bluuebunny/crossref_metadata_2025 huggingface dataset:
Thyroid hormones promote transcriptional activation of the nuclear gene coding for mitochondrial β‐F1ATPase in rat liver
Authors: Izquierdo José M. , Cuezva José M.
Published: July 2002

Thyroid hormones acutely regulate gene expression of the β‐catalytic subunit of the mitochondrial F1‐ATPase complex in the liver of hypothyroid rat neonates at either a transcriptional and/or post‐transcriptional level [(1990) J. Biol. Chem. 265, 9090‐9097]. Administration at birth of various thyroid hormone doses to hypothyroid newborn rats promote a rapid (1 h) increase in liver steady‐state amounts of both β‐F1‐ATPase protein and mRNA. Induction of the β‐F1‐ATPase mRNA is coincident with an elevation in gene transcription detected using nascent RNA chains synthesized by isolated nuclei. These results suggest that thyroid hormones induction of postnatal mitochondrial differentiation in the liver of hypothyroid rat neonates is mostly triggered by transcriptional regulation of β‐F1‐ATPase gene.
Purification of a vasoactive peptide related to lysyl‐bradykinin from trout plasma
Authors: Conlon J.Michael , Olson Kenneth R.
Published: July 2002

Incubation of plasma from the steelhead trout, Oncorhynchus mykiss with porcine pancreatic glandular kallikrein generated bradykinin‐like immunoreactivity. The primary structure of the immunoreactive peptide was established as: Lys‐Arg‐Pro‐Pro‐Gly‐Trp‐Ser‐Pro‐Leu‐Arg. This sequence shows two amino acid substitutions (Phe6→Trp and Phe9→Leu) compared with mammalian lysyl‐bradykinin (kallidin). Bolus intra‐arterial injection of the purified peptide produced a strong and sustained vasopressor response in the unanaesthetized trout. The data demonstrate that the kallikrein—kinin system predates the appearance of tetrapods and suggest a role for this system in cardiovascular regulation in fish.
Immunopurification of a subcomplex of the NAD(P)H‐plastoquinone‐oxidoreductase from the cyanobacterium Synechocystis sp. PCC6803
Authors: Berger Susanne , Ellersiek Ulrike , Kinzelt Dagmar , Steinmüller Klaus
Published: July 2002

An antibody against the NDH‐K subunit of the NAD(P)H‐dehydrogenase from the cyanobacterium Synechocystis sp. PCC6803 was used to isolate a subcomplex of the enzyme from Triton X‐100 solubilized total membranes by immunoaffinity chromatography. The isolated subcomplex consisted of seven major polypeptides with molecular masses of 43, 27, 24, 21, 18, 14 and 7 kDa. The amino‐terminal amino acid sequences of the polypeptides were determined. By comparing the sequences with the amino acid sequences deduced from DNA, three proteins were identified as NDH‐H (43 kDa), NDH‐K (27 kDa) and NDH‐I (24 kDa). A fourth subunit (NDH‐J, 21 kDa) was identified by Western blot analysis with an NDH‐J antibody.
Cardiac hypertrophy in juvenile visceral steatosis (jvs) mice with systemic carnitine deficiency
Authors: Horiuchi Masahisa , Yoshida Hiroki , Kobayashi Keiko , Kuriwaki Kazumi , Yoshimine Kosei , Tomomura Mineko , Koizumi Tsutomu , Nikaido Hiroko , Hayakawa Junichiro , Kuwajima Masamichi , Saheki Takeyori
Published: July 2002

We have reported the clinical and biochemical findings in juvenile visceral steatosis (jvs) mice with systemic carnitine deficiency. This paper is the first report about cardiomyopathy in jvs mice. Adult jvs mice (at the age of 2 3 months) show cardiac hypertrophy which is caused by enlargement of the cardiac muscle cell associated with increases of non‐collagen protein and DNA content. Carnitine administration (2 mg/head, twice a day, from 1 month of age) significantly suppresses the cardiac hypertrophy, showing that carnitine deficiency plays an important role in the development of the cardiac hypertrophy. The discovery of cardiac hypertrophy in carnitine‐deficient jvs mice will lead to clarification of the pathophysiology of cardiomyopathy in systemic carnitine deficiency in human beings.
Activity oscillations predicted for pyruvate dehydrogenase complexes
Authors: Selivanov V.A. , Zakrzhevskaya D.T. , Goldstein B.N.
Published: July 2002

A kinetic model for the pyruvate dehydrogenase complex is analyzed. The model takes into account intermediate channeling through the lipoyl network attached to the complex core, as well as inter‐related regulatory effects of protein X acetylation and enzyme phosphorylation. The model predicts undamped oscillations of enzyme activity.
Identification of a putative membrane‐interacting domain of CTP: Phosphocholine cytidylyltransferase from rat liver
Authors: Wieder Thomas , Geilen Christoph C. , Wieprecht Marcus , Becker Andreas , Orfanos Constantin E.
Published: July 2002

A putative membrane‐interacting domain of CTP:phosphocholine cytidylyltransferase (CT) was identified using two peptide‐specific antibodies. One antibody (SA2) was raised against the N‐terminus of CT (amino acid residues 1–17) and the other antibody (SA209) against an α‐helical domain of the enzyme (amino acid residues 247–257). Both antibodies quantitatively immunoprecipitated CT from rat liver cytosol and showed specificity towards CT when octylglucoside extracts of rat liver cytosol were assessed by Western blot analysis. However, further experiments revealed that the antibodies had different characteristics. Whereas the antibody directed against the N‐terminus of CT (SA2) did not influence CT/membrane interaction, the new antibody (SA209) against the α‐helical domain of the enzyme interfered with this interaction. Our results provide experimental evidence that the α‐helical domain (amino acid residues 228–287) of CT may serve as a membrane‐interacting domain.
Purification, and some molecular and enzymatic features of a novel ccb‐type cytochrome c oxidase from a microaerobic denitrifier, Magnetospirillum magnetotacticum
Authors: Tamegai Hideyuki , Fukumori Yoshihiro
Published: July 2002

