bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of the polycation‐mediated DNA uptake and cell transfection with Pluronic P85 block copolymer
Authors: Astafieva Irina , Maksimova Irina , Lukanidin Eugenii , Alakhov Valery , Kabanov Alexander
Published: July 2002

Polyelectrolyte complexes formed between DNA and poly(N‐ethyl‐4‐vinylpyridinium) cations were shown to effectively transfect mammalian cells [7]. This work suggests that the polycation‐mediated uptake of the plasmid DNA and cell transfection are significantly enhanced when these complexes are administered simultaneously with a poly(ethylene oxide)block‐poly(propylene oxide)‐block‐poly(ethylene oxide) copolymer, Pluronic P85. The uptake studies were performed using radioactively labeled pRSV CAT plasmid on NIH 3T3, MDCK, and Jurkat cell lines. The transfection was investigated by chloramphenicol acetyltransferase assay using 3T3 cells as a model. The effects reported may be useful for the enhancement of the polycation‐mediated cell transfection.
LAMININ STIMULATES EXPRESSION OF TWO MITOCHONDRIAL PROTEINS DURING NEURITE OUTGROWTH
Authors: Weeks Benjamin S. , Burbelo Peter , Jucker Mathias , Weiner Matthew A. , Roque Eva , Kleinman Hynda K.
Published: July 2002

Differential hybridization was utilized with mRNA from NG108‐15 cells cultured on either tissue culture plastic or laminin for 4 hr to identify genes whose mRNA was increased by laminin, a potent stimulator of neurite outgrowth. Two of the 16 laminin‐induced clones, cytochrome b and chargerin II, are mitochondrial proteins. Northern blotting confirmed that laminin increased the mRNA levels of cytochrome b and chargerin II several fold. Antibody to chargerin II stained both processes and cell bodies of the cerebellar Purkinje cells and localized in the mitochondria of NG108‐15 cells, which also showed increased protein levels in the presence of laminin. In addition, higher levels of chargerin II protein were detected in the newborn brain compared to the adult. However, inhibitors of mitochondrial protein synthesis did not affect laminin‐mediated neurite outgrowth. These data suggest that the increased synthesis of mitochondrial enzymes observed with laminin treatment may not be necessary for the formation of neurites.
A mutation in the second intracellular loop of the pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation
Authors: Cao Yong-Jiang , Gimpl Gerald , Fahrenholz Falk
Published: July 2002

The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor belongs to the glucagon/secretin/vasoactive intestinal polypeptide (VIP) receptor family. We mutated and deleted an amino acid residue (E261) which is located within the second intracellular loop of the rat PACAP type I receptor and which is highly conserved among the receptor family. The wild‐type receptor and the mutant receptors were efficiently expressed at the surface of COS‐7 cells at nearly the same level and revealed the same high affinity for the agonist PACAP‐27. The cAMP contents of COS cells transfected with the E261A, E261Q, and the deletion mutant receptor were 4.6‐, 5.7‐, and 6.7‐fold higher as compared with COS cells transfected with the wild‐type receptor. Thus, all the mutant PACAP receptors were constitutively active. The data suggest that the glutamic acid in the second intracellular loop of the PACAP receptor may be a key residue to constrain the receptor in the inactive conformation with respect to its coupling to Gs proteins.
The G protein β3 subunit 825T allele is a genetic marker for enhanced T cell response
Authors: Lindemann Monika , Virchow Sebastian , Ramann Frank , Barsegian Vahé , Kreuzfelder Ernst , Siffert Winfried , Müller Norbert , Grosse-Wilde Hans
Published: July 2002

The G protein β3 subunit (GNB3) 825T allele is predictive of enhanced Gi protein activation. Studying the influence of C825T allele status on cellular in vitro immune responses towards recall antigens and interleukin‐2 stimulation we observed a 2–4‐fold, significantly increased proliferation in homozygous 825T (TT) vs. C825 allele (CC) carriers. Furthermore, lymphocyte chemotaxis and CD4+ T cell counts of individuals with TT+TC genotypes were significantly enhanced compared to the CC genotype. In summary, it appears that C825T allele status is highly predictive of immunocompetence and could be a candidate gene in disorders associated with inadequate immune response.
Heme‐copper oxidases with modified D‐ and K‐pathways are yet efficient proton pumps
Authors: Gomes Cláudio M. , Backgren Camilla , Teixeira Miguel , Puustinen Anne , Verkhovskaya Marina L. , Wikström Mårten , Verkhovsky Michael I.
Published: July 2002

The cytochrome aa
3‐type quinol oxidase from the archaeon Acidianus ambivalens and the ba3
‐type cytochrome c oxidase from Thermus thermophilus are divergent members of the heme‐copper oxidase superfamily of enzymes. In particular they lack most of the key residues involved in the proposed proton transfer pathways. The pumping capability of the A. ambivalens enzyme was investigated and found to occur with the same efficiency as the canonical enzymes. This is the first demonstration of pumping of 1 H+/electron in a heme‐copper oxidase that lacks most residues of the K‐ and D‐channels. Also, the structure of the ba3
oxidase from T. thermophilus was simulated by mutating Phe274 to threonine and Glu278 to isoleucine in the D‐pathway of the Paracoccus denitrificans cytochrome c oxidase. This modification resulted in full efficiency of proton translocation albeit with a substantially lowered turnover. Together, these findings show that multiple structural solutions for efficient proton conduction arose during evolution of the respiratory oxidases, and that very few residues remain invariant among these enzymes to function in a common proton‐pumping mechanism.
The position of the cell penetrating peptide penetratin in SDS micelles determined by NMR
Authors: Lindberg Mattias , Gräslund Astrid
Published: July 2002

