bluuebunny/crossref_metadata_2025 huggingface dataset:
Reoperation for Renal Hyperparathyroidism
Authors: Hibi Yatsuka , Tominaga Yoshihiro , Sato Tetsuhiko , Katayama Akio , Haba Toshihito , Uchida Kazuharu , Ichimori Toshihiro , Numano Masahiro , Tanaka Yuji , Takagi Hiroshi , Imai Tsuneo , Funahashi Hiroomi , Nakao Akimasa
Published: October 2002

Reoperation for secondary hyperparathyroidism (HPT) due to uremia (2HPT) may be required among patients with persistent renal failure if not all parathyroid glands are removed at the initial operation. Between March 1981 and July 2001,
altogether 1110 patients underwent total parathyroidectomy with forearm autograft for advanced 2HPT in our department. In this study, we evaluated the clinical features of patients who required reoperation and classified them into persistent HPT [the lowest intact parathyroid
(PTH) level after initial operation remained higher than 60 pg/ml] and recurrent HPT (the lowest intact PTH level was normalized after surgery but reelevated became high enough to require reoperation). Removal of residual glands was indicated in 30 (2.7%) cases for persistent or recurrent HPT. All remaining glands were detected by preoperative imaging diagnoses. In 44 (4.0%) patients persistent HPT was recognized and in 15 of them (1.4% of all cases) reoperation was required. In 11
cases, the responsible glands were supernumerary ones removed from the mediastinum. In 4 cases, the glands were resected from the neck. In 15
cases (1.4%), reoperation was performed for recurrent HPT when residual glands were left either in the neck or in the thymic tongue.
In all but one case, the missed glands were supernumerary. This study reveals that it is often difficult to avoid persistent HPT induced by mediastinal supernumerary glands and recurrent HPT caused by small glands left in the neck. Our findings indicate that patients with uremia should be closely followed considering the possibility that persistent or recurrent HPT may occur after parathyroidectomy.
Regulation of nuclear import by light‐induced activation of caged nuclear localization signal in living cells
Authors: Watai Yoriko , Sase Ichiro , Shiono Hirofumi , Nakano Yoshitaro
Published: October 2002

A novel fluorescence probe suitable for the study of nuclear import in living cells has been developed. The lysine‐128 residue in SV40 T‐antigen nuclear localization signal (NLS) was converted to a caged lysine with the amino acid blocked by a photocleavable protecting group. Following irradiation of ultraviolet (UV) light, the caged NLS conjugate translocated into and accumulated in the nucleus within 20 min similar to uncaged NLS conjugate. Maximum import rate saturated approximately 4.78±0.21% per minute when the duration of irradiation was more than 1/15 s (22 mW/cm2). Caged NLS conjugate tended to distribute near the surface of the nucleus, and this association became stronger after UV irradiation. The caged conjugate enabled us to regulate the initial state of the reaction, both spatially and temporally.
ATP synthase: constrained stoichiometry of the transmembrane rotor
Authors: Müller Daniel J. , Dencher Norbert A. , Meier Thomas , Dimroth Peter , Suda Kitaru , Stahlberg Henning , Engel Andreas , Seelert Holger , Matthey Ulrich
Published: October 2002

Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation‐powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native‐like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch‐shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions.
The anion conductance of the glutamate transporter EAAC1 depends on the direction of glutamate transport
Authors: Watzke Natalie , Grewer Christof
Published: October 2002

The steady‐state and pre‐steady‐state kinetics of glutamate transport by the neuronal glutamate transporter EAAC1 were determined under conditions of outward glutamate transport and compared to those found for the inward transport mode. In both transport modes, the glutamate‐induced current is composed of two components, the coupled transport current and the uncoupled anion current, and inhibited by a specific non‐transportable inhibitor. Furthermore, the glutamate‐independent leak current is observed in both transport modes. Upon a glutamate concentration jump outward transport currents show a distinct transient phase that deactivates within 15 ms. The results demonstrate that the general properties of EAAC1 are symmetric, but the rates of substrate transport and anion flux are asymmetric with respect to the orientation of the substrate binding site in the membrane. Therefore, the EAAC1 anion conductance differs from normal ligand‐gated ion channels in that it can be activated by glutamate and Na+ from both sides of the membrane.
Specific collagenolysis by gelatinase A, MMP‐2, is determined by the hemopexin domain and not the fibronectin‐like domain
Authors: Patterson Margaret L , Atkinson Susan J , Knäuper Vera , Murphy Gillian
Published: October 2002

