bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of HIV‐1 wild‐type protease and characterization of proteolytically inactive HIV‐1 protease mutants by pepstatin A affinity chromatography
Authors: Wondrak Ewald M. , Louis John M. , Mora Peter T. , Oroszlan Stephen
Published: July 2002

Recombinant wild‐type protease of human immunodeficiency virus, type [(HIV‐1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88‐fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/μg. Two proteolytically inactive HIV‐1 mutant proteases (Arg‐87 → Lys; Asn‐88 → Glu) were found to bind to pepstatin A agarose, and they were purified as the wild‐type protease. A third mutant protease (Arg‐87 → Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV‐1 proteases.
The primary structure of a PYY‐related peptide from chicken intestine suggests an anomalous site of cleavage of the signal peptide in preproPYY
Authors: Conlon J.Michael , O'Harte Finbarr
Published: July 2002

Although the amino acid sequence of members of the pancreatic polypeptide (PP)‐family of regulatory peptides has been poorly conserved during vertebrate evolution, the overall length of the peptides (36 amino acid residues) has remained constant. Nucleotide sequence analysis of cloned cDNAs and/or genomic fragments has shown the PP‐related sequence immediately follows the signal peptide in the prepropeptides. A peptide tyrosine—tyrosine (PYY)‐related peptide with 37 residues has been isolated from the chicken intestine, and its primary structure was established as: Ala‐Tyr‐Pro‐Pro‐Lys‐Pro‐Glu‐Ser‐Pro‐Gly10‐Asp‐Ala‐Ala‐Ser‐P ro‐Glu‐Glu‐Ile‐Ala‐Gln20‐Tyr‐Phe‐Ser‐Ala‐Leu‐Arg‐His‐Tyr‐Il e‐Asn30‐Leu‐Val‐Thr‐Arg‐Gln‐Arg‐Tyr,CONH2. The presence of an additional alanine residue at the NH2‐terminus of the peptide suggests that the site of cleavage of the signal peptide in chicken preproPYY is different from the site of cleavage in other PP‐family prepropeptides.
Identification of zinc‐binding ligands in the Class II fructose‐ 1,6‐bisphosphate aldolase of Escherichia coli
Authors: Berry Alan , Marshall Karen E.
Published: July 2002

An expression and mutagenesis system for the E. coli Class II fructose‐1,6‐bisphosphate aldolase has been created by modification of the vector pKfda (Biochem. J. 257 (1989) 529‐534). Large amounts of Class II aldolase (about 1 g/l in crude extracts), with properties consistent with those previously reported for the naturally occurring enzyme (Biochem. J. 169 (1978) 633‐641) are obtained. The enzyme contains 2 zinc ions per enzyme dimer. We have investigated the nature of the zinc‐binding site of the enzyme by site‐directed mutagenesis. His‐108, His‐111, Cys‐112 and His‐142 were identified as possible zinc‐binding ligands by sequence alignments and comparisons with other known zinc‐containing enzymes. Mutation of these residues identified His‐108 and His‐111 as two of the ligands directly responsible for the tight binding of zinc. Mutation of the other two residues results in only a small effect on the amount of zinc bound per monomer and a corresponding change in specific activity. These residues are, therefore, unlikely to be directly involved in zinc binding, but may be indirectly involved in some manner in the zinc‐binding environment.
Dephosphorylation of tyrosine phosphorylated synthetic peptides by rat liver phosphotyrosine protein phosphatase isoenzymes
Authors: Stefani Massimo , Caselli Anna , Bucciantini Monica , Pazzagli Luigia , Dolfi Fabrizio , Camici Guido , Manao Giampaolo , Ramponi Giampietro
Published: July 2002

Five phosphotyrosine‐containing peptides have been synthesized by FMOC solid‐phase peptide synthesis. These peptides correspond to the 411–419 sequence of the Xenopus src oncogene, to the 1191–1220 sequence of the human EGF receptor precursor, to the 1146–1158 sequence of the human insulin receptor, to the 856–865 sequence of the human, β‐PDGF receptor, and to the 5–16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (AcP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, performed in microtiter E1A plates using Malachite green to determine the released phosphate, was rapid, reproducible, and sensitive. Both PTPase isoforms were able to hydrolyse all synthesized peptides, though with different affinity and rate. The main kinetic parameters were compared and discussed with respect to the role of the two enzymes in the cell.
Pyrimidine tract binding protein strongly stimulates in vitro encephalomyocarditis virus RNA translation at the level of preinitiation complex formation
Authors: Borovjagin Anton , Pestova Tatyana , Shatsky Ivan
Published: July 2002

Cellular protein p57/58, now known to be identical to polypyrimidine tract binding protein (PTB), has earlier been shown to specifically bind to the internal ribosome entry sites (IRES) of encephalomyocarditis virus (EMCV) and some other picornaviral RNAs. To elucidate its relevance to the internal initiation, the effect of cloned purified PTB on EMCV IRES directed translation was studied in cytoplasmic extracts of Krebs‐2 ascites carcinoma cells partially depleted of endogenous PTB. Addition of PTB to such extracts resulted in a strong stimulation of translation of a β‐glucuronidase (GUS) reporter cistron fused to the EMCV IRES, but had no effect on translation of capped mRNAs, such as β‐globin, and tobacco mosaic virus (TMV) RNAs. PTB was found to exert its effect at the level of 48S pre‐initiation complex formation.
A new additive for protein crystallization
Authors: Vuillard Laurent , Rabilloud Thierry , Leberman Reuben , Berthet-Colominas Carmen , Cusack Stephen
Published: July 2002

