bluuebunny/crossref_metadata_2025 huggingface dataset:
A simple method for extraction of DNA from fungi and yeasts with anhydrous hydrogen fluoride
Authors: Oshiro Satoru , Katsura Nobuhiko , Kitada Kunio , Gunge Norio
Published: July 2002

A novel method is described for the extraction of DNAs from fungi and yeasts. Anhydrous hydrogen fluoride (HF) selectively cleaves their cell walls under mild conditions (for 5 min at 0°C), enabling the effective extraction of DNAs from organisms with a cell wall. A possible mechanism for this method concerning the selective cleavage of O‐glycosidic linkages in cell walls has been described previously [(1977) Anal. Biochem. 82, 289–309]. The extracted DNA is intact: in fact, the yeast DNA is directly applicable for restriction analysis and transformation of Escherichia coli.
The effect of selenium deficiency on peroxidative injury in the house fly, Musca domestica
Authors: Simmons Thomas W. , Jamall I.Siraj , Lockshin Richard A.
Published: July 2002

Selenium‐dependent glutathione peroxidase activity is documented for the first time in insects. Reduction in glutathione peroxidase activity in the cytosol of adult house flies by lowering selenium in the diet results in significant increases in peroxidative injury. Catalase activity, while higher in low‐selenium flies than in selenium‐supplemented flies, does not prevent lipid peroxidation. The discovery of glutathione peroxidase activity in insects eliminates an anomaly which partially limited the usefulness of these animals as models for the study of the antioxidant defense system.
Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein
Authors: Ponnambalam Sreenivasan , Spassky Annick , Busby Stephen
Published: July 2002

We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, S1 and S2. In the presence of this point mutation, transcription initiates at a third start point bp downstream of S1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initiation in the gal operon is normally regulated by the cylic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to P1. With the point mutation, CRP binding switches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.
A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets
Authors: Kattelman E.J. , Arora S.K. , Lim C.T. , Venton D.L. , Le Breton G.C.
Published: July 2002

A photoactive iodoarylazide derivative (I‐APA‐PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13‐azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor‐binding studies using [3H]U46619, I‐APA‐PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I‐APA‐PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding radio‐iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2‐binding protein in human platelets.
Initial and sustained calcium mobilizations in the parietal cell during stimulations with gastrin, inositol trisphosphate, phorbol ester and erogenous diacylglycerol
Authors: Tsunoda Yasuhiro , Takeda Hiroshi , Asaka Masahiro , Nakagaki Ikuko , Sasaki Sadao
Published: July 2002

Electron probe X‐ray microanalysis revealed that cytoplasmic Ca2+ concentration increased in the restricted apical cytoplasm during stimulation of isolated guinea pig parietal cells with gastrin. Furthermore, this study, using 45Ca2+, aequorin and fura‐2, revealed the mechanism involved in intracellular Ca2+ shifts caused by gastrin and the involvements of inositol 1,4,5‐trisphosphate (IP3) and diacylglycerol in producing those shifts. The gastrin‐mediated and IP3‐sensitive Ca2+ pool was located in the smooth‐surfaced membrane‐enriched areas and released Ca2+ in the initial phase. Gastrin‐mediated Ca2+ mobilization was also evoked by diacylglycerol, comprising an intracellular Ca2+ mobilization followed by a late, sustained and more localised Ca2+ entry from the extracellular space.
Synthesis of a photoaffinity‐spin‐labeled derivative of ATP and its first application to F1‐ATPase
Authors: Vogel-Claude Pia , Schäfer Günter , Trommer Wolfgang E.
Published: July 2002

The synthesis of an ATP derivative is described, in which a spin‐label is attached to the 3′‐position of the ribose moiety and an azido group to C8 of the adenine ring (SL‐N3‐ATP). Irradiation of this compound at 350 nm generates a nitrene, which will react with any functional group in its vicinity. SL‐N3‐ATP exhibits a strongly immobilized ESR spectrum in a complex with F1‐ATPase from beef heart mitochondria. It was covalently incorporated into this enzyme. SL‐N3‐ATP may be employed in ESR investigations under conditions in which non‐covalent interactions are too weak for motionally restricted species to be easily observed.
Eicosapentaenoic acid and docosahexaenoic acid are antagonists at the thromboxane A2/prostaglandin H2 receptor in human platelets
Authors: Swann Patrick G. , Venton Duane L. , Le Breton Guy C.
Published: July 2002

The present study investigated the mechanism by which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibit platelet activation induced by thromboxane A2. DHA was found to be more potent than EPA in blocking platelet aggregation induced by the stable thromboxane A2 mimetic, U46619. Furthermore, this inhibition by DHA or EPA was competitive. Binding studies using 3H‐U46619 demonstrated that both EPA and DHA interact with the platelet thromboxane receptor. The potency of the inhibition of binding corresponded with that seen for the inhibition of aggregation. These results suggest that thromboxane receptor antagonism may be an important mechanism by which EPA and DHA modulate platelet reactivity in vivo.
Conserved amino acids in F‐helix of bacteriorhodopsin form part of a retinal binding pocket
Authors: Rothschild K.J. , Braiman M.S. , Mogi T. , Stern L.J. , Khorana H.G.
Published: July 2002

