bluuebunny/crossref_metadata_2025 huggingface dataset:
A leucine‐rich repeat peptide derived from the Drosophila Toll receptor forms extended filaments with a β‐sheet structure
Authors: Gay Nicholas J. , Packman Leonard C. , Weldon Michael A. , Barna Jennifer C.J.
Published: July 2002

Leucine‐rich repeats (LRRs) are 22–28 amino acid‐long sequence motifs found in a family of cytoplasmic, membrane and extracellular proteins. There is evidence that LRRs function in signal transduction, cellular adhesion and protein‐protein interactions. Here we report unusual properties of a synthetic LRR peptide derived from the sequence of the Drosophila membrane receptor Toll. In neutral solution the peptide forms a gel revealed by electron microscopy to consist of extended filaments approximately 8 nm in thickness. As the gel forms, the circular dichroism spectrum of the peptide solution changes from one characteristic of random coil to one associated with β‐sheet structures. Molecular modelling suggests that the peptides form an amphipathic structure with a predominantly apolar and charged surface. Based on these results, models for the gross structure of the peptide filaments and a possible molecular mechanism for cellular adhesion are proposed.
Uncoupling protein is expressed in liver mitochondria of cold‐exposed and newborn rats
Authors: Shinohara Yasuo , Shima Atsushi , Kamida Makio , Terada Hiroshi
Published: July 2002

Uncoupling protein has been thought to be expressed only in the brown adipose tissue mitochondria of mammals. However. mRNA encoding mitochondrial uncoupling protein was detected in the liver of newborn rats and adult rats after cold exposure, although not in the liver of untreated adult rats.
Effective activation of the proenzyme form of the urokinase‐type plasminogen activator (pro‐uPA) by the cysteine protease cathepsin L
Authors: Goretzki Lothar , Schmitt Manfred , Mann Karlheinz , Calvete Juan , Chucholowski Nicolaus , Kramer Michael , Günzler Wolfgang A. , Jänicke Fritz , Graeff Henner
Published: July 2002

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase‐type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single‐chain proenzyme (pro‐uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro‐uPA. Enzymatic assays, SDS‐PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro‐uPA. As determined by N‐terminal amino acid sequence analysis, activation of pro‐uPA by cathepsin L is achieved by cleavage or the Lys158‐lle159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147‐5152) cleavage of pro‐uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro‐uPA of 1:2,000). Nevertheless, even at pH 7.0, pro‐uPA was activated by cathepsin L, although a 10‐fold higher concentration of cathepsin L was required. As tumor cells may produce both pro‐uPA and cathepsin L, implications for the activation of tumor cell‐derived pro‐uPA by cathepsin L may be considered. Different pathways activation of pro‐uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro‐uPA; (ii) activation by serine proteases (plasmin, kallikrein. Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).
Identification of an isoform with an extremely large Cys‐rich region of PC6, a Kex2‐like processing endoprotease
Authors: Nakagawa Tsutomu , Murakami Kazuo , Nakayama Kazuhisa
Published: July 2002

In the previous study [1993, J. Biochem. (Tokyo) 113, 132‐135] we identified PC6, a member of the Kex2 family of processing endoproteases. In this study, we identified another cDNA encoding an isoform of PC6, and designated it as PC6B and redesignated the originally identified PC6 as PC6A. PC6B had a very large Cys‐rich region consisting of 22‐times repeats of a Cys‐rich motif, and a putative transmembrane domain which is not present in PC6A. A PC6B transcript was found mainly in the intestine, while PC6A transcripts were in various tissues. These results suggest distinct roles of PC6A and PC6B in endoproteolytic processing of precursor proteins.
Increased production of endothelin‐1 in the hypertrophied rat heart due to pressure overload
Authors: Yorikane Ryosuke , Sakai Satoshi , Miyauchi Takashi , Sakurai Takeshi , Sugishita Yasuro , Goto Katsutoshi
Published: July 2002

Endothelin‐1 (ET‐1) has been demonstrated to induce hypertrophy in cultured cardiac myocytes. We investigated the production of ET‐1 in the heart of aorta‐banded rats in vivo. Seven days after the banding of the abdominal aorta, rats developed a significant left ventricular hypertrophy. The tissue content of mature ET‐1 and the level of expression of prepro ET‐1 mRNA were higher in the left ventricle of aorta‐banded rats than in those of sham‐operated rats. The expression of prepro ET‐1 mRNA in the right ventricle was not different between the two groups. These findings indicate that the production of ET‐1 increased in the hypertrophied left ventricle, thereby suggesting the possible involvement of endogenous ET‐1 in the development of cardiac hypertrophy due to pressure overload.
The effect of sugars on the morphology of the bacterial flagellum
Authors: Seville Mark , Ikeda Takeshi , Hotani Hirokazu
Published: July 2002

Using dark‐field microscopy, we have found that certain sugars cause the normal‐to‐curly helical transition of bacterial flagella. Titration of flagella isolated from Salmonella typhimwium with 16 different carbohydrates showed that: (i) only certain sugars cause the transition. There is no obvious relationship between the simple physico‐chemical properties of the sugar and whether the sugar causes the transition or not; (ii) the efficacies of sugars that do cause the transition differ markedly. For these sugars there is a relationship between efficacy and molecular size. These results suggest that the specific, though weak, binding of sugars to sites on flagella cause the morphological transition.
Differentiation potentiates oxidant injury to mitochondria by hydrogen peroxide in Friend's erythroleukemia cells
Authors: Comelli Marina , Lippe Giovanna , Mavelli Irene
Published: July 2002