The potential usefulness of the new zwitterionic solubilizing agent, dimethyl ethylammonium propane sulfonate (NDSB195), in protein crystallization was shown using hen egg‐white lysozyme. In the presence of this agent, highly diffracting crystals were obtained using ammonium sulphate as a precipitant, whereas in its absence only amorphous precipitates were obtained. The crystals possess a triclinic unit cell not previously described and diffract to a resolution of 2 Å. To ascertain that the new reagent had not produced significant changes in the protein fold the structure was determined to a resolution of 2.6 Å. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).
Tricyclics and malignant syndrome
Authors: Abbar M , Carlander B , Castelnau D
Published: July 2002

A clinical and biological pattern similar to that of neuroleptic malignant syndrome is reported in a depressed patient treated with trimipramine, without any concomitant use of neuroleptics. The antidopaminergic properties of this drug, one of the tricyclic antidepressants, may account for this uncommon side effect.
Endogenous nitric oxide synthase inhibitors are responsible for the L‐arginine paradox
Authors: Tsikas Dimitrios , Böger Rainer H , Sandmann Jörg , Bode-Böger Stefanie M , Frölich Jürgen C
Published: July 2002

L‐Arginine, the substrate of nitric oxide (NO) synthases (NOSs), is found in the mammalian organism at concentrations by far exceeding K
M values of these enzymes. Therefore, additional L‐arginine should not enhance NO formation. In vivo, however, increasing L‐arginine concentration in plasma has been shown repeatedly to increase NO production. This phenomenon has been named the L‐arginine paradox; it has found no satisfactory explanation so far. In the present work, evidence for the hypothesis that the endogenous NOS inhibitors methylarginines, asymmetric dimethylarginine being the most powerful (IC50 1.5 μM), are responsible for the L‐arginine paradox is presented.
The GS‐GOGAT pathway is not operative in the heterocysts. Cloning and expression of glsF gene from the cyanobacterium Anabaena sp. PCC 7120
Authors: Martı́n-Figueroa Eugenio , Navarro Francisco , Florencio Francisco J.
Published: July 2002

The gene encoding the ferredoxin‐dependent glutamate synthase (Fd‐GOGAT), glsF, from the heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120, has been cloned and sequenced. Unlike other cyanobacteria, Anabaena 7120 contains only Fd‐GOGAT, lacking NADH‐GOGAT. The amount of glsF transcript and Fd‐GOGAT activity were similar under all the nitrogen growth conditions tested. Enzyme activity, Western and Northern blot analyses indicated that Fd‐GOGAT is absent in the heterocysts, while glutamine synthetase (GS) and NADP‐isocitrate dehydrogenase (IDH) were present in these specialised cells. Our results clearly indicate that the GS‐GOGAT pathway is not operative in the heterocysts, and hence glutamate must be imported from the adjacent vegetative cells, to sustain GS activity. Heterocysts probably export glutamine or another nitrogen rich compound like arginine to the vegetative cells.
A fluorescent reporter gene as a marker for ventricular specification in ES‐derived cardiac cells
Authors: Meyer Nathalie , Jaconi Marisa , Landopoulou Angela , Fort Philippe , Pucéat Michel
Published: July 2002

We have established a CGR8 embryonic stem (ES) cell clone (MLC2ECFP) which expresses the enhanced cyan variant of Aequorea victoria green fluorescent protein (ECFP) under the transcriptional control of the ventricular myosin light chain 2 (MLC2v) promoter. Using epifluorescence imaging of vital embryoid bodies (EB) and reverse transcription‐polymerase chain reaction (RT‐PCR), we found that the MLC2v promoter is switched on as early as day 7 and is accompanied by formation of cell clusters featuring a bright ECFP blue fluorescence. The fluorescent areas within the EBs were all beating on day 8. MLC2ECFP ES cells showed the same time course of cardiac differentiation as mock ES cells as assessed by RT‐PCR of genes encoding cardiac‐specific transcription factors and contractile proteins. The MLC2v promoter conferred ventricular specificity to ECFP expression within the EB as revealed by MLC2v co‐staining of ECFP fluorescent cells. MLC2ECFP‐derived cardiac cells still undergo cell division on day 12 after isolation from EBs but withdraw from the cell cycle on day 16. This ES cell clone provides a powerful cell model to study the signalling roads of factors regulating cardiac cell proliferation and terminal differentiation with a view to using them for experimental cell therapy.
The protein kinase C‐related kinase PRK2 interacts with the protein tyrosine phosphatase PTP‐BL via a novel PDZ domain binding motif
Authors: Gross Christina , Heumann Rolf , Erdmann Kai S
Published: July 2002

Protein tyrosine phosphatase‐basophil like (PTP‐BL) is a large non‐transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP‐BL with the protein kinase C‐related kinase 2 (PRK2), a serine/threonine kinase regulated by the G‐protein rho. This interaction is mediated by the PSD‐95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP‐BL and the extreme C‐terminus of PRK2 as shown by yeast two‐hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C‐terminal cysteine of PRK2 is indispensable for the interaction with PTP‐BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP‐BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP‐BL in the regulation of the actin cytoskeleton.
RBI, a one‐domain α‐amylase/trypsin inhibitor with completely independent binding sites
Authors: Maskos Klaus , Huber-Wunderlich Martina , Glockshuber Rudi
Published: July 2002