In view of the essential role of the hemopexin domain of the traditional interstitial collagenases, MMP‐1, ‐8, ‐13 and MT1‐MMP (MMP‐14), in determining specific collagen cleavage we have studied the function of this domain in MMP‐2, relative to that of the fibronectin‐like domain that promotes gelatinolysis. Although the fibronectin‐like domain promotes avid binding to collagen, our data demonstrate that the catalytic and hemopexin domains of MMP‐2 are sufficient to effect the critical step in cleavage of rat type I collagen into 3/4 and 1/4 fragments. The mechanism of MMP‐2 cleavage of collagen proceeds in two phases, the first resembling that of the interstitial collagenases, followed by gelatinolysis, promoted by the fibronectin‐like domain.
Proteasome inhibitors and immunosuppressive drugs promote the cleavage of eIF4GI and eIF4GII by caspase‐8‐independent mechanisms in Jurkat T cell lines
Authors: Morley Simon J , Pain Virginia M
Published: October 2002

Previously, we have shown that translation eukaryotic initiation factor (eIF) 4GI is cleaved during anti‐Fas‐mediated apoptosis. Here, we have investigated the effects of the proteasome inhibitors, MG132 and lactacystin, and the immunosuppressants, 2‐amino‐2[2‐(4‐octylphenyl)ethyl]‐1,3,propane diol (FTY720) and cyclosporin A, on the integrity of eIF4GI and eIF4GII in T cells. Using wild‐type Jurkat T cells, we show that the proteasome inhibitors MG132 and lactacystin promote the cleavage of eIF4G, activate caspase‐8 and caspase‐3‐like activities and decrease cell viability. Furthermore, MG132 also promotes the cleavage of eIF4G and the activation of caspase‐3‐like activity in a caspase‐8‐deficient Jurkat cell line which is resistant to anti‐Fas‐mediated apoptosis. Using specific anti‐peptide antisera, we show that both eIF4GI and eIF4GII are cleaved in either cell line in response to MG132 and lactacystin. In response to such treatments, we demonstrate that the fragments of eIF4GI generated include those previously observed with anti‐Fas antiserum together with a novel product which lacks the ability to interact with eIF4E. In contrast, cells treated with the immunosuppressants FTY720 and cyclosporin A appear to contain only the novel cleavage fragment of eIF4GI and to lack those characteristic of cells treated with anti‐Fas antiserum. These data suggest that caspase‐8 activation is not required for apoptosis and eIF4G cleavage mediated by proteasome inhibitors and immunosuppressants in human T cells.
Inter‐subunit rotation and elastic power transmission in F0F1‐ATPase
Authors: Junge Wolfgang , Pänke Oliver , Cherepanov Dmitry A , Gumbiowski Karin , Müller Martin , Engelbrecht Siegfried
Published: October 2002

ATP synthase (F‐ATPase) produces ATP at the expense of ion‐motive force or vice versa. It is composed from two motor/generators, the ATPase (F1) and the ion translocator (F0), which both are rotary steppers. They are mechanically coupled by 360° rotary motion of subunits against each other. The rotor, subunits γϵc
10–14, moves against the stator, (αβ)3δab
2. The enzyme copes with symmetry mismatch (C3 versus C10–14) between its two motors, and it operates robustly in chimeric constructs or with drastically modified subunits. We scrutinized whether an elastic power transmission accounts for these properties. We used the curvature of fluorescent actin filaments, attached to the rotating c ring, as a spring balance (flexural rigidity of 8·10−26 N m2) to gauge the angular profile of the output torque at F0 during ATP hydrolysis by F1. The large average output torque (56 pN nm) proved the absence of any slip. Angular variations of the torque were small, so that the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the three‐fold stepping and high activation barrier (>40 kJ/mol) of the driving motor (F1) itself, the rather constant output torque seen by F0 implied a soft elastic power transmission between F1 and F0. It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate under load of the two counteracting and stepping motors/generators.
Cell type‐dependent regulation of human DNA topoisomerase IIIα gene expression by upstream stimulatory factor 2
Authors: Han Sang Yup , Kim Jun Chul , Suh Jae Myoung , Chung In Kwon
Published: October 2002