A novel ccb‐type cytochrome c oxidase was purified from the magnetic bacterium, Magnetospirillum magnetotacticum MS‐1. The enzyme was composed of three subunits with M
r's of 43,000, 34,000 and 28,000, respectively, and contained 0.91 mol of protoheme, 2.0 mol of heme c and 0.70 g atom of copper per mol of minimal structural unit. One mol of enzyme oxidized 187 mol of horse heart ferrocytochrome c and 34.4 mol of M.
magnetotacticum ferrocytochrome c
550/s. The cytochrome c oxidase activity of the enzyme was 50% inhibited by 12 μM KCN. The enzyme seems to function as the terminal oxidase in microaerobic respiration.
Isolation and structural characterization of a novel peptide related to γ‐melanocyte stimulating hormone from the brain of the leech Theromyzon tessulatum
Authors: Salzet Michel , Wattez Christian , Bulet Philippe , Malecha Jean
Published: July 2002

This paper reports the purification of a novel pro‐opiomelanocortin derivative peptide (a γ‐melanocyte stimulating hormone‐like (γ‐MSH‐like) molecule) from the brain of the leech Theromyzon tessulatum. After reverse‐phase HPLC purification, the sequence of the γ‐MSH‐like peptide (YVMGHFRWDKFamide) was established by a combination of automated Edman degradation, electrospray mass spectrometry measurement, enzymatic treatment and co‐elution experiments in reverse‐phase HPLC with synthetic peptides.
Inhibition of reverse transcriptase of human immunodeficiency virus type 1 and chimeric enzymes of human immunodeficiency viruses types 1 and 2 by two novel non‐nucleoside inhibitors
Authors: Rubinek Tami , McMahon James B. , Hizi Amnon
Published: July 2002

We have studied the effects of two non‐nucleoside reverse transcriptase inhibitors (NNRTI), nitrophenyl phenyl sulfone (NPPS) and a potent derivative of oxathiin carboxanilide (UC‐38), on enzymatically active molecular chimeras composed of complementary segments of the reverse transcriptases (RTs) of human immunodeficiency virus type 1 (HIV‐1) and ‐2 (HIV‐2). The substances inhibit only the DNA polymerase activity of HIV‐1 RT with no effect on HIV‐2 RT. The results suggest that there is a protein segment located between residues 158 and 190 that is critical for the inhibition by both compounds. However, there is probably a second segment that resides between residues 192 and 202, as in the case of NPPS, or residues 203 and 224, as in the case of UC‐38, that is also crucial for the sensitivity of HIV‐1 RT to both inhibitors.
Secretory cleavage site of Alzheimer amyloid precursor protein is heterogeneous in Down's syndrome brain
Authors: Kametania Fuyuki , Tanaka Kikuko , Tokuda Takahiko , Ikeda Shu-ichi
Published: July 2002

Aβ (β/A4) is the major constituent of brain amyloid in Alzheimer's disease (AD), Down's syndrome (DS) and normal aged persons. This protein is presumably derived by normal proteolysis from a precursor protein (APP). In this study, C‐terminal fragments of APP in a Tris/Triton soluble fraction were partially purified from DS brain by heparin‐affinity and reverse phase chromatography, and analyzed by N‐terminal amino acid sequencing after SDS polyacrylamide gel electrophoresis and Western blotting. We found at least six different C‐terminal fragments including those with the entire Aβ region. These results suggest that secretory processing of APP is heterogeneous and generates amyloidogenic C‐terminal fragments.
The bacteriochlorophyll biosynthesis gene, bchM, of Rhodobacter sphaeroides encodes S‐adenosyl‐L‐methionine: Mg protoporphyrin IX methyltransferase
Authors: Gibson Lucien C.D. , Hunter C.Neil
Published: July 2002

The bchM gene of Rhodobacter sphaeroides has been sequenced and then overexpressed in E. coli producing a protein of M
r approximately 27,500. Cell‐free extracts of the transformed E. coli strain are able to methylate added Mg protoporphyrin, resulting in the formation of Mg protoporphyrin monomethyl ester. The identity of this product was verified by HPLC. The bchM gene product is therefore assigned to the methyltransferase step in bacteriochlorophyll biosynthesis.
A comparison and analysis of the toxicity and receptor binding properties of Bacillus thuringiensis CryIC ∂‐endotoxin on Spodoptera littoralis and Bombyx mori
Authors: Sanchis Vincent , Chaufaux Josette , Pauron David
Published: July 2002

The binding of l‐[35S]methionine in vivo labelled CryIC toxin to its receptor in brush border membrane vesicle (BBMV's) prepared from Spodoptera littoralis and Bombyx mori was studied. Both insect species were highly susceptible to the CryIC toxin in bioassays, B. mori being 7‐fold more sensitive to CryIC than S. littoralis (LC50's of 10 ng/cm2 and 70 ng/cm2, respectively). Competition and direct binding experiments revealed saturable high‐affinity binding sites on BBMV's from both insects which had similar binding characteristics for the CryIC toxin (K
d = 10 nM, B
max = 8 to 9 pmol/mg BBMV's and IC50 = 37 nM for both insect species). Thus a specific receptor for the CryIC toxin is present in both insect species and the 7‐fold greater potency of CryIC towards B. mori is not due to qualitative or quantitative differences in binding affinity or receptor site concentration. Dissociation experiments also indicated that the binding of [35S]CryIC to B. mori BBMV's is partially reversible.
Spectroscopic evidence for an intramolecular RNA triple helix
Authors: Klinck R. , Guittet E. , Liquier J. , Taillandier E. , Gouyette C. , Huynh-Dinh T.
Published: July 2002