Penetratin is a 16 residue peptide, RQI KIWFQ NRRMK WKK‐amide, with the ability to penetrate cell membranes and a sequence taken from the homeodomain of the Drosophila Antennapedia transcription factor. 600 MHz 1H‐nuclear magnetic resonance has been used to study the structure and location of penetratin interacting with a sodium dodecyl sulphate micelle. The positioning of penetratin in the micelle was studied by adding paramagnetic probes (Mn2+ ions, 5‐doxyl and 12‐doxyl stearic acid) to the solvent. The results show that the peptide is a straight helix positioned with its C‐terminus deep inside the micelle and its N‐terminus near the surface of the micelle.
Visualizing detergent resistant domains in model membranes with atomic force microscopy
Authors: Rinia Hilde A. , Snel Margot M.E. , van der Eerden Jan P.J.M. , de Kruijff Ben
Published: July 2002

Evidence is accumulating that in cell membranes microdomains exist, also referred to as rafts or detergent resistant membranes. In this study, atomic force microscopy is used to study supported lipid bilayers, consisting of a fluid phosphatidylcholine, sphingomyelin and cholesterol. Domains were visualized of which the morphology and size depended on the cholesterol concentration. The presence of cholesterol was found to induce bilayer coupling. At 30 mol% cholesterol, a change in percolation phase was observed, and at 50 mol%, when both fluid lipids and solid lipids are saturated with cholesterol, phase separation was still observed. In addition, we were able to directly visualize the resistance of domains against non‐ionic detergent.
Regulation of apoptosis by respiration: cytochrome c release by respiratory substrates
Authors: Nishimura Goshi , Proske Rita J. , Doyama Hiroko , Higuchi Masahiro
Published: July 2002

Cytochrome c release from mitochondria is essential for apoptosis. Using human myelogenous leukemia ML‐1a, its respiration‐deficient and reconstituted cells, we demonstrated that respiratory function is essential for tumor necrosis factor‐induced cytochrome c release. In a cell free system using mitochondrial fraction from ML‐1a, initiation of respiration by substrates for complexes I, II, and III but not IV released cytochrome c, suggesting that reduction of coenzyme Q or complex III is essential for cytochrome c release. In the same system, disruption of mitochondrial outer membrane was neither enough nor the cause for cytochrome c release by succinate. These observations define an early pathway in which a change in respiration releases cytochrome c.
A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29
Authors: Testi Maria Grazia , Croce Roberta , Laureto Patrizia Polverino-De , Bassi Roberto
Published: July 2002

Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light‐harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of the electron carriers. A previously unknown reversible phosphorylation event has recently been described on the CP29 subunit which leads to conformational changes and protection from cold stress (Bergantino, E., Dainese, P., Cerovic, Z. Sechi, S. and Bassi, R. (1995) J. Biol Chem. 270, 8474–8481). In this study, we have identified the phosphorylation site on the N‐terminal, stroma‐exposed domain, showing that it is located in a sequence not homologous to the other members of the Lhc family. The phosphorylated sequence is unique in chloroplast membranes since it meets the requirements for CK2 (casein kinase II) kinases. The possibility that this phosphorylation is involved in a signal transduction pathway is discussed.
NMR evidence for a novel asparagine‐linked oligosaccharide on cellobiohydrolase I from Trichoderma reesei RUTC 30
Authors: De Bruyn André , Maras Marleen , Schraml Jan , Herdewijn Piet , Contreras Roland
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Identification and localization of the sod2 gene product in fission yeast
Authors: Dibrov Pavel , Smith Jennifer J , Young Paul G , Fliegel Larry
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Fluorescence spectral characteristics of novel asymmetric monomethine cyanine dyes in nucleic acid solutions
Authors: Timcheva Iliana I , Maximova Vera A , Deligeorgiev Todor G , Gadjev Nikolai I , Sabnis Ram W , Ivanov Ivan G
Published: July 2002

Six new asymmetric monomethine cyanine dyes have been synthesized and their fluorescence characteristics in the presence of nucleic acids studied. The new dyes have no fluorescence of their own in water solutions upon excitation at 480 nm but they become strongly fluorescent in the presence of nucleic acids. The fluorescence maxima of the investigated dyes are found at 525–545 nm when bound to dsDNA and around 600 nm upon binding to RNA and ssDNA. Fluorescence quenching studies with increasing concentrations of NaCl indicate that the cyanine dyes have a mixed (intercalating and groove binding) type of interaction with dsDNA. © 1997 Federation of European Biochemical Societies.
The complete inventory of the yeast Saccharomyces cerevisiae P‐type transport ATPases
Authors: Catty Patrice , de Kerchove d'Exaerde Alban , Goffeau André
Published: July 2002

A total of sixteen open reading frames encoding for P‐type ATPases have been identified in the complete genome sequence of Saccharomyces cerevisiae. Phylogenetic analysis distinguishes 6 distinct families. Topology predictions, identification of aminoacid sequence motifs and phenotype analysis of the available mutants suggest that these families correspond to ATPases transporting either H+ (2 members), Ca2+ (2 members), Na+ (3 members), heavy metals (2 members), possibly aminophospholipids (5 members including 4 new ones) or unknown substrates (2 new members). It is proposed that the latter family which has homologs in Tetrahymena thermophila, Plasmodium falciparum and Caenorhabditis elegans constitutes a new group called P4‐ATPases with characteristic topology and aminoacid signatures.
A recombinant polypeptide model of the second nucleotide‐binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase
Authors: Randak Christoph , Neth Peter , Auerswald Ennes A , Eckerskorn Christoph , Assfalg-Machleidt Irmgard , Machleidt Werner
Published: July 2002

