bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of sites for alkylation by N‐ethylmaleimide and pertussis toxin‐catalyzed ADP‐ribosylation on GTP‐binding proteins
Authors: Hoshino Shin-ichi , Kikkawa Satoshi , Takahashi Katsunobu , Itoh Hiroshi , Kaziro Yoshito , Kawasaki Hiroshi , Suzuki Koichi , Katada Toshiaki , Ui Michio
Published: July 2002

An αβγ‐trimeric GTP‐binding protein (Go) serving as the substrate of pertussis toxin‐ (IAP) catalyzed ADP‐ribosylation was purified from rat brain membranes. The constituent α‐subunit (αo) was alkylated with .N‐ethylmaleimide (NEM), and the functionally important sulfhydryl groups were investigated. There were at least two cysteine residues highly reactive to NEM on the GDP‐bound form of αo. These alkylations resulted in loss of its ability to be ADP‐ribosylated by IAP and to associate with βγ, but leaving the GTP‐binding site of αo intact. The reacted cysteine residues were identified by the sequencing of tryptic fragments of αo. One of the alkylation sites was Cys‐351, which was four amino acid residues away from the carboxyl‐terminus of the molecule. The Cys‐351 was proven to be also a site for IAP‐catalyzed ADP‐ribosylation. Possible roles of cysteine residues on the α‐subunit of Go are discussed in the functions of the signal transducing protein.
Apis mellifera cytoplasmic elongation factor 1α (EF‐1α) is closely related to Drosophila melanogaster EF‐1α
Authors: Walldorf Uwe , Hovemann Bernhard T.
Published: July 2002

Using low stringency hybridisation with a Drosophila melanogaster EF‐1α gene fragment we have isolated a genomic DNA clone encoding elongation factor 1α (EF‐1α) from Apis mellifera. The hybridising Apis mellifera sequence could be delineated to two small EcoRI fragments that were also revealed by genomic Southern hybridisation. By comparison with the corresponding Drosophila melanogaster data the complete translational reading frame has been deduced. It is interrupted by two intervening sequences of 220 and about 790 nucleotides. Comparison with known eucaryotic EF‐1α sequences further confirms that certain amino acid sequences seem to be invariable within the EF‐1α protein family.
Norepinephrine evokes a marked Mg2+ efflux from liver cells
Authors: Romani A. , Scarpa A.
Published: July 2002

The addition of norepinephrine to perfused rat livers and to collagenase isolated hepatocytes induced a marked and dose‐dependent magnesium efflux. The addition of β‐adrenergic receptor antagonists, but not α‐antagonists, completely blocked the Mg2+ efflux. The Mg2+ efflux could also be induced by forskolin and by permeable cAMP analogues. By contrast, the addition of carbachol or vasopressin induced a Mg2+ influx into isolated hepatocytes. These results indicate that a significant Mg2+ efflux from liver cells can be induced through the β‐adrenergic receptors and that it is mediated through the cytosolic cAMP levels.
Interleukin‐1 induces rapid and transient expression of the c‐fos proto‐oncogene in isolated pancreatic islets and in purified β‐cells
Authors: Hughes Jonathan H. , Watson Mark A. , Easom Richard A. , Turk John , McDaniel Michael L.
Published: July 2002

The effect of interleukin‐1β (IL‐1) on expression of c‐fos mRNA in isolated rat pancreatic islets was examined. Accumulation of c‐fos mRNA was demonstrable after 30 min of exposure to IL‐1, peaked by 60 min, and declined thereafter. Fluorescence‐activated cell sorting (FACS) of dispersed islet cells was employed to localize the accumulation of c‐fos mRNA to the β‐cell. Cycloheximide did not influence the induction of c‐fos mRNA by IL‐1. Accumulation of c‐fos mRNA therefore appears to be an early signal transduction event in the β‐cell and a component of the cellular mechanism(s) by which IL‐1 influences β‐cell function.
Purification and characterization of human platelet phospholipase A2 which preferentially hydrolyzes an arachidonoyl residue
Authors: Takayama Kiyoshi , Kudo Ichiro , Kim Dae Kyong , Nagata Koichi , Nozawa Yoshinori , Inoue Keizo
Published: July 2002

A phospholipase A2 with an arachidonoyl residue preference was purified about 11 700‐fold from human platelet soluble fraction to near homogeneity. The purified phospholipase A2 exhibited a molecular mass of about 90 kDa on SDS polycrylamide gel electrophoresis and hydrolyzed phospholipids with a arachidonoyl residue more effectively than those with a linoleoyl residue. The catalytic activity of the purified enzyme detected with phosphatidylcholine as a substrate increased sharply between 3 × 10−7 and 10−6 M free calcium ion. Thus, the 90‐kDa phospholipase A2 is considered to be a novel enzyme, distinct from the 14‐kDa one previously purified from human platelets. The 90‐kDa phospholipase A2 may participate mainly in arachidonate metabolism of platelets.
Autoimmune antigen Ku is enriched on oligonucleotide columns distinct from those containing the octamer binding protein DNA consensus sequence
Authors: Quinn J.P. , Farina A.R.
Published: July 2002

During purification of the AP1 complex from the T cell line MLA144 we enriched for a complex which bound to an oligonucleotide column containing the AP1 DNA consensus sequence and co‐eluted with a fraction required for AP1 binding activity. This complex although co‐eluting with AP1 binding activity had previously been determined to be non‐specific in its DNA binding properties. Further investigation determined that the complex was a heterodimer of 85 and 70 kDa which was antigenically related to the autoimmune antigen Ku. It is important to be aware of the abundance and avidity of the Ku complex to bind oligonucleotide columns when purifying sequence specific binding proteins.
Inhibition of nerve growth factor‐induced neurite outgrowth of PC12 cells by a protein kinase inhibitor which does not permeate the cell membrane
Authors: Nagashima Ken , Nakanishi Satoshi , Matsuda Yuzuru
Published: July 2002