Oxidative damage to mitochondrial functions was investigated upon non‐lethal treatment with H2O2 of Friend's erythroleukemia cells induced to differentiate, in comparison with the parental cell line. Both respiration and maximal ATP synthase capacity were more severely diminished by H2O2 in induced cells. The effects were mediated by intracellular redox‐active iron and OH. radicals. Specifically, the mechanisms of the selective oxidant injury to F0F1 ATP synthase observed in differentiating cells likely involved impairment of F0—F1 coupling sensitive to oligomycin. We suggest a Fenton‐like reaction of H2O2 with iron ions, more available in the differentiating cells, as occurring at the surface and/or in the lipid bulk phase of the inner mitochondrial membrane, thus injuring subunits responsible for the coupling of F0F1 ATP synthase through generation in situ of the actual damaging species. Besides, we propose heme iron as the most likely candidate for such reaction in induced cells actively synthesizing heme. In accordance, pretreatment of uninduced cells with hemin made H2O2‐damage qualitatively identical.
The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a Ping‐Pong mechanism
Authors: Nakamura Akira , Haga Keiko , Yamane Kunio
Published: July 2002

A new photometric assay of the disproportionation activity of cyclodextrin glucanotransferase (CGTase) using 3‐ketobutylidene‐β‐2‐chloro‐4‐nitrophenyl ‐maltopentaoside as the donor, proved that the transglycosylation reaction of CGTase was operated by a Ping‐Pong Bi Bi mechanism. The values of the k
cat/K
m
acceptor proved that the same configurations of free hydroxyl groups with those of d‐glucopyranose at C2, C3 and C4 positions were required for the acceptors used by CGTase. The structure around C6 on acceptors was not essential for acceptor function, but it was recognized by CGTase, since the values of k
cat/K
m for d‐xylose were smaller than that for d‐glucose. The value of k
cat/K
mfor maltose was about 20‐times larger than that for d‐glucose, indicating that at least two glucopyranosyl rings are recognized by the acceptor binding sites.
Novel avidin and streptavidin binding sequences found in synthetic peptide libraries
Authors: Østergaard Soren , Hansen Per Hertz , Olsen Marianne , Holm Arne
Published: July 2002

Synthetic resin‐bound peptide libraries made of protein l‐amino acids have been synthesized. During screening of the libraries, peptides that bind to avidin have been identified containing a novel motif with two histidines separated by one residue. A sub‐library was synthesized and screened, and new critical residues appeared surrounding the two histidines. Additionally peptide libraries made of d‐amino acids have been screened with avidin and streptavidin and novel motifs have been found.
Recombinant NeutraLite Avidin: a non‐glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non‐specific binding properties
Authors: Marttila Ari T. , Laitinen Olli H. , Airenne Kari J. , Kulik Tikva , Bayer Edward A. , Wilchek Meir , Kulomaa Markku S.
Published: July 2002

A recombinant non‐glycosylated and acidic form of avidin was designed and expressed in soluble form in baculovirus‐infected insect cells. The mutations were based on the same principles that guided the design of the chemically and enzymatically modified avidin derivative, known as NeutraLite Avidin. In this novel recombinant avidin derivative, five out of the eight arginine residues were replaced with neutral amino acids, and two of the lysine residues were replaced by glutamic acid. In addition, the carbohydrate‐bearing asparagine‐17 residue was altered to an isoleucine, according to the known sequences of avidin‐related genes. The resultant mutant protein, termed recombinant NeutraLite Avidin, exhibited superior properties compared to those of avidin, streptavidin and the conventional NeutraLite Avidin, prepared by chemo‐enzymatic means. In this context, the recombinant mutant is a single molecular species, which possesses strong biotin‐binding characteristics. Due to its acidic pI, it is relatively free from non‐specific binding to DNA and cells. The recombinant NeutraLite Avidin retains seven lysines per subunit, which are available for further conjugation and derivatization.
States and transitions during forced unfolding of a single spectrin repeat
Authors: Lenne P.-F , Raae A.J , Altmann S.M , Saraste M , Hörber J.K.H
Published: July 2002

Spectrin is a vital and abundant protein of the cytoskeleton. It has an elongated structure that is made by a chain of so‐called spectrin repeats. Each repeat contains three antiparallel α‐helices that form a coiled‐coil structure. Spectrin forms an oligomeric structure that is able to cross‐link actin filaments. In red cells, the spectrin/actin meshwork underlying cell membrane is thought to be responsible for special elastic properties of the cell. In order to determine mechanical unfolding properties of the spectrin repeat, we have used single molecule force spectroscopy to study the states of unfolding of an engineered polymeric protein consisting of identical spectrin domains. We demonstrate that the unfolding of spectrin domains can occur in a stepwise fashion during stretching. The force–extension patterns exhibit features that are compatible with the existence of at least one intermediate between the folded and the completely unfolded conformation. Only those polypeptides that still contain multiple intact repeats display intermediates, indicating a stabilisation effect. Precise force spectroscopy measurements on single molecules using engineered protein constructs reveal states and transitions during the mechanical unfolding of spectrin. Single molecule force spectroscopy appears to open a new window for the analysis of transition probabilities between different conformational states.
Characterization of dehydrodolichyl diphosphate synthase of Arabidopsis thaliana, a key enzyme in dolichol biosynthesis
Authors: Cunillera Núria , Arró Montserrat , Forés Oriol , Manzano David , Ferrer Albert
Published: July 2002

