bluuebunny/crossref_metadata_2025 huggingface dataset:
A model for transmembrane helix with a cis‐Proline in the middle
Authors: Iyer Lakshmanan K. , Vishveshwara Saraswathi
Published: April 2003

The presence of a higher percentage of Proline in the transmembrane helices of transport proteins indicates that they are involved in the function of these integral membrane proteins (IMPs). In many cases, the possible involvement of cis‐trans isomerization in function/folding of IMPs has been suggested. The introduction of cis‐Pro in an ideal α‐helix results in a heli‐turn‐helix motif. A molecular dynamics (MD) simulation is carried out on the sequence ACE‐(ALA)10‐cis‐Pro‐(ALA)10‐NME with ideal α‐helical structure to investigate if and how a straight helix can accommodate a cis‐Pro. The analysis of the conformations accessed during MD simulation showed that the residues near cis‐Pro can adopt alternate conformations other than the right‐handed helical conformation such that an almost straight helix is obtained. This may have implications in the involvement of cis‐trans isomerization in folding and/or function of IMPs.
Alcohol dehydrogenase of class III: consistent patterns of structural and functional conservation in relation to class I and other proteins
Authors: Hjelmqvist Lars , Shafqat Jawed , Siddiqi Abdur Rehman , Jörnvall Hans
Published: April 2003

Class III alcohol dehydrogenase from the lizard Uromastix hardwickii has been characterized. This non‐mammalian, gnathostomatous vertebrate class III form allows correlations of structures and functions of this class, the traditional class I alcohol dehydrogenase, and other well‐studied proteins. Catalytically, results show similar recoveries and activities of all vertebrate class III forms independent of source, similar activities also in invertebrates but in lower amounts, and considerably higher specific activities in microorganisms. Structurally, variability patterns are consistent throughout the vertebrate system with a ratio in accepted point mutations versus class I of 0.4. This ratio between different classes of a zinc enzyme is comparable to that between different heme proteins (cytochrome c and myoglobin), suggesting defined but non‐identical functions also for the alcohol dehydrogenase classes.
Specific neurotrophin binding to leucine‐rich motif peptides of TrkA and TrkB
Authors: Windisch Jörg M. , Auer Bernhard , Marksteiner Rainer , Lang Monika E. , Schneider Rainer
Published: April 2003

The extracellular domains of the TrkA and TrkB neurotrophin receptors contain defined structural modules such as immunoglobulin‐like domains and leucine‐rich motifs (LRMs) [Schneider and Schweiger, Oncogene 6 (1991) 1807–1811]. Recently, the second LRM of TrkA was identified as a functional nerve growth factor (NGF) binding site [Windisch et al, J. Biol. Chem. (1995) in press]. A peptide corresponding to this region effectively bound NGF and blocked binding of NGF to the recombinant extracellular domain of TrkA. The corresponding TrkB peptide exhibited the same effects with respect to brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4 (NT‐4), indicating that all three TrkB ligands utilize this same binding site. Isolated LRMs therefore embody independent functional entities.
Multiple gene products are produced from a novel protein kinase transcription region
Authors: Martin Watterson D , Collinge Mark , Lukas Thomas J , Van Eldik Linda J , Birukov Konstantin G , Stepanova Olga V , Shirinsky Vladimir P
Published: April 2003

The nonmuscle/smooth muscle myosin light chain kinase (MLCK) and the kinase related protein (KRP) that lacks protein kinase activity are myosin II binding proteins encoded in the vertebrate genome by a true gene within a gene relationship. The genomic organization and expression result in the same amino acid sequence in different molecular contexts from two different sizes of mRNA. We report here the identification and characterization of a third size class of gene products. The protein appears to be a higher molecular weight form of MLCK with additional amino terminal tail sequence which might provide differential subcellular targeting characteristics.
The role of the skeletal muscle myosin light chains N‐terminal fragments
Authors: Stȩpkowski Dariusz
Published: April 2003

The myosin regulatory and essential light chains in skeletal muscle do not play a role as significant as in scallop or smooth muscle, however, there are some data suggesting that the skeletal myosin light chains and their N‐terminal parts may have a modulatory function in the interaction of actin with myosin heads. In this paper four conformational states of the myosin head with respect to the regulatory light chain bound cation (magnesium or calcium) and phosphorylation are proposed. Communication between regulatory and essential light chains and putative binding of the N‐terminus of A1 essential light chain to actin is discussed.
Cathelicidins: a novel protein family with a common proregion and a variable C‐terminal antimicrobial domain
Authors: Zanetti Margherita , Gennaro Renato , Romeo Domenico
Published: April 2003

A novel protein family, showing a conserved proregion and a variable C‐terminal antimicrobial domain, and named0 cathelicidin, has been identified in mammalian myeloid cells. The conserved proregion shows sequence similarity to members of the cystatin superfamily of cysteine proteinase inhibitors. Cathelicidins are stored in the cytoplasmic granules of neutrophil leukocytes and release the antimicrobial peptides upon leukocyte activation. Some of these peptides can assume an α‐helical conformation, others contain one or two disulfide bonds, still others are Pro‐ and Arg‐rich, or Trp‐rich. In addition to bacterial killing, some of these peptides exert additional functions related to host defense such as LPS‐neutralization and promotion of wound healing.
Loss of allosteric behaviour in recombinant hemoglobin α2β29r)F8)His→Ala: Restoration upon addition of strong effectors
Authors: Dumoulin A. , Kiger L. , Jiang R. , Baudin V. , Vasseur C. , Sligar S.G. , Marden M.C. , Pagnier J. , Poyart C.
Published: April 2003