K‐252a, a protein kinase inhibitor of microbial origin, has proven to be a specific inhibitor of nerve growth factor. In this study, the effects of K‐252b, the 9‐carboxylic acid derivative of K‐252a, on nerve growth factor‐induced neurite outgrowth in PC12 cells was examined. K‐252b is hydrophilic and does not permeate the cell membrane of PC12 cells, whereas K‐252a clearly does. K‐252b is, however, as potent as K‐252a itself in inhibiting the nerve growth factor‐induced neurite outgrowth. These results can be interpreted to suggest that effects of K‐252b may be through surface‐bound/anchored K‐252b‐sensitive molecules on PC12 cells.
Cytotoxic action of IL‐1β against pancreatic islets is mediated via nitric oxide formation and is inhibited by NG‐monomethyl‐l‐arginine
Authors: Bergmann Lars , Kröncke Klaus-Dietrich , Suschek Christoph , Kolb Hubert , Kolb-Bachofern Victoria
Published: July 2002

IL‐1β has been previously shown to act as a cytotoxic agent in islets. Here we show by electron microscopy of alginate encapsulated islets, that islet cell lysis is induced by culturing islets for 24 or 48 h in the presence of IL‐1β. The extent of lysis depends on the IL‐1β concentration and is slightly enhanced by the addition of TNF‐α. Cells can be protected from lysis by N
G‐monomethyl‐l‐arginine. Lysis is paralleled by an increase in nitrite concentration in culture supernatants of whole islets but not in supernatants of isolated endocrine cells. The results indicate that IL‐1β toxicity occurs via inducing in non‐endocrine islet cells the synthesis and release of nitric oxide, which has been shown earlier to be highly toxic for islet cells.
Time‐varying and static magnetic fields act in combination to alter calcium signal transduction in the lymphocyte
Authors: Yost M.G. , Liburdy R.P.
Published: July 2002

We have tested the hypothesis that extremely low frequency (ELF) time‐varying magnetic fields act in combination with static magnetic fields to alter calcium signalling in the lymphocyte. Results indicate that a 60‐min exposure of thymic lymphocytes at 37 ± 0.05°C to a 16 Hz, 421 mG (42.1 μT) magnetic field simultaneously with a colinear static magnetic field of 234 mG (23.4 μT) (a.c./d.c. field intensity ratio = 1.8) inhibits calcium influx triggered by the mitogen Concanavalin A. Significantly, resting lymphocytes do not respond to the fields, thus, only mitogen‐activated cells undergoing calcium signalling exhibit a field response. These results indicate that signal transduction involving calcium is an important biological constraint which operates to mediate this field interaction. Additional split field exposures show that the presence of the a.c. field or the d.c. field alone does not produce an effect. This is consistent with a proposed parametric resonance theory of interaction of low intensity magnetic fields with biological systems (L.L. Lednev (1991) Bioelectromagnetics 12, 71–75), which predicts the occurrance or biological effects at specific values for the frequency and field intensity of the ELF and static magnetic fields.
Aerobic heat shock activates trehalose synthesis in embryos of Artemia franciscana
Authors: Clegg James S. , Jackson Susan A.
Published: July 2002

Encysted embryos (cysts) of the brine shrimp, Artemia franciscana, contain large amount of trehalose which they use as a major substrate for energy metabolism and biosynthesis for Development under aerobic conditions at 25°C. When cysts are placed at 42°C (heat shock) these pathways stop, and the cysts re‐synthesize the trehalose that was utilized during the previous incubation at 25°C, Glycogen and glycerol, produced from trehalose at 25°C, appear to be substrates for trehalose synthesis during heat shock. Anoxia prevents trehalose synthesis in cysts undergoing heat shock. These results are consistent with the view that trehalose may play a protective role in cells exposed to heat shock, and other environmental insults, in addition to being a storage form of energy and organic carbon for development.
Synthesis of homocysteine thiolactone by methionyl‐tRNA synthetase in cultured mammalian cells
Authors: Jakubowski Hieronim , Goldman Emanuel
Published: July 2002

Homoeysteine thiolactone is a product of an error‐editing reaction, catalyzed by Escherichia coli and Saccharomyces cerevisiae methionyl‐tRNA synthetases, which prevents incorporation of homocysteine into tRNA and protein both in vitro and in vivo. Here, homocysteine thiolactone is also shown to be synthesized by cultured mammalian cells such as human cervical carcinoma (HeLa), mouse renal adenocarcinoma (RAG), and Chinese hamster ovary (CHO) cells labeled with [35S]methionine, but not by normal human and mouse (Balb/c 3T3) fibroblasts. A temperature‐sensitive methionyl‐tRNA synthetase mutant of CHO cells, Met‐1, does not make the thiolactone at the non‐permissive temperature. The data indicate that methionyl‐tRNA synthetase is involved in synthesis of homocysteine thiolactone in CHO cells, thereby extending this important proofreading mechanism to mammalian cells.
Predicting the conformation of proteins man versus machine
Authors: Benner Steven A. , Gerloff Dietlind L.
Published: July 2002

Two types of approaches for predicting the conformation of proteins from sequence data have lately received attention: ‘black box’ tools that generate fully automated predictions of secondary structure from a set of homologous protein sequences, and methods involving the expertise of a human biochemist who is assisted, but not replaced, by computer tools. A friendly controversy has emerged as to which approach offers a brighter future. In fact, both are necessary. Nevertheless, a snapshot of the controversy at this instant offers much insight into the structure prediction problem itself.
Crystallization and crystallographic investigations of ribonucleotide reductase protein R1 from Escherichia coli
Authors: Uhlin Ulla , Uhlin Tomas , Eklund Hans
Published: July 2002