The bifunctional inhibitor from Ragi (Eleusine coracana Gaertneri) (RBI) is the only member of the α‐amylase/trypsin inhibitor family that inhibits both trypsin and α‐amylase. Here, we show that both enzymes simultaneously and independently bind to RBI. The recently solved three‐dimensional NMR structure of RBI has revealed that the inhibitor possesses a hitherto unknown fold for serine proteinase and α‐amylase inhibitors. Despite its different fold, RBI obeys the standard mechanism observed for most protein inhibitors of serine proteinases and is a strong, competitive inhibitor of bovine trypsin (K

i
= 1.2 ± 0.2 nM). RBI is also a competitive inhibitor of porcine α‐amylase (K

i
= 11 ± 2 nM) when a disaccharide is used as a substrate of α‐amylase. However, the inhibition mode becomes complex when larger (≥7 saccharide units) α‐amylase substrates are used. A second saccharide binding site on porcine α‐amylase may enable larger oligosaccharides to displace RBI from its binding site in an intramolecular reaction.
Tissue‐specific expression of splice variants of the mouse voltage‐gated calcium channel α2/δ subunit
Authors: Angelotti Timothy , Hofmann Franz
Published: July 2002

Five different splice variants of mouse α2/δ subunit isoforms (α2/a‐e), which arose from various combinations of three alternatively spliced regions, were cloned with a combination of cDNA library screening and RT‐PCR. Expression patterns and relative abundance of the various isoforms in mouse tissues were determined with an RNAse protection assay. Skeletal muscle and brain expressed single isoforms, α2a and α2b, respectively; however, the cardiovascular system expressed all five isoforms. Heart expressed mainly isoforms α2c and α2d while, in contrast to other species, aorta expressed predominantly α2a, the ‘skeletal muscle’ isoform. Smooth muscle‐containing tissues expressed α2d and α2e. Thus, α2/δ isoforms are restricted in their tissue expression, suggesting an important functional role for the differentially spliced variants.
Identification and characterization of homologues of the Exocyst component Sec10p
Authors: Guo Wei , Roth Dagmar , Gatti Evelina , De Camilli Pietro , Novick Peter
Published: July 2002

© 1997 Federation of European Biochemical Societies.
A single serine residue confers tetrodotoxin insensitivity on the rat sensory‐neuron‐specific sodium channel SNS
Authors: Sivilotti L , Okuse K , Akopian A.N , Moss S , Wood J.N
Published: July 2002

Sensory neurons express a sodium channel (SNS) that is highly resistant to block by tetrodotoxin (IC50=60 μM). SNS is 65% homologous to the cardiac sodium channel, in which a single hydrophilic residue in the SS2 segment is critical for tetrodotoxin resistance. By site‐directed mutagenesis, we have substituted phenylalanine for serine at the equivalent position in SNS: this mutated (S356F) SNS channel is functionally similar to wild‐type SNS when expressed inXenopusoocytes, but is potently blocked by tetrodotoxin and saxitoxin with IC50s of 2.8 nM and 8.2 nM, respectively. These data provide clues to the rational design of selective blockers of SNS with potential as analgesic drugs.
RIN14B: a pancreatic δ‐cell line that maintains functional ATP‐dependent K+ channels and capability to secrete insulin under conditions where it no longer secretes somatostatin
Authors: Bränström Robert , Höög Anders , Wahl Martin A. , Berggren Per-Olof , Larsson Olof
Published: July 2002

The δ‐cell line RIN14B was characterized with regard to ATP‐regulated K+ (KATP) channel activity and hormone release. By applying the patch‐clamp technique, dose–response curves for ATP and the sulfonylurea tolbutamide were obtained in inside‐out patches. The concentration causing half‐maximal KATP channel inhibition was found to be 23.7 and 27.6 μM for ATP and tolbutamide, respectively. ADP and diazoxide stimulated KATP channel activity, an effect dependent on the presence of intracellular Mg2+. The stimulatory effect of diazoxide also required the presence of ATP. The kinetic properties of the KATP channel were analysed in the presence of ATP, a combination of ADP and ATP and in nucleotide‐free solutions. The distribution of KATP channel open time could be described by a single exponential function with a time constant of approximately 30 ms in nucleotide‐free and in ATP‐containing solutions. The presence of both ATP and ADP resulted in the appearance of an additional time constant of >150 ms. Single‐channel unitary current–voltage (i‐V) relation was characterised for the KATP channel present in RIN14B cells. The slope conductance, measured at the reversal potential was found to be 19.1±2.4 pS. The permeability for K+ ions was calculated to be 0.31×10−13 cm3·s−1. We have not been able to confirm the somatostatin releasing profile of the RIN14B cells using radioimmunoassays, nor could we find positive somatostatin stain with immunocytochemical techniques. We conclude that the RIN14B cell line, previously characterized as a somatostatin‐secreting cell line, contains KATP channels with properties closely resembling the KATP channel described in the pancreatic β‐cell. However, the cell line appears to have dedifferentiated with regard to the ability to secrete somatostatin, maintaining the highly differentiated function of both insulin biosynthesis and exocytosis.
A single nucleotide is a sufficient 5′ untranslated region for translation in an eukaryotic in vitro system
Authors: Hughes Martin J.G , Andrews David W
Published: July 2002