A 29‐base RNA oligomer has been chemically synthesized and shown to form an intramolecular triple helix in solution at acidic pH. Assignment of the majority of the exchangeable proton NMR resonances demonstrated the Watson—Crick and Hoogsteen base pairings consistent with folding to form pyrimidine—purine—pyrimidine base triplets. FTIR spectroscopy provided independent evidence of base triplet formation, and indicated a predominately C3′‐endo sugar pucker. UV absorption as a function of temperature suggested monophasic melting behaviour, which was confirmed by NMR of the imino protons.
The 26S proteasome degrades mouse and yeast ornithine decarboxylase in yeast cells
Authors: Mamroud-Kidron Emanuelle , Kahana Chaim
Published: July 2002

Eukaryotic cells possess two high‐molecular‐mass proteases, the 700 kDa, 20S proteasome, as well as the even larger 1,400 kDa, 26S proteasome. It has been demonstrated that ornithine decarboxylase is degraded, in vitro, by the 26S proteasome that contains the 20S protease as its catalytic core, but not by the free 20S proteasome. Recently, by demonstrating severe inhibition of mouse and yeast ODC degradation in a mutant yeast cell line, defective in the chymotripsin‐like activity of the yeast 20S proteasome, we implicated the 20S proteasome in the degradation of ODC, in vivo, in yeast cells. Here we show that the degradation of ODC is also severely inhibited in the mutant yeast cell lines, cim3‐1 and cim5‐1 containing a specific lesion in subunits that are unique to the yeast 26S proteasome. We therefore, conclude, that as illustrated in vitro, also in intact cells, it is the 26S proteasome, not the free 20S proteasome, that degrades ODC. We also demonstrate, that while deficiency in the proteasome chymotrypsine‐like activity (in the yeast pre1‐1 mutant) inhibits the degradation of both yeast and mouse ODCS, deficiency in the peptidyl‐glutamyl‐peptide‐hydrolyzing (PGPH) activity inhibits only yeast ODC degradation. Similarly, we have noted that whereas the putative ATPase activity of both the CIM3 and CIM5 subunits is essential for the degradation of mouse ODC, only that of the CIM3 subunit is required for the degradation of yeast ODC. These results suggest differential utilization of individual proteasomal subunits in the recognition and degradation of individual short‐lived proteins.
Light‐dependent in vivo phosphorylation of an inhibitory subunit of cGMP‐phosphodiesterase in frog rod photoreceptor outer segments
Authors: Hayashi Fumio
Published: July 2002

In vivo phosphorylation of Pγ, an inhibitory subunit of cGMP‐phosphodiesterase of frog (Rana catesbeiana) photoreceptor rod outer segments, was investigated using a quick‐freezing technique and a newly developed method for the preparation of rod outer segments. Light‐dependent phosphorylation of Pγ was observed. Okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, enhanced the apparent incorporation of 32P into Pγ suggesting that Pγ is in equilibrium between phosphorylation and dephosphorylation. Neither phorbol ester, a potent activator of protein kinase C, nor changes in the extracellular Ca2+ concentration affected the in vivo phosphorylation of Pγ.
Cyclic AMP downregulates c‐myc expression by inhibition of transcript initiation in human B‐precursor Reh cells
Authors: Andersson Kristin Brevik , Taskén Kjetil , Blomhoff Heidi Kill
Published: July 2002

In the human pre‐B cell line Reh, activation of the cyclic AMP signal transduction pathway induces a rapid, transient 10‐fold down‐regulation of steady‐state c‐myc mRNA. We have investigated the mechanisms involved in this cAMP‐mediated regulation of c‐myc expression. Forskolin did not alter c‐myc mRNA stability. Initiation of c‐myc transcripts was strongly inhibited after 1 h of forskolin treatment, as measured by nuclear run‐on assays. Reinitiation of c‐myc transcription was apparent after 3–4 h, and full transcriptional elongation was detected after 8 h of forskolin treatment. These data suggest that cyclic AMP downregulates c‐myc expression by inhibition of transcriptional initiation.
Light is required for efficient translation elongation and subsequent integration of the D1‐protein into Photosystem II
Authors: van Wijk Klaas J. , Eichacker Lutz
Published: July 2002

The light dependence of translation and successive assembly of the D1 reaction center protein into Photosystem II subcomplexes was followed in fully developed chloroplasts isolated from the dark phase of diurnally grown spinach. The incorporation of synthesized D1 protein into Photosystem II (PSII) was analyzed by fractionation of radiolabeled unassembled protein and PSII (sub)complexes on sucrose density gradients. The ribosomes with attached nascent chains were recovered as pellets in the same gradients, and nascent chains of the D1 protein were immunoprecipitated. The analysis showed that absence of light during translation leads to an increased accumulation of polysome‐bound D1 translation intermediates, indicating that light is required for efficient elongation of the D1 protein. The accumulation of the D1 protein and CP43 decreased three‐fold in darkness, whereas accumulation of the D2 reaction center protein was not affected by light. In addition, light was also required for efficient incorporation of the D1 protein into the PSII core complex. In darkness, the newly synthesized D1 protein accumulated predominantly as unassembled protein or in PSII subcomplexes smaller than 100 kDa.
MBA1 encodes a mitochondrial membrane‐associated protein required for biogenesis of the respiratory chain
Authors: Rep Martijn , Grivell Leslie A.
Published: July 2002