Crystals of Escherichia coli ribonucleotide reductase protein R1 have been grown in complex with a synthetic peptide corresponding to the carboxyl end of protein R2. Good quality crystals could only be obtained after improvement of the purification protocol and are of the space group R32 with hexagonal cell axes a = b = 226 Å and c = 341 Å. They contain 3 subunits per asymmetric unit and diffract to 2.5 Å resolution in synchrotron radiation. A multiple isomorphous replacement map at 5.5 Å, improved by solvent flattening, shows that the dimeric molecules are elongated, about 110 Å long. The dimer is thin in the middle around the molecular two‐fold axis. The subunit is shaped like a bowl, probably with the active site in its center.
11‐Hydroxythromboxane B2 dehydrogenase is identical to cytosolic aldehyde dehydrogenase
Authors: Westlund Pär , Fylling Ann Catrin , Cederlund Ella , Jörnvall Hans
Published: July 2002

11 ‐Hydroxythromboxane B2 dehydrogenase purified from porcine kidney has been identified as cytosolic aldehyde dehydrogenase (EC 1.2.1.3). This identification is based on protein characteristics, sequence analysis of one proteolytic digest, blocked N‐terminus, subunit molecular mass of 55 kDa, and enzymatic activities. The sequence difference with the human enzyme is 7.5% in the fragments analyzed (29 exchanges of 388 positions, corresponding to the expected species variability for cytosolic aldehyde dehydrogenase The substrate thromboxane B2 contains a hemiacetal in its ring structure, but the reaction most likely proceeds via the aldehyde form of the substrate. This finding is in agreement with the proposed metabolism of 4‐hydroxycyclophosphamide and highlights the possibility that molecules containing a hemiacetal structure can function as substrates for aldehyde dehydrogenase.
A pulsed field gradient isotope‐filtered 3D 13C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes
Authors: Lee Woentae , Revington Matthew J. , Arrowsmith Cheryl , Kay Lewis E.
Published: July 2002

A pulsed field gradient three‐dimensional isotope‐filtered 13C HMQC‐NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly 13C labeled trp‐repressor, its natural abundance co‐repressor, l‐tryptophan, and natural abundance operator DNA. The pulse scheme makes use of pulsed field gradients for the removal of artifacts and dephasing of unwanted magnetization during isotope filtering, and employs a strategy to minimize the time that magnetization resides in the transverse plane. The experiment provides solely intermolecular NOE contacts between protons of the labeled protein and protons of the unlabeled species, and has proven to be especially useful in eliminating ambiguities between intra‐ and intermolecular NOEs in the isotope‐edited 3D 13C HMQC‐NOESY spectrum of the complex.
src‐related protein tyrosine kinases are physically associated with the surface antigen CD36 in human dermal microvascular endothelial cells
Authors: Bull Helen A. , Brickell Paul M. , Dowd Pauline M.
Published: July 2002

src‐related cytoplasmic PTKs are physically and functionally associated with cell surface receptors and are involved in signal transduction. In this paper we report the identification of src‐related proteins p59
fyn
, pp60c‐scr
and p62
yes
in human microvascular endothelial cells cultured from normal human skin and their physical association with the thrombospondin receptor CD36. Such an association represents a potential signalling pathway by which thrombospondin may regulate angiogenesis.
Protein‐chromophore interactions in α‐crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus a study by 13C magic angle spinning NMR
Authors: Weesie R.J , Askin D , Jansen F.J.H.M , de Groot H.J.M , Lugtenburg J , Britton G
Published: July 2002

MAS (magic angle spinning) 13C NMR has been used to study protein‐chromophore interactions in α‐crustacyanin, the blue astaxanthin‐binding carotenoprotein of the lobster, Homarus gammarus, reconstituted with astaxanthins labelled with 13C at the 14,14′ or 15,15′ positions. Two signals are seen for α‐crustacyanin containing [14,14′‐13C2]astaxanthin, shifted 6.9 and 4.0 ppm downfield from the 134.1 ppm signal of uncomplexed astaxanthin in the solid state. With α‐crustacyanin containing [15,15′‐13C2]astaxanthin, one essentially unshifted broad signal is seen. Hence binding to the protein causes a decrease in electronic charge density, providing the first experimental evidence that a charge redistribution mechanism contributes to the bathochromic shift of the astaxanthin in α‐crustacyanin, in agreement with inferences based on resonance Raman data [Salares, et al. (1979) Biochim. Biophys. Acta 576, 176–191]. The splitting of the 14 and 14′ signals provides evidence for asymmetric binding of each astaxanthin molecule by the protein.
mtDNA‐depleted U937 cells are sensitive to TNF and Fas‐mediated cytototxicity
Authors: Gamen Susana , Anel Alberto , Montoya Julio , Marzo Isabel , Piñeiro Andrés , Naval Javier
Published: July 2002

It has been proposed that TNF cytotoxicity is mediated by reactive oxygen intermediates generated by uncoupling of mitochondrial respiration. We have compared sensitive U937 cells and derived cell lines depleted of mtDNA for their ability to undergo TNF‐ and Fas‐induced apoptosis. Cells lacking around 98% of mtDNA were still sensitive to TNF‐induced apoptosis. U937 cells devoid of mtDNA (U937‐ϱ°) were resistant to TNF, but this was due to the loss of its 55 kDa receptor. U937‐ϱ° cells were also resistant to docosahexaenoic acid, which causes U937 cell death by lipid peroxidation. These cells were sensitive to anti‐Fas toxicity. The results indicate that TNF and Fas‐induced toxicity occurs by a mechanism mostly independent of mitochondrial free radical generation.
Differences in specificity for the interactions of stefins A, B and D with cysteine proteinases
Authors: Lenarčič Brigita , Križaj Igor , Žunec Petra , Turk Vito
Published: July 2002