CFTR–NBF‐2 expressed and purified in fusion with the maltose‐binding protein was shown to catalyse the reaction ATP→ADP+Pi by three different assays, monitoring ATP turnover, formation of ADP and release of Pi (K
m 86 μM, rate constant 0.37 min−1). The reaction product ADP inhibits this ATPase activity. In a similar manner the hydrolysis of GTP to GDP and Pi was demonstrated (K
m 40 μM, rate constant 0.29 min−1). In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP. As typical for adenylate kinases a distinct AMP‐binding site could be verified for CFTR–NBF‐2 by the inability of TNP–ATP and AMP to compete for binding. All three enzymatic activities were inhibited by the symmetric double‐substrate‐mimicking inhibitor Ap5A. As NBF‐2 plays a central role in CFTR channel opening and closing the results reported here are fundamental in understanding mechanisms of CFTR channel activity regulation.
Overlapping sites on the Link module of human TSG‐6 mediate binding to hyaluronan and chondroitin‐4‐sulphate
Authors: Parkar Ashfaq A , Day Anthony J
Published: July 2002

Link modules are hyaluronan‐binding domains that are involved in the formation and stability of extracellular matrix and cell migration. We have examined the glycosaminoglycan specificity of the Link module from the arthritis‐associated protein, human TSG‐6, by microtitre plate‐based assays employing biotinylated‐hyaluronan or mono‐biotinylated Link module. This domain was found to interact specifically with chondroitin‐4‐sulphate (C4S), with similar affinity to hyaluronan, but not with chondroitin‐6‐sulphate or heparin. Competition experiments indicate that C4S and hyaluronan have overlapping binding surfaces on the TSG‐6 Link module. Disease‐associated changes in C4S expression may influence the localisation and biological role of TSG‐6.
Stable expression of the human large‐conductance Ca2+‐activated K+ channel α‐ and β‐subunits in HEK293 cells
Authors: Ahring Philip K. , Strøbæk Dorte , Christophersen Palle , Olesen Søren-Peter , Johansen Teit E.
Published: July 2002

We have generated HEK293 cell lines stably expressing high levels of either the human BK channel α‐subunit alone or the BK channel α‐subunit and β‐subunit together. For co‐expression a plasmid with three expression cassettes was constructed. Patch‐clamp recordings on inside‐out patches from the transfected cells resulted in macroscopic currents reflecting the expression of 200–800 BK channels per patch. No decrease in channel expression could be detected in cells grown for more than 50 passages. The α‐subunit when expressed alone conducted currents which were sensitive to intracellular Ca2+ in the physiological range. In the presence of the β‐subunit the steady‐state activation curves were shifted by −20 to −30 mV and channel deactivation kinetics were slowed. The BK channel opener NS1608 (10 μM) shifted the steady‐state activation curves for the α‐subunit as well as for the αβ‐subunits by −40 to −50 mV.
The mouse relaxin‐like factor gene and its promoter are located within the 3′ region of the JAK3 genomic sequence
Authors: Koskimies Pasi , Spiess Andrej-Nikolai , Lahti Petra , Huhtaniemi Ilpo , Ivell Richard
Published: July 2002

Isolation and sequencing of a genomic clone encoding the mouse gene for the relaxin‐like factor (RLF), which is endogenously expressed to a high level exclusively in Leydig cells, indicated that similar sequences were also present at the 3′ end of the mouse JAK3 gene, a gene expressed predominantly in lymphoid tissues. More extensive Southern blot, polymerase chain reaction and sequencing analyses showed that the published mouse sequence for exon 23 of the JAK3 gene in fact comprises two exons, 23A and 23B, separated by an additional novel intron of 2.2 kb, and that within this intron the promoter and exon 1 of the mouse RLF gene are encoded. The two overlapping transcripts appear to use different polyadenylation signals in the common 3′ untranslated region of exon 23B. Transient transfection of different RLF promoter reporter constructs into Leydig, Sertoli, granulosa and kidney cell lines indicate that as little as 0.7 kb of the region upstream of exon 1 of the RLF gene, and within the novel intron 22 of the JAK3 gene, is sufficient to account for cell‐specific expression of the RLF gene. This promoter region is specifically hypomethylated in Leydig cells compared to non‐expressing tissues.
Differences in F9 and 5.51 cell elasticity determined by cell poking and atomic force microscopy
Authors: Goldmann Wolfgang H , Galneder Reinhard , Ludwig Markus , Kromm Alexander , Ezzell Robert M
Published: July 2002

We studied the elasticity of both a wild type (F9) mouse embryonic carcinoma and a vinculin‐deficient (5.51) cell line, which was produced by chemical mutagenesis. Using cell poking, we measured the effects of loss of vinculin on the elastic properties of these cells. F9 cells were about 20% more resistant to indentation by the cell poker (a glass stylus) than were 5.51 cells. Using the atomic force microscope to map the elasticity of wild type and vinculin‐deficient cells by 128×128 force scans, we observed a correlation of elasticity with cell poking elastometric measurements. These findings, as well as previous atomic force, rheologic, and magnetometric measurements [Goldmann and Ezzell, Exp. Cell Res. 226 (1996) 234–237; Ezzell et al., Exp. Cell Res. 231 (1997) 14–26], indicate that vinculin is an integral part of the cytoskeletal network.
Features of replicative senescence induced by direct addition of antennapedia‐p16INK4A fusion protein to human diploid fibroblasts
Authors: Kato Daishiro , Miyazawa Kazuhiro , Ruas Marugarida , Starborg Maria , Wada Ikuo , Oka Takahiro , Sakai Toshiyuki , Peters Gordon , Hara Eiji
Published: July 2002