The yeast MBA1 gene (ulti‐copy ypass of
FG3) is one of three genes whose overexpression suppresses afg3‐null and rcal‐null mutations. Bypass of AFG3 and RCA1, whose products are essential for assembly of mitochondrial inner membrane enzyme complexes, suggests a related role for MBA1. The predicted translation product is a 30 kDa hydrophilic protein with a putative mitochondrial targeting sequence and no homology to any sequence in protein or EST databases. Gene disruption leads to a partial respiratory growth defect, which is more pronounced at temperatures above 30°C. Concomitantly, amounts of cytochromes b and aa3 are reduced. A C‐terminal c‐myc‐tagged MBA1 gene product is functional and is found associated with the mitochondrial inner membrane, from which it can be extracted by carbonate, but not by high salt. These observations give further support to a role of MBA1 in assembly of the respiratory chain.
Involvement of phospholipase D in store‐operated calcium influx in vascular smooth muscle cells
Authors: Walter Michael , Tepel Martin , Nofer Jerzy-Roch , Neusser Markus , Assmann Gerd , Zidek Walter
Published: July 2002

In non‐excitable cells, sustained intracellular Ca2+ increase critically depends on influx of extracellular Ca2+. Such Ca2+ influx is thought to occur by a ‘store‐operated’ mechanism, i.e. the signal for Ca2+ entry is believed to result from the initial release of Ca2+ from inositol 1,4,5‐trisphosphate‐sensitive intracellular stores. Here we show that the depletion of cellular Ca2+ stores by thapsigargin or bradykinin is functionally linked to a phosphoinositide‐specific phospholipase D (PLD) activity in cultured vascular smooth muscle cells (VSMC), and that phosphatidic acid formed via PLD enhances sustained calcium entry in this cell type. These results suggest a regulatory role for PLD in store‐operated Ca2+ entry in VSMC.
Agonist‐induced long‐term desensitization of the human prostacyclin receptor
Authors: Nilius Sigrid M. , Hasse Andreas , Kuger Petra , Schrör Karsten , Meyer-Kirchrath Jutta
Published: July 2002

Phosphorylation of the human prostacyclin (PGI2) receptor (hIP‐R) by diacylglycerol‐regulated protein kinase C (PKC) has been reported to be responsible for its rapid desensitization in HEK293 cells. In this study we demonstrate, that human fibroblasts reveal a much slower hIP‐R desensitization kinetics, which was neither affected by stimulation nor inhibition of PKC by either phorbol 12‐myristate‐13‐acetate or GF‐109203X suggesting a different cellular mechanism. Although agonist‐promoted sequestration of a C‐terminally green fluorescent protein‐tagged hIP‐R was demonstrated, it did not account for the long‐term desensitization. Concanavalin A did not abolish, but accelerated receptor desensitization kinetics. Resensitization of hIP‐R involved receptor recycling and/or de novo synthesis of receptor protein, depending on the duration of prior desensitization. This is the first study investigating the mechanisms of hIP‐R desensitization in intact human cells naturally expressing hIP‐R. Our data suggest, that a hitherto unknown mechanism of hIP‐R long‐term desensitization, which is independent of receptor phosphorylation by conventional and novel type PKC isoforms or endocytosis, is a key event in regulating the cellular responsiveness to PGI2.
A conserved proline residue is present in the transmembrane‐spanning domain of Tom7 and other tail‐anchored protein subunits of the TOM translocase
Authors: Allen Renae , Egan Billie , Gabriel Kipros , Beilharz Traude , Lithgow Trevor
Published: July 2002

The TOM translocase consists of several integral membrane proteins organised around the channel forming protein Tom40. Here we show that one of these protein subunits, Tom7, is a tail‐anchored protein. The carboxy‐terminal 33 amino acids of Tom7 contain the information for targeting the protein to the mitochondrial outer membrane, and a conserved proline residue within the transmembrane segment is required for efficient targeting of Tom7 to the outer membrane. An equivalent proline residue is important in targeting each of the other three tail‐anchored proteins that associate with Tom40 to form the core of the TOM translocase.
Mechanism of hydride transfer during the reduction of 3‐acetylpyridine adenine dinucleotide by NADH catalyzed by the pyridine nucleotide transhydrogenase of Escherichia coli
Authors: Bragg Philip D.
Published: July 2002

The pyridine nucleotide transhydrogenase is a proton pump which catalyzes the reversible transfer of a hydride ion equivalent between NAD+ and NADP+ coupled to translocation of protons across the cytoplasmic membrane. The enzyme also catalyzes the reduction of the NAD+ analog 3‐acetylpyridine adenine dinucleotide (AcPyAD+) by NADH. It has been proposed (Hutton et al. (1994) Eur. J. Biochem. 219, 1041–1051) that this reaction requires NADP(H) as an intermediate. Thus, NADP+ bound at the NADP(H)‐binding site on the transhydrogenase would be reduced by NADH and reoxidized by AcPyAD+ binding alternately to the NAD(H)‐binding site. The reduction of AcPyAD+ by NADPH would be a partial reaction in the reduction of AcPyAD+ by NADH. Using cytoplasmic membrane vesicles from mutants having elevated activities for transhydrogenation of AcPyAD+ by NADH in the absence of added NADP(H), the kinetics of reduction of AcPyAD+ by NADH and NADPH have been compared. The K

m
values for the reductants NADPH and NADH over a range of mutants, and for the non‐mutant enzyme, differed to a much lesser degree than the K

m
for AcPyAD+ in the two reactions. The K

m

AcPyAD
values for the transhydrogenation of AcPyAD+ by NADH were over an order of magnitude greater than those for the transhydrogenation of AcPyAD+ by NADPH. It is unlikely that AcPyAD+ binds at the same site in both reactions. A plausible explanation is that this substrate binds to the NADP(H)‐binding site for transhydrogenation by NADH. Thus, a hydride equivalent can be transferred directly between NADH and AcPyAD+ under these conditions.
Carbohydrate induced modulation of cell membrane. VI. Binding of exogenous lectin induces susceptibility of erythrocytes to free radical damage: a spin label study
Authors: Hajela Krishnan , Pande Abhay H , Sumati
Published: July 2002