Four different stefin‐type cysteine proteinase inhibitors have been isolated from porcine thymus and skin. Amino acid sequence determination revealed the presence of stefin A and stefin B type inhibitors and two new inhibitors, designated as porcine stefin D1 and stefin D2. Stefin D1 was identified as PLCPI, an inhibitor recently characterized from porcine polymorphonuclear leukocytes [Lenarčič et al. (1993) FEBS Lett. 336, 289–292]. Stefin A is composed of 101 amino acids and has an M
r of 11391 while stefin B contains 98 amino acids, has an M
r of 11174 and is N‐terminally blocked. All inhibitors were found to be fast‐acting inhibitors of papain, cathepsin L and cathepsin S (K
i = 0.009–0.161 nM). Stefins A and B also bind tightly and rapidly to cathepsin H (K
i = 0.027 and 0.069 nM, respectively), while stefins D1 and D2 have been shown to be very poor inhibitors of cathepsin H (K
i = 102–150 nM). he decreased affinity of these inhibitors toward cathepsin B (K
i = 2–1700 nM) was shown to be mainly due to the low second order association rate constants. The presence of a highly negatively charged N‐terminus on stefin D1 constitutes a likely structural determinant of inhibitor specificity.
An iterative local constraints based analysis for solving the resource constrained project scheduling problem
Authors: Özdamar Linet , Ulusoy Gündüz
Published: July 2002

In this paper, a new heuristic iterative scheduling approach is proposed for the resource constrained project scheduling problem. Well‐known dispatching rules and a decision‐making process called Local Constraint Based Analysis (LCBA), previously tested in a single pass forward parallel scheduling algorithm, are imbedded in the iterative scheduling algorithm. LCBA evaluates and selects the activities competing for scarce resources by considering the currently active temporal and resource constraints imposed by the network/resource characteristics. The iterative scheduling algorithm proposed here makes consecutive forward/backward scheduling passes which improve the project duration obtained by the single forward scheduling pass. Extensive experimentation has been done on a set of 188 benchmark problems from the literature. The results demonstrate that all the methods imbedded in the iterative algorithm improved the project duration significantly. It is observed that LCBA's average project duration deviation from the optimal duration is of the magnitude of 1% using the iterative scheduling algorithm. Among all the methods which have been tested, LCBA and a list rule, Weighted Resource Utilization and Precedence (WRUP), are the ones which have benefited most from the iterative algorithm. It is also observed that for various cash flow schemes the project Net Present Value (NPV) is improved through the use of the iterative scheduling algorithm.
Neural activity and the levels of high energy phosphates during deprivation of oxygen and/or glucose in hippocampal slices of immature and adult rats
Authors: Nabetani M. , Okada Y. , Kawai S. , Nakamura H.
Published: July 2002

To investigate the relationship between neural activity and cerebral energy metabolism during anoxia or ischemia in neural tissue of different ages, hippocampal slices were prepared from four‐, seven‐ and 10‐day‐old and adult rats. For the index of the neural activity, the population spikes were recorded in the pyramidal cell layer of the CA3 area. ATP and phosphocreatine levels inthe slices were measured during oxygen and/or glucose deprivation.

After deprivation of both oxygen and glucose, population spikes of the slices from four, seven‐ and 10‐day‐old and adult rats ceased completely in 14.2, 11.8, 9.4 and 5.3 min, respectively. The level of ATP at the time of cessation of population spike in four‐, seven‐ and 10‐day‐old and adult rats was 37.4, 30.2, 28.5 and 56.4% of the original concentrations. After deprivation of glucose only, the decay time of the population spikes of the slices from four‐, seven‐ and 10‐day‐old and adult rats was 17.8, 14.5, 9.0 and 10.0 min and at the time of population spikes cessation the level of ATP was 99.8, 84.2, 79.3 and 49%, respectively. After deprivation of oxygen only, population spikes of the slices from four, seven‐ and 10‐day old and adult rats ceased completely in 257, 283, 109 and 8.5 min, respectively. The level of ATP at the time of population spikes cessation was 50, 40, 36.6 and 94.4% of the initial values, respectively.

These results indicate that the immature rat is extremely resistant to oxygen deprivation from a functional and a metabolic view, whereas in the adult rat, preservation of neural activity depends much on both oxygen and glucose. During glucose deprivation, population spikes of the slices of immature and mature rats ceased rapidly although the level of ATP is preserved at high levels. This suggests that glucose plays an important role in the preservation of neural activity in addition to its major function as an energy substrate especially in immature animals.
Effect of genomic and subgenomic leader sequences of potato leafroll virus on gene expression
Authors: Juszczuk Marek , Pączkowska Elżbieta , Sadowy Ewa , Zagórski Włodzimierz , Hulanicka Danuta M.
Published: July 2002

The effect of the genomic and subgenomic leader sequence of potato leafroll polerovirus on the efficiency of translation of the downstream located genes has been studied. The results obtained in vitro and in vivo indicate that neither leader sequence functions as translational enhancer, a generally important feature of leader sequences. Deletion analyses demonstrated that both leader sequences not only decrease translation of the downstream located genes but also alter the ratio of the synthesized proteins. A correlation between the in vitro and in vivo results can be established in the case of the subgenomic leader sequence.
Effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin
Authors: Holland Carrie A , Henry Alexis T , Whinna Herbert C , Church Frank C
Published: July 2002