A 3‐dimensional model for the retinal binding pocket in the light‐driven proton pump, bacteriorhodopsin, is proposed on the basis of spectroscopic studies of bacteriorhodopsin mutants. In this model Trp‐182, Pro‐186 and Trp‐189 surround the polyene chain while Tyr‐185 is positioned close to the retinylidene Schiff base. This model is supported by sequence homologies in the F‐helices of bacteriorhodopsin and the related retinal proteins, halorhodopsin and rhodopsins.
Gene structure and 5'‐upstream sequence of rat cathepsin L
Authors: Ishidoh Kazumi , Kominami Eiki , Suzuki Koichi , Katunuma Nobuhiko
Published: July 2002

The structure of rat cathepsin L gene has been determined. The gene spans 8.5 kilobase pairs comprising 8 exons, and has an intron located near the active site cysteine residue. The gene structure does not correspond well to the functional units of the proteinase. These characteristics are found to be in common with the cysteine proteinase gene family. In the 5'‐upstream region, one CAAT‐box and four SP‐1 binding sites, together with two AP‐2 binding sites and CRE, but no typical TATA‐box are found. Further, SP‐1 and AP‐2 binding sites and an octamer motif are also found in the 1st intron, suggesting a complex regulatory mechanism for the expression of the cathepsin L gene.
Highly selective affinity labeling of the primer‐binding site of E.coli DNA polymerase I
Authors: Mitina R.L. , Mustaev A.A. , Zaychikov E.F. , Khomov V.V. , Lavrik O.I.
Published: July 2002

Highly selective affinity labeling of the primer site of E.coli DNA polymerase I was performed with the 5'‐reactive derivatives of oligothymidylate in the presence of poly(dA) template. Subtilysine cleavage proved that the site of affinity modification belonged to the ‘Klenow’ part of DNA polymerase I. If taken separately, Klenow fragment was not labeled by these oligonucleotide derivatives. The sites of affinity labeling were tested in the structure of DNA polymerase I by hydroxylamine cleavage. At least two sites of labeling were revealed. The main one was localized between Gly‐833 and His‐928.
The NH2‐terminal residues of rat liver proteasome (multicatalytic proteinase complex) subunits, C2, C3 and C8, are Nα‐acetylated
Authors: Tokunaga Fuminori , Aruga Rie , Iwanaga Sadaaki , Tanaka Keiji , Ichihara Akira , Takao Toshifumi , Shimonishi Yasutsugu
Published: July 2002

Rat liver proteasome (multicatalytic proteinase complex) is a 20S‐ring shaped particle having a molecular mass of 750 kDa, and iscomposed of at least 13 non‐identical components ranging from 21 to 31 kDa in size. We found here that the NH2‐terminal residues of all the known 13 components, except for C5, are not reactive to phenylisothiocyanate. Among them, components C2, C3 and C8 are blocked in their NH2‐termini with Nα‐acetyl‐Met, Nα‐acetyl‐Ala, and Nα‐acetyl‐Ser, respectively. The NH2‐terminal portions of C2, C3, and C8 exhibit sequence similarity to one another, but that of the non‐blocked component C5 differs from those of C2, C3, and C8.
B–Z DNA reversible conformation changes effected by high pressure
Authors: Krzyźaniak Andrzej , Salański Piotr , Jurczak Janusz , Barciszewski Jan
Published: July 2002

There are numerous data showing that a DNA molecule with alternate pirymidine‐purine sequence can adopt a left‐handed, double‐helical Z‐DNA conformation. Such structural changes of DNA occur as a consequence of environmental conditions (e.g. 4 M NaCl) or chemical modification (e.g. methylation or bromination of bases). In this paper, we found for the first time that high pressure (several kilobars) can change the DNA conformation from the B to the Z form. When the pressure is reduced to an atmospheric one, DNA conformation returns back to the B‐form. The Z‐DNA structure formation was confirmed by circular dichroism (CD) and ultraviolet (UV) measurements. However, we found, that the values of the ratio of absorbance at the wavelenghts 295 and 257 nm in the range of 0.3–0.4 is not a fully conclusive proof for the Z‐DNA conformation. Although the ratio is typical for Z‐DNA form, it is not obvious that the negative band in CD spectrum will be observed. On the other hand, methylated DNA does not undergo B→Z DNA transitions at the high pressure. These conformational changes of DNA molecules could be interpreted as the effect of different hydration of various DNA forms.
Molecular cloning and primary structure of Thermoactinomyces vulgaris carboxypeptidase T A metalloenzyme endowed with dual substrate specificity
Authors: Smulevitch Sergey V. , Osterman Andrey L. , Galperina Olga V. , Matz Mikhail V. , Zagnitko Olga P. , Kadyrov Rafail M. , Tsaplina Iraida A. , Grishin Nikolai V. , Chestukhina Galina G. , Stepanov Valentin M.
Published: July 2002

A gene coding for an extracellular Zn‐carboxypeptidase of Thermoactinomyces vulgaris has been cloned and sequenced (EMBL X56901). This enzyme named carboxypeptidase T reveals simultaneously both types of substrate specificity characteristic of mammalian carboxypeptidases A and B. The carboxypeptidase T gene is primarily expressed in E. coli as a non‐active preproenzyme with an additional 98 amino acid residues at the N‐terminus. Primary structure alignment or mature carboxypeptidase T and mammalian metallocarboxypeptidases demonstrated 25–30% overall identity but a full preservation of presumed catalytically important residues. These observations imply a basic uniformity of the general catalytic mechanism for enzymes of that class produced by evolutionarily remote organisms.
Expansion of the mammalian 3β‐hydroxysteroid dehydrogenase/plant dihydroflavonol reductase superfamily to include a bacterial cholesterol dehydrogenase, a bacterial UDP‐galactose‐4‐epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus
Authors: Baker Michael E. , Blasco Rafael
Published: July 2002