In the stereochemical model proposed by Perutz [1], the Fe‐His(F8) bond plays a significant role in the allosteric transition in hemoglobin and the resulting cooperativity in ligand binding. When this bond is ruptured, there is a loss in the transmission of the information concerning ligand binding; examples are Hb(NO)4 in the presence of inositol hexakisphosphate (IHP), or nickel substituted Hb hybrids which, despite being liganded, exhibit deoxy‐like properties. To study the effects of the loss of the iron proximal histidine bond, we have engineered the α
2
β
2(F8)H92A recombinant Hb. The replacement of the highly conserved proximal histidine F8 residue by an alanine results in a low affinity for the heme group and a loss of the allosteric properties; kinetics of CO recombination after photodissociation show only the rapid bimolecular phase, characteristic of the high affinity R‐state. However, a significant amount of deoxy (T‐state) kinetics are observed after addition of external effectors such as IHP. The iron‐histidine bond is apparently crucial for the heme‐heme interaction, but the allosteric equilibrium may still be influenced by external constraints.
Functional complementation of yeast phosphofructokinase mutants by the non‐allosteric enzyme from Dictyostelium discoideum
Authors: Estévez Antonio M. , Heinisch Jürgen J. , Aragón Juan J.
Published: April 2003

Phosphofructokinase (PFK) from yeast has been replaced by the non‐allosteric isozyme from the slime mold Dictyostelium discoideum. This has been achieved by overexpression of the latter in a PFK‐deficient strain of Saccharomyces cerevisiae under the control of the PFK2 promoter. Transformants complemented the glucose‐negative growth phenotype exhibiting generation times on glucose‐containing media similar to those of an untransformed strain being wild‐type for yeast PFK genes. The PFK produced reacted with an antibody against D. discoideum PFK. It exhibited the same subunit size, quaternary structure and kinetic parameters than those of the wild‐type enzyme, and was also devoid of specific regulatory properties.
Stimulatory effect of ouabain on VCAM‐1 and iNOS expression in murine endothelial cells: involvement of NF‐κB
Authors: Bereta Joanna , Cohen Marion C. , Bereta Michał
Published: April 2003

Endothelial cells play a pivotal role in the development of atherosclerosis. An ‘activated’ phenotype of these cells is manifested by signal transduction‐dependent expression of genes encoding cytokines, pro‐ and anticoagulant factors, and cell adhesion molecules. In the current study we examined the effect of ouabain, an inhibitor of Na+/K+‐ATPase, on the process of endothelial cell activation. We demonstrated that ouabain was able to stimulate VCAM‐1 expression and potentiate the effect of IFN‐γ on this process. Moreover, ouabain provided a complementary signal for either TNF or IFN‐γ in inducing iNOS expression. Our data also show, for the first time, that inhibition of Na+/K+‐ATPase led to activation of the transcription factor, NF‐κB, which may provide an explanation for the effects of ouabain on endothelial cells.
Close evolutionary relatedness among functionally distantly related members of the (α/β)8‐barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region
Authors: Janeček Štefan
Published: April 2003

A short conserved sequence equivalent to the fifth conserved sequence region of α‐amylases (173_LPDLD, Aspergillus oryzae α‐amylase) comprising the calcium‐ligand aspartate, Asp‐175, was identified in the amino acid sequences of several members of the family of (α/β)8‐barrel glycosyl hydrolases. Despite the fact that the aspartate is not invariantly conserved, the stretch can be easily recognised in all sequences to be positioned 26–28 amino acid residues in front of the well‐known catalytic aspartate (Asp‐206, A. oryzae α‐amylase) located in the β4‐strand of the barrel. The identification of this region revealed remarkable similarities between some α‐amylases (those from Bacillus megaterium, Bacillus subtilis and Dictyoglomus thermophilum) on the one hand and several different enzyme specificities (such as oligo‐1,6‐glucosidase, amylomaltase and neopullulanase, respectively) on the other hand. The most interesting example was offered by B. subtilis α‐amylase and potato amylomaltase with the regions LYDWN and LYDWK, respectively. These observations support the idea that all members of the family of glycosyl hydrolases adopting the structure of the α‐amylase‐type (α/β)8‐barrel are mutually closely related and the strict evolutionary borders separating the individual enzyme specificities can be hardly defined.
Mg2+ activation of Escherichia coli inorganic pyrophosphatase
Authors: Avaeva Svetlana M. , Rodina Elena V. , Kurilova Svetlana A. , Nazarova Tatjana I. , Vorobyeva Natalja N. , Harutyunyan Emil H. , Oganessyan Vaheh Yu.
Published: April 2003

Further refinement of X‐ray data on Escherichia coli inorganic pyrophosphatase [Oganessyan et al. (1994) FEBS Lett. 348, 301–304] to 2.2 Å reveals a system of noncovalent interactions involving Tyr55 and Tyr141 in the active site. The pKa for one of the eight Tyr residues in wild‐type pyrophosphatase is as low as 9.1 and further decreases to 8.1 upon Mg2+ binding, generating characteristic changes in the absorption spectrum. These effects are lost in a Y55F but not in a Y141F variant. It is suggested that the lower‐affinity site for Mg2+ in the enzyme is formed by Tyr55 and Asp70, which are in close proximity in the apo‐enzyme structure.
Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma ‘anandamide amidohydrolase’
Authors: Maurelli Stefano , Bisogno Tiziana , De Petrocellis Luciano , Di Luccia Aldo , Marino Gennaro , Di Marzo Vincenzo
Published: April 2003