The 5′ untranslated region of an RNA molecule is thought to play an important role in the regulation of translation. Following a recent report that a single nucleotide is sufficient to act in this role in the unicellular organism Giardia, we show that this is also the case for a mammalian in vitro system. These results also demonstrate that an RNA can initiate translation from a start codon where an ideal translational consensus sequence is impossible.
Inhibition of MAP kinase blocks insulin‐mediated DNA synthesis and transcriptional activation of c‐fos by Elk‐1 in vascular smooth muscle cells
Authors: Xi Xiao-Ping , Graf Kristof , Goetze Stephan , Hsueh Willa A , Law Ronald E
Published: July 2002

Insulin‐stimulated DNA synthesis, MAP kinase (MAPK) activity and c‐fos expression in vascular smooth muscle cells (VSMCs) was blocked by the MAPK inhibitor PD 98059. Regulation of c‐fos expression by the transcription factor Elk‐1 at the serum response element (SRE) is dependent on its phosphorylation by MAPK. PD 98059 also suppressed insulin‐induced Elk‐1 transcriptional activity through the SRE. These data show that MAPK plays a critical role in both insulin‐mediated growth and Elk‐1‐dependent induction of c‐fos in VSMCs.
G protein‐coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions
Authors: Aragay Anna M. , Ruiz-Gómez Ana , Penela Petronila , Sarnago Susana , Elorza Ana , Jiménez-Sainz M.Carmen , Mayor, Jr Federico
Published: July 2002

G protein‐coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein‐coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.
Constitutive and inducible type 1 ribosome‐inactivating proteins (RIPs) in elderberry (Sambucus nigra L.)
Authors: de Benito Fernando M , Iglesias Rosario , Ferreras José Miguel , Citores Lucı́a , Camafeita Emilio , Méndez Enrique , Girbés Tomás
Published: July 2002

Two novel highly basic type 1 (single chain) ribosome‐inactivating proteins (RIPs) with N‐glycosidase activity have been found in elderberries (the fruits of Sambucus nigra L.). Mass spectrometry of these RIPs, which we named nigritins f1 and f2, gave M
r values of 24 095 and 23 565, respectively. Both proteins strongly inhibited protein synthesis in rabbit reticulocyte lysates but were inactive against plant ribosomes. Both nigritins have a similar topological activity on pBlueScript SK+ DNA as that displayed by dianthin 30. Nigritin f1 is a constitutive RIP since it is present in both green and mature intact elderberries at nearly the same proportion with respect to total fruit protein. By contrast, nigritin f2 is inducible and only appeared in mature intact elderberries. Elderberries also contain two isoforms of a basic nigrin equivalent to the recently found basic nigrin b in elder bark (De Benito et al., FEBS Letters 413 (1997) 85–91). Our results indicate that probably not all plant RIPs exert the same biological function and that this may be determined by the physiological state of the tissue.
Mosaic Qβ coats as a new presentation model
Authors: Vasiljeva Inta , Kozlovska Tatjana , Cielens Indulis , Strelnikova Anna , Kazaks Andris , Ose Velta , Pumpens Paul
Published: July 2002

The new protein carrier was developed on the basis of recombinant RNA phage Qβ capsid. C‐terminal UGA extension of the short form of Qβ coat, so‐called A1 extension, served as a target for presentation of foreign peptides on the outer surface of mosaic Qβ particles. In conditions of enhanced UGA suppression, the proportion of A1‐extended to short coats in mosaic particles dropped from 48% to 14%, with an increase of the length of A1 extension. A model insertion, short preS1 epitope 31‐DPAFR‐35 of hepatitis B surface antigen, demonstrated superficial location on the mosaic Qβ particles and ensured specific antigenicity and immunogenicity.
The 49‐kDa subunit of NADH‐ubiquinone oxidoreductase (Complex I) is involved in the binding of piericidin and rotenone, two quinone‐related inhibitors
Authors: Darrouzet Elisabeth , Issartel Jean-Paul , Lunardi Joël , Dupuis Alain
Published: July 2002

Piericidin is a potent inhibitor of the mitochondrial and bacterial type I NADH‐ubiquinone oxidoreductases (Complex I) and is considered to bind at or close to the ubiquinone binding site(s) of the enzyme. Piericidin‐resistant mutants of the bacterium Rhodobacter capsulatus have been isolated and the present work demonstrates that a single missense mutation at the level of the gene encoding the peripheral 49‐kDa/NUOD subunit of Complex I is definitely associated with this resistance. Based on this original observation, we propose a model locating the binding site for piericidin (and quinone) at the interface between the hydrophilic and hydrophobic domains of Complex I.
Evolution of new protein function: recombinational enhancer Fis originated by horizontal gene transfer from the transcriptional regulator NtrC
Authors: Morett Enrique , Bork Peer
Published: July 2002

New protein function is thought to evolve mostly by gene duplication and divergence. Here we present phylogenetic evidence that the multifunctional protein Fis of the γ proteobacterial species derived from the COOH‐terminal domain of an ancestral α proteobacterial NtrC transcriptional regulatory protein. All of the known enterobacterial fis genes are preceded by an open reading frame, named yhdG, that is highly similar to nifR3, a gene that forms an operon with ntrC in several α proteobacterial species. Thus, we propose that yhdG and fis were acquired by a lineage ancestral to the γ proteobacteria in a single horizontal gene transfer event, and later diverged to their present functions.
Sequence specific cleavage of the HIV‐1 coreceptor CCR5 gene by a hammer‐head ribozyme and a DNA‐enzyme: inhibition of the coreceptor function by DNA‐enzyme
Authors: Goila Ritu , Banerjea Akhil C
Published: July 2002