The enzyme dehydrodolichyl diphosphate (dedol‐PP) synthase is a cis‐prenyltransferase that catalyzes the synthesis of dedol‐PP, the long‐chain polyprenyl diphosphate used as a precursor for the synthesis of dolichyl phosphate. Here we report the cloning and characterization of a cDNA from Arabidopsis thaliana encoding dedol‐PP synthase. The identity of the cloned enzyme was confirmed by functional complementation of a yeast mutant strain defective in dedol‐PP synthase activity together with the detection of high levels of dedol‐PP synthase activity in the transformed yeast mutant. The A. thaliana dedol‐PP synthase mRNA was detected at high levels in roots but was hardly detected in flowers, leaves, stems and in A. thaliana suspension‐cultured cells.
Expression of different coding sequences in cell‐free bacterial and eukaryotic systems indicates translational pausing onEscherichia coliribosomes
Authors: Ramachandiran Vasanthi , Kramer Gisela , Hardesty Boyd
Published: July 2002

Five different coding sequences of bacterial or eukaryotic origin in plasmids under the T7 promoter were expressed in a cell‐free system derived fromEscherichia coli. Translation onE. coliribosomes resulted in a full‐length product only in four of the five coding sequences tested. A unique pattern of less than full‐length polypeptides was generated in each case. Many of these polypeptides onE. coliribosomes reacted with a puromycin derivative, cytidylic acid‐puromycin, which was radioactively labeled. Thus these incomplete polypeptides can be defined as nascent peptides bound to the ribosomal P site. Certain nascent peptides could be shifted into full‐length protein indicating that they resulted from translational pausing. In contrast to these results, expression of the same coding sequences in a wheat germ or reticulocyte cell‐free system resulted in a 80–90% full‐length product with no evidence for nascent polypeptides and translational pausing.
Pantetheinase activity of membrane‐bound Vanin‐1: lack of free cysteamine in tissues of Vanin‐1 deficient mice
Authors: Pitari Giuseppina , Malergue Fabrice , Martin Florent , Philippe Jean Marc , Massucci Maria Teresa , Chabret Claude , Maras Bruno , Duprè Silvestro , Naquet Philippe , Galland Franck
Published: July 2002

Pantetheinase (EC 3.5.1.‐) is an ubiquitous enzyme which in vitro has been shown to recycle pantothenic acid (vitamin B5) and to produce cysteamine, a potent anti‐oxidant. We show that the Vanin‐1 gene encodes pantetheinase widely expressed in mouse tissues: (1) a pantetheinase activity is specifically expressed by Vanin‐1 transfectants and is immunodepleted by specific antibodies; (2) Vanin‐1 is a GPI‐anchored pantetheinase, and consequently an ectoenzyme; (3) Vanin‐1 null mice are deficient in membrane‐bound pantetheinase activity in kidney and liver; (4) in these organs, a major metabolic consequence is the absence of detectable free cysteamine; this demonstrates that membrane‐bound pantetheinase is the main source of cysteamine in tissues under physiological conditions. Since the Vanin‐1 molecule was previously shown to be involved in the control of thymus reconstitution following sublethal irradiation in vivo, this raises the possibility that Vanin/pantetheinase might be involved in the regulation of some immune functions maybe in the context of the response to oxidative stress.
Iron stress restricts photosynthetic intersystem electron transport in Synechococcus sp. PCC 7942
Authors: Ivanov A.G. , Park Y.-I. , Miskiewicz E. , Raven J.A. , Huner N.P.A. , Öquist G.
Published: July 2002

Although exposure of Synechococcus sp. PCC 7942 to iron stress induced the accumulation of the isiA gene product (CP43′) compared with non‐stressed controls, immunodetection of the N‐terminus of cytochrome (Cyt) f indicated that iron stress not only reduced the content of the 40 kDa, heme‐binding, Cyt f polypeptide by 32% but it also specifically induced the accumulation of a new, 23 kDa, non‐heme‐binding, putative Cyt f polypeptide. Concomitantly, iron stress restricted intersystem electron transport based on the in vivo reduction of P700+, monitored as ΔA
820/A
820 in the presence and absence of electron transport inhibitors, as well as the inhibition of the Emerson enhancement effect on O2 evolution. However, iron stress appeared to be associated with enhanced rates of PS I cyclic electron transport, low rates of PS I‐driven photoreduction of NADP+ but comparable rates for PS II+PS I photoreduction of NADP+ relative to controls. We hypothesize that Synechococcus sp. PCC 7942 exhibits a dynamic capacity to uncouple PS II and PS I electron transport, which may allow for the higher than expected growth rates observed during iron stress.
Non‐thermal effects of microwaves on proteins: thermophilic enzymes as model system
Authors: Porcelli Marina , Cacciapuoti Giovanna , Fusco Stefania , Massa Rita , d'Ambrosio Guglielmo , Bertoldo Costanzo , De Rosa Mario , Zappia Vincenzo
Published: July 2002