The endogenous cannabimimetic substance, anandamide (N‐arachidonoyl‐ethanolamine) and the recently isolated sleep‐inducing factor, oleoyl‐amide (cis‐9,10‐octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of ‘anandamide amidohydrolase’ from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]anandamide was found in both microsomal and 10,000 × g pellet fractions. The latter fractions, which displayed the highest V
max for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl‐amide, with an apparent K
m of 9.0 ± 2.2 μM. [14C]anandamide‐ and [14C]oleoyl‐amide‐hydrolysing activities: (i) exhibited identical pH‐ and temperature‐dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl‐trifluoromethyl‐ketone with the same IC50 (3 μM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid‐ethanolamides) and oleoyl‐amide. Proteins solubilised from 10,000 × g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]anandamide and [14C]oleoyl‐amide. These data suggest that ‘anandamide amidohydrolase’ enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep‐inducing factor oleoyl‐amide.
Betaine is an osmolyte in RAW 264.7 mouse macrophages
Authors: Warskulat Ulrich , Wettstein Matthias , Häussinger Dieter
Published: April 2003

Hyperosmotic (405 mosmol/l) exposure of RAW 264.7 mouse macrophages led to a stimulation of betaine uptake and an increase in betaine transporter (BGT‐1) mRNA levels. Conversely, hypoosmotic (205 mosmol/l) exposure decreased betaine uptake and diminished BGT‐1 mRNA levels. Betaine uptake was Na+‐dependent and was inhibited by about 90% by GABA, whereas inhibition by methylaminoisobutyrate and myoinositol was less than 15%. Addition of betaine strongly diminished BGT‐1 mRNA levels in cells exposed to normoosmotic or hyperosmotic media. When mouse macrophages were preloaded with betaine, lowering of the extracellular osmolarity was followed by a rapid betaine efflux from the cells. This study identifies a constitutively expressed and osmosensitive betaine transporter in RAW 264.7 macrophages and the use of betaine as an osmolyte in these cells.
4‐Picoline‐2,2′:6′,2″‐terpyridine‐platinum(II) — A potent intercalator of DNA
Authors: McCoubrey Angela , Latham Harriet C. , Cook Peter R. , Rodger Alison , Lowe Gordon
Published: April 2003

4‐Picoline‐2,2′:6′,2″‐terpyridine‐platinum(II) is shown in a ligation assay to unwind and so intercalate into DNA. Circular dichroism is used to determine an equilibrium binding constant of approximately 2 × 107 M−1 for the most stable binding mode of 4‐picoline‐2,2′:6′,2″‐terpyridine‐platinum(II) to poly[d(A‐T)2] with a site size of about 4 base pairs, and about 1 × 106 M−1 for a second binding mode with a site size of about 2 base pairs. Fluorescence spectroscopy provides further evidence for the strong equilibrium binding constant of 4‐picoline‐2,2′:6′,2″‐terpyridine‐platinum(II) in that it displaces ethidium bromide bound to DNA. The double positive charge on 4‐picoline‐2,2′:6′,2″‐terpyridine‐platinum(II), together with the intercalative binding mode is probably responsible for the large binding constant.
The 170 kDa glucose regulated stress protein is a large HSP70‐ HSP110‐like protein of the endoplasmic reticulum
Authors: Chen Xing , Easton Douglas , Oh Hyun-Ju , Lee-Yoon Dong-Sin , Liu Xiaoguang , Subjeck John
Published: April 2003

The existence of a family of unusually large and highly diverged hsp70‐like proteins (the hsp110/SSE family) has recently been described. The 170 kDa glucose regulated stress protein (grp170) is a retained endoplasmic reticulum glycoprotein that may be involved in immunoglobulin folding and/or assembly. We describe here the cloning of the cDNA for grp170 and show that it, like hsp110, is a large and highly diverged hsp70‐like polypeptide which shares specific features with hsp70 (the dnaK family) and the hsp110/SSE family, while also differing from both. Grp170 contains an ATP binding domain and binds ATP, it possesses a carboxyl terminal NDEL sequence, and its mRNA is anoxia inducible.
UV‐B‐ and oxidative stress‐induced increase in nicotinamide and trigonelline and inhibition of defensive metabolism induction by poly(ADP‐ribose)polymerase inhibitor in plant tissue
Authors: Berglund Torkel , Kalbin Georgi , Strid Åke , Rydström Jan , Ohlsson Anna B.
Published: April 2003

Nicotinamide and trigonelline contents increased in Catharanthus roseus tissue culture after exposure to 2,2′azobis(2‐amidinopropane) dihydrochloride (AAPH) or vanadylsulfate and in Pisum sativum leaves after exposure to UV‐B radiation. Vanadylsulfate increased phenylalanine ammonia‐lyase (PAL) activity and the content of reduced and oxidized glutathione in C. roseus tissue culture. The increases in PAL activity caused by 2 mM AAPH or 0.2 mM vanadylsulfate were prevented by 0.1 mM 3‐aminobenzamide (3‐AB), an inhibitor of poly(ADP‐ribose)polymerase. Present results support the hypothesis [Berglund T., FEBS Lett. (1994) 351, 145–149] that nicotinamide and/or its metabolites may function as signal transmittors in the response to oxidative stress in plants and that poly(ADP‐ribose)polymerase has a function in the induction of defensive metabolism.
Binding of matrix attachment regions to nuclear lamin is mediated by the rod domain and depends on the lamin polymerization state
Authors: Zhao Kun , Harel Amnon , Stuurman Nico , Guedalia Dina , Gruenbaum Yosef
Published: April 2003