‘Thrombin aptamers’ are based on the 15‐nucleotide consensus sequence of d(GGTTGGTGTGGTTGG) that binds specifically to thrombin's anion‐binding exosite‐I. The effect of aptamer–thrombin interactions during inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) and antithrombin (AT) has not been described. Thrombin inhibition by HCII without glycosaminoglycan was decreased ∼two‐fold by the aptamer. In contrast, the aptamer dramatically reduced thrombin inhibition by >200‐fold and 30‐fold for HCII–heparin and HCII–dermatan sulfate, respectively. The aptamer had essentially no effect on thrombin inhibition by AT with or without heparin. These results add to our understanding of thrombin aptamer activity for potential clinical application, and they further demonstrate the importance of thrombin exosite‐I during inhibition by HCII–glycosaminoglycans.
Antioxidant actions of ovothiol‐derived 4‐mercaptoimidazoles: glutathione peroxidase activity and protection against peroxynitrite‐induced damage
Authors: Bailly Fabrice , Zoete Vincent , Vamecq Joseph , Catteau Jean-Pierre , Bernier Jean-Luc
Published: July 2002

4‐Mercaptoimidazoles derived from the naturally occurring antioxidants, ovothiols, were tested for their glutathione peroxidase‐like (GSH Px‐like) activity and protection against peroxynitrite‐induced damage. All the thiol compounds displayed similar significant GSH Px‐like activities, which are however weaker than that of the reference compound, ebselen. The inhibitions of the peroxynitrite‐dependent oxidation of Evans blue dye and dihydrorhodamine 123 showed that the thiol compounds substituted on position 5 of the imidazole ring were nearly as effective as ebselen while the C‐2 substituted ones were less effective. Both assays corroborate the large superiority of mercaptoimidazoles over glutathione as inhibitors of peroxynitrite‐dependent oxidation.
GPI‐anchored proteins and glycoconjugates segregate into lipid rafts in Kinetoplastida
Authors: Denny Paul W , Field Mark C , Smith Deborah F
Published: July 2002

The plasma membranes of the divergent eukaryotic parasites, Leishmania and Trypanosoma, are highly specialised, with a thick coat of glycoconjugates and glycoproteins playing a central role in virulence. Unusually, the majority of these surface macro‐molecules are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. In mammalian cells and yeast, many GPI‐anchored molecules associate with sphingolipid and cholesterol‐rich detergent‐resistant membranes, known as lipid rafts. Here we show that GPI‐anchored parasite macro‐molecules (but not the dual acylated Leishmania surface protein (hydrophilic acylated surface protein) or a subset of the GPI‐anchored glycoinositol phospholipid glycolipids) are enriched in a sphingolipid/sterol‐rich fraction resistant to cold detergent extraction. This observation is consistent with the presence of functional lipid rafts in these ancient, highly polarised organisms.
Hydrophobic analogues of the winter flounder ‘antifreeze’ protein
Authors: Haymet A.D.J. , Ward Leanne G. , Harding Margaret M.
Published: July 2002

The synthesis, solution conformation and ice‐growth inhibition properties of four new analogues of the type I 37‐residue winter flounder ‘antifreeze’ protein are reported. All four analogues contain two extra salt bridges to facilitate comparison of results with previously published data. In two analogues, all four threonine residues in the native polypeptide were mutated to 2‐amino butyric acid (an unnatural amino acid) and isoleucine, respectively. The butyric acid analogue was ∼85% helical at 3°C, modified the shape of ice growth, and exhibited reduced hysteresis compared to the native protein (9% at 4 mM). These results show that the γ‐methyl group of threonine, which is present in the sidechain of 2‐amino butyric acid, is not sufficient for activity. The isoleucine analogue, in which the threonine hydroxyl group is replaced by an ethyl group, was 100% helical at 3°C, showed no hysteresis but was able to modify the shape of ice crystal growth. In the third and fourth analogues, mutations of the aspartic acids 1 and 5 to alanine, and asparagines 16 and 27 to leucine in the threonine‐ and valine‐substituted analogues did not affect the helicity of the polypeptides, but removed the ability to inhibit ice growth.
Conformational states of annexin VI in solution induced by acidic pH
Authors: Golczak Marcin , Kirilenko Aneta , Bandorowicz-Pikula Joanna , Pikula Slawomir
Published: July 2002

Acidic pH‐induced folding of annexin (Anx)VI in solution was investigated in order to study the mechanism of formation of ion channels by the protein in membranes. Using 2‐(p‐toluidino)naphthalene‐6‐sulfonic acid as a hydrophobic probe, it was demonstrated that AnxVI exerts a large change in hydrophobicity at acidic pH. Moreover, circular dichroism spectra indicated that the native state of AnxVI changes at acidic pH towards a state characterized by a significant loss of α‐helix content and appearance of new β‐structures. These changes are reversible upon an increase of pH. It is postulated that the structural folding of AnxVI could explain how a soluble protein may undergo transition into a molecule able to penetrate the membrane hydrophobic region. The physiological significance of these observations is discussed.
Molecular mechanism of translocation through nuclear pore complexes during nuclear protein import
Authors: Stewart Murray , Baker Rosanna P , Bayliss Richard , Clayton Lesley , Grant Richard P , Littlewood Trevor , Matsuura Yoshiyuki
Published: July 2002