Two thermophilic and thermostable enzymes, isolated from Sulfolobus solfataricus, S‐adenosylhomocysteine hydrolase and 5′‐methylthioadenosine phosphorylase, were exposed to 10.4 GHz microwave radiation in order to discriminate between thermal and non‐thermal microwave effects. The exposure causes a non‐thermal, irreversible and time‐dependent inactivation of both enzymes; the inactivation rate is related to the energy absorbed and is independent of the enzyme concentration. The influence of salts on enzyme inactivation has also been investigated. Conformational changes of S‐adenosylhomocysteine hydrolase, detected by fluorescence and circular dichroism techniques, suggest that microwaves induce protein structural rearrangements not related to temperature.
Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation
Authors: Kinto Naoki , Iwamoto Masahiro , Enomoto-Iwamoto Motomi , Noji Sumihare , Ohuchi Hideyo , Yoshioka Hidefumi , Kataoka Hiroko , Wada Yasuhiro , Yuhao Gao , Takahashi Hideaki E , Yoshiki Shusaku , Yamaguchi Akira
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Osmoregulation and protein expression in a pbs2Δ mutant of Saccharomyces cerevisiae during adaptation to hypersaline stress
Authors: Akhtar Noreen , Blomberg Anders , Adler Lennart
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Direct physical interaction between the Caenorhabditis elegans ‘death proteins’ CED‐3 and CED‐4
Authors: Irmler Martin , Hofmann Kay , Vaux David , Tschopp Jürg
Published: July 2002

The two genes CED‐4 and CED‐3 (the nematode homologue of interleukin‐1β converting enzyme, ICE) of Caenorhabditis elegans are implicated in the control of cell death, but the mechanism by which this occurs is unknown. Here we provide evidence that CED‐3 and CED‐4 both contain sequences with homology to a domain present in RAIDD and the prodomain of certain ICE‐like proteases (caspases). This domain is known to establish an interaction between RAIDD and these caspases. Similarly, CED‐4 was found to interact with CED‐3. Thus, the activity of the death protease CED‐3 appears to be controlled by CED‐4 through a direct physical interaction.
The L7/L12 ribosomal domain of the ribosome: structural and functional studies
Authors: Gudkov A.T.
Published: July 2002

The L7/L12 protein forms a functionally important domain in the ribosome. This domain is involved in interaction with translation factors during protein biosynthesis. The tertiary and quaternary structure of the L7/L12 protein was established as a result of intensive studies in solution and in the ribosome. The conformational changes of L7/L12, the elongation factors Tu and G and other ribosomal proteins were traced by different experimental techniques. These changes occur upon interaction of the ribosome with the elongation factors and depend on GTP hydrolysis in accordance with the functional states of the ribosome.
Redox chemistry of cobalamin and iron‐sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus
Authors: Schumacher Wolfram , Holliger Christof , Zehnder Alexander J.B , Hagen Wilfred R
Published: July 2002

Respiration of Dehalobacter restrictus is based on reductive dechlorination of tetrachloroethene. The terminal component of the respiratory chain is the membrane‐bound tetrachloroethene reductase. The metal prosthetic groups of the purified enzyme have been studied by optical and EPR spectroscopy. The 60‐kDa monomer contains one cobalamin with E
m(Co1+/2+)=−350 mV and E
m(Co2+/3+)>150 mV and two electron‐transferring [4Fe–4S](2+;1+) clusters with rather low redox potentials of E
m≈−480 mV. The cob(II)alamin is present in the base‐off configuration. A completely reduced enzyme sample reacted very rapidly with tetrachloroethene yielding base‐off cob(II)alamin rather than trichlorovinyl‐cob(III)alamin.
Oligomer formation of histamine H2 receptors expressed in Sf9 and COS7 cells1
Authors: Fukushima Yasushi , Asano Tomoichiro , Saitoh Toshihito , Anai Motonobu , Funaki Makoto , Ogihara Takehide , Katagiri Hideki , Matsuhashi Nobuyuki , Yazaki Yoshio , Sugano Kentaro
Published: July 2002

A histamine H2 receptor, which had been mutated at its glycosylation site and tagged at its N‐terminus with an HA tag (HA‐H2 receptor), was expressed in Sf9 cells and COS7 cells. Immunoprecipitation and immunoblotting of HA–H2 receptors with αHA antibody revealed four bands of 31.5±2.5 kDa, 59.0±6.0 kDa, 80.5±4.5 kDa and 120 kDa. These bands were also detected by immunoblot using anti‐H2 receptor serum (C‐terminus). In addition, H2 receptors without the HA‐tag co‐immunoprecipitated with HA‐tagged H2 receptors devoid of the 51 C‐terminal amino acids, via immunoprecipitation with αHA antibody, when the two receptors were coexpressed. These results suggest that H2 receptors are present as receptor oligomers and that the C‐terminal portion is not involved in the formation of these oligomers.
Transfer RNAPhe isoacceptors possess non‐identical set of identity elements at high and low Mg2+ concentration
Authors: Kholod Natalia S , Pan'kova Natalia V , Mayorov Sergey G , Krutilina Antonina I , Shlyapnikov Michael G , Kisselev Lev L , Ksenzenko Vladimir N
Published: July 2002