Mammalian 3β‐hydroxysteroid dehydrogenase and plant dihydroflavonol reductases are descended from a common ancestor. Here we present evidence that Nocardia cholesterol dehydrogenase, E. coli UDP‐galactose‐4 epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus are homologous to 3β‐hydroxysteroid dehydrogenase and dehydroflavonol reductase. Analysis of a multiple alignment of these sequences indicates that viral ORFs are most closely related to the mammalian 3β‐hydroxysteroid dehydrogenases. The ancestral protein of this superfamily is likely to be one that metabolized sugar nucleotides. The sequence similarity between 3β‐hydroxysteroid dehydrogenase and the viral ORFs is sufficient to suggest that these ORFs have an activity that is similar to 3β‐hydroxysteroid dehydrogenase or cholesterol dehydrogenase, although the putative substrates are not yet known.
Stoichiometric binding of 2′(or 3′)‐O‐(2,4,6‐trinitrophenyl)adenosine 5′‐triphosphate to bovine heart cytochrome c oxidase
Authors: Reimann Achim , Kadenbach Bernhard
Published: July 2002

The binding of 2′(or 3′)‐O‐(2,4,6‐trinitrophenyl)adenosine 5′‐triphosphate (TNP‐ATP) to isolated bovine heart cytochrome c oxidase (COX) was studied by following its specific spectral change at 510 nm. The quantitative titration revealed two binding sites for TNP‐ATP per monomer COX with a K
d of 1.6 μM.
Three‐dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 Å resolution
Authors: Murshudov G.N. , Melik-Adamyan W.R. , Grebenko A.I. , Barynin V.V. , Vagin A.A. , Vainshtein B.K. , Dauter Z. , Wilson K.S.
Published: July 2002

The three‐dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 Å resolution. The subunit of the tetrameric molecule of 222 symmetry consists of a single polypeptide chain of about 500 amino acid residues and one haem group. The crystals belong to space group P42212 with unit cell parameters a = b = 106,7 Å, c = 106,3 Å, and there is one subunit of the tetramer. per asymmetric unit. The amino acid sequence has been tentatively determined by computer graphics model building and comparison with the known three‐dimensional structure of beef liver catalase and sequences of several other catalases. The atomic model has been refined by Hendrickson and Konnert's least‐squares minimisation against 94,315 reflections between 8 Å and 1.5 Å. The final model consists or 3,977 non‐hydrogen atoms of the protein and haem group, 426 water molecules and ones sulphate ion. The secondary and tertiary sructures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made.
Effect of metal ions on the activity of cascein kinase II from Xenopus laevis
Authors: Gatica Marta , Hinrichs María V. , Jedlicki Ana , Allende Catherine C. , Allende Jorge E.
Published: July 2002

CaSein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant α and β subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7–10 mM for Mg2+, 0.5–0.7 mM for Mn2+ and 1–2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence ofMg2+. The apparent K
m values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 μM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and α subunit. Inhibition of the holoenzyme by 400 μM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the α subunit.
Histone acetylation and gene induction in human cells
Authors: Clayton Alison L. , Hebbes Tim R. , Thorne Alan W. , Crane-Robinson Colyn
Published: July 2002

An antibody recognising acetylated core histones was used to immunoprecipitate chromatin fragments from proliferating human K562 cells and from cells induced to differentiate with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). The DNA of the acetylated chromatin was probed with sequences of platelet derived growth factor B chain (PDGF‐B), a gene which is induced to strong expression upon differentiation. A high level of acetylation was observed before gene induction and no change seen following induction. This implies that core histone acetylation is an essential precondition for transcription.
Positively charged residues influence the degree of SecA dependence in protein translocation across the E. coli inner membrane
Authors: Andersson Helena , von Heijne Gunnar
Published: July 2002

The sec machinery catalyzes the translocation of nascent polypeptide chains across the inner membrane of E. coli, yet some inner membrane proteins depend only weakly or not at all on an intact sec function for membrane insertion even though they have stretches of chain protruding into the periplasmic space. Earlier work has demonstrated that the length of a periplasmic loop correlates with its degree of sec‐dependence. We now show that the content of positively charged residues in a translocated loop also correlates with the degree of dependence on SecA function, suggesting that arginines and lysines may be inherently difficult to move across the membrane during sec‐dependent translocation.
The role of water in the mechanism of muscular contraction
Authors: Oplatka Avraham
Published: July 2002

Twenty‐five years after its proposal, the swinging theory of muscular contraction, in which the majority of scientists in the field have blindly believed, has not yet been verified. Rapidly growing experimental evidence indicates that the myosin heads do not swing. It is time to look for an alternative mechanism. Data is presented indicating that water is liberated during tension development and the extent to which it is released appears to affect the degree of tension. Since water can move (because of acquired extra energy, involvement in hydration forces etc.), it might cause protein movement.
Antibacterial activity of peptides homologous to a loop region in human lactoferrin
Authors: Odell Edward W. , Sarra Robert , Foxworthy Morwenna , Chapple Daniel S. , Evans Robert W.
Published: July 2002