The p16INK4A cyclin‐dependent kinase (Cdk) inhibitor is now recognized as a major tumor suppressor that is inactivated by a variety of mechanisms in a wide range of human cancers. It is also implicated in the mechanisms underlying replicative senescence since p16INK4A RNA and protein accumulate as cells approach their proscribed limit of population doublings in tissue culture. To obtain further evidence of its role in senescence, we have sought ways of overexpressing p16INK4A in primary human diploid fibroblasts (HDF). To circumvent the low transfection efficiency of primary cells we have exploited a recombinant form of the full‐length p16INK4A protein fused to a 16 amino acid peptide from the Drosophila antennapedia protein. This peptide has the capacity to cross both cytoplasmic and nuclear membranes allowing the direct introduction of the active protein to primary cells. Here, we show that antennapedia‐tagged wild‐type p16INK4A protein, but not a functionally compromised tumor‐specific variant, causes G1 arrest in early passage HDFs by inhibiting the phosphorylation of the retinoblastoma protein. Significantly, the arrested cells display several phenotypic features that are considered characteristic of senescent cells. These data support a role for p16INK4A in replicative senescence and raise the possibility of using the antennapedia‐tagged protein therapeutically.
Mobility of cell surface receptors: a re‐evaluation
Authors: Cherry Richard J , Smith Patricia R , Morrison Ian E.G , Fernandez Nelson
Published: July 2002

It has long been known from fluorescence recovery after photobleaching experiments that the mobility of most cell surface receptors is much smaller than expected for free diffusion of proteins in a fluid lipid bilayer. Single‐particle tracking experiments are currently revealing the complexity of the constraints to free diffusion. Evidence has been obtained for several different processes: domain‐limited diffusion, temporary confinement and anomalous diffusion. The type of motion exhibited by a given receptor will profoundly influence the rate of any functional process which requires movement in the plane of the membrane. In particular, anomalous diffusion greatly reduces the distance travelled by a receptor on a time scale of minutes.
Voltage gating is a fundamental feature of porin and toxin β‐barrel membrane channels
Authors: Bainbridge Graeme , Gokce Isa , Lakey Jeremy H
Published: July 2002

Beta‐barrel pores are found in outer membrane porins of Gram‐negative bacteria, bacterial toxins and mitochondrial channels. Apart from the β‐barrel the three groups show no close sequence or structural homology but these pores exhibit symmetrical voltage gating when reconstituted into planar lipid bilayers. The structures of several of these are known and many site‐directed mutants have been examined. As a result it seems evident that the gating is a common characteristic of these unrelated large pores and is not generated by specialised structures in the pore lumen.
Electrical current generation and proton pumping catalyzed by the ba3‐type cytochrome c oxidase from Thermus thermophilus
Authors: Kannt Aimo , Soulimane Tewfik , Buse Gerhard , Becker Anja , Bamberg Ernst , Michel Hartmut
Published: July 2002

Several amino acid residues that have been shown to be essential for proton transfer in most cytochrome c oxidases are not conserved in the ba
3‐type cytochrome c oxidase from the thermophilic eubacterium Thermus thermophilus. So far, it has been unclear whether the Th. thermophilus ba
3‐type cytochrome c oxidase can nevertheless function as an electrogenic proton pump. In this study, we have combined charge translocation measurements on a lipid bilayer with two independent methods of proton pumping measurements to show that enzymatic turnover of the Th. thermophilus cytochrome c oxidase is indeed coupled to the generation of an electrocurrent and proton pumping across the membrane. In addition to a ‘vectorial’ consumption of 1.0 H+/e− for water formation, proton pumping with a stoichiometry of 0.4–0.5 H+/e− was observed. The implications of these findings for the mechanism of redox‐coupled proton transfer in this unusual cytochrome c oxidase are discussed.
ATP synthesis by the F1Fo ATP synthase of Escherichia coli is obligatorily dependent on the electric potential
Authors: Kaim Georg , Dimroth Peter
Published: July 2002

The H+‐translocating F1Fo ATP synthase of Escherichia coli was purified and reconstituted into proteoliposomes. This system catalyzed ATP synthesis when energized by an acid/base transition (pHin=5.0; pHout=8.3) with succinate, malonate or maleinate but not with MES as the acidic buffer. Under these experimental conditions an electric potential of 125–130 mV is generated by the diffusion of succinate, probably the monoanionic species, whereas with MES buffer the measured potential was at background level (∼5 mV). ATP was also synthesized at pH 7.2 in the absence of a ΔpH by applying a K+/valinomycin diffusion potential. The rate of ATP synthesis increased with the potential in an exponential manner with an inflection point at about 70 mV. We conclude from these results that ΔpH and ΔΨ are kinetically unequivalent driving forces for ATP synthesis by the E. coli ATP synthase and that ΔΨ is a mandatory force for this synthesis. The significance of these findings for the mechanism of ATP synthesis in general is discussed.
C‐terminal truncation of yeast SerRS is toxic for Saccharomyces cerevisiae due to altered mechanism of substrate recognition
Authors: Lenhard Boris , Prætorius-Ibba Mette , Filipic Sanda , Söll Dieter , Weygand-Durasevic Ivana
Published: July 2002