The nuclear matrix maintains specific interactions with genomic DNA at sites known as matrix attachment regions (M/SARs). M/SARs bind in vitro to lamin polymers. We show that the polymerized α‐helical rod domain of lamin Dm0 provides by itself the specific binding to the ftz M/SAR. In contrast, unpolymerized rod domain does not bind specifically to this M/SAR. Non‐specific binding to DNA is also observed with Dm0 containing a point mutation that impairs its ability to polymerize or with the isolated tail domain. These data suggests that the specific binding of lamins to M/SARs requires the rod domain and depends on the lamin polymerization state.
Promoter and expression studies on an Arabidopsis thaliana dehydrin gene
Authors: Rouse Dean T. , Marotta Rosetta , Parish Roger W.
Published: April 2003

A genomic clone of a group 2 lealrabldehydrin gene from Arabidopsis thaliana, Xero2llti30, was cloned and sequenced. Promoter‐GUS fusions were introduced into plants to analyse the promoter and determine expression patterns. Using root cultures, GUS expression was found to be moderately stimulated by abscisic acid (ABA), wounding, cold and dehydration. Results with an ABA‐deficient mutant suggested endogenous ABA is required for these responses. Promoter deletion studies indicated multiple cis‐acting elements are involved in the induction of the gene. GUS expression occurred in desiccated seeds, in all tissues of young seedlings and in roots (with the exception of the root tip), desiccated pollen grains, trichomes and the vascular tissues of leaves and stems in mature plants.
A water channel closely related to rat brain aquaporin 4 is expressed in acid‐ and pepsinogen‐secretory cells of human stomach
Authors: Misaka Takumi , Abe Keiko , Iwabuchi Kyoko , Kusakabe Yuko , Ichinose Masao , Miki Kazumasa , Emori Yasufumi , Arai Soichi
Published: April 2003

We isolated a cDNA clone encoding a water channel protein, aquaporin (AQP), from human stomach. The encoded protein consisted of 323 amino acid residues, containing six putative transmembrane domains. The protein was designated human aquaporin 4 (hAQP4) because of its 94% sequence similarity to rat brain AQP4. Expression of hAQP4 cRNA in Xenopus oocytes resulted in a significant increase in osmotic water permeability, indicating that this protein functions as a water channel. Northern blot analysis demonstrated a strong signal of hAQP4 mRNA in brain, lung, and skeletal muscle as well as in stomach. Immunohistochemical experiments with human stomach tissues showed that hAQP4 as a protein is expressed mainly in cells located in the glandular portion of the fundic mucosa. These include chief cells which secrete pepsinogen and parietal cells which secrete hydrochloric acid. These results strongly indicate that hAQP4 is a principal factor involved in the osmotic regulation of pepsinogen and acid secretion in the stomach.
Tyrosinase and related proteins in mammalian pigmentation
Authors: del Marmol Véronique , Beermann Friedrich
Published: April 2003

Tyrosinase is the key enzyme in pigment synthesis, initiating a cascade of reactions which convert the amino acid tyrosine to the melanin biopolymer. Two other tyrosinase‐related proteins (TRP) are known, TRP‐1 (probably DHICAoxidase) and TRP‐2 (DOPAchrome tautomerase). These proteins show about 40% homology, and recent results have indicated that the genes might be derived from a common ancestor. We will discuss recent findings on genomic organization, and on the proteins and their presumed function, which is important for eumelanin synthesis in mouse and man.
In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase Cμ
Authors: Dieterich Sabine , Herget Thomas , Link Gisela , Böttinger Heiner , Pfizenmaier Klaus , Johannes Franz-Josef
Published: April 2003

To study enzymatic activity and activation conditions of the recently identified novel protein kinase C μ (PKCμ) subtype, epitope tagged PKCμ was propagated in the baculovirus expression system and was purified to homogeneity. PKCμ displays high affinity phorbol ester binding (K
d = 7 nM) resulting in enhanced phosphatidylserine‐dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns‐4,5‐P2, were found to promote PKCμ kinase activity. Two peptides derived from the glycogen synthase, GS‐peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine‐rich C‐kinase substrate) served as an in vitro substrate for PKCμ too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKCμ.
DNA affinity to biological membranes is enhanced due to complexation with hydrophobized polycation
Authors: Yaroslavov A.A. , Sukhishvili S.A. , Obolsky O.L. , Yaroslavova E.G. , Kabanov A.V. , Kabanov V.A.
Published: April 2003

The interaction of negatively charged liquid phosphatidylcholine/cardiolipin liposomes with water‐soluble negatively charged DNA/cetylpyridinium bromide and DNA/poly(N‐alkyl‐4‐vinylpyridinium bromide) complexes was studied. It is shown that the DNA/cetylpyridinium bromide complex while interacting with the liposomes is destroyed, so that the cetylpyridinium cation is incorporated into the liposomal membrane and DNA remains in the solution. The DNA/poly‐(N‐ethyl‐4‐vinylpyridinium bromide) complex does not interact at all with the liposomes. On the contrary, the complex of DNA with the poly(vinylpyridinium) cation carrying a small amount of N‐cetyl groups is adsorbed on the membrane as a whole. The data obtained indicate that complexation of DNA with hydrophobized polycations can be used for enhancing DNA affinity to biological membranes.
A20, an inhibitor of cell death, self‐associates by its zinc finger domain
Authors: De Valck Dirk , Heyninck Karen , Van Criekinge Wim , Contreras Roland , Beyaert Rudi , Fiers Walter
Published: April 2003