Primary structures of phage T5‐ and Escherichia coli‐encoded tRNAPhe are distinct at four out of 11 positions known as identity elements for E. coli phenylalanyl‐tRNA synthetase (FRS). In order to reveal structural requirements for FRS recognition, aminoacylation of wild‐type phage T5 tRNAPhe gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNAPhe gene transcript. The wild‐type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E. coli counterpart. However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg2+ concentrations: 4 and 15 mM, respectively. Aminoacylation assays with tRNAPhe mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg2+ but not at optimal 4 mM Mg2+; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg2+ whereas G34A mutation in the E. coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg2+; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg2+ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS. Collectively, the results indicate the non‐identical mode of tRNAPhe recognition by E. coli FRS at low and high Mg2+ concentrations. Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS.
Peroxynitrite‐induced hemolysis of human erythrocytes and its inhibition by antioxidants
Authors: Kondo Hiroshi , Takahashi Mareyuki , Niki Etsuo
Published: July 2002

It was found that human erythrocytes underwent hemolysis when incubated with peroxynitrite at 37°C under air. The extent of hemolysis increased with increasing peroxynitrite concentration and decreasing hematocrit. The peroxynitrite‐induced hemolysis was suppressed only partially by a radical scavenging antioxidant such as uric acid and Trolox, a water‐soluble vitamin E analogue, but reduced glutathione, N‐acetylcysteine and albumin efficiently inhibited the hemolysis. A selenium‐containing organic compound, ebselen, also suppressed the hemolysis. On the other hand, nitric oxide and superoxide generated concomitantly from 3‐morpholinosydnonimine (SIN‐1) did not induce appreciable hemolysis, while it converted hemoglobin to methemoglobin extensively.
Alu sequences
Authors: Mighell A.J. , Markham A.F. , Robinson P.A.
Published: July 2002

Alu sequences are frequently encountered during study of human genomic nucleic acid and form a major component of repetitive DNA. This review describes the origin of Alu sequences and their subsequent amplification and evolution into distinct subfamilies. In recent years a number of different functional roles for Alu sequences have been described. The multiple influences of Alu sequences on RNA polymerase II‐mediated gene expression and the presence of Alu sequences in RNA polymerase III‐generated transcripts are discussed.
Identification of sucrose synthase as an actin‐binding protein
Authors: Winter Heike , L. Huber Joan , Huber Steven C
Published: July 2002

Several lines of evidence indicate that sucrose synthase (SuSy) binds both G‐ and F‐actin: (i) presence of SuSy in the Triton X‐100‐insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co‐immunoprecipitation of actin with anti‐SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin‐stabilized F‐actin filaments; and (iv) direct binding to F‐actin, polymerized in vitro. Aldolase, well known to interact with F‐actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.
A conserved domain for glycogen binding in protein phosphatase‐1 targeting subunits
Authors: Wu Jun , Liu Jun , Thompson Irene , Oliver Carey J , Shenolikar Shirish , Brautigan David L
Published: July 2002

The skeletal muscle glycogen‐binding subunit (GM) of protein phosphatase‐1 (PP1) is the founding member of a family of proteins that tether the PP1 catalytic subunit (PP1C) to glycogen and promote the dephosphorylation of glycogen synthase. A hydrophobic sequence (called here the VFV motif) is conserved among GM, the liver subunit GL, and the widely expressed subunits, PTG, R5 and U5. This study analyzed the role of this VFV motif in binding to glycogen and PP1C. Glutathione S‐transferase (GST) fusions with the N‐terminal domain of GM (GST‐GM(1–240)) and with the full length R5 protein (GST‐R5) both bound to glycogen in a co‐sedimentation assay. In contrast, GST itself did not bind to glycogen. A single residue substitution in GST‐GM(1–240), F155A, reduced glycogen binding by 40%. Double residue substitutions V150A/F155A and F155A/V159A resulted in greater reductions (60–70%) in glycogen binding, showing these hydrophobic residues influenced the protein‐glycogen interaction. The wild type and V150A/F155A fusion proteins were digested by trypsin into the same sized fragments at the same rate. Furthermore, the wild type and mutated GST‐GM proteins as well as GST‐R5 bound equivalent amounts of PP1C, in either pull‐down or far‐Western assays. These results demonstrated retention of overall tertiary structure by the mutated fusion proteins, and indicated that glycogen and PP1C binding are independent of one another. A 68 residue segment of R5 encompassing the VFV motif was sufficient to produce glycogen binding when fused to GST. This motif, that is in bacterial and fungal starch metabolizing enzymes, probably has been conserved during evolution as a functional domain for binding glycogen and starch.
Moesin, the major ERM protein of lymphocytes and platelets, differs from ezrin in its insensitivity to calpain
Authors: Shcherbina Anna , Bretscher Anthony , Kenney Dianne M. , Remold-O'Donnell Eileen
Published: July 2002