Like all other eukaryal cytosolic seryl‐tRNA synthetase (SerRS) enzymes, Saccharomyces cerevisiae SerRS contains a C‐terminal extension not found in the enzymes of eubacterial and archaeal origin. Overexpression of C‐terminally truncated SerRS lacking the 20‐amino acid appended domain (SerRSC20) is toxic to S. cerevisiae possibly because of altered substrate recognition. Compared to wild‐type SerRS the truncated enzyme displays impaired tRNA‐dependent serine recognition and is less stable. This suggests that the C‐terminal peptide is important for the formation or maintenance of the enzyme structure optimal for substrate binding and catalysis.
Purification of histidine tagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sensory rhodopsin II functionally expressed in Escherichia coli
Authors: Hohenfeld Ilja P , Wegener Ansgar A , Engelhard Martin
Published: July 2002

Bacteriorhodopsin (BR) from Halobacterium salinarum as well as halorhodopsin (pHR) and sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were functionally expressed in E. coli using the method of Shimono et al. [FEBS Lett. (1997) 420, 54–56]. The histidine tagged proteins were purified with yields up to 1.0 mg/l cell culture and characterized by ESI mass spectrometry and their photocycle. The pSRII and pHR photocycles were indistinguishable from the wild type proteins. The BR photocycle was considerably prolonged. pSOII is located in the cytoplasmic membrane and the C‐terminus is oriented towards the cytoplasm as determined by immunogold labelling.
Absence in amphotericin B‐spiked human plasma of the free monomeric drug, as detected by SERS
Authors: Ridente Yara , Aubard Jean , Bolard Jacques
Published: July 2002

Using surface enhanced Raman spectroscopy (SERS) which enables us to specifically detect traces of monomeric amphotericin B (AmB), we were able to show that in a 10−5 M AmB suspension, the concentration of free drug was below 10−8 M in the presence of low density lipoproteins (LDL) or plasma. The affinity constant of AmB for LDL determined from electronic absorption data, was found to be 4×106 M−1. Therefore, since AmB appears to be in the majority bound to lipoproteins under in vivo conditions, its toxicity should not result from the induction of host‐cell transmembrane permeability but rather from the internalization of the AmB‐LDL complex.
Spectroscopic resolution of the picosecond reduction kinetics of the secondary electron acceptor A1 in photosystem I
Authors: Brettel Klaus , H. Vos Marten
Published: July 2002

Forward electron transfer in photosystem I from Synechocystis sp. PCC 6803 has been studied in the picosecond time range with transient absorption spectroscopy in the blue and near‐UV spectral regions. From the direct measurement, at 380–390 nm, of the reduction kinetics of the phylloquinone secondary acceptor A1 and from the absence of spectral evolution between 100 ps and 2 ns, we conclude that electron transfer, from the chlorophyll a primary acceptor A0, to A1 occurs directly and completely with a time constant of about 30 ps.
Discrimination between ubiquitin‐dependent and ubiquitin‐independent proteolytic pathways by the 26S proteasome subunit 5a
Authors: Mahaffey David , Rechsteiner Martin
Published: July 2002

The 26S proteasome subunit 5a binds polyubiquitin chains and has previously been shown to inhibit the degradation of mitotic cyclins. Presumably inhibition results from S5a binding and preventing recognition of Ub‐cyclin conjugates by the 26S proteasome. Here we show that S5a does not inhibit the degradation of full‐length ornithine decarboxylase (ODC) consistent with previous reports that the enzyme is degraded in an antizyme‐dependent, but ubiquitin‐independent reaction. S5a does, however, inhibit degradation of short ODC translation products generated by internal initiation events. Because in vitro translation often produces some shortened products, the existence of ubiquitin conjugated to a 35S‐labeled protein is not necessarily evidence that the full‐length protein is a substrate of the Ub‐dependent proteolytic pathway.
Regulation of UCP3 by nucleotides is different from regulation of UCP1
Authors: Echtay Karim Salim , Liu Qingyun , Caskey Tom , Winkler Edith , Frischmuth Karina , Bienengräber Martin , Klingenberg Martin
Published: July 2002

UCP3 is an isoform of UCP1, expressed primarily in skeletal muscle. Functional properties of UCP3 are still largely unknown. Here, we report about the expression of UCP3 and of UCP1 in inclusion bodies of Escherichia coli. On solubilization and reconstitution into proteoliposomes, both UCP3 and UCP1 transport Cl− at rates equal to the reconstituted native UCP1. Cl− transport is inhibited by low concentrations of ATP, ADP, GTP and GDP. However, no H+ transport activity is found possibly due to the lack of a cofactor presents in UCP from mitochondria. The specificity of inhibition by nucleoside tri‐ and diphosphate is different between UCP1 and UCP3. UCP1 is more sensitive to tri‐ than diphosphate whereas in UCP3, the gradient is reverse. These results show a new paradigm for the regulation of thermogenesis at various tissues by the ATP/ADP ratio. In brown adipose tissue, the thermogenesis is correlated with a low ATP/ADP whereas in skeletal muscle, non‐shivering thermogenesis is active at a high ATP/ADP ratio, i.e. in the resting state.
VacA from Helicobacter pylori: a hexameric chloride channel
Authors: Iwamoto Hideki , Czajkowsky Daniel M. , Cover Timothy L. , Szabo Gabor , Shao Zhifeng
Published: July 2002