Human lactoferrin contains a 46 residue sequence named lactoferricin H thought to be responsible for its antimicrobial properties. Synthetic peptides HLT1, corresponding to the loop region of human lactoferricin (FQWQRNMRKVRGPPVS) and HLT2, corresponding to its charged portion (FQWQRNMRKVR), exerted significant antibacterial effects against E. coli serotype O111 strains NCTC 8007 and ML35. The corresponding sequences in native human lactoferrin were shown to adopt a charged helix and hydrophobic tall within the N‐lobe remote from the iron binding site. Sequence similarities between lactoferricin and dermaseptin and magainins suggest that lactoferricin may act as an amphipathic alpha helix.
Functional roles of the tyrosine within the NP(X)nY motif and the cysteines in the C‐terminal juxtamembrane region of the CB2 cannabinoid receptor
Authors: Feng Wenke , Song Z.H
Published: July 2002

In G protein‐coupled receptors, a NP(X)
n
Y motif in the seventh transmembrane domain and cysteine residues in the C‐terminal juxtamembrane region are conserved. In the current study, the roles of Y299 within the NPVIY motif and C313 and C320 in the C‐terminal juxtamembrane region of the human CB2 cannabinoid receptor were investigated by site‐directed mutagenesis. Replacing Y299 with alanine resulted in a complete loss of ligand binding and a severe impairment of cannabinoid‐induced inhibition of forskolin‐stimulated cAMP accumulation. The C313A and C320A mutations markedly reduced functional coupling to adenylate cyclase, but had no effect on ligand binding and agonist‐induced receptor desensitization.
The primary structure of BSP‐30K, a major lipid‐, gelatin‐, and heparin‐binding glycoprotein of bovine seminal plasma
Authors: Calvete Juan J. , Mann Karlheinz , Sanz Libia , Raida Manfred , Töpfer-Petersen Edda
Published: July 2002

BSP‐30K is a major acidic glycoprotein of bovine seminal plasma. It displays heparin‐, gelatin‐, and phospholipid‐binding activities. BSP‐30K binds to spermatozoa upon ejaculation and is thought to play a role in sperm capacitation. We have determined its amino acid sequence, disulfide bonds, and O‐glycosylation sites. BSP‐30K consists of 158 amino acids arranged in a mosaic structure. BSP‐30K has a unique 48‐residue N‐terminal extension which includes three 7–8‐ amino acid repeats and the six O‐glycosylated threonine residues. The polypeptide stretch 49–71 is homologous to type ‘A’ domains found in heparin‐binding proteins from other mammalian species. The C‐terminal portion of BSP‐30K is organized in a tandem of 40–44‐residue domains each sharing the consensus pattern of the gelatin‐binding fibronectin type II module. The mosaic structure of BSP‐30K suggests that this glycoprotein might be a factor contributing to the different sperm‐capacitatiog effects exerted by heparin in different mammalian species.
NMDA receptors in cultured radial glia
Authors: López Tomás , López-Colomé Ana Marı́a , Ortega Arturo
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Effect of repeated experience of victory and defeat in daily agonistic confrontations on brain tryptophan hydroxylase activity
Authors: Amstislavskaya Tamara G , Kudryavtseva Natalia N
Published: July 2002

The rate‐limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), was studied in brain areas of male mice with repeated experience of victory (winners) or defeat (losers) gained in 10 daily agonistic confrontations. A reduction of TPH activity in the midbrain and an increase in the hypothalamus was demonstrated for winners compared with controls. In contrast, repeated defeat in social confrontations was associated with higher TPH activity in the striatum and hypothalamus in losers compared with controls. Agonistic interactions did not affect TPH activity in the amygdala, nucleus accumbens or hippocampus in either winners or losers. The sensory contact technique used in this work for generating winners and losers may be productive in the analysis of TPH gene regulation.
Seryl‐phosphorylation of soybean nodule sucrose synthase (nodulin‐100) by a Ca2+‐dependent protein kinase
Authors: Zhang Xiu-Qing , Chollet Raymond
Published: July 2002

Sucrose synthase (SS; EC 2.4.1.13) was radiolabeled in situ by incubating detached soybean nodules with 32Pi. Phosphoamino acid analysis indicated that SS was phosphorylated on a serine residue(s). In‐vitro phosphorylation of purified nodule SS by desalted nodule extracts was Ca2+‐dependent. This SS‐kinase was partially purified (∼2200‐fold) from nodules harvested from illuminated plants. The molecular mass of the SS‐kinase was about 55 000 on a Superdex 75 size‐exclusion column or in a denaturing autophosphorylation gel. With either purified nodule SS or Syntide 2 as substrate, exogenous calmodulin and phosphatidylserine showed little or no effect on the in‐vitro activity of this partially purified protein kinase. However, its activity was inhibited by W‐7. The purified nodule SS‐kinase (or CDPK) phosphorylated nodule PEP carboxylase (PEPC; EC 4.1.1.31) in the presence of Ca2+. In contrast, a partially purified nodule PEPC‐kinase preparation was incapable of phosphorylating nodule SS. Unlike nodule PEPC [Zhang et al. (1995) Plant Physiol. 108, 1561–1568], the phosphorylation state of SS is not likely modulated in planta by photosynthate supply from the shoots.
Quail cystatin: Isolation and characterisation of a new member of the cystatin family and its hypothetical interaction with cathepsin B
Authors: Gerhartz Bernd , Engh Richard A , Mentele Reinhard , Eckerskorn Christoph , Torquato Richardo , Wittmann Josef , Kolb Helmut J , Machleidt Werner , Fritz Hans , Auerswald Ennes A
Published: July 2002