The chemokine receptor CCR5 is used as a major coreceptor for fusion and entry by non‐syncytia inducing macrophage tropic isolates of HIV‐1, which is mainly involved in transmission. Individuals who are homozygous for the Δ32 allele of CCR5 are usually resistant to HIV‐1 infection and continue to lead a normal healthy life. Thus this gene is dispensable and is, therefore, an attractive target in the host cell for interfering specifically with the virus‐host interaction. With the aim to develop a specific antiviral approach at the molecular level, we have synthesized a hammer‐head ribozyme and a DNA‐enzyme. Both ribozyme and DNA‐enzyme cleaved the CCR5 RNA in a sequence specific manner. This cleavage was protein independent but Mg2+ dependent. The extent of cleavage increased with increasing concentration of magnesium chloride. DNA‐enzyme was more effective in cleaving a full length (1376 bases) in vitro generated transcript of CCR5 gene. In this communication, we show that the DNA‐enzyme when introduced into a mammalian cell, results in decreased CD4‐CCR5‐gp160 mediated fusion of cell membranes. Potential applications of these trans acting molecules are discussed.
The structural flexibility of the preferredoxin transit peptide
Authors: Wienk Hans L.J. , Czisch Michael , de Kruijff Ben
Published: July 2002

In order to obtain insight into the structural flexibility of chloroplast targeting sequences, the Silene pratensis preferredoxin transit peptide was studied by circular dichroism and nuclear magnetic resonance spectroscopy. In water, the peptide is unstructured, with a minor propensity towards helix formation from Val‐9 to Ser‐12 and from Gly‐30 to Ser‐40. In 50% (v/v) trifluoroethanol, structurally independent N‐ and C‐terminal helices are stabilized. The N‐terminal helix appears to be amphipathic, with hydrophobic and hydroxylated amino acids on opposite sides. The C‐terminal helix comprises amino acids Met‐29–Gly‐50 and is destabilized at Gly‐39. No ordered tertiary structure was observed. The results are discussed in terms of protein import into chloroplasts, in which the possible interactions between the transit peptide and lipids are emphasized.
A 6 bp Z‐DNA hairpin binds two Zα domains from the human RNA editing enzyme ADAR1
Authors: Schade Markus , Behlke Joachim , Lowenhaupt Ky , Herbert Alan , Rich Alexander , Oschkinat Hartmut
Published: July 2002

The Zα domain of the human RNA editing enzyme double‐stranded RNA deaminase I (ADAR1) binds to left‐handed Z‐DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two Zα domains bind to one d(CG)3T4(CG)3 hairpin which contains a stem of six base pairs in the Z‐DNA conformation. Both wild‐type Zα and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (≥6 bp) segments of Z‐DNA within a gene are able to recruit two ADAR1 enzymes to that particular site.
Sequence‐specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides
Authors: Hosoya Takeshi , Takeuchi Hiroaki , Kanesaka Yoshiyuki , Yamakawa Hidefumi , Miyano-Kurosaki Naoko , Takai Kazuyuki , Yamamoto Naoki , Takaku Hiroshi
Published: July 2002

The inhibition of specific transcription regulatory proteins is a new approach to control gene expression. The transcriptional activities of DNA‐binding proteins can be inhibited by the use of double‐stranded oligonucleotides that compete for the binding to their specific target sequences in promoters and enhancers. We used nicked (NDODN‐κB) and circular (CDODN‐κB) dumbbell DNA oligonucleotides containing a NF‐κB binding site to analyze the inhibition of the NF‐κB‐dependent activation of the human immunodeficiency virus type‐1 (HIV‐1) enhancer. The dumbbell DNA oligonucleotides are stable, short segments of double‐stranded DNA with closed nucleotide loops on each end, which confer resistance to exonucleases. The dumbbell and other oligonucleotides (decoys) with the NF‐κB sequence were found to compete with the native strand for NF‐κB binding. The circular dumbbell and double‐stranded phosphorothioate oligonucleotides competed with the native strand for binding to the NF‐κB binding proteins, while the nicked NF‐κB dumbbell was a less effective competitor. In Jurkat T‐cells, the dumbbell and other oligonucleotides were tested for their ability to block the activation of the plasmid HIV‐NL4‐3 Luc. The CDODN‐κB strongly inhibits the specific transcriptional regulatory proteins, as compared with the NDODN‐κB and the double stranded phosphodiester oligonucleotides. On the other hand, the double stranded phosphorothioate oligonucleotides could also block this activation, but the effect was non‐specific. The circular (CDODN) dumbbell oligonucleotides may efficiently compete for the binding of specific transcription factors within cells, thus providing anti‐HIV‐1 or other therapeutic effects.
Static and magic angle spinning 31P NMR spectroscopy of two natural plasma membranes
Authors: Moreau C. , Le Floch M. , Segalen J. , Leray G. , Metzinger L. , de Certaines J.D. , Le Rumeur E.
Published: July 2002

Static and magic angle spinning 31P NMR spectroscopy was used for the first time in natural plasma membranes from erythrocytes and skeletal muscle to study phospholipid arrangement and composition. Typical static powder‐like spectra were obtained showing that phospholipids were in a bilayer arrangement. Magic angle spinning narrowed spectra into two components. The first one corresponded to phosphatidylcholine and the second one to the other phospholipids with intensities in agreement with the known phospholipid composition. These findings show that NMR data previously acquired using model membranes can be transposed to studies on phospholipids in their natural environment.
Kinetic mechanism and ATP‐binding site reactivity of p38γ MAP kinase
Authors: Fox Ted , Fitzgibbon Matthew J , Fleming Mark A , Hsiao Hsun-Mei , Brummel Christopher L , Su Michael S-S
Published: July 2002