The oxidation of erythrocyte membrane has been widely used as a model to study the damage of biomembranes by free radicals. Whether binding of lectin to erythrocytes has any effect on peroxidant injury has never been studied. This study reports for the first time that crosslinking of erythrocyte surface glycoprotein by an exogenous lectin significantly enhances the susceptibility to membrane damage by free radicals, as evidenced by the increase in membrane fluidity measured by EPR using spin label and the increase in the amount of oxyhemoglobin liberated due to cell lysis.
Asp‐193 and Glu‐218 of subunit II are involved in the Mn2+‐binding of Paracoccus denitrificans cytochrome c oxidase
Authors: Witt Heike , Wittershagen Axel , Bill Eckhard , Kolbesen B.O , Ludwig Bernd
Published: July 2002

Cytochrome c oxidase contains a binding site for a non‐redox‐active metal at the interface of subunits I and II, usually a magnesium ion. In Paracoccus denitrificans oxidase, typically 20% may be replaced by manganese, using standard growth media. Site‐directed mutants were constructed in subunit II (D193N and E218Q), and the isolated enzymes analyzed by total‐reflection X‐ray fluorescence spectrometry and EPR. Both mutants show a strong reduction of the manganese stoichiometry and a diminished electron transfer activity, demonstrating that D193 and E218 are involved in the binding of a manganese/magnesium ion in this site.
Highly efficient control of iron‐containing nitrile hydratases by stoichiometric amounts of nitric oxide and light
Authors: Bonnet Didier , Artaud Isabelle , Moali Catherine , Pétré Dominique , Mansuy Daniel
Published: July 2002

The reaction of two iron‐containing nitrile hydratases (NHase) with NO has been studied: NHase from Rhodococcus sp. R312, which is probably similar to the photosensitive N771 NHase, and the new NHase from Comamonas testosteroni NI1 whose aminoacid sequence is quite different from those of BR312 and N771 NHases. Both enzymes are equally inactivated after addition of stoichiometric amounts of NO added as an anaerobic solution or produced in situ under physiological conditions by a rat brain NO‐synthase. Both enzymes are reactivated by photoirradiation, and two cycles of NO inactivation/photoactivation can be performed without significant loss of activity. Both iron‐containing NHases have a high affinity for NO, similar to that of methemoglobin.
The β1,6‐GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site‐specific branch formation on a long polylactosamine backbone
Authors: Helin J , Penttilä L , Leppänen A , Maaheimo H , Lauri S , Costello C.E , Renkonen O
Published: July 2002

We find that the β1,6‐GlcNAc transferase activity present in hog gastric mucosal microsomes converts the linear pentasaccharide GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAc (1) in a site‐specific way to the branch‐bearing hexasaccharide GlcNAcβ1‐3(GlcNAcβ1‐6)Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAc (2). The product is a positional isomer of GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3(GlcNAcβ1‐6)Galβ1‐4GlcNAc (3), reportedly formed from 1 by another polylactosamine β1,6‐GlcNAc transferase activity present in human serum (Leppänen et al., Biochemistry, 30 (1991) 9287). Combined use of the two kinds of activities gave in the present experiments the heptasaccharide GlcNAcβ1‐3(GlcNAcβ1‐6)Galβ1‐4GlcNAcβ1‐3(GlcNAcβ1‐6)Galβ1‐4GlcNAc (4), in which one of the branches occupies the position of the branch in 2 and the other the position of the branch in 3.
Mouse sperm patch‐clamp recordings reveal single Cl− channels sensitive to niflumic acid, a blocker of the sperm acrosome reaction
Authors: Espinosa F , de la Vega-Beltrán J.L , López-González I , Delgado R , Labarca P , Darszon A
Published: July 2002

Ion channels lie at the heart of gamete signaling. Understanding their regulation will improve our knowledge of sperm physiology, and may lead to novel contraceptive strategies. Sperm are tiny (∼3 μm diameter) and, until now, direct evidence of ion channel activity in these cells was lacking. Using patch‐clamp recording we document here, for the first time, the presence of cationic and anionic channels in mouse sperm. Anion selective channels were blocked by niflumic acid (NA) (IC50=11 μM). The blocker was effective also in inhibiting the acrosome reaction induced by the zona pellucida, GABA or progesterone. These observations suggest that Cl− channels participate in the sperm acrosome reaction in mammals.
Importance of the N‐glycan in the V3 loop of HIV‐1 envelope protein for CXCR‐4‐ but not CCR‐5‐dependent fusion
Authors: Nakayama Emi E , Shioda Tatsuo , Tatsumi Masashi , Xin Xiaomi , Yu Deshan , Ohgimoto Shinji , Kato Atsushi , Sakai Yuko , Ohnishi Yukano , Nagai Yoshiyuki
Published: July 2002

The V3 region of HIV‐1 envelope protein possesses a single N‐linked sugar chain, which is conserved in most HIV‐1 strains. We studied its role in the life cycle of HIV‐1 strains with different co‐receptor usage. Removal of the glycan appeared to cause a marked reduction of CXCR‐4‐ but not CCR‐5‐dependent virus entry. A basic amino acid substitution at the 11th position of V3 markedly compensated for the removal of the N‐glycan. These results indicate that the N‐glycan plays an important role for CXCR‐4‐dependent virus entry and that this role is exerted in a particular context of the peptide backbone.
Purification and characterization of alkaline phosphatase containing phosphotyrosyl phosphatase activity from the bacterium Prevotella intermedia
Authors: Ansai Toshihiro , Awano Shuji , Chen Xiaochi , Fuchi Tomoko , Arimoto Takafumi , Akifusa Sumio , Takehara Tadamichi
Published: July 2002