A20 is a primary response gene which is induced after monocyte adherence or cytokine stimulation of a variety of cells. The A20 protein belongs to a novel class of Cys2/Cys2 zinc finger proteins, and has been characterized as an inhibitor of both apoptotic and necrotic cell death. In order to clarify its molecular mechanism of action, we used the yeast‐based‐two‐hybrid system to screen for A20‐associated proteins. Here we report that A20 is able to self‐associate, and demonstrate that the latter interaction is mediated by its zinc finger domain.
The significance of Ser1029 of the heat‐stable enterotoxin receptor (STaR): Relation of STa‐mediated guanylyl cyclase activation and signaling by phorbol myristate acetate
Authors: Akihiro Wada  , Makoto Hasegawa  , Kazuya Matsumoto  , Takuro Niidome  , Yuki Kawano  , Yuji Hidaka  , Ian Padilla Philip , Hisao Kurazono  , Yasutsugu Shimonishi  , Toshiya Hirayama
Published: April 2003

To characterize Ser1029 in STaR at a consensus sequence of phosphorylation site by PKC, two mutants of mS1029A with replacement of Ser1029 to Ala1029 and CΔ1029 lacking 22 amino acids including Ser1029 were prepared. Preincubation of the wild type‐STaR (wt‐STaR) transfectant with 1 μM PMA caused additional STa‐mediated guanylyl cyclase (GC) activation compared to control, whereas the mS1029A‐ and CΔ1029‐transfected cells did not show a similar enhanced GC activation by PMA. After metabolic labeling with [32P]phosphate, transfected cells with wt‐STaR and mutants were incubated with 1 μM PMA. Subsequent 32P‐radiolabeled proteins were immunoprecipitated using anti‐STaR antibody, and analyzed by autoradiography after separation on SDS‐PAGE. The immunoprecipitated wt‐STaR but not mS1029A and CΔ1029 had a significant radioactivity. These results suggest that the effect of PMA on wt‐STaR transfectants may be caused by phosphorylation of Ser1029. The CΔ1012 mutant, with further truncation (Gln1012‐Phe1050) of the carboxy terminus, did not show STa‐mediated GC activation. Based on these data, these 17 amino acids (Gln1012‐Ala1028), essential for signaling of GC activation by STa, have an abundance of basic amino acids which might be functionally influenced by phosphorylation of Ser1029.
A potential site of functional modulation by protein kinase A in the cardiac Ca2+ channel α1C subunit
Authors: Perets Tuvia , Blumenstein Yakov , Shistik Elena , Lotan Ilana , Dascal Nathan
Published: April 2003

The well‐characterized enhancement of the cardiac Ca2+ L‐type current by protein kinase A (PKA) is not observed when the corresponding channel is expressed in Xenopus oocytes, possibly because it is fully phosphorylated in the basal state. However, the activity of the expressed channel is reduced by PKA inhibitors. Using this paradigm as an assay to search for PKA sites relevant to channel modulation, we have found that mutation of serine 1928 of the α
1C subunit to alanine abolishes the modulation of the expressed channel by PKA inhibitors. This effect was independent of the presence of the β subunit. Phosphorylation of serine 1928 of α
1C may mediate the modulatory effect of PKA on the cardiac voltage‐dependent Ca2+ channel.
Folding and characterization of the amino‐terminal domain of human tissue inhibitor of metalloproteinases‐1 (TIMP‐1) expressed at high yield in E. coli
Authors: Huang Wen , Suzuki Ko , Nagase Hideaki , Arumugan S. , Van Doren Steven R. , Brew Keith
Published: April 2003

Methods are described for producing an active amino‐terminal domain of tissue inhibitor of metalloproteinases‐1 (N‐TIMP‐1) from inactive protein expressed as inclusion bodies in E. coli. Yields exceed 20 mg per litre of bacterial culture. Activity measurements, CD spectroscopy and NMR spectroscopy of the 15N‐labeled protein show that it is fully active, homogeneous in conformation and suitable for high‐resolution structural analysis. The affinity of N‐TIMP‐1 for matrix metalloproteinases 1, 2 and 3 is 6–8‐fold less than that of the recombinant full‐length protein, indicating that deletion of the C‐terminal domain reduces the free energy of interaction by < 10%.
Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae
Authors: Sasaki Kazuhiro , Abid Ruhul , Miyazaki Masazumi
Published: April 2003

Deoxyhypusine synthase catalyzes the first of two steps in the biosynthesis of hypusine, a modification of a specific lysine residue in the precursor of eukaryotic translation initiation factor 5A. We have purified deoxyhypusine synthase from yeast, and cloned and sequenced the corresponding gene encoding a 387‐amino acid protein from Saccharomyces cerevisiae. Gene disruption experiments indicated that the deoxyhypusine synthase gene is essential for cell growth in yeast. This gene was shown to be an intron‐free, single‐copy gene, and its product can catalyze the synthesis of deoxyhypusine equally in two precursor forms of eIF‐5A, derived from two distinct genes of yeast.
Localization of the specific binding site for magnesium(II) ions in factor IX
Authors: Sekiya Fujio , Yoshida Misa , Yamashita Toshiko , Morita Takashi
Published: April 2003