The trafficking of macromolecules between cytoplasm and nucleus through nuclear pore complexes is mediated by specific carrier molecules such as members of the importin‐β family. Nuclear pore proteins (nucleoporins) frequently contain sequence repeats based on FG cores and carriers appear to move their cargo through the pores by hopping between successive FG cores. A major question is why some macromolecules are transported while others are not. This selectivity may be generated by the ability to bind FG repeats, a local concentration of carrier–cargo complexes near the entrance to the pore channel, and steric hindrance produced by high concentrations of nucleoporins in the channel.
‘Empty’ minicircles and petB/atpA and psbD/psbE (cytb559 α) genes in tandem in Amphidinium carterae plastid DNA1
Authors: Hiller Roger G.
Published: July 2002

Amphidinium carterae minicircle chloroplast DNA was separated from total DNA by centrifugation through a sucrose/NaCl gradient. Sequences of minicircles with psbA and 23S rRNA contained a common region of 67 bp. Primers designed from this generated numerous polymerase chain reaction products of 1.5–2.6 kb. These contained psaA and psaB as one gene/circle, and petB/atpA and psbD/psbE as two genes/circle. ‘Empty’ minicircles of 1.7–2.5 kb containing no identifiable genes or parts of genes were more abundant than gene‐containing circles. From 15 minicircles a minimum common region of 48 bp was identified, with little identity to that from other dinoflagellate minicircles.
Fish testicular 11β‐hydroxylase : cDNA cloning and mRNA expression during spermatogenesis
Authors: Jianga Jian-Qiao , Kobayashi Tohru , Ge Wei , Kobayashi Hiroko , Tanaka Minoru , Okamoto Mitsuhiro , Nonaka Yasuki , Nagahama Yoshitaka
Published: July 2002

We isolated and characterized a cDNA encoding testicular 11β‐hydroxylase, cytochrome P450(11β) from the Japanese eel (Anguilla japonica) testis. The cDNA contains an open reading frame that encodes a protein of 511 amino acids. The predicted amino acid sequence shares 38–48% homology with those of adrenal P450(11β) from mammals and frog. Transient expression in COS 1 cells confirmed that the protein encoded by this cDNA had P450(11β) activity. Northern blotting revealed a single 1.8 kb long transcript of P450(11β). This transcript was not found in immature eel testes prior to an injection with human chorionic gonadotropin (hCG), but it was present in eel testes after hCG injection.
Immune localization of prolactin receptor in the mitochondria‐rich cells of the euryhaline teleost (Oreochromis mossambicus) gill
Authors: Weng Ching-Feng , Lee Tsung-Han , Hwang Pung-Pung
Published: July 2002

© 1997 Federation of European Biochemical Societies.
GroES binding regulates GroEL chaperonin activity under heat shock
Authors: Goloubinoff Pierre , Diamant Sophia , Weiss Celeste , Azem Abdussalam
Published: July 2002

Chaperonins GroEL14 and GroES7 are heat‐shock proteins implicated in the molecular response to stress. Protein fluorescence, crosslinking and kinetic analysis revealed that the bond between the two otherwise thermoresistant oligomers is regulated by temperature. As temperature increased, the affinity of GroES7 and the release of bound proteins from the chaperonin concomitantly decreased. After heat shock, GroES7 rebinding to GroEL14 and GroEL14GroES7 particles correlated with the restoration of optimal protein folding/release activity. Chaperonins thus behave as a molecular thermometer which can inhibit the release of aggregation‐prone proteins during heat shock and restore protein folding and release after heat shock.
DNA helix destabilization by proline and betaine: possible role in the salinity tolerance process
Authors: Rajendrakumar Chadalavada S.V , Suryanarayana Tangirala , Reddy Arjula R
Published: July 2002

Evidence is provided for the ability of proline, a salinity induced osmoprotectant, to destabilize the double helix and lower the T
m of DNA in a concentration dependent manner. At the reported salinity‐adaptive bio‐accumulation of 1 M and above, proline could considerably decrease the T
m and partially counteract the effect of sodium chloride and spermidine on DNA stability. On the contrary, several other amino acids tested did not show any such destabilizing effect on DNA helix. Enhanced susceptibility to S1 nuclease and insensitivity to DNase I in presence of increasing proline concentrations have further suggested a clear destabilization of the double helix. Such an effect is somewhat reminiscent of the interaction between betaine, another salinity induced osmolyte, and DNA resulting in decreased T
m values. These interactions may be significant in view of the abundance of such osmolytes in cells under salinity stress‐adapted conditions, with many a bacterial mutant accumulating them exhibiting improved tolerance to salinity.
Genomic organization and functional characterization of the mouse GalR1 galanin receptor
Authors: Wang Suke , He Chaogang , Maguire Maureen T , Clemmons Anthony L , Burrier Robert E , Guzzi Mario F , Strader Catherine D , Parker Eric M , Bayne Marvin L
Published: July 2002

Galanin mediates diverse physiological functions in digestive, endocrine, and central nervous systems through G‐protein‐coupled receptors. Two galanin receptors have been cloned but the gene structures are unknown. We report genomic and cDNA cloning of the mouse GalR1 galanin receptor and demonstrate that the coding sequence is uniquely divided into three exons encoding the N‐terminal portion through the fifth transmebrane domain, the third intracellular loop, and the sixth transmembrane domain through the C‐terminus. Functional analysis of the encoded cDNA revealed active ligand binding and intracellular signaling. The expression is detected in brain, spinal cord, heart and skeletal muscle.
The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus
Authors: Allen Mark D , Perham Richard N
Published: July 2002