A novel alkaline phosphatase, designated PiALP, has been purified and characterized from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease. The enzyme was a homodimer of apparently identical subunits of M
r 54 kDa. Thiol‐reducing agents completely inhibited the purified enzyme. The enzyme was highly stable even at 80°C. It exhibited substantial activity against tyrosine‐phosphate‐containing Raytide. The phosphatase activity was sensitive to orthovanadate and Zn2+ but highly resistant to okadaic acid. The amino acid sequence of peptides derived from PiALP showed a high degree of identity (65%) with alkaline phosphatases from Zymomonas mobilis and Synechococcus. The present results imply that PiALP might represent a new family of alkaline phosphotyrosyl phosphatases which has not been described previously.
All three surface tryptophans in Type IIa cellulose binding domains play a pivotal role in binding both soluble and insoluble ligands
Authors: Nagy Tibor , Simpson Peter , Williamson Michael P. , Hazlewood Geoffrey P. , Gilbert Harry J. , Orosz Laszlo
Published: July 2002

The three surface tryptophans of the Type IIa cellulose binding domain of Pseudomonas fluorescens subsp. cellulosa xylanase A (CBDXYLA) were independently mutated to alanine, to create the mutants W13A, W49A and W66A. The three mutant proteins were purified, and their capacity to bind to a variety of ligands was determined. The mutant proteins have native‐like structures but exhibited much weaker affinity for crystalline and amorphous cellulose and for cellohexaose than the wild type. These data indicate that all three tryptophans are important for binding to cellulose, and support a model in which the three tryptophans form an aromatic strip on the surface of the protein that binds to a single cellulose.
Mapping of MCP‐1 functional domains by peptide analysis and site‐directed mutagenesis
Authors: Steitz Susan A , Hasegawa Ko , Chiang Shiu-Lan , Cobb Ronald R , Castro Mary A , Lobl Thomas J , Yamada Masaki , Lazarides Elias , Cardarelli Pina M
Published: July 2002

Monocyte chemoattractant protein‐1 (MCP‐1) is a member of the β chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP‐1 protein which are important for its biological activity, we have synthesized domain‐specific peptides and tested their ability to antagonize MCP‐1 binding and chemotaxis in THP‐1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13–35 inhibits MCP‐1 binding and chemotactic activity, while peptides representing the amino‐terminus (amino acids 1–10), second loop (amino acids 37–51), and carboxy‐terminus (amino acids 56–71) of MCP‐1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C‐terminus of the peptide by amidation increases the activity of this peptide to block MCP‐1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP‐1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP‐1 recombinant proteins. While baculovirus produced wild type and R18A MCP‐1 proteins are indistinguishable in their ability to induce THP‐1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP‐1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP‐1 will assist in the design of MCP‐1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.
A putative HCO−3 transporter in the cyanobacterium Synechococcus sp. strain PCC 79421
Authors: Bonfil David J , Ronen-Tarazi Michal , Sültemeyer Dieter , Lieman-Hurwitz Judy , Schatz Daniella , Kaplan Aaron
Published: July 2002

Cyanobacteria possess an inducible mechanism which enables them to concentrate inorganic carbon (Ci) within the cells. An inactivation library was used to raise the high‐CO2‐requiring mutant of Synechococcus PCC 7942, IL‐2, impaired in HCO−
3 transport. Analysis of the relevant genomic DNA detected several modifications, probably due to the single crossover recombination, leading to inactivation of ORF467 (designated ictB) in IL‐2. IctB contains 10 trans‐membrane regions and is homologous to several transport‐related proteins from various organisms. Kinetic analyses of HCO−
3 uptake in the wild type and IL‐2 suggested the presence of two or three HCO−
3 carriers exhibiting different affinities to HCO−
3.
cis‐Parinaric acid is a ligand for the human peroxisome proliferator activated receptor γ: development of a novel spectrophotometric assay for the discovery of PPARγ ligands
Authors: Palmer Colin N.A , Wolf C.Roland
Published: July 2002

Peroxisome proliferator activated receptor γ (PPARγ) is the subject of intense investigation as a target for drugs against diabetes, atherosclerosis and cancer. For this reason there is considerable interest in the spectrum of compounds that bind this receptor. In this paper we have identified cis‐parinaric acid (CPA) as a novel hPPARγ ligand. The binding of this fatty acid to the receptor increases its fluorescence and causes a shift in the UV spectrum. This spectral shift is reversible by competition with other known ligands for PPARγ. This report represents the first direct demonstration of a fatty acid binding to PPARγ.
The effect of the lipid A analog E5531 on phospholipid membrane properties
Authors: Asai Yasuyuki , Iwamoto Kiyoshi , Watanabe Sumio
Published: July 2002

In order to determine the effect of the lipid A analog, E5531, on phospholipid membranes, we used dipalmitoylphosphatidylcholine (DPPC) and investigated the physicochemical interaction between E5531 and DPPC membranes. E5531 and DPPC are miscible in the bulk phase at 25°C. Within the E5531 mole fraction range (X
E5531) of 0–0.5, E5531 decreased the zeta potentials of DPPC membranes but did not change the size of the DPPC liposomes. E5531/DPPC mixtures formed liposome‐like structures. E5531 increased the fluidity of the DPPC membrane and decreased pyrene diffusion in the membrane. E5531 decreased the phase transition temperature and the cooperative interactions between DPPC molecules. These effects of E5531 on phospholipid membranes were different from those of lipid A from Escherichia coli and Salmonella minnesota.
FTDP‐17 mutations N279K and S305N in tau produce increased splicing of exon 10
Authors: Hasegawa Masato , Smith Michael J , Iijima Masaaki , Tabira Takeshi , Goedert Michel
Published: July 2002