VacA is a unique protein toxin secreted by the human pathogen Helicobacter pylori. At a neutral pH, the cytotoxin self‐associates into predominantly dodecameric complexes. In this report, we show that at an acidic pH, VacA forms anion selective channels in planar phospholipid bilayers. Similar to several other chloride channels, the VacA channel exhibits a moderate selectivity for anions over cations (PCl:PNa=4.2:1), inhibition by the blocker 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid and a permeability sequence, SCN−≫I−>Br−>Cl−>F, consistent with a ‘weak field strength’ binding site for the permeant anion. Single channel recordings reveal rapid transitions (486 s−1) between the closed state and a single open state of 24 pS (+60 mV, 1.5 M NaCl). Evaluation of the rate of increase in macroscopic current as well as atomic force microscopy suggest that this VacA channel is a hexamer, formed by the assembly of membrane‐bound monomers. Not only are these VacA channels likely to play an important role in the pathological activity of this toxin, but they may also serve as a model system to further investigate the mechanism of anion selectivity in general.
Images of oligomeric Kvβ2, a modulatory subunit of potassium channels
Authors: van Huizen Rika , Czajkowsky Daniel M. , Shi Dan , Shao Zhifeng , Li Min
Published: July 2002

The Shaker type voltage‐gated potassium (K+) channel consists of four pore‐forming Kvα subunits. The channel expression and kinetic properties can be modulated by auxiliary hydrophilic Kvβ subunits via formation of heteromultimeric Kvα‐Kvβ complexes. Because each (Kvα)4 could recruit more than one Kvβ subunit and different Kvβ subunits could potentially interact, the stoichiometry of α‐β and β‐β complexes is therefore critical for understanding the functional regulation of Shaker type potassium channels. We expressed and purified Kvβ2 subunit in Sf9 insect cells. The purified Kvβ2, examined by atomic force and electron microscopy techniques, is found predominately as a square‐shaped tetrameric complex with side dimensions of 100×100 Å2 and height of 51 Å. Thus, Kvβ2 is capable of forming a tetramer in the absence of pore‐forming α subunits. The center of the Kvβ2 complex was observed to be the most heavily stained region, suggesting that this region could be part of an extended tubular structure connecting the inner mouth of the ion permeation pathway to the cytoplasmic environment.
Comparison of ΔpH‐ and Δφ‐driven ATP synthesis catalyzed by the H+‐ATPases from Escherichia coli or chloroplasts reconstituted into liposomes
Authors: Fischer Susanne , Gräber Peter
Published: July 2002

The H+‐ATPases from Escherichia coli, EF0F1, and from chloroplasts, CF0F1, were reconstituted in liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid‐base transition and a K+/valinomycin diffusion potential and the initial rate of ATP synthesis was measured as a function of the transmembrane pH difference, ΔpH, and the electric potential difference, Δφ. With EF0F1, a rate of 80 s−1 is observed at ΔpH=4.1 and Δφ≈140 mV. The rate decreases sigmoidally with Δφ and at Δφ≈0 mV, the rate is about 1 s−1 although ΔpH is still 4.1. Under the same conditions with CF0F1, a rate of 280 s−1 is observed which decreases to 190 s−1 when Δφ is abolished, i.e. ATP synthesis catalyzed by EF0F1 and CF0F1 depends in a different way on ΔpH and Δφ. EF0F1‐catalyzed ATP synthesis was measured as a function of ΔpH at a constant Δφ. The rate depends sigmoidally on ΔpH reaching a maximal rate which cannot be further increased by increasing ΔpH. However, this maximal rate depends on Δφ, i.e. ΔpH and Δφ are not kinetically equivalent in driving ATP synthesis. We assume that EF0F1 must be converted into a metastable, active state before it catalyzes proton transport‐coupled ATP synthesis. For EF0F1, this activation step depends only on Δφ, whereas for CF0F1, the activation depends on ΔpH and Δφ.
Binding of the third Na+ ion to the cytoplasmic side of the Na,K‐ATPase is electrogenic
Authors: Domaszewicz Wolfgang , Apell Hans-Jürgen
Published: July 2002

A new experimental setup was constructed to allow parallel measurements of total internal reflection fluorescence and of capacitance changes in Na,K‐ATPase‐containing membranes. Effects correlated with cytoplasmic sodium binding to Na,K‐ATPase were investigated. Ion binding‐induced fluorescence changes of the electrochromic dye RH421 in membrane fragments adsorbed on a transparent capacitative electrode corresponded perfectly to capacitance increases detected by a lock‐in technique. From these electric measurements it was possible to estimate a dielectric coefficient of about 0.25 for the electrogenic binding of the third Na+ ion. Binding of K+ to cytoplasmic sites was electroneutral.
Zinc binding reverses the calcium‐induced arachidonic acid‐binding capacity of the S100A8/A9 protein complex
Authors: Kerkhoff Claus , Vogl Thomas , Nacken Wolfgang , Sopalla Claudia , Sorg Clemens
Published: July 2002

Analysis of the calcium‐induced arachidonic acid (AA) binding to S100A8/A9 revealed that maximal AA binding was achieved at molar ratios of 1 mol S100A8 and 1 mol S100A9 and for values greater than 3 calciums per EF‐hand. The AA binding capacity was not induced by the binding of other bivalent cations, such as Zn2+, Cu2+, and Mg2+, to the protein complex. In contrast, the binding of AA was prevented by the addition of either Zn2+ or Cu2+ in the presence of calcium, whereas Mg2+ failed to abrogate the AA binding capacity. The inhibitory effect was not due to blocking the formation of S100A8/A9 as demonstrated by a protein‐protein interaction assay. Fluorescence measurements gave evidence that both Zn2+ and Cu2+ induce different conformational changes thereby affecting the calcium‐induced formation of the AA binding pocket within the protein complex. Due to the fact that the inhibitory effect of Zn2+ was present at physiological serum concentrations, it is assumed that released S100A8/A9 may carry AA at inflammatory lesions, but not within the blood compartment.
The importance of C‐terminal residues of vertebrate and invertebrate tachykinins for their contractile activities in gut tissues
Authors: Ikeda Tetsuya , Minakata Hiroyuki , Nomoto Kyosuke
Published: July 2002