We demonstrated recently that coagulation factor IX has a specific binding site(s) for Mg2+ ions, independent of the Ca2+‐binding sites, and that binding of Mg2+ ions is very important for expression of the functional conformation of this protein. We report here the localization of this Mg2+‐specific binding site. We prepared three Gla‐containing fragments of bovine factor IX, namely GlaEGFNC (residues 1–144+286–296), GlaEGFN (1–83) and the Gla domain peptide (1–46). Fragments GlaEGFNC and GlaEGFN retained the ability to undergo a conformational change upon binding of Mg2+ ions in the presence of excess Ca2+ ions. This change could be detected by a conformation‐specific antibody. Furthermore, the Gla domain peptide was capable of binding Mg2+ ions, as determined by the metal ion‐induced quenching of the intrinsic fluorescence. It appears that the Mg2+‐specific binding site of factor IX is located in the N‐terminal Gla domain.
A comparison of the substrate specificity of MAPKAP kinase‐2 and MAPKAP kinase‐3 and their activation by cytokines and cellular stress
Authors: Clifton Andrew D. , Young Peter R. , Cohen Philip
Published: April 2003

MAPKAP kinase‐2 and MAPKAP kinase‐3 were both activated in response to cellular stress, interleukin‐1 and tumour necrosis factor in KB and HeLa cells, and with identical kinetics. Activation of MAPKAP kinase‐3, like MAPKAP kinase‐2, was prevented by SB 203580, a specific inhibitor of SAPK‐2, the upstream activator of MAPKAP kinase‐2. MAPKAP kinase‐3 and MAPKAP kinase‐2 phosphorylated peptide substrates with similar kinetic constants and phosphorylated the same serine residues in HSP27 at the same relative rates. These results establish that MAPKAP kinase‐3 lies ‘downstream’ of SAPK‐2 and that it is likely to have overlapping or identical substrates to MAPKAP kinase‐2 in vivo.
Characterisation of secondary metabolites associated with neutrophil apoptosis
Authors: Nunn Alistair V.W. , Barnard Maria L. , Bhakoo Kishore , Murray Joanna , Chilvers Edwin J. , Bell Jimmy D.
Published: April 2003

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFα) stimulated apoptosis by a combination of high‐performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFα and the phosphatidylinositol‐3‐kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFα, compared to TNFα by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non‐invasive quantification of apoptosis.
DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum
Authors: Faix Jan , Dittrich Werner
Published: April 2003

A protein accumulated in the cortical region of Dictyostelium discoideum cells proved to be a homologue of GTPase activating proteins that are responsible for the inactivation of ras in yeast and man. Elimination of this protein, DGAP1, by gene replacement resulted in an increased rate of growth of D. discoideum cells on bacterial lawns, and in the formation of aberrant, multi‐tipped fruiting bodies. Overexpression of DGAP1 caused the cells to become multi‐nucleated since chromosome segregation during mitosis was not reliably followed by cleavage of the cells. These results suggest that in D. discoideum, ras or a related small GTP‐binding protein is involved in regulating growth based on the phagocytosis of bacteria, and in coupling activities of the cell cortex to the organization of spindle and asters in mitotic cells.
Cloning and sequencing of the proximal promoter of the rat iNOS gene: activation of NFκB is not sufficient for transcription of the iNOS gene in rat mesangial cells
Authors: Beck Karl-Friedrich , Bernd Sterzel R.
Published: April 2003

It has previously been shown that expression of the inducible form of NO synthase (EC 1.14.23) is controlled at the transcriptional level and that induction of iNOS transcription is dependent on activation of transcription factors of the NFκB family. TNF‐α and IL‐1β synergistically stimulate iNOS transcription in rat glomerular mesangial cells. We have recently reported that endothelin‐1 completely blocks cytokine‐induced iNOS expression at the transcriptional level. To further investigate the molecular mechanisms and the role of NFκB in cytokine‐elicited iNOS transcription, we cloned a 661 bp genomic rat DNA fragment, which contains 497 bp of the proximal iNOS promoter. An NFκB‐binding site identical to that described for the murine sequence was identified and used for electrophoretic mobility shift experiments. We found that binding of NFκB is strongly induced in mesangial cells by both IL‐1β and TNF‐α. While endothelin‐1 blocks cytokine‐induced iNOS expression, it has no influence on the binding pattern of NFκB. We conclude from these data that transcription of iNOS in mesangial cells requires additional signals besides activation of NFκB.
Sensitivity of human pancreatic islets to peroxynitrite‐induced cell dysfunction and death
Authors: Delaney Carol A. , Tyrberg Björn , Bouwens Luc , Vaghef Hamid , Hellman Björn , Eizirik Décio L.
Published: April 2003

Nitric oxide and peroxynitrite (generated by the reaction of nitric oxide with the superoxide anion) may both be mediators of β‐cell damage in early insulin‐dependent diabetes mellitus. We observed that acute exposure of primary cultured human pancreatic islets to peroxynitrite results in a significant decrease in glucose oxidation and islet retrieval. DNA strand breaks in single human and rat islet cells are detectable after acute peroxynitrite exposure, followed by a decrease in islet cell survival after 1 h and 24 h. Cell death appeared to occur via a toxic cell death mechanism (necrosis) rather than apoptosis, as suggested by vital staining and ultrastructural evidence of early membrane and organelle degradation, mitochondrial swelling and loss of matrix. This study demonstrates for the first time that cultured human pancreatic islets are susceptible to the noxious effects of peroxynitrite.
IL‐10 induces DNA binding activity of three STAT proteins (Stat1, Stat3, and Stat5) and their distinct combinatorial assembly in the promoters of selected genes
Authors: Wehinger Jens , Gouilleux Fabrice , Groner Bernd , Finke Juergen , Mertelsmann Roland , Maria Weber-Nordt Renate
Published: April 2003