The ERM proteins, ezrin, radixin and moesin, provide regulated linkage of the cytoskeleton with the plasma membrane, particularly in cell surface projections. Ezrin and moesin were found co‐expressed, and radixin was not detected, in human blood lymphocytes, monocytes and neutrophils. Moesin is the quantitatively dominant ERM protein in these cells and the only one in platelets. Because Ca2+ signaling pathways involving calpain cleavages are important in blood cells, we examined ERM protein sensitivity to this protease. A striking difference was discovered: sensitivity of ezrin and resistance of moesin (and radixin) to calpain. In intact stimulated lymphocytes, ezrin was cleaved, while moesin was not, strongly suggesting that differential sensitivity to calpain contributes to specialized functions of these proteins.
Differential expression and phosphorylation of CTCF, a c‐myc transcriptional regulator, during differentiation of human myeloid cells
Authors: Delgado M.Dolores , Chernukhin Igor V. , Bigas Anna , Klenova Elena M. , León Javier
Published: July 2002

CTCF is a transcriptional repressor of the c‐myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down‐regulated during terminal differentiation in most cell lines tested; (ii) CTCF down‐regulation is retarded and less pronounced than that of c‐myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post‐transcriptional levels.
The non‐enzymatic microbicidal activity of lysozymes
Authors: Düring Klaus , Porsch Petra , Mahn Andreas , Brinkmann Olaf , Gieffers Werner
Published: July 2002

T4 lysozyme was thought to destroy bacteria by its muramidase activity. However, we demonstrate here that amphipathic helix stretches in the C‐terminus of T4 lysozyme mediate its bactericidal and fungistatic activities. In heat‐denatured T4 lysozyme, the enzymatic activity is completely abolished but unexpectedly, the antimicrobial functions remain preserved. Small synthetic peptides corresponding to amphipathic C‐terminal domains of T4 lysozyme show a microbicidal activity. Its membrane disturbing activity was directly demonstrated for bacterial, fungal and plant cells but not in a hemolysis assay. Comparable results were obtained with hen egg white lysozyme. This opens up many new opportunities for optimization of lysozymes as antimicrobial agents in various applications by protein engineering.
The energy conserving methyltetrahydromethanopterin:coenzyme M methyltransferase complex from methanogenic archaea: function of the subunit MtrH
Authors: Hippler Birgit , Thauer Rudolf K
Published: July 2002

In methanogenic archaea the transfer of the methyl group of N
5‐methyltetrahydromethanopterin to coenzyme M is coupled with energy conservation. The reaction is catalyzed by a membrane associated multienzyme complex composed of eight different subunits MtrA–H. The 23 kDa subunit MtrA harbors a corrinoid prosthetic group which is methylated and demethylated in the catalytic cycle. We report here that the 34 kDa subunit MtrH catalyzes the methylation reaction. MtrH was purified and shown to exhibit methyltetrahydromethanopterin:cob(I)alamin methyltransferase activity. Sequence comparison revealed similarity of MtrH with MetH from Escherichia coli and AcsE from Clostridium thermoaceticum: both enzymes exhibit methyltetrahydrofolate:cob(I)alamin methyltransferase activity.
Site‐specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening
Authors: Oikawa Shinji , Kawanishi Shosuke
Published: July 2002

Telomere shortening during human aging has been reported to be accelerated by oxidative stress. We investigated the mechanism of telomere shortening by oxidative stress. H2O2 plus Cu(II) caused predominant DNA damage at the 5′ site of 5′‐GGG‐3′ in the telomere sequence. Furthermore, H2O2 plus Cu(II) induced 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) formation in telomere sequences more efficiently than that in non‐telomere sequences. NO plus O2
− efficiently caused base alteration at the 5′ site of 5′‐GGG‐3′ in the telomere sequence. It is concluded that the site‐specific DNA damage at the GGG sequence by oxidative stress may play an important role in increasing the rate of telomere shortening with aging.
Nitrite reductase activity is a novel function of mammalian mitochondria
Authors: Kozlov Andrey V , Staniek Katrin , Nohl Hans
Published: July 2002

Nitrite, which is the major stable degradation product of nitric oxide, exists in all tissues capable of nitric oxide synthesis fromL‐arginine. The present study provides experimental evidence that nitrite in contact with respiring mitochondria accepts reducing equivalents from the ubiquinone cycle of the respiratory chain. Univalent reduction of nitrite was totally inhibited by myxothiazol. We therefore conclude on the involvement of redox cycling that ubisemiquinone is associated with the bc1complex. Recycling of nitric oxide degradation products via these electron carriers may become a threat to energy‐linked respiration since nitric oxide in direct contact with mitochondria was shown to slow the energy‐linked respiration down and to trigger a mitochondrial source for superoxide radicals. Until now, the existence of nitrite reductase activity was only demonstrated in plants and bacteria. In addition, the present observation elucidates the existence of a nitric oxide synthase‐independent nitric oxide source.
Peptide models for inherited neurodegenerative disorders: conformation and aggregation properties of long polyglutamine peptides with and without interruptions
Authors: Sharma Deepak , Sharma Sunita , Pasha Santosh , Brahmachari Samir K
Published: July 2002

