‐B (Leu3→Ala) and 9MP‐B (Trp9→Ala). Other MPs including mastoparan 7 also stimulated the PLD activity, but inactive mastoparan 17 did not. The stimulatory effect of various MP analogs could be correlated with their α‐helical contents. The PLD activity stimulated by MP‐B was not affected by G‐protein blocking chemicals. The extent of PLD stimulation by various MP‐Bs, as well as by digitonin and β‐escin, correlated with the permeability of the membrane to ethidium bromide. These results suggest that the stimulation of PLD activity by MP‐B in L1210 cells is probably coupled with membrane perturbation brought about by the peptide.
Rapid degradation of polyadenylated oop RNA
Authors: Szalewska-Pałasz Agnieszka , Wróbel Borys , Węgrzyn Grzegorz
Published: July 2002

The oop RNA is a short (77 nucleotides (nt)) transcript encoded by bacteriophage λ which acts as an antisense RNA for λ cII gene expression. Recently we demonstrated that oop RNA is specifically polyadenylated at its 3′ end by poly(A) polymerase I (PAP I), the pcnB gene product. Here we demonstrate that the half life of oop RNA is 3 times longer in the pcnB mutant relative to the pcnB
+ host, indicating that polyadenylation of this transcript causes its accelerated degradation. Although it was proposed that polyadenylation of RNAs in bacteria leads to their enhanced degradation, in most cases stabilization of these molecules was observed only when other mutations (pnp, rnb and rne) were present in the pcnB
− strain. Therefore it seems that oop RNA may serve as a very useful model in further studies on molecular mechanisms of RNA polyadenylation and degradation in bacteria. Analysis of oop RNA and its degradation product isolated from Escherichia coli cells suggests that both polyadenylated and non‐modified oop transcripts can act as antisense RNA.
Escherichia coli SecA shape and dimensions
Authors: Shilton Brian , Svergun Dmitri I , Volkov Vladimir V , Koch Michel H.J , Cusack Stephen , Economou Anastassios
Published: July 2002

SecA shape and conformational flexibility in solution were studied by small angle X‐ray scattering. Dimeric SecA is a very elongated molecule, 15 nm long and 8 nm wide. SecA is therefore four times as long as the membrane is wide. The two globular protomers are distinctly separated and share limited surface of intermolecular contacts. ATP, ADP or adenylyl‐imidodiphosphate (AMP‐PNP) binding does not alter the SecA radius of gyration. A SecA mutant that catalyzes multiple rounds of ATP hydrolysis does not undergo conformational changes detectable by small angle X‐ray scattering (SAXS). We conclude that SecA conformational alterations observed biochemically during nucleotide interaction are only small‐scale and localized. The ramifications of these findings on SecA/SecYEG interaction are discussed.
Molecular characterization of an exceptionally acidic lysozyme‐like protein from the protozoon Entamoeba histolytica
Authors: Nickel Rosa , Jacobs Thomas , Leippe Matthias
Published: July 2002

The protozoan parasite Entamoeba histolytica contains a second antibacterial protein with lysozyme‐like properties. The newly recognized bacteriolytic protein was purified from extracts of amoebic trophozoites to allow amino‐terminal sequencing. Subsequent molecular cloning revealed that it is an isoform of the amoeba lysozyme described previously but also demonstrated a substantial sequence divergence of the two forms. As lysozymes typically are basic proteins, the novel amoebic protein differs markedly in having a pI of 4.5. There is no significant similarity of both amoeba lysozymes with any bacteriolytic protein of other organisms reported so far; however, striking sequence identity is found with predicted gene products of unknown function derived from the bacteria‐feeding nematode Caenorhabditis elegans.