The C‐terminal residues of mammalian tachykinins and urechistachykinins (Uru‐TKs), tachykinin‐related peptides of echiuroid worm origin, were substituted for each other. Their contractile effects were assayed on the cockroach hindgut and the guinea pig ileum. [Met10] substitution of Uru‐TKs caused a 1000 times lower activity on the hindgut, but a 1000 times higher activity on the ileum. In contrast, [Arg11]substance P (SP) was 100 times more and 400 times less potent than SP on the hindgut and ileum, respectively. A SP antagonist blocked these Uru‐TK activities on the hindgut. These results demonstrated that the C‐terminal Met‐NH2 is necessary for ileum contraction and the Arg‐NH2 is required for hindgut contraction, which was caused by binding to the cockroach's neurokinin‐like receptor.
Complementary packing of α‐helices in proteins
Authors: Efimov Alexander V
Published: July 2002

The packing of α‐helices in proteins is restricted by both the principle of close packing and the chemical nature of side chains. As a result, (1) α‐helical surfaces forming the interface should be complementary to each other, (2) hydrophobic stripes of the α‐helices should fit together like pieces of a jigsaw puzzle, and (3) buried polar side chains (if there are any) should be arranged in a complementary fashion.
The γ subunit in chloroplast F1‐ATPase can rotate in a unidirectional and counter‐clockwise manner
Authors: Hisabori Toru , Kondoh Aiko , Yoshida Masasuke
Published: July 2002

Rotation of the γ subunit in chloroplast F1‐ATPase (CF1) was investigated by using a single molecule observation technique, which is developed by Noji et al. to observe the rotation of a central γ subunit portion in the α3β3γ sub‐complex of F1‐ATPase from thermophilic Bacillus PS3 (TF1) during ATP hydrolysis [Noji, H. et al. (1997) Nature 386, 299–302]. We used two cysteines of the γ subunit (Cys‐199 and Cys‐205) of CF1‐ATPase, which are involved in the regulation of this enzyme, to fix the fluorochrome‐labeled actin filament. Then we successfully observed a unidirectional, counter‐clockwise rotation of the actin filament with the fluorescent microscope indicating the rotation of the γ subunit in CF1‐ATPase. We conclude that the rotation of the γ subunit in the F1‐motor is a ubiquitous phenomenon in all F1‐ATPases in prokaryotes as well as in eukaryotes.
A validation study of maternal self reports of obstetrical complications: implications for health surveys
Authors: Seoane G , Castrillo M , O'Rourke K
Published: July 2002

Objective: The purpose of this study was to determine the validity of maternal self reports of obstetrical complications, which are commonly used in estimating the prevalence of complications. These estimates vary greatly between countries and could either reflect true differences or reporting bias. Methods: A cross‐sectional study was conducted among 1027 women in two Bolivian maternity hospitals. A questionnaire recorded mothers' perceptions of obstetrical complications while hospital medical records and physical examinations established their clinical condition. Sensitivity, specificity, predictive values, and percent agreement were obtained for obstetrical conditions. Results: In general, women's reports of obstetrical complications did not match medical diagnoses. The highest agreement was obtained for reporting eclampsia, with less agreement for labor disorders, postpartum hemorrhage and malpresentation. Conclusions: Maternal self reports in this study did not provide a valid estimate of the prevalence of obstetrical complications. Health surveys based on maternal self reports must be interpreted with consideration of this limitation.
Description of the fully mature oocyte of Drosophila melanogaster
Authors: Hatsumi Machiko , Watanabe Yasuhiro , Sawa Masami
Published: July 2002

Summry— We detected fully mature undescribed oocytes in Drosophila melanogaster ovaries. The fully mature oocytes were rehydrate in ovaries. In the oocyte of stage 14, chromosomes make globular mass, while the chromosome shows meiotic metaphase I in fully mature oocytes. Both mature oocytes and stage 14 oocytes were activated by hypotonic treatment. When the mature oocytes or the stage 14 oocytes were activated, telophase II figures were observed in former oocytes but meiosis in latter oocyte stopped at late metaphase I, suggesting the stage 14 activated oocyte cannot pass the second checkpoint of meiosis.
Service quality along the supply chain: implications for purchasing
Authors: Stanley L.L. , Wisner J.D.
Published: July 2002

The overall objective of this research was to explore the association between implementation of cooperative purchasing/supplier relationships, internal service quality, and an organization’s ability to provide quality products and services to its customers. Specifically, purchasing‐related factors influencing internal and external product and service quality were identified from the literature and an internal service quality model was developed and then tested using empirical data. Survey data were collected from 118 US purchasing executives in a wide range of industries. The findings from this study indicate the existence of strong positive relationships between implementation of cooperative purchasing/supplier relationships, internal service quality, and the service and product quality provided to external customers. Additionally, the key role of purchasing in the integration and communication of quality expectations and quality performance throughout the firm is demonstrated.
Effect of service supplier performance on satisfaction and loyalty of store managers in the fast food industry
Authors: Stank Theodore P , Goldsby Thomas J , Vickery Shawnee K
Published: July 2002