Several neurodegenerative diseases are caused by expansion of polyglutamine repeats in the affected proteins. In spino‐cerebellar ataxia type 1 (SCA1), histidine interruptions have been reported to mitigate the pathological effects of long glutamine stretches. To understand this phenomenon, we investigated the conformational preferences of peptides containing both the uninterrupted polyglutamine stretches and those with histidine interruption(s) as seen in SCA1 normals. Our study suggests that substitution of histidines by glutamines induces a conformational change which results in decreased solubility and increased aggregation. Our findings also suggest that all the polyglutamine peptides with and without interruption(s) adopt a β‐structure and not random coil.
Occurrence of an HIV‐1 gp160 endoproteolytic activity in low‐density vesicles and evidence for a distinct density distribution from endogenously expressed furin and PC7/LPC convertases
Authors: Wouters Sandrine , Decroly Etienne , Vandenbranden Michel , Shober Daniela , Fuchs Renate , Morel Valérie , Leruth Michèle , Seidah Nabil G. , Courtoy Pierre J. , Ruysschaert Jean-Marie
Published: July 2002

Human immunodeficiency virus (HIV) glycoprotein (gp) 160 processing by host cell proteinases is an essential step for viral fusion and infectivity. We have identified a rat liver subcellular fraction which specifically processes gp160 into gp120 and gp41. Using equilibration of microsomes in sucrose gradients, the gp160 cleavage activity was associated with particles equilibrating at low densities, well‐separated from the endoplasmic reticulum, cis‐Golgi network, Golgi stacks, lysosomes and plasma membrane. Its density distribution was compatible with light secretory vesicles derived from the trans‐Golgi network (TGN) or to endosomes, but association with endosomes was not supported by free flow electrophoresis. Although furin and pro‐protein convertase (PC) 7/LPC have been proposed as the major gp160 processing convertases, the rat liver microsomal gp160 processing activity was essentially resolved from furin and only partially overlapped PC7/LPC. These data suggest that proteinase(s) other than furin and PC7/LPC, presumably located in TGN‐derived vesicles, may participate in the gp160 processing into gp120 and gp41.
Calcium‐induced calcium release mediated by a voltage‐activated cation channel in vacuolar vesicles from red beet
Authors: Bewell Michael A , Maathuis Frans J.M , Allen Gethyn J , Sanders Dale
Published: July 2002

Little is known about the mechanisms underlying calcium‐induced Ca2+ release (CICR) in plants. The slow‐activating vacuolar (SV) channel is both permeable to, and activated by Ca2+, and is therefore a prime candidate for a role in CICR. Cytosol‐side‐out vacuolar membrane vesicles loaded with 45Ca2+ showed voltage‐ and Ca2+‐dependent Ca2+ release, which was sensitive to the SV channel modulators DIDS, protein phosphatase 2B and calmodulin. Significantly, voltage‐dependent Ca2+ release strongly depended on cytoplasmic Ca2+ concentrations. The results support the notion that CICR occurs in plant cells and that the process can be catalysed by the SV channel on the vacuolar membrane.
The delta subunit of rod specific cyclic GMP phosphodiesterase, PDE δ, interacts with the Arf‐like protein Arl3 in a GTP specific manner
Authors: Linari Marco , Hanzal-Bayer Michael , Becker Jörg
Published: July 2002

Recently, we have shown that the δ subunit of the cGMP phosphodiesterase (PDE δ) interacts with the retinitis pigmentosa guanine regulator (RPGR). Here, using the two‐hybrid system, we identify a member of the Arf‐like protein family of Ras‐related GTP‐binding proteins, Arl3, that interacts with PDE δ. The interaction was verified by fluorescence spectroscopy and co‐immunoprecipitation. Arl3 features an unusually low affinity for guanine nucleotides, with a K
D of 24 nM for GDP and 48 μM for GTP. Fluorescence spectroscopy shows that PDE δ binds and specifically stabilizes the GTP‐bound form of Arl3 by strongly decreasing the dissociation rate of GTP. Thus, PDE δ is an effector of Arl3 and could provide a novel nucleotide exchange mechanism by which PDE δ stabilizes Arl3 in its active GTP‐bound form.
BH‐protocadherin‐c, a member of the cadherin superfamily, interacts with protein phosphatase 1 alpha through its intracellular domain
Authors: Yoshida Kenichi , Watanabe Manabu , Kato Hiroyuki , Dutta Anindya , Sugano Sumio
Published: July 2002

Using a yeast two‐hybrid system, we isolated eight cDNA clones which interacted with BH‐protocadherin‐c (BH‐Pcdh‐c) from the human brain cDNA library. One clone encoded protein phosphatase type 1 isoform α (PP1α) and another two PP1α2. PP1α was co‐immunoprecipitated from the extract of a gastric adenocarcinoma cell line MKN‐28 with anti‐BH‐Pcdh‐c antibody. PP1α activity towards glycogen phosphorylase was inhibited by the intracellular domain of BH‐Pcdh‐c. Inhibition of the phosphatase required more than the minimal domain of BH‐Pcdh‐c which could associate with PP1α. In situ hybridization revealed that BH‐Pcdh‐c mRNA was predominantly expressed in cerebral cortex neurons in the adult mouse brain.
FTDP‐17 tau mutations decrease the susceptibility of tau to calpain I digestion
Authors: Yen Samuel , Easson Colin , Nacharaju Parimala , Hutton Michael , Yen Shu-Hui
Published: July 2002