Missense mutations and intronic mutations in the tau gene cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP‐17). Known missense mutations reduce the ability of tau to promote microtubule assembly. Intronic mutations lead to increased mRNA splicing of the alternatively spliced exon 10, resulting in an overproduction of tau isoforms with four microtubule‐binding repeats. We show here that the recently identified FTDP‐17 missense mutations N279K and S305N do not reduce the ability of tau to promote microtubule assembly. Instead they lead to increased splicing of exon 10, like the intronic mutations. The N279K and S305N mutations define a class of missense mutations in tau whose primary effects are at the RNA level.
Structural changes in the recombinant, NADP(H)‐binding component of proton translocating transhydrogenase revealed by NMR spectroscopy
Authors: Quirk Philip G. , Jeeves Mark , Cotton Nick P.J. , Smith John K. , Jackson Baz J.
Published: July 2002

We have analysed 1H, 15N‐HSQC spectra of the recombinant, NADP(H)‐binding component of transhydrogenase in the context of the emerging three dimensional structure of the protein. Chemical shift perturbations of amino acid residues following replacement of NADP+ with NADPH were observed in both the adenosine and nicotinamide parts of the dinucleotide binding site and in a region which straddles the protein. These observations reflect the structural changes resulting from hydride transfer. The interactions between the recombinant, NADP(H)‐binding component and its partner, NAD(H)‐binding protein, are complicated. Helix B of the recombinant, NADP(H)‐binding component may play an important role in the binding process.
Defective binding of IRFs to the initiator element of interleukin‐1β‐converting enzyme (ICE) promoter in an interferon‐resistant Daudi subline
Authors: Iwase Satsuki , Furukawa Yusuke , Kikuchi Jiro , Saito Shinobu , Nakamura Mitsuru , Nakayama Ritsuko , Horiguchi-Yamada Junko , Yamada Hisashi
Published: July 2002

To investigate mechanisms of interferon (IFN) resistance, we have established an IFN‐resistant Daudi subline (Daudires), which is 1×104 times more resistant to IFN‐α than parental cells. Among the IFN‐inducible genes examined, only ICE mRNA expression was deficient in Daudires cells. We then analyzed the regulatory mechanisms of ICE transcription, and found that IFN‐induced activation of the ICE promoter was dependent on the binding of IRFs to its initiator (Inr) element. Inr binding of IRFs was markedly diminished in Daudires cells, and forced expression of IRF‐1 was able to activate the ICE promoter to the level of parental cells. These results suggest that IRFs and their target genes, as represented by ICE in this study, are involved in IFN resistance.
Kinetics of peroxidases in guinea pig bone marrow under immunostimulation
Authors: Keyhani E , Zarei M.A , Lashgarblooki-Livani T
Published: July 2002

Eosinophil peroxidase and myeloperoxidase play an important role in the host defense. Both enzymes are present in bone marrow, synthesized by blood progenitor cells. This research investigated the kinetic properties of peroxidases under immunostimulation in guinea pig bone marrow. Results suggest that there are at least two myeloperoxidase isozymes and at least three eosinophil peroxidase isozymes in guinea pig bone marrow and that some of these isozymes are expressed upon immunostimulation.
Diastereoselective reduction of protein‐bound methionine sulfoxide by methionine sulfoxide reductase
Authors: Sharov Victor S. , Ferrington Deborah A. , Squier Thomas C. , Schöneich Christian
Published: July 2002

Methionine sulfoxide (MetSO) in calmodulin (CaM) was previously shown to be a substrate for bovine liver peptide methionine sulfoxide reductase (pMSR, EC 1.8.4.6), which can partially recover protein structure and function of oxidized CaM in vitro. Here, we report for the first time that pMSR selectively reduces the D‐sulfoxide diastereomer of CaM‐bound L‐MetSO (L‐Met‐D‐SO). After exhaustive reduction by pMSR, the ratio of L‐Met‐D‐SO to L‐Met‐L‐SO decreased to about 1:25 for hydrogen peroxide‐oxidized CaM, and to about 1:10 for free MetSO. The accumulation of MetSO upon oxidative stress and aging in vivo may be related to incomplete, diastereoselective, repair by pMSR.
Increased leukotriene A4 hydrolase expression in the heart of angiotensin II‐induced hypertensive rat
Authors: Ishizaka Nobukazu , Nakao Akihide , Ohishi Nobuya , Suzuki Masatoshi , Aizawa Toru , Taguchi Jun-ichi , Nagai Ryozo , Shimizu Takao , Ohno Minoru
Published: July 2002

Leukotriene A4 (LTA4) hydrolase is essential for the conversion of LTA4 to LTB4, an inflammatory lipid mediator. We investigated whether LTA4 hydrolase was regulated in the heart by angiotensin II (ang II) infusion. Continuous ang II infusion via an osmotic minipump for up to 7 days upregulated mRNA and protein levels of LTA4 hydrolase (∼3.5‐fold of control) in the heart in a pressor‐dependent manner. Immunohistochemistry demonstrated intense LTA4 hydrolase staining in the myofibroblast as well as migrated monocytes/macrophages. These data suggest that the cardiac LTA4 hydrolase‐LTB4 system plays a positive role in the promotion of cardiac inflammation in hypertension.
Different transcriptional properties of mSim‐1 and mSim‐2
Authors: Moffett Peter , Pelletier Jerry
Published: July 2002

The mSim‐1 and mSim‐2 gene products are mammalian homologues of the Drosophila Sim gene. The dSim gene product transactivates through a DNA binding site known as the CNS midline enhancer (CME) element. We have investigated the transcriptional properties of mSIM‐1 and mSIM‐2 mediated through the CME element in concert with their dimerization partners, ARNT and ARNT‐2. The mSIM‐1/ARNT heterodimer transactivates reporter constructs via the ARNT carboxy‐terminus. However, mSIM‐2 quenches ARNT transactivation. We find that mSIM‐2 competes with mSIM‐1 for binding to ARNT, suggesting a possible antagonism between these transcription factors.
Integrated approach to women's health
Authors: Pinotti J.A. , Tojal M.L.V.A.A. , Nisida A.C.T. , Pinotti M.
Published: July 2002