Activated p38γ MAP kinase exhibited significant basal ATPase activity in the absence of a kinase substrate, and addition of a phosphoacceptor substrate increased k
cat/K
m>20‐fold. AMP‐PCP was competitive with ATP binding and non‐competitive with phosphoacceptor substrate binding. The nucleotide binding site affinity label 5′‐(p‐fluorosulfonylbenzoyl)adenosine (FSBA) bound stoichiometrically at Lys‐56 in the ATP site of both unphosphorylated and activated p38γ. AMP‐PCP only protected the activated enzyme from FSBA inactivation, implying that AMP‐PCP does not bind unphosphorylated p38γ. Basal ATPase activities were also observed for activated p38α, ERK2 and JNK3 suggesting that the enzymatic mechanism may be similar for all classes of MAP kinases.
The E358S mutant of Agrobacterium sp. β‐glucosidase is a greatly improved glycosynthase
Authors: Mayer Christoph , Zechel David L. , Reid Stephen P. , Warren R.Antony J. , Withers Stephen G.
Published: July 2002

Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizing oligosaccharides. The ‘original’ glycosynthase, derived from Agrobacterium sp. β‐glucosidase (Abg) by mutating the nucleophile glutamate to alanine (E358A), synthesizes oligosaccharides in yields exceeding 90% [Mackenzie, L.F., Wang, Q., Warren, R.A.J. and Withers, S.G. (1998) J. Am. Chem. Soc. 120, 5583–5584]. This mutant has now been re‐cloned with a His6‐tag into a pET‐29b(+) vector, allowing gram scale production and single step chromatographic purification. A dramatic, 24‐fold, improvement in synthetic rates has also been achieved by substituting the nucleophile with serine, resulting in improved product yields, reduced reaction times and an enhanced synthetic repertoire. Thus poor acceptors for Abg E358A, such as PNP‐GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP‐β‐LacNAc. The increased glycosylation activity of Abg E358S likely originates from a stabilizing interaction between the Ser hydroxyl group and the departing anomeric fluorine of the α‐glycosyl fluoride.
Intraperitoneal cisplatin‐based chemotherapy vs. intravenous cisplatin‐based chemotherapy for stage III optimally cytoreduced epithelial ovarian cancer
Authors: Yen M.‐S. , Juang C.‐M. , Lai C.‐R. , Chao G.‐C. , Ng H.‐T. , Yuan C.‐C.
Published: July 2002

Objective: To compare the survival between intraperitoneal cisplatin‐based chemotherapy (IPCT) and intravenous cisplatin‐based chemotherapy (IVCT) in stage III epithelial ovarian cancer with minimal residual disease (<1 cm) after primary debulking surgery. Method: One hundred and thirty‐two patients with stage III epithelial ovarian cancer after optimal primary debulking surgery with minimal residual disease between April 1990 and March 1995 were entered into a randomized clinical trial in which IPCT or IVCT was administered at 3‐week intervals. Patients in the IPCT arm received cisplatin‐based (100 mg/m2) intraperitoneal chemotherapy. Patients in the IVCT arm received cisplatin‐based (50 mg/m2) intravenous chemotherapy. The tumor response was assessed every 3 months. The hematological toxicity using the South West Oncology Group (SWOG) toxicity criteria was assessed. Catheter complications associated with intraperitoneal chemotherapy were also analyzed. Result: The estimated median survival in the IPCT group was 43 months (95% confidence interval, 34–54) and IVCT group was 48 months (95% confidence interval, 37–59). The hazard ratio of death was not statistically significant between IPCT and IVCT (hazard ratio, 1.13; 95% CI, 0.69–1.86; P=0.317). The frequencies of hematological toxic effects were significantly lower in the IPCT group than in the IVCT group. Conclusion: Intravenous and intraperitoneal chemotherapy are associated with equivalent survival in patients with minimal residual stage III epithelial ovarian cancer after optimal cytoreductive surgery.
The scientific theory‐building process: a primer using the case of TQM
Authors: Handfield Robert B , Melnyk Steven A
Published: July 2002

As Operations Management (OM) researchers begin to undertake and publish more empirical research, there is a need to understand the nature of the scientific theory‐building process implicit in this activity. This tutorial presents a process map approach to this process. We begin by defining the nature of scientific knowledge, and proceed through the stages of the theory‐building process, using illustrations from OM research in Total Quality Management. The tutorial ends with a discussion of the criteria for OM journal reviewers to consider in evaluating theory‐driven empirical research, and suggests a number of OM topic areas that require greater theory development.
A role for an AP‐1‐like site in the expression of the myelin basic protein gene during differentiation
Authors: Miskimins Robin , Miskimins W.Keith
Published: July 2002

Differentiation of oligodendrocyte progenitors into mature oligodendrocytes involves the timely, cell‐type specific expression of a number of different genes. Among these, the expression of the myelin basic protein (MBP) gene closely parallels the course of oligodendrocyte differentiation. To understand how transcription of the myelin basic protein gene is controlled, binding to the distal end of the 5′ flanking sequence of the MBP gene was investigated. Specific protein‐DNA complexes were localized to an AP‐1‐like element located between −1230 and −1240. The protein–DNA complexes formed at this site were shown to change as the cells differentiated. In undifferentiated cells two complexes were formed but, as the cells differentiated, binding was nearly completely lost. One of the two complexes was shown to contain a member of the fos family of transcription factors but no jun family members were involved. Mutation of the AP‐1‐like site resulted in loss of the complex and a change in expression of a reporter construct driven by the mutated promoter sequence. These results demonstrate a role for the AP‐1‐like site in repression of MBP gene expression in oligodendrocyte progenitor cells.
Adjunctive gabapentin treatment of bipolar disorder
Authors: Vieta E , Martinez-Arán A , Nieto E , Colom F , Reinares M , Benabarre A , Gastó C
Published: July 2002

Introduction

The aim of this study was to analyze the effectiveness of gabapentin administration to bipolar patients who had an incomplete response to other mood stabilizers.