Frontal temporal dementia and Parkinsonism linked to chromosome 17 (FTDP‐17) is caused by splice site and missense mutations in the tau gene, and characterized by the accumulation of filamentous tau in cerebral neurons and glia. The missense mutations reduce the ability of tau to promote microtubule assembly and increase the ability of tau to form filaments. In this report we demonstrate that mutants V337M and R406W are less susceptible than mutant P301L or corresponding wild type tau to degradation by calpain I. The differences were at least in part due to changes in accessibility of a cleavage site located about 100 amino acids off the carboxy‐terminus. The results suggest that the pathogenesis of some forms of FTDP‐17 may involve tau accumulation due to decreased proteolytic degradation.
Endogenous mutations in human uncoupling protein 3 alter its functional properties
Authors: Brown Angela M. , Dolan Joseph W. , Willi Steven M. , Garvey W.Timothy , Argyropoulos George
Published: July 2002

Human uncoupling protein 3 (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative phosphorylation and is a candidate gene for obesity. Expression of native human UCP3 mutations in yeast showed complete loss (R70W), significant reduction (R143X), or no effect (V102I and IVS6+1G>A) on the uncoupling activity of UCP3. It is concluded that certain mutations in UCP3 alter its functional impact on membrane potential (ΔΨ), possibly conferring susceptibility to develop metabolic diseases.
Human papillomavirus with co‐existing vulvar vestibulitis syndrome and vestibular papillomatosis
Authors: Origoni M , Rossi M , Ferrari D , Lillo F , Ferrari A.G
Published: July 2002

Objective: The role of HPV infection in cases of vulvar papillomatosis and vulvar vestibulitis syndrome is still unclear and data from the literature is controversial. In this study we intended to investigate the prevalence of viral infection, with a multidisciplinary approach, in cases with a co‐existence of the two patterns. Method: Sixteen consecutive cases with diagnosis of vulvar vestibulitis syndrome and co‐existence of vestibular papillomatosis were enrolled in the study and investigated by the means of vulvar cytology, vulvoscopy, histology, ViraPap and Polymerase Chain Reaction. Result: Cytology, vulvoscopy and histology did not demonstrate suitable accuracy for the diagnosis. Viral DNA identification revealed two (12.50%) positive cases using PCR and one (6.25%) positive case with ViraPap. Conclusion: The results of the present investigation indicate that even in cases of co‐existing vulvar papillomatosis and severe vulvar vestibulitis syndrome, the prevalence of HPV infection is too low to be considered causal.
Fracture resistance of gamma radiation sterilized cortical bone allografts
Authors: Akkus Ozan , Rimnac Clare M.
Published: July 2002

Gamma radiation is widely used for sterilization of human cortical bone allografts. Previous studies have reported that cortical bone becomes brittle due to gamma radiation sterilization. This embrittlement raises concern about the performance of a radiation sterilized allograft in the presence of a stress concentration that might be surgically introduced or biologically induced. The purpose of this study was to investigate the effect of gamma radiation sterilization on the fracture resistance of human femoral cortical bone in the presence of a stress concentration. Fracture toughness tests of specimens sterilized at a dose of 27.5 kGy and control specimens were conducted transverse and longitudinal to the osteonal orientation of the bone tissue. The formation of damage was monitored with acoustic emission (AE) during testing and was histologically observed following testing. There was a significant decrease in fracture toughness due to irradiation in both crack growth directions. The work‐to‐fracture was also significantly reduced. It was observed that the ability of bone tissue to undergo damage in the form of microcracks and diffuse damage was significantly impaired due to radiation sterilization as evidenced by decreased AE activity and histological observations. The results of this study suggest that, for cortical bone irradiated at 27.5 kGy, it is easier to initiate and propagate a macrocrack from a stress concentration due to the inhibition of damage formation at and near the crack tip. © 2001 Orthopaedic Research Society. Punlished by Elsevier Science Ltd. All rights reserved.
Corticosterone release in response to repeated, short episodes of neonatal isolation : evidence of sensitization
Authors: McCormick Cheryl M. , Kehoe Priscilla , Kovacs Sandra
Published: July 2002

Repeated isolation of neonatal rats produces persistent changes in physiology and behavior. In Experiment 1, we examined changes in plasma corticosterone (CORT) levels as a possible mechanism for the effects of isolation. Pups that were isolated from their mother and the nest for 1 h per day on postnatal days (PND) 2–9 were compared to control litters of pups that were either nonhandled or handled but not isolated. On PND 2, compared to nonhandled pups, handled pups had elevated CORT levels that returned to baseline levels within 30 to 60 min of return to the home cage. No significant elevation of CORT levels were found in handled pups on PND 9. The CORT levels of isolated pups were over twice those of nonhandled pups on PND 2 and four times those of nonhandled pups on PND 9. In Experiment 2, we investigated whether the increased CORT release in response to isolation on PND 9 was the result of the pups treatment on the previous six days as against an effect of maturation. Plasma CORT levels were measured in rat pups that were either isolated, handled or nonhandled on PNDs 2–8 during the conditions of isolation, handling and nonhandling on PND 9. There were no differences among the groups in basal plasma levels of CORT. Handling on PND 9 did not result in elevated CORT levels in any of the groups. All three groups showed a significant increase in plasma CORT levels after isolation on PND 9. However, the CORT response to isolation of pups previously isolated on PND 2–8 were significantly higher than pups that were either handled or nonhandled on PNDs 2–8. Thus, daily episodes of isolation potentiate the hypothalamic‐pituitary‐adrenal response to stress.