Quail cystatin, a new cysteine proteinase inhibitor protein of the cystatin superfamily, was purified from egg albumen of Japanese quail Coturnix coturnix japonica. Amino acid sequencing and mass spectrometry revealed the complete 116 amino acid residue primary structure of a phosphorylated form (13 173 Da). The inhibitor has a 90% sequence identity with chicken cystatin. Its interaction with papain is rapid and tight (K
i=4.4 pM; k
on=1.8×107 M−1 s−1; k
off=0.8×10−4 s−1) and very similar to that of chicken cystatin. Surprisingly, however, cathepsin B was inhibited 15‐fold more strongly by quail cystatin (K
i=47 pM; k
on=19×107 M−1 s−1; k
off=9×10−4 s−1) than by chicken cystatin (K
i=784 pM; k
on=2.9×107 M−1 s−1; k
off=24×10−4 s−1). Intuitive comparative conformational inspection of related inhibitors and of cognate enzymes suggest that: (i) the 3D structure of quail cystatin is nearly identical to that of chicken cystatin, (ii) quail cystatin can interact with cathepsin B analogous to the stefin B–papain interaction, if the `occluding loop' of cathepsin B possesses an `open' conformation, (iii) the greater inhibition of cathepsin B by quail cystatin compared to chicken cystatins probably arises from two additional ionic interactions between residues Arg15 and Lys112 of the inhibitor and Glu194 and Asp124 of the enzyme, respectively. The two potential salt bridges are located outside of the known contact regions between cystatins and peptidases of the papain family.
Neutral trehalase Nth1p of Saccharomyces cerevisiae encoded by the NTH1 gene is a multiple stress responsive protein
Authors: Zähringer Harald , Burgert Markus , Holzer Helmut , Nwaka Solomon
Published: July 2002

We have shown previously that expression of the NTH1 gene is increased at heat stress (40°C) both at the mRNA and enzymatic activity levels. This increased expression was correlated to the requirement of the NTH1 gene for recovery after heat shock at 50°C and the presence of stress responsive elements STRE (CCCCT) 3 times in its promoter region [S. Nwaka et al., FEBS Lett. 360 (1995) 286–290; S. Nwaka et al., J. Biol. Chem. 270 (1995) 10193–10198]. We show here that expression of the NTH1 gene and its product, neutral trehalase (Nth1p), are also induced by other stressors such as H2O2, CuSO4, NaAsO2, and cycloheximide (CHX). Heat‐induced expression of the NTH1 gene is shown to be accompanied by accumulation of trehalose. In contrast, the chemical stressors which also induce the expression of NTH1 did not lead to accumulation of trehalose under similar conditions. Our data suggest that: (1) heat‐ and chemical stress‐induced expression of neutral trehalase is largely due to de novo protein synthesis, and (2) different mechanisms may control the heat‐ and chemical stress‐induced expression of NTH1 at the transcriptional level. Participation of neutral trehalase (Nth1p) in multiple stress response dependent and independent on trehalose is discussed.
Unexpected influence of a C‐terminal‐fused His‐tag on the processing of an enzyme and on the kinetic and folding parameters
Authors: Ledent Philippe , Duez Colette , Vanhove Marc , Lejeune Annabelle , Fonzé Eveline , Charlier Paulette , Rhazi-Filali Fouzia , Thamm Iris , Guillaume Gilliane , Samyn Bart , Devreese Bart , Van Beeumen Jozef , Lamotte-Brasseur Josette , Frère Jean-Marie
Published: July 2002

The addition of a poly‐His C‐terminal extension, designed to facilitate the purification of the protein, to the β‐lactamase of a thermophilic Bacillus licheniformis strain modified the site of action of the signal peptidase. This resulted in the secretion of a protein with a different N‐terminus, showing that this type of protein engineering might not always be as `neutral' as generally assumed.
Aqueous extracts of cigarette smoke promote the oxidation of low density lipoprotein by peroxidases
Authors: Santanam Nalini , Sanchez Robert , Hendler Sheldon , Parthasarathy Sampath
Published: July 2002

Oxidation of low density lipoprotein (LDL) by cigarette smoke has been considered a potential mechanism by which smoking may promote atherosclerosis. We report in this study that cigarette smoke extract (CSE) inhibited copper‐induced oxidation of LDL suggesting the presence of antioxidants in CSE. It is currently believed that peroxidases may oxidize LDL in vivo and during such oxidations antioxidants become pro‐oxidants. Accordingly, when LDL was oxidized by peroxidase in the presence of CSE there was an increase in the oxidation of LDL. This is the first study suggesting that smoking may promote atherosclerosis by enhancing peroxidase‐catalyzed lipid peroxidation.
Bombesin and zinc enhance the synergistic mitogenic effects of insulin and phosphocholine by a MAP kinase‐dependent mechanism in Swiss 3T3 cells
Authors: Kiss Zoltan , Crilly Karan S. , Tomono Masahiro
Published: July 2002