Subjects and methods

Twenty-two RDC bipolar 1 and II patients were assessed by means of the SADS and entered if they gave their consent to participate. All them had suffered from frequent relapses, subsyndromal features (mostly depressive) and incomplete response to other drugs. They all received open-label increasing doses of gabapentin until clinical response. The patients were assessed through the CGI-BP and a specific questionnaire at baseline and at 12 weeks of follow-up.

Results

Six out of the 22 patients dropped out for various reasons (four because of relapse, one because of side effects and one more because of poor compliance). Eight of the 16 patients that completed the 12-week follow-up showed at least two stages of improvement in the CGI. Using the last observation-carried forward analysis, the improvement was statistically significant for the depression subscale, and apparently related to social functioning, irritability and anxiety. Only one patient dropped out because of intolerance (mild rash). The mean dose of gabapentin was 1,310 mg/day.

Conclusion

Gabapentin may be a useful drug for the add-on treatment of bipolar patients with poor response to other mood stabilizers. Gabapentin may improve depressive residual symptoms such as irritability, social withdrawal or anxiety. These results should be confirmed in randomized clinical trials.
Psychotic versus nonpsychotic bipolar outpatient depression
Authors: Benazzi F.
Published: July 2002

Psychotic bipolar depression was compared with nonpsychotic bipolar depression. Psychotic (n = 59) and nonpsychotic (n = 176) bipolar depressed outpatients were SCID-DSM-IV interviewed. Psychotic bipolar depression had significantly higher severity, more chronicity, fewer atypical features and axis I co-morbidity, more bipolar I, and fewer bipolar II patients. Age at onset, duration of illness, gender, and recurrences, were not significantly different.
Global FKRP Registry: observations in more than 300 patients with Limb Girdle Muscular Dystrophy R9
Authors: Murphy Lindsay B. , Schreiber‐Katz Olivia , Rafferty Karen , Robertson Agata , Topf Ana , Willis Tracey A. , Heidemann Marcel , Thiele Simone , Bindoff Laurence , Laurent Jean‐Pierre , Lochmüller Hanns , Mathews Katherine , Mitchell Claudia , Stevenson John Herbert , Vissing John , Woods Lacey , Walter Maggie C. , Straub Volker
Published: April 2020

Objective

The Global FKRP Registry is a database for individuals with conditions caused by mutations in the Fukutin‐Related Protein (FKRP) gene: limb girdle muscular dystrophy R9 (LGMDR9, formerly LGMD2I) and congenital muscular dystrophies MDC1C, Muscle–Eye–Brain Disease and Walker–Warburg Syndrome. The registry seeks to further understand the natural history and prevalence of FKRP‐related conditions; aid the rapid identification of eligible patients for clinical studies; and provide a source of information to clinical and academic communities.

Methods

Registration is patient‐initiated through a secure online portal. Data, reported by both patients and their clinicians, include: age of onset, presenting symptoms, family history, motor function and muscle strength, respiratory and cardiac function, medication, quality of life and pain.

Results

Of 663 registered participants, 305 were genetically confirmed LGMDR9 patients from 23 countries. A majority of LGMDR9 patients carried the common mutation c.826C > A on one or both alleles; 67.9% were homozygous and 28.5% were compound heterozygous for this mutation. The mean ages of symptom onset and disease diagnosis were higher in individuals homozygous for c.826C > A compared with individuals heterozygous for c.826C > A. This divergence was replicated in ages of loss of running ability, wheelchair‐dependence and ventilation assistance; consistent with the milder phenotype associated with individuals homozygous for c.826C > A. In LGMDR9 patients, 75.1% were currently ambulant and 24.6%, nonambulant (unreported in 0.3%). Cardiac impairment was reported in 23.2% (30/129).

Interpretation

The Global FKRP Registry enables the collection of patient natural history data, which informs academics, healthcare professionals and industry. It represents a trial‐ready cohort of individuals and is centrally placed to facilitate recruitment to clinical studies.
Trypsin‐Instructed Self‐Assembly on Endoplasmic Reticulum for Selectively Inhibiting Cancer Cells
Authors: Kim Beom Jin , Fang Yu , He Hongjian , Xu Bing
Published: April 2020