The paper presents research that examines the complex relationship between service quality and profitability in service operations. Specifically, the effects of operational performance and relational performance on customer satisfaction and loyalty in the unique context of industrial services are examined. A model of service performance effects on customer satisfaction and loyalty is introduced and tested in the fast food service industry using customer perceptions of provider performance rather than relying on providers' self‐reported performance indicators. The research revealed that the benefit of establishing customer relationships emerges from the enhanced insight the supplier is able to gain regarding customer needs and wants. Upon learning of these needs and wants, the service provider can focus on operational means of meeting the needs and wants, and doing so in the lowest cost manner.
Predictability of dropout in unipolar depressed outpatients
Authors: Hoencamp E , Haffmans PMJ , Duivenvoorden HJ
Published: July 2002

In a three phase sequential treatment strategy study involving 119 depressed outpatients, a total of 31 patients (26.7%) stopped treatment prematurely due to side-effects (21/31), aggravation of symptoms (3/31), non-compliance (4/31), and non-treatment related events (3/31). At baseline, no significant differences were found on sociodemographic and psychiatric data between patients who did or did not drop out. As predictors of dropout eight domains of data concerning psychiatric history, premorbid history, symptomatology, personality, and social adjustment were used. Using a logistic linear regression analysis, only three variables were related to dropout. Patients with a history of alcohol use or poor social functioning according to axis V of the Diagnostic and Statistical Manual (DSM)-III-R had a higher chance to drop out, while patients with a sleep disturbance according to the Symptom Checklist (SCL)-90 had a smaller chance to drop out.
Up‐regulation of ‘clearance’ receptors in patients with chronic heart failure: a possible explanation for the resistance to biological effects of cardiac natriuretic hormones
Authors: Andreassi Maria Grazia , Del Ry Silvia , Palmieri Cataldo , Clerico Aldo , Biagini Andrea , Giannessi Daniela
Published: July 2002

Background:

Three specific receptors for the cardiac natriuretic peptide system have been identified to date. Down‐regulation of the biologically active binding sites (i.e. NPR‐A and NPR‐B) could explain the blunted response to cardiac natriuretic hormones observed in heart failure (HF), but not the increased metabolic clearance rate. Variations in the ratio between biological and clearance (NPR‐C) receptors in target tissue may explain this increase.

Aim:

The aim of this study was to investigate the regulation of NPR‐C receptors on platelets, in patients with HF.

Methods:

Eighteen patients with HF (NYHA class: I–II, n ‐ 8; III–IV, n ‐ 10) and 18 age‐matched healthy subjects were studied. The affinity constant (Kd) and density (Bmax) of binding sites were derived by saturation assays on platelet suspensions using 125I‐ANP as radioligand.

Results:

Bmax increased as a function of the severity of disease: 21.3±3.3 fmol/109 cells in class III–IV, 11.7±2.2 in class I–II, and 11.6±1.1 in controls, respectively (P ‐ 0.0179 for class III–IV vs. controls and P ‐ 0.0451 vs. NYHA I–II).

Conclusions:

The increase in density of ‘clearance’ receptors in severe HF is theoretically consistent with the reduction in cardiac natriuretic peptide biological activity, as well as the increase in metabolic clearance rate. This suggests that clearance receptor blockade may be of potential therapeutic value in HF.
Wide‐open conformation of UDP‐MurNc‐tripeptide ligase revealed by the substrate‐free structure of MurE from Acinetobacter baumannii
Authors: Jung Kyoung Ho , Kim Yeon‐Gil , Kim Chang Min , Ha Hyun Ji , Lee Chang Sup , Lee Jun Hyuck , Park Hyun Ho
Published: November 2020

MurE ligase catalyzes the attachment of meso‐diaminopimelic acid to the UDP‐MurNAc‐l‐Ala‐d‐Glu using ATP and producing UDP‐MurNAc‐l‐Ala‐d‐Glu‐meso‐A2pm during bacterial cell wall biosynthesis. Owing to the critical role of this enzyme, MurE is considered an attractive target for antibacterial drugs. Despite extensive studies on MurE ligase, the structural dynamics of its conformational changes are still elusive. In this study, we present the substrate‐free structure of MurE from Acinetobacter baumannii, which is an antibiotic‐resistant superbacterium that has threatened global public health. The structure revealed that MurE has a wide‐open conformation and undergoes wide‐open, intermediately closed, and fully closed dynamic conformational transition. Unveiling structural dynamics of MurE will help to understand the working mechanism of this ligase and to design next‐generation antibiotics targeting MurE.
Autophagy activation and SREBP‐1 induction contribute to fatty acid metabolic reprogramming by leptin in breast cancer cells
Authors: Pham Duc‐Vinh , Tilija Pun Nirmala , Park Pil‐Hoon
Published: November 2020

Leptin, a hormone predominantly derived from adipose tissue, is well known to induce growth of breast cancer cells. However, its underlying mechanisms remain unclear. In this study, we examined the role of reprogramming of lipid metabolism and autophagy in leptin‐induced growth of breast cancer cells. Herein, leptin induced significant increase in fatty acid oxidation‐dependent ATP production in estrogen receptor‐positive breast cancer cells. Furthermore, leptin induced both free fatty acid release and intracellular lipid accumulation, indicating a multifaceted effect of leptin in fatty acid metabolism. These findings were further validated in an MCF‐7 tumor xenograft mouse model. Importantly, all the aforementioned metabolic effects of leptin were mediated via autophagy activation. In addition, SREBP‐1 induction driven by autophagy and fatty acid synthase induction, which is mediated by SREBP‐1, plays crucial roles in leptin‐stimulated metabolic reprogramming and are required for growth of breast cancer cell, suggesting a pivotal contribution of fatty acid metabolic reprogramming to tumor growth by leptin. Taken together, these results highlighted a crucial role of autophagy in leptin‐induced cancer cell‐specific metabolism, which is mediated, at least in part, via SREBP‐1 induction.