Simultaneous treatment of serum‐starved (24 h) Swiss 3T3 cells with insulin (500 nM) and phosphocholine (PCho) (0.25–1 mM) resulted in synergistic stimulation of DNA synthesis via a mitogen activated protein (MAP) kinase‐independent rapamycin‐sensitive mechanism. Co‐treatment of cells with bombesin (10 nM) or zinc (25 μM) enhanced the combined mitogenic effects of insulin and PCho 2–3‐fold; however, in the presence of bombesin or zinc the combined effects of insulin and PCho were not inhibited by rapamycin. The potentiating effects of bombesin and zinc on insulin plus PCho‐induced DNA synthesis were accompanied by large stimulation of p42 MAP kinase activity. The results indicate that in Swiss 3T3 cell cultures, synergistic stimulation of DNA synthesis by extracellular insulin and PCho via a p42 MAP kinase‐dependent mechanism requires the presence of other growth regulatory agents, such as bombesin or zinc.
Extensive cellular uptake into endothelial cells of an amphipathic β‐sheet forming peptide
Authors: Oehlke Johannes , Krause Eberhard , Wiesner Burkhard , Beyermann Michael , Bienert Michael
Published: July 2002

Extensive internalization into endothelial cells has been found for a water soluble amphipathic 26‐mer β‐sheet peptide (FLUOS‐DPKGDPKGVTVTVTVTVTGKGDPKPD‐NH2; VT5). With the d‐Val13,d‐Thr14 di‐d‐amino acid analog of VT5 (DD‐VT5), exhibiting an identical primary structure but no propensity to adopt a β‐sheet conformation, only about 5% of the cellular uptake of VT5 was found. The mechanism of entry of VT5 into the cells remained unclear, but proved to be energy, temperature and pH dependent and, therefore, clearly distinct from that reported for helical amphipathic peptides. No detectable cytotoxicity, high solubility in water and the found extensive entry into endothelial cells make VT5 appear a good lead for developing new types of vectors for delivering oligonucleotides and peptides into intact cells.
CD146: biosynthesis and production of a soluble form in human cultured endothelial cells
Authors: Bardin N , Francès V , Combes V , Sampol J , Dignat-George F
Published: July 2002

We previously identified the S‐Endo 1‐associated antigen (CD146), an endothelial member of the immunoglobulin superfamily with a characteristic V‐V‐C2‐C2‐C2 Ig domain structure. In cultured human endothelial cells, we investigated its biosynthesis by immunoprecipitation and pulse‐chase labeling. CD146 was synthesized as a 100 kDa precursor form, which was processed into a 120 kDa mature form. In the culture media of endothelial cells, we observed a CD146 soluble form that was about 10 kDa smaller than cell‐associated CD146. In parallel with soluble forms of other members of the immunoglobulin superfamily, soluble CD146 could modulate and control the functions of the molecule.
Two‐step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop
Authors: Nycander Maria , Estrada Sergio , Mort John S , Abrahamson Magnus , Björk Ingemar
Published: July 2002

Stopped‐flow kinetics showed that the inhibition of the lysosomal cysteine proteinase, cathepsin B, by its endogenous inhibitor, cystatin C, occurs by a two‐step mechanism, in which an initial, weak interaction is followed by a conformational change. The initial interaction most likely involves binding of the N‐terminal region of the inhibitor to the proteinase. Considerable evidence indicates that the subsequent conformational change is due to the inhibitor displacing the occluding loop of the proteinase that partially obscures the active site. The presence of this loop, which allows the enzyme to function as an exopeptidase, thus complicates the inhibition mechanism, rendering cathepsin B much less susceptible than other cysteine proteinases to inhibition by cystatins.
Involvement of the IRF family transcription factor IRF‐3 in virus‐induced activation of the IFN‐β gene
Authors: Sato Mitsuharu , Tanaka Nobuyuki , Hata Naoki , Oda Eri , Taniguchi Tadatsugu
Published: July 2002

The virus‐induced activation of interferon α/β (IFN‐α/β) gene transcription is essential for host defense. The IFN‐β promoter is controlled primarily by the virus‐inducible enhancer elements, the IRF‐Es. Here we show that IRF‐3, an IRF family transcription factor, translocates to the nucleus from the cytoplasm upon virus infection in NIH/3T3 cells. The nuclear IRF‐3 is phosphorylated, interacts with the co‐activators CBP/p300, and binds specifically to the IFN‐β IRF‐E. Furthermore, overexpression of IRF‐3 causes a marked increase in virus‐induced IFN‐β mRNA expression. Thus, IRF‐3 is a candidate transcription factor mediating the activation of the IFN‐β gene.
Characterization of a gene encoding an abscisic acid‐inducible type‐2 lipid transfer protein from rice
Authors: Garcı́a-Garrido José Manuel , Menossi Marcelo , Puigdoménech Pére , Martı́nez-Izquierdo José Antonio , Delseny Michel
Published: July 2002

The cloning and sequence analysis of a novel gene that encodes a type 2 non‐specific lipid transfer‐like protein (LTP) from rice is reported. Sequence analysis revealed an ORF encoding a protein showing characteristics of the LTP proteins. However, rice LTP2 is more similar to heterologous LTPs than to rice LTP1, supporting the existence of two distinct families of plant LTPs.Ltp2mRNA is accumulated only in mature seeds. In vegetative tissues, mRNA was only detected after treatment with abscisic acid (ABA), mannitol or NaCl. Transient expression experiments that the 61 nucleotides upstream of the TATA box, containing two ACGT boxes and the motif I, are sufficient for ABA responsiveness of theLtpgene.
Endogenous extracellular purine nucleotides redirect α2‐adrenoceptor signaling
Authors: Åkerman Karl E.O , Näsman Johnny , Lund Per-Eric , Shariatmadari Ramin , Kukkonen Jyrki P
Published: July 2002