Selectively targeting the endoplasmic reticulum (ER) of cancer cells, though promising a new strategy for cancer therapy, remains underdeveloped. Enzyme‐instructed self‐assembly (EISA) is emerging as a promising approach for selectively targeting ER of cancer cells. This work reports an easily accessible branched peptide that consists of a D‐tetrapeptide backbone and a branch with the sequence of KYDKKKKDG (K: lysine; Y: tyrosine; D: aspratic acid; G: glycine), being an EISA substrate of typsin‐1 (PRSS1), selectively inhibits cancer cells. Depending on the type of cells, the level of PRSS1 expression dictates the cytotoxicity of the branched peptide. Moreover, immunostaining and fluorescent imaging reveal that PRSS1 overexpresses on the ER of a high‐grade serous ovarian cancer cell line (OVSAHO). The overexpression of PRSS1 renders the branched peptide to exhibit high selectivity against OVSAHO by the in situ formation of the peptide assemblies on the ER of OVSAHO cells, which causes ER stress and eventual cell death. This work, illustrating trypsin‐guided EISA for inhibiting cancer cells by enzymatic reaction on ER for the first time, offers a new way to target the subcellular organelles of cancer cells for potential cancer therapy.
Immune Modulation by Design: Using Topography to Control Human Monocyte Attachment and Macrophage Differentiation
Authors: Vassey Matthew J. , Figueredo Grazziela P. , Scurr David J. , Vasilevich Aliaksei S. , Vermeulen Steven , Carlier Aurélie , Luckett Jeni , Beijer Nick R. M. , Williams Paul , Winkler David A. , de Boer Jan , Ghaemmaghami Amir M. , Alexander Morgan R.
Published: April 2020

Macrophages play a central role in orchestrating immune responses to foreign materials, which are often responsible for the failure of implanted medical devices. Material topography is known to influence macrophage attachment and phenotype, providing opportunities for the rational design of “immune‐instructive” topographies to modulate macrophage function and thus foreign body responses to biomaterials. However, no generalizable understanding of the inter‐relationship between topography and cell response exists. A high throughput screening approach is therefore utilized to investigate the relationship between topography and human monocyte–derived macrophage attachment and phenotype, using a diverse library of 2176 micropatterns generated by an algorithm. This reveals that micropillars 5–10 µm in diameter play a dominant role in driving macrophage attachment compared to the many other topographies screened, an observation that aligns with studies of the interaction of macrophages with particles. Combining the pillar size with the micropillar density is found to be key in modulation of cell phenotype from pro to anti‐inflammatory states. Machine learning is used to successfully build a model that correlates cell attachment and phenotype with a selection of descriptors, illustrating that materials can potentially be designed to modulate inflammatory responses for future applications in the fight against foreign body rejection of medical devices.
Schein's Remand Decision: Should Scotus Review The Provider Rule Incorporation‐by‐Reference Issue?
Authors: Faulkner Richard D. , Loree Philip J.
Published: April 2020

This article follows on co‐author Philip J. Loree Jr.'s article, “Schein's Remand Decision Goes Back To the Supreme Court. What's Next?” 38 Alternatives 54 (April 2020) (available at https://bit.ly/3aYy7Sg) (referred to below as last month's article), which discussed how Henry Schein Inc., a Melville, N.Y.‐based dental equipment distributor—the petitioner—is seeking to arbitrate disputes pursuant to a written agreement.
Now Plaguing Dispute Resolution Processes: Proceeding in ADR Without the Handshakes
Authors: Samuel Adam
Published: April 2020

The optimist in me hopes that, as you are reading this, the coronavirus will be a matter of medical history.
Still no Arbitration: In its Latest Jock Decision, Second Circuit Reverses for more Contract Interpretation
Authors: Muenchinger Anne
Published: April 2020

A long‐running Second U.S. Circuit Court of Appeals arbitration case is now before the U.S. Supreme Court on a petition for certiorari, after four visits at the New York federal appeals court.
Robust Atomistic Modeling of Materials, Organometallic, and Biochemical Systems
Authors: Spicher Sebastian , Grimme Stefan
Published: April 2020

Modern chemistry seems to be unlimited in molecular size and elemental composition. Metal‐organic frameworks or biological macromolecules involve complex architectures and a large variety of elements. Yet, a general and broadly applicable theoretical method to describe the structures and interactions of molecules beyond the 1000‐atom size regime semi‐quantitatively is not self‐evident. For this purpose, a generic force field named GFN‐FF is presented, which is completely newly developed to enable fast structure optimizations and molecular‐dynamics simulations for basically any chemical structure consisting of elements up to radon. The freely available computer program requires only starting coordinates and elemental composition as input from which, fully automatically, all potential‐energy terms are constructed. GFN‐FF outperforms other force fields in terms of generality and accuracy, approaching the performance of much more elaborate quantum‐mechanical methods in many cases.
Robust DNA‐Bridged Memristor for Textile Chips
Authors: Xu Xiaojie , Zhou Xufeng , Wang Tianyu , Shi Xiang , Liu Ya , Zuo Yong , Xu Limin , Wang Mengying , Hu Xiaofeng , Yang Xinju , Chen Jiaxin , Yang Xiubo , Chen Lin , Chen Peining , Peng Huisheng
Published: April 2020

Electronic textiles may revolutionize many fields, such as communication, health care and artificial intelligence. To date, unfortunately, computing with them is not yet possible. Memristors are compatible with the interwoven structure and manufacturing process in textiles because of its two‐terminal crossbar configuration. However, it remains a challenge to realize textile memristors owing to the difficulties in designing advanced memristive materials and achieving high‐quality active layers on fiber electrodes. Herein we report a robust textile memristor based on an electrophoretic‐deposited active layer of deoxyribonucleic acid (DNA) on fiber electrodes. The unique architecture and orientation of DNA molecules with the incorporation of Ag nanoparticles offer the best‐in‐class performances, e.g., both ultra‐low operation voltage of 0.3 V and power consumption of 100 pW and high switching speed of 20 ns. Fundamental logic calculations such as implication and NAND are demonstrated as functions of textile chips, and it has been thus integrated with power‐supplying and light emitting modules to demonstrate an all‐fabric information processing system.