The objective of this study was to demonstrate the importance of the practical application of the concept of reproductive health in São Paulo, Brazil, from 1991 to 1998 at the Pérola Byington Hospital with a new model of Primary Health Care (PHC) in which 2000 women/day, separated into two groups and over 45 years, was attended by nurses trained to detect the most frequently occurring gynecological problems supervised by doctors, who finalized the visit of each patient. The results demonstrated the advantages and viability of this strategy and also the bad health conditions of the women. Based on the high incidence of different kind of diseases detected, programs were set up for the diagnosis and treatment of gynecological cancer, STD, AIDS, hypertension, diabetes, etc. The results of two of these control programs, cervical cancer and breast cancer, demonstrated a significant increase in the diagnosis of early lesions. An economic study demonstrated an obvious and significant impact of this model not only in saving lives, but also in decreasing financial expenses in health.
Prenatal diagnosis of Osteogenesis imperfecta type II
Authors: Tongsong T. , Wanapirak C. , Siriangkul S.
Published: July 2002

Objective: To characterize the prenatal sonographic features of Osteogenesis imperfecta (OI) type II. Design: Descriptive (case series). Setting: Department of Obstetrics and Gynecology, Faculty of Medicine, Maharaj Nakorn Chiang Mai Hospital, Chiang Mai University. Subjects: Six fetuses with prenatal diagnosis of OI were evaluated. Results: Six fetuses were prenatally diagnosed as OI type II in five mothers without familial history of the disease. One mother had two consecutive pregnancies complicated with this condition. The first five cases were classified as OI type IIA, while the last one was OI type IIB. All of subtype A exhibited typical triad of bone shortening, diffuse hypomineralization and multiple fractures of long bones including beaded ribs whereas the subtype B showed shortening of only femurs, normal bone echodensity and isolated fractures of long bones. The postnatal radiography and autopsy confirmed the prenatal diagnosis in all cases. Other findings may occasionally be found, including polyhydramnios, oligohydramnios, hydrop fetalis and small for gestational age. Conclusion: The triad of bone shortening, decreased bone density and numerous fractures including beaded ribs permits a confident diagnosis of OI type IIA. Furthermore, sonographic features may differentiate the subtype of OI type II, depending on degree of bone shortening and echodensity.
Today's treatments: how do you choose?
Authors: Barlow D
Published: July 2002

Objective: To provide a view on how the clinician can select appropriate treatment when managing individual patients with endometriosis. Methods: Review of randomized controlled trials and personal experience. Results: The main determinants of therapy choice are personal experience and patient acceptability. Placebo‐controlled trial results support the use of naproxen, dydrogesterone, danazol and leuprolide for pain relief. Laser laparoscopy is more effective than expectant management for pain relief. In direct comparisons, oral contraceptives, Zoladex, danazol, gestrinone, nafarelin and leuprolide have similar efficacies in relieving pain, but have different side‐effect profiles. In controlled trials, only laser laparoscopy was shown to improve fertility in minimal/mild disease. The physiological response of bone metabolism to GnRH agonist therapy should be seen in context and the place of add‐back regimens understood. The general medical history of the patient must be considered when choosing therapy. Conclusions: The clinician must provide the patient with appropriate information on the treatment options to allow her to make an informed choice.
Prenatal protein restriction alters synaptic mechanisms of callosal connections in the rat visual cortex
Authors: Soto‐Moyano R. , Alarcón S. , Belmar J. , Kusch C. , Pérez H. , Ruiz S. , Hernández A.
Published: July 2002

Mild prenatal protein malnutrition, induced by reduction of the casein content of the maternal diet from 25 to 8%, calorically compensated by the addition of excess carbohydrates, leads to so‐called “hidden” malnutrition in the rat. This form of malnutrition results in normal body and brain weights of pups at birth, but in significant alterations of their central nervous system neurochemical profiles. Since severe forms of prenatal malnutrition induce morpho‐functional deficits on callosal interhemispheric communication together with brain neurochemical disturbances, we evaluated, in rats born from mothers submitted to an 8% casein diet, the potassium‐induced release of [3H]‐noradrenaline in visual cortex slices, as well as functional properties of callosal‐cortical synapses by determining cerebral cortical excitability to callosal inputs and fatigability and temporal summation of transcallosal evoked responses. Rats born from mothers submitted to a 25% casein diet served as controls. At birth prenatally malnourished pups had significantly higher cortical percent net noradrenaline release (14.79±1.11) than controls (9.14±1.26). At 45–50 days of age, rehabilitated previously malnourished rats showed, when compared to controls : (i) significantly reduced percent net noradrenaline release in the visual cortex (4.50±0.52 vs 11.31±1.14) ; (ii) decreased cortical excitability to callosal inputs as revealed by significantly increased chronaxie (607.2±82.8 μs vs 351.3±47.7 μs) ; (iii) enhanced fatigability of transcallosal evoked responses as revealed by significantly decreased stimulus frequency required to fatigate the responses (4.9±0.8 Hz vs 9.2±1.3 Hz) ; and (iv) decreased ability of callosal‐cortical synapses to perform temporal summation, as revealed by significantly reduced percent response increment to double‐shock (54.2±6.2 vs 83.0±11.0, for a 3.2‐ms interstimulus time interval). These changes, resulting from mild prenatal protein restriction, are discussed in relationship to developmental processes leading to the formation of synaptic contacts between callosal axons and their appropriate cortical target during perinatal age.