Many receptors coupled to inhibitory Go/Gi‐type G proteins often also produce stimulatory signals like Ca2+ mobilisation. When expressed in CHO cells the α2‐adrenoceptor subtypes α2A, α2B and α2C mobilised Ca2+. These responses were strongly reduced by the P2Y‐purinoceptor antagonist suramin. A large proportion of the total pool of purine nucleotides was found extracellularly. Removal of extracellular nucleotides with apyrase or by constant perfusion had a similar effect as suramin. These treatments did not affect the α2‐adrenoceptor‐mediated inhibition of cAMP production. This indicates that cells may be primed or their signaling pathways redirected towards Ca2+ mobilisation by `autocrine' release of nucleotides.
The Chlamydomonas reinhardtii MoCo carrier protein is multimeric and stabilizes molybdopterin cofactor in a molybdate charged form
Authors: Witte Claus-Peter , Igeño M.Isabel , Mendel Ralf , Schwarz Günter , Fernández Emilio
Published: July 2002

In Chlamydomonas reinhardtii, molybdopterin cofactor (MoCo) able to reconstitute active nitrate reductase (NR) with apoenzyme from the Neurospora crassa mutant nit‐1 was found mostly bound to a carrier protein (CP). This protein is scarce in the algal free extracts and has been purified 520‐fold. MoCoCP is a protein of 64 kDa with subunits of 16.5 kDa and an isoelectric point of 4.5. In contrast to free MoCo, MoCo bound to CP was remarkably protected against inactivation under both aerobic conditions and basic pH. MocoCP transferred active MoCo to apoNR in vitro without addition of molybdate, though reconstituted activity was 20% higher in the presence of molybdate. Incubation with tungstate specifically inhibited MoCoCP activity but had no effect on the activity of free MoCo released from milk xanthine oxidase. MoCoCP did not charge molybdate unless in the presence of N. crassa extracts. Our data support that MoCoCP stabilizes MoCo in an active form charged with molybdate to provide MoCo to apomolybdoenzymes.
Human nerve growth factor beta (hNGF‐β): mammary gland specific expression and production in transgenic rabbits
Authors: Coulibaly S , Besenfelder U , Fleischmann M , Zinovieva N , Grossmann A , Wozny M , Bartke I , Tögel M , Müller M , Brem G
Published: July 2002

Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF‐β) cDNA were generated. Expression of hNGF‐β mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF‐β was purified from the milk by a two‐step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF‐β depicted a molecular weight of 13 261 Da per subunit. The biological activity of the hNGF‐β was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF‐β demonstrated full biological activity when compared to commercial recombinant hNGF‐β.
Murine 12(R)‐lipoxygenase: functional expression, genomic structure and chromosomal localization1
Authors: Krieg Peter , Siebert Malte , Kinzig Andreas , Bettenhausen Rainer , Marks Friedrich , Fürstenberger Gerhard
Published: July 2002

A cDNA, recently cloned (by Krieg et al. (1998)) from mouse skin, was shown to encode a 12(R)‐lipoxygenase. When expressed in HEK cells, the recombinant protein converted methyl arachidonate into the corresponding 12‐HETE ester which was shown to be the R‐enantiomer by chiral phase chromatography. Neither arachidonic acid nor linoleic acid were substrates for the recombinant protein. The structure of the 12(R)‐lipoxygenase gene is unique among all animal lipoxygenases in that it is divided into 15 exons and 14 introns spanning approximately 12.5 kb. By interspecific backcross analysis, the 12(R)‐lipoxygenase gene was localized to the central region of mouse chromosome 11.
Evidence suggesting protein tyrosine phosphorylation in plants depends on the developmental conditions
Authors: Barizza Elisabetta , Lo Schiavo Fiorella , Terzi Mario , Filippini Francesco
Published: July 2002

Protein tyrosine phosphorylation plays a central role in a variety of signal transduction pathways regulating animal cell growth and differentiation, but its relevance and role in plants are controversial and still largely unknown. We report here that a large number of proteins from all plant subcellular fractions are recognized by recombinant, highly specific, anti‐phosphotyrosine antibodies. Protein tyrosine phosphorylation patterns vary among different adult plant tissues or somatic embryo stages and somatic embryogenesis is blocked in vivo by a cell‐permeable tyrosyl‐phosphorylation inhibitor, demonstrating the involvement of protein tyrosine phosphorylation in control of specific steps in plant development.
Gender differences in expression of organic cation transporter OCT2 in rat kidney
Authors: Urakami Yumiko , Nakamura Nobuhiko , Takahashi Kazushige , Okuda Masahiro , Saito Hideyuki , Hashimoto Yukiya , Inui Ken-ichi
Published: July 2002

The organic cation transporter (OCT) mediates translocation of various cationic molecules including drugs, toxins and endogenous substances. We examined gender differences in the expression of rat (r) OCT2 in the kidney. Slices and basolateral membrane vesicles of male rat kidney showed a higher transport activity for tetraethylammonium than those of female rat kidney. The expression levels of rOCT2 mRNA and protein in the kidney of males were much higher than those in females. There was no gender difference in mRNA expression of rOCT1 and rOCT3. These findings suggest that rOCT2 is responsible for the gender differences in renal basolateral membrane organic cation transport activity.
Fish can't see water: the need to humanize birth
Authors: Wagner M
Published: July 2002