Interaction of IL‐10 with its receptor leads to the activation of STAT transcription factors. Herein we report the IL‐10 dependent simultaneous activation of three STAT transcription factors: Stat1, Stat3, and Stat5. Upon IL‐10 treatment multiple Stat proteins become simultaneously activated, and bind to different promoters with equal kinetics but form distinct homo‐ and heterodimeric transcriptionally active complexes depending on the STAT‐consensus elements of a selected gene promoter. Upon IL‐10 treatment Stat1, 3, and 5 bind to the GRR of the FcγRI gene, activated Stat1, and 3 bind to the SIE sequence of the c‐fos promoter and transcriptionally active Stat5 assembles at the PRL‐STAT consensus sequence of the β‐casein gene. Thus, functionally relevant STAT dimerization is influenced by the activated cytokine receptor as well as the specific STAT consensus sequence present in a specific gene promoter.
Structural and serological studies of the O‐specific polysaccharide of the bacterium Proteus mirabilis O10 containing L‐altruronic acid, a new component of O‐antigens
Authors: St. Swierzko Anna , Shashkov Alexander S. , Senchenkova Sof'ya N. , Toukach Filip V. , Ziolkowski Andrzej , Cedzynski Maciej , Paramonov Nikolay A. , Kaca W. , Knirel Yuriy A.
Published: April 2003

An acidic O‐specific polysaccharide from the lipopolysaccharide of Proteus mirabilis O10 contains 2‐acetamido‐2deoxy‐d‐glucose, 2‐acetamido‐2‐deoxy‐d‐galactose, d‐galacturonic acid, and l‐altruronic acid, the last‐named sugar having not been found hitherto in O‐antigens. Structure of a branched tetrasaccharide repeating unit of the polysaccharide was established by 1H and 13C NMR spectroscopy, including two‐dimensional COSY and rotating‐frame NOE spectroscopy. The lateral l‐altruronic acid residue plays the immunodominant role in manifestation of the O10 specificity of Proteus, whereas a disaccharide fragment of the main chain in common with the O‐specific polysaccharide of P. mirabilis O43 provides the one‐way serological cross‐reactivity between anti‐O10 serum and O43‐antigen.
Characterisation of a human homologue of a yeast cell division cycle gene, MCM6, located adjacent to the 5′ end of the lactase gene on chromosome 2q21
Authors: Harvey Clare B. , Yangxi Wang  , Darmoul Dalila , Phillips Alan , Mantei Ned , Swallow Dallas M.
Published: April 2003

Four exons of a human homologue of a yeast cell division cycle gene (MCM6/mis5, which is thought to encode a DNA replication licensing factor) have been identified 3.3 kb upstream from the start of transcription of the intestinal lactase gene on human chromosome 2q21, initially by similarity to a rat ‘intestinal crypt‐cell replication factor’. RT‐PCR analysis shows, that unlike lactase, MCM6 is not restricted in its tissue distribution and does not show person‐to‐person variation in the level of expression in adult intestine.
Uroguanylin gene expression in the alimentary tract and extra‐gastrointestinal tissues
Authors: Miyazato Mikiya , Nakazato Masamitsu , Matsukura Shigeru , Kangawa Kenji , Matsuo Hisayuki
Published: April 2003

Uroguanylin, a member of the guanylin peptide family, is a novel peptide regulator for intestinal salt and water transport. A cDNA encoding a precursor for rat uroguanylin was cloned from a rat jejunum cDNA library and sequenced. The precursor was 106 amino acids long and included a 21 residue putative signal peptide at the N‐terminus. Rat uroguanylin consisted of 15 amino acids similar to, but distinct from human uroguanylin; the C‐terminal leucine residue was deleted and 3 residues were substituted compared to those in the human peptide. Synthetic rat uroguanylin‐15 dose‐dependently increased the cyclic GMP level in cultured T84 cells. RNA blot analysis showed that rat uroguanylin mRNA is expressed not only in the gastrointestinal tract but also in the lung, pancreas and kidney. Evidence for uroguanylin expression in extra‐gastrointestinal tissues indicates the possible existence of a novel system for water and electrolyte homeostasis, and a more global effect of uroguanylin on epithelial cell function.
Glucocorticoid‐mediated inhibition of RANTES expression in human T lymphocytes
Authors: Wingett Denise , Forcier Kristin , Nielson Christopher P.
Published: April 2003

The chemokine RANTES has been implicated in the pathogenesis of allergic inflammatory diseases including asthma and rhinitis which are frequently treated with glucocorticoids. We observed that dexamethasone dramatically inhibited RANTES mRNA expression dose dependently in anti‐CD3 activated Hut‐78 T cells and human PBMCs. Inhibition of RANTES expression did not appear to be secondary to IL‐2 inhibition and required binding to the intracellular glucocorticoid receptor. The down‐regulation of RANTES expression by glucocorticoids in T cells may directly contribute to the efficacy of these agents in suppressing cellular infiltration and to their anti‐inflammatory properties.
Identification and characterization of a substrate specific for the T cell protein tyrosine kinase ZAP‐70
Authors: Watts J.D. , Brabb T. , Bures E.J. , Wange R.L. , Samelson L.E. , Aebersold R.
Published: April 2003

ZAP‐70 is a protein tyrosine kinase (PTK) that plays a critical role in T cell activation. To study the role of ZAP‐70 catalytic activity in this process, a substrate capable of distinguishing between the activities of ZAP‐70 and other PTKs would be useful, especially since it has recently been shown that ZAP‐70 interacts with another T cell PTK, Lck. We have thus identified a site of phosphorylation on the cytoplasmic fragment of the erythrocyte band 3 protein that is recognized by ZAP‐70, but not Lck. A synthetic peptide based on this site has been demonstrated to be a good in vitro substrate for ZAP‐70 and a poor substrate for the T cell PTKs Lck and Itk. This peptide molecule should thus prove useful to many investigators working in the field of T cell activation.
Human 2‐D PAGE databases for proteome analysis in health and disease: http ://biobase.dk/cgi‐bin/celis
Authors: Celis Julio E. , Gromov Pavel , Østergaard Morten , Madsen Peder , Honoré Bent , Dejgaard Kurt , Olsen Eydfinnur , Vorum Henrik , Kristensen Dan Bach , Gromova Irina , Haunsø Anders , Van Damme Josef , Puype Magda , Vandekerckhove Joel , Holm Rasmussen Hanne
Published: April 2003