A sub‐gene encoding the catalytic (acetyltransferase) domain (E2pCD) comprising residues 173–427 of the dihydrolipoyl acetyltransferase (E2p) chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was expressed in Escherichia coli. The product assembled to form the characteristic icosahedral (60‐mer) core structure with full catalytic activity. The K
m values for dihydrolipoamide and acetyl‐CoA were 1.2 mM and 13 μM, respectively. Dissociation of the icosahedral E2pCD into monomers by exposure to guanidine hydrochloride and the subsequent reassociation by gradual removal of the denaturing agent demonstrated the ability of the polypeptide chain to fold and reassemble in the absence of chaperonins.
Expression of protein‐tyrosine phosphatases in the major insulin target tissues
Authors: Norris Kjeld , Norris Fanny , Kono Dwight H , Vestergaard Henrik , Pedersen Oluf , Theofilopoulos Argyrios N , Møller Niels Peter H
Published: July 2002

Protein‐tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a representative picture, all tissues were analyzed by PCR using three different primer sets corresponding to conserved regions of known PTPs. A total of 24 different PTPs were identified. A multiprobe RNase protection assay was developed to obtain a semi‐quantitative measure of the expression levels of selected PTPs. Surprisingly, PTP‐LAR, previously suggested to be a major regulator of the insulin receptor tyrosine kinase, was expressed in extremely low levels in skeletal muscle, whereas the related receptor‐type PTP‐σ and PTP‐α were expressed in relatively high levels in all four tissues. The low levels of LAR PTP mRNA in skeletal muscle were further confirmed by Northern blot analysis.
Growth phase dependent stop codon readthrough and shift of translation reading frame in Escherichia coli
Authors: Wenthzel Anne-Marie K , Stancek Martin , Isaksson Leif A
Published: July 2002

Nonsense codon readthrough and changed translational reading frame were measured in different growth phases in E. coli. The strains used carry plasmid constructs with a translation assay reporter gene. This reporter gene contains an internal stop codon or a run of U‐residues. Termination or frameshifting give rise to stable proteins that can be physically quantified on gels along with the complete protein products. Readthrough of the stop codon UGA by a nearcognate tRNA is several fold higher in active growth than in late exponential phase. In early exponential phase, about 7% of −1 frameshift at a U9 slippery sequence is detectable; upon entry to stationary phase this frameshifting increases to about 40% followed by a decrease in stationary phase. A similar increase is observed in the case of +1 reading frameshift at the U9 sequence, which increases from 13% in early exponential growth phase up to 38% at the beginning of stationary phase followed by a decrease. Thus, the levels of both stop codon readthrough and frameshifting are growth phase dependent, though not in an identical fashion.
Identification of six subunits constituting Na+‐translocating NADH‐quinone reductase from the marine Vibrio alginolyticus
Authors: Nakayama Yuji , Hayashi Maki , Unemoto Tsutomu
Published: July 2002

We previously reported that the purified Na+‐translocating NADH‐quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of three major subunits, α, β and γ. NQR operon was sequenced and was found to be composed of 6 structural genes. Among these genes, nqr1, nqr3 and nqr6 were identified to code for α‐, γ‐ and β‐subunits, respectively. The protein products from nqr2, nqr4 and nqr5, however, were not reported. The sequence data predicted that these three proteins are very hydrophobic and may be unusual in mobility and staining on SDS‐PAGE. By modifying the detection method of proteins on SDS‐PAGE, we could detect all six subunits encoded by NQR operon in the purified NQR complex. The open reading frame of each subunit was identified from its N‐terminal amino acid sequence.
Cloning, expression analysis and chromosomal localization of the human nuclear receptor gene GCNF
Authors: Agoulnik I.Y , Cho Y , Niederberger C , Kieback D.G , Cooney A.J
Published: July 2002

Germ cell nuclear factor (GCNF) is an orphan member of the nuclear receptor gene superfamily. We report the cloning of a cDNA encoding a new variant of human GCNF from human testis and its expression analysis. Southern blot analysis of the human genomic DNA indicates that the GCNF gene is not closely related to other members within the nuclear receptor superfamily. Chromosomal localization of the GCNF gene shows that the gene is located on chromosome 9 at the locus q33–34.1. In situ hybridization analysis of GCNF expression in the testis shows that human GCNF is expressed exclusively in germ cells.
The oxidation of naphthalene and pyrene by cytochrome P450cam
Authors: England Paul A , Harford-Cross Charles F , Stevenson Julie-Anne , Rouch Duncan A , Wong Luet-Lok
Published: July 2002

Mutants of the heme monooxygenase cytochrome P450cam in which Y96 had been replaced with hydrophobic residues, have been shown to oxidise naphthalene and pyrene with rates one to two orders of magnitude faster than the wild‐type. Naphthalene was oxidised to 1‐ and 2‐naphthol, probably via the 1,2‐oxide intermediate. In the case of the Y96F mutant, naphthalene was oxidised at a rate comparable to camphor. Pyrene oxidation gave 1,6‐ and 1,8‐pyrenequinone with no evidence for attack at the K‐region, in contrast to mammalian enzymes. The results show that the Y96 residue plays a key role in controlling the substrate range of P450cam.
Involvement of microsomal fatty aldehyde dehydrogenase in the α‐oxidation of phytanic acid
Authors: Verhoeven N.M. , Jakobs C. , Carney G. , Somers M.P. , Wanders R.J.A. , Rizzo W.B.
Published: July 2002