Humanized birth puts the woman in the center and in control, focuses on community based primary maternity care with midwives, nurses and doctors working together in harmony as equals, and has evidence based services. Western, medicalized, high tech maternity care under obstetric control usually dehumanizes, often leads to unnecessary, costly, dangerous, invasive obstetric interventions and should never be exported to developing countries. Midwives and planned out‐of‐hospital births are perfectly safe for low‐risk births.
Mouse peripherin isoforms
Authors: Landon Françoise , Wolff Annie , de Néchaud Béatrice
Published: July 2002

Three distinct mRNAs have been shown to be produced by alternative splicing from the unique mouse peripherin gene. They generate three translation products, one major form, Pe—58, and two minor forms, Pe—56 which possess a shorter C‐terminal sequence, and Pe—61 in which an additional sequence has been inserted in the central rod domain (Landon et al., 1989, EMBO J. 8, 1719–1726). In this study, the simultaneous occurrence of multiple transcripts in murine nervous tissues and neuroblastoma cell lines was shown by PCR amplification of fragments overlapping the sites of alternative splicing. Recombinant peripherin isoforms were purified from E. coli expressing full‐length cDNAs. Rabbit antisera were raised against synthetic peptides mimicking parts of the two C‐terminal sequences and of the inserted sequence of Pe—61 and were immunoadsorbed until they became monoreactive. By western blot analysis, the peripherin isoforms were localised in neuroblastoma NB2a cell lysates and detergent insoluble fractions separated by two‐dimensional electrophoresis. In addition, each isoform was resolved into several charge variants. At the cellular level, each antibody decorated the filament array of the NB2a cells, suggesting the participation of the minor peripherin isoforms in the intermediate filament network.
Verbal perseveration after right-unilateral ECT
Authors: Adler G. , Achenbach C.
Published: July 2002

After electroconvulsive therapy (ECT), many patients experience a decrement in their mnestic capacity. We studied episodic memory in eight severely depressed patients treated with a course of right-unilateral ECT. For this purpose, a testing instrument was constructed by the authors. It was made of paper cards that held four pieces of information, namely a word, a number, a figure, and the color of the card. One of the cards was presented to the patients and the respective information was asked for on the subsequent day. Patients were tested every morning during the first two weeks of the ECT course. About half of the responses were correct. Patients did best in recalling the color; they did worst in recalling the number. Seven of the patients showed verbal perseverations. This is in accordance with the literature on perseveration in patients with neurologic deficits, especially in proactive-inhibitory tasks. Perseveration may be attributed to a deficit in selective attention, producing an arousal of irrelevant cues.
Sleep panic attacks in patients with panic disorder: the association with major depression
Authors: Ağargün MY , Kara H
Published: July 2002

The purpose of this study was to examine the relationship of sleep panic to major depression in patients with panic disorder. We found that the patients with sleep panic had a higher prevalence of major depression than subjects of other panic disorders.
Practical recommendations for the use of ACE inhibitors, beta‐blockers and spironolactone in heart failure: putting guidelines into practice
Authors: John McMurray , Alain Cohen‐Solal , Rainer Dietz , Eric Eichhorn , Leif Erhardt , Richard Hobbs , Aldo Maggioni , Ileana Pina , Jordi Soler‐Soler , Karl Swedberg
Published: July 2002

Surveys of prescribing in both hospitals and primary care have shown delays in translating improved survival data from clinical trials into clinical practice thereby denying patients the benefits of proven treatments, such as the angiotensin converting enzyme inhibitors. This may be due to unfamiliarity with clinical guidelines and concerns about adverse events. Recent trials have shown that substantial improvements in survival are associated with spironolactone and beta‐blocker therapy. In order to accelerate the uptake of these treatments, and to ensure that all eligible patients should receive the most appropriate medications, a clear and concise set of clinical recommendations has been prepared by a group of clinicians with practical expertise in the management of heart failure. The objective of these recommendations is to provide practical guidance for non‐specialists in order to support the implementation of evidenced‐based therapy for heart failure. These practical recommendations are meant to supplement rather than replace existing guidelines.
Development of a Sensing Array for Human Breath Analysis Based on SWCNT Layers Functionalized with Semiconductor Organic Molecules
Authors: Freddi Sonia , Emelianov Aleksei V. , Bobrinetskiy Ivan I. , Drera Giovanni , Pagliara Stefania , Kopylova Daria S. , Chiesa Maria , Santini Giuseppe , Mores Nadia , Moscato Umberto , Nasibulin Albert G. , Montuschi Paolo , Sangaletti Luigi
Published: May 2020

A sensor array based on heterojunctions between semiconducting organic layers and single walled carbon nanotube (SWCNT) films is produced to explore applications in breathomics, the molecular analysis of exhaled breath. The array is exposed to gas/volatiles relevant to specific diseases (ammonia, ethanol, acetone, 2‐propanol, sodium hypochlorite, benzene, hydrogen sulfide, and nitrogen dioxide). Then, to evaluate its capability to operate with real relevant biological samples the array is exposed to human breath exhaled from healthy subjects. Finally, to provide a proof of concept of its diagnostic potential, the array is exposed to exhaled breath samples collected from subjects with chronic obstructive pulmonary disease (COPD), an airway chronic inflammatory disease not yet investigated with CNT‐based sensor arrays, and breathprints are compared with those obtained from of healthy subjects. Principal component analysis shows that the sensor array is able to detect various target gas/volatiles with a clear fingerprint on a 2D subspace, is suitable for breath profiling in exhaled human breath, and is able to distinguish subjects with COPD from healthy subjects based on their breathprints. This classification ability is further improved by selecting the most responsive sensors to nitrogen dioxide, a potential biomarker of COPD.