Human 2‐D PAGE Databases established at the Danish Centre for Human Genome Research are now available on the World Wide Web (http://biobase.dk/cgi‐bin/celis). The databanks, which offer a comprehensive approach to the analysis of the human proteome both in health and disease, contain data on known and unknown proteins recorded in various IEF and NEPHGE 2‐D PAGE reference maps (non‐cultured keratinocytes, non‐cultured transitional cell carcinomas, MRC‐5 fibroblasts and urine). One can display names and information on specific protein spots by clicking on the image of the gel representing the 2‐D gel map in which one is interested. In addition, the database can be searched by protein name, keywords or organelle or cellular component. The entry files contain links to other databases such as Medline, Swiss‐Prot, PIR, PDB, CySPID, OMIM, Methabolic pathways, etc. The on‐line information is updated regularly.
Core mutants of the immunoglobulin binding domain of streptococcal protein G: Stability and structural integrity
Authors: Gronenborn Angela M. , Frank M.Kirsten , Clore G.Marius
Published: April 2003

A library of core mutants of the GB1 domain of streptococcal protein G was created, and the structure and stability of selected members was assessed by 1H‐15N heteronuclear correlation NMR spectroscopy and fluorescence. All mutants comprised changes in β‐sheet residues, with sidechains at positions 5 (Leu), 7 (Leu), 52 (Phe) and 54 (Val) forming the β‐sheet side of the sheet‐helix core interface. A solvent exposed position Ile‐6 was chosen as a control. Randomization of bases at codon positions 1 and 3 with thymine at position 2 introduces five possible hydrophobic amino acids, namely Leu, Val, Ile, Phe, and Met. The distribution of encoded amino acids at all five positions is approximately as expected theoretically and indicates that no major bias was introduced towards particular residues. The overall structural integrity of several mutants, as assessed by NMR, ranges from very close to wild type to fully unfolded. Interestingly, the stability of the mutants is not strictly correlated with the number of changes or residue volume.
Assessment of glucose tolerance and pregnancy outcome of polycystic ovary patients
Authors: Turhan N.Ö. , Seçkin N.C. , Aybar F. , Inegöl I.
Published: April 2003

Objectives: To determine the impact of polycystic ovary syndrome (PCOS) on glucose tolerance during pregnancy and perinatal outcome. Methods: Pregnancy records of 38 PCOS patients were compared retrospectively with 136 non‐PCOS patients randomly. Patients with glucose challenge tests values of >130 mg/dl were referred for the 3‐h, 100‐g oral glucose tolerance test (OGTT). Results: A family history of diabetes mellitus, pre‐pregnancy body mass index (BMI), gestational weight gain was significantly higher in PCOS patients than controls. The prevalence of gestational diabetes mellitus (GDM) was similar in both groups. Impaired glucose tolerance (IGT) was observed in 18.4% of PCOS patients vs. 5.1% of controls. The main predictor of GDM was found pre‐pregnancy BMI >25 while main predictor of IGT was found as PCOS. Mean gestational age at delivery, prevalence of preterm labor, modes of delivery, mean birthweight, mean Apgar score at 5 min, proportion of babies admitted to the neonatal intensive care unit (NICU) were similar in both groups. Conclusions: Higher IGT prevalence in PCOS patients might be related to maternal obesity and excess gestational weight gain and does not affect perinatal outcome.
A comparison between a 75‐g and 100‐g oral glucose tolerance test in pregnant women
Authors: Soonthornpun S. , Soonthornpun K. , Aksonteing J. , Thamprasit A.
Published: April 2003

Objectives: To test the validity of a 75‐g, 2‐h oral glucose tolerance test (OGTT) for diagnosing gestational diabetes mellitus (GDM) using the criteria and reference values suggested by the American Diabetes Association for the 100‐g, 3‐h OGTT. Methods: The results of a 75‐g, 2‐h OGTT were compared with those of a 100‐g, 3‐h OGTT in 42 pregnant women. The women's mean±S.D. age and gestational age were 33.6±5.4 years and 28.2±4.2 weeks, respectively. Each subject was randomly scheduled within 1 week for both the 75‐g and 100‐g OGTTs. Results: The mean plasma glucose concentrations at 1, 2, and 3 h during the 100‐g OGTT were significantly higher than those during the 75‐g OGTT. Using the Carpenter and Coustan criteria, the prevalence of GDM was 21.4% when using the 100‐g, 3‐h OGTT, whereas it was found to be at only 7.1% when using the 75‐g, 2‐h OGTT. Conclusions: Plasma glucose responses during the 75‐g OGTT were found to be lower than those during the 100‐g OGTT. When using the same diagnostic criteria, the prevalence of GDM was also found lower using the 75‐g glucose load. It would therefore not be appropriate to use the 75‐g OGTT for diagnosing GDM using the criteria and reference values of the 100‐g OGTT. To give a comparable prevalence of GDM, the threshold of abnormal plasma glucose levels of the 75‐g OGTT would need to be lower than that of the 100‐g OGTT.