We investigated the role of microsomal fatty aldehyde dehydrogenase (FALDH) in the conversion of pristanal into pristanic acid. Cultured skin fibroblasts from controls and patients with Sjögren‐Larsson syndrome (SLS) who are genetically deficient in FALDH activity were incubated with [2,3‐3H]phytanic acid. The release of aqueous‐soluble radioactivity by the SLS cells was decreased to 25% of normal, consistent with an intact formation of pristanal but a deficiency of further oxidation. SLS cells also accumulated four‐fold more radioactivity in N‐alkyl‐phosphatidyl ethanolamine, which arises from incorporation of free aldehyde into phosphatidyl ethanolamine. Recombinant human FALDH expressed in Chinese hamster ovary cells readily oxidized pristanal and cultured fibroblasts from SLS patients showed a severe deficiency in FALDH activity (13% of normal) when pristanal was used as substrate. Nevertheless, SLS patients did not accumulate phytanic acid in their plasma. We conclude that FALDH is involved in the oxidation of pristanal to pristanic acid and that this reaction is deficient in patients with SLS.
Structure and organization of the cardiotoxin genes in Naja naja sputatrix1
Authors: Lachumanan Ramkumar , Armugam Arunmozhiarasi , Tan Chee-Hong , Kandiah Jeyaseelan
Published: July 2002

We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding cardiotoxins (CTX) of Naja naja sputatrix. The cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP‐2, NFIL‐6/C/EBP, NF‐κB and PuF have been identified in the 5′‐region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three‐finger loop structure of the cardiotoxin molecule. The diverse functions of cardiotoxins together with our findings suggest that the cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.
1α,25‐Dihydroxyvitamin D3 decreases DNA binding of nuclear factor‐κB in human fibroblasts
Authors: Harant Hanna , Wolff Barbara , Lindley Ivan J.D
Published: July 2002

1α,25‐Dihydroxyvitamin D3 (1,25‐(OH)2‐D3), the active metabolite of vitamin D, can inhibit NF‐κB activity in human MRC‐5 fibroblasts, targeting DNA binding of NF‐κB but not translocation of its subunits p50 and p65. The partial inhibition of NF‐κB DNA binding by 1,25‐(OH)2‐D3 is dependent on de novo protein synthesis, suggesting that 1,25‐(OH)2‐D3 may regulate expression of cellular factors which contribute to reduced DNA binding of NF‐κB. Although NF‐κB binding is decreased by 1,25‐(OH)2‐D3 in MRC‐5 cells, IL‐8 and IL‐6 mRNA levels are only moderately downregulated, demonstrating that inhibition of NF‐κB DNA binding alone is not sufficient for optimal downregulation of these genes.
Receptor activity modifying proteins regulate the activity of a calcitonin gene‐related peptide receptor in rabbit aortic endothelial cells
Authors: Muff Roman , Leuthäuser Kerstin , Bühlmann Nicole , Foord Steven M. , Fischer Jan A. , Born Walter
Published: July 2002

In Xenopus oocytes with an endogenous calcitonin gene‐related peptide (CGRP) receptor, a receptor activity modifying protein (RAMP1) enhancing CGRP stimulated chloride currents of the cystic fibrosis transmembrane regulator was recently cloned [McLatchie, L.M. et al. (1998) Nature 393, 333–339]. Here, transient expression of RAMP1 in rabbit aortic endothelial cells (RAEC) brought about stimulation of cAMP accumulation by human (h) αCGRP with an EC50 of 0.41 nM. This was antagonized by a CGRP receptor antagonist αCGRP(8–37). Co‐expression of RAMP3 together with RAMP1 reduced the maximal cAMP response to hαCGRP by 47% (P<0.05). The cells also express RAMP2 encoding mRNA and an adrenomedullin (ADM) receptor coupled to stimulation of cAMP formation by hADM (EC50 0.18 nM). The latter was antagonized by an ADM receptor antagonist hADM(22–52). In conclusion, expression of a CGRP receptor in RAEC requires RAMP1. The same receptor presumably recognizes ADM making use of endogenous RAMP2. The results reveal competition between the different RAMPs in the regulation of CGRP/ADM receptor activity.
Protein phosphatase 1 is involved in the dissociation of Ca2+/calmodulin‐dependent protein kinase II from postsynaptic densities
Authors: Yoshimura Yoshiyuki , Sogawa Yoshimi , Yamauchi Takashi
Published: July 2002

Autophosphorylation‐dependent translocation of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) to postsynaptic densities (PSDs) from cytosol may be a physiologically important process during synaptic activation. We investigated a protein phosphatase responsible for dephosphorylation of the kinase. CaM kinase II was shown to be targeted to two sites using the gel overlay method in two‐dimensional gel electrophoresis. Protein phosphatase 1 (PP1) was identified to dephosphorylate CaM kinase II from its complex with PSDs using phosphatase inhibitors and activators, and purified phosphatases. The kinase was released from PSDs after its dephosphorylation by PP1.
A phosphoglycerate to inorganic phosphate ratio is the major factor in controlling starch levels in chloroplasts via ADP‐glucose pyrophosphorylase regulation
Authors: Kleczkowski Leszek A
Published: July 2002

Purified barley leaf ADP‐glucose pyrophosphorylase, a key enzyme of the starch synthesis in the chloroplast stroma, was analysed with respect to its possible regulation by factors defining the metabolic/effector status of the chloroplast during light and dark conditions. The enzyme required 3‐phosphoglyceric acid for the maximal activity and was inhibited by inorganic phosphate. The optimal pH for the enzyme was at circa 7.0, regardless of the presence or absence of 3‐phosphoglyceric acid, whereas the maximal activation by 3‐phosphoglyceric acid was observed at pH 8.5 and higher. Changes in the concentration of Mg2+ and dithiothreitol had little or no effect on the enzymatic activity of AGPase. It has been directly demonstrated for the first time that a 3‐phosphoglyceric acid/inorganic phosphate ratio, a crucial regulatory parameter, could be directly related to a defined activation state of the enzyme, allowing the prediction of a relative AGPase activity under given conditions. The predicted changes in the enzyme activity were directly correlated with earlier reported responses of starch levels to the 3‐phosphoglyceric acid/inorganic phosphate ratio in chloroplasts. Consequences of this for the starch biosynthesis are discussed.