Here, we report that an E‐box element located within the human topoisomerase IIIα (hTOP3α) gene promoter acts as a cell type‐specific enhancer. The upstream stimulatory factor (USF) was shown to specifically recognize the mutationally sensitive E‐box element. When assayed by transient transfection with hTOP3α promoter‐dependent reporter genes, USF is transcriptionally active in HeLa cells but lacks transcriptional activity in Saos‐2 cells. The hTOP3α mRNA level in Saos‐2 cells was reduced to about 30% of the level observed for HeLa cells, suggesting that the inactivity of USF in hTOP3α promoter activity may be the cause of the marked reduction of hTOP3α mRNA levels in Saos‐2 cells. Using transient transfection assays in HeLa cells, we demonstrated that ectopically expressed USF2, but not USF1, was capable of activating hTOP3α transcription through the E‐box element. However, USF2 did not stimulate hTOP3α promoter activity in Saos‐2 cells. This cell type‐specific regulation of promoter activity by USF2 may provide a mechanism for the differential expression of hTOP3α in various tissues and during developmental stages.
Interaction of bepridil with the cardiac troponin C/troponin I complex
Authors: Abusamhadneh Ekram , Abbott M.Bret , Dvoretsky Alex , Finley Natosha , Sasi Soumya , Rosevear Paul R.
Published: October 2002

We have investigated the binding of bepridil to calcium‐saturated cardiac troponin C in a cardiac troponin C/troponin I complex. Nuclear magnetic resonance spectroscopy and [15N,2H]cardiac troponin C permitted the mapping of bepridil‐induced amide proton chemical shifts. A single bepridil‐binding site in the regulatory domain was found with an affinity constant of ∼140 μM−1. In the presence of cardiac troponin I, bepridil binding to the C domain of cardiac troponin C was not detected. The pattern of bepridil‐induced chemical shifts is consistent with stabilization of more open regulatory domain conformational states. A similar pattern of chemical shift perturbations was observed for interaction of the troponin I cardiac‐specific amino‐terminus with the cardiac troponin C regulatory domain. These results suggest that both bepridil and the cardiac‐specific amino‐terminus may mediate an increase in calcium affinity by interacting with and stabilizing open regulatory domain conformations. Chemical shift mapping suggests a possible role for inactive calcium‐binding site I in the modulation of calcium affinity.
Novel EPR signals associated with FeMoco centres of MoFe protein in MgADP‐inhibited turnover of nitrogenase
Authors: Maritano Silvana , Fairhurst Shirley A , Eady Robert R
Published: October 2002

Two novel electron paramagnetic resonance (EPR) signals arising from the [1Mo–7Fe–9S‐homocitrate] (FeMoco) centres of MoFe protein of Klebsiella pneumoniae nitrogenase (Kp1) were observed following turnover under MgATP‐limited conditions. The combination of the nitrogenase Fe protein of Clostridium pasteurianum showed similar signals. The accumulation of MgADP under these conditions causes the normal EPR signal of dithionite‐reduced Kp1 (with g=4.3, 3.6, 2.01) to be slowly converted to novel signals with g=4.74, 3.32, 2.00 and g=4.58, 3.50, 1.99. These signals do not form in incubation of protein mixtures containing only MgADP, thus they may be associated with trapped intermediates of the catalytic cycle.
Effects of histamine and interleukin‐4 synthesized in arterial intima on phagocytosis by monocytes/macrophages in relation to atherosclerosis
Authors: Higuchi Satoshi , Tanimoto Akihide , Arima Nobuyuki , Xu Hui , Murata Yoshitaka , Hamada Tetsuo , Makishima Kazumi , Sasaguri Yasuyuki
Published: October 2002

We investigated the localization of histidine decarboxylase (HDC), which is the rate‐limiting enzyme that generates histamine from histidine, in human aorta/coronary artery. RT‐PCR and immunohistochemical staining revealed that the HDC gene was expressed in monocytes/macrophages and T cells in the arterial intima but not in smooth muscle cells in either the arterial intima or the media. A luciferase promoter assay with U937 and Jurkat cells demonstrated that interleukin‐4 (IL‐4) inhibited the expression of the HDC gene. In contrast, among a scavenger receptor family, IL‐4 as well as histamine up‐regulated U937 cells to express the LOX‐1 gene but not the SR‐A gene, which genes encode receptors that scavenge oxidized lipids. These findings suggest that histamine synthesized in the arterial wall participates in the initiation and progression of atherosclerosis and that IL‐4 can act as an important inhibitory and/or stimulatory factor in the function of monocytes/macrophages modulated by histamine in relation to the process of atherosclerosis.
Polycystin‐1: immunoaffinity isolation and characterisation by mass spectrometry
Authors: Malhas Ashraf N. , Abuknesha Ramadan A. , Price Robert G.
Published: October 2002

Polycystin‐1 is a putative 460 kDa membrane protein with a unique structure and is possibly representative of a new family of proteins. Its structure suggests an involvement in cell signalling and cell–matrix interactions. The amino acid sequence of polycystin‐1 has to date been predicted from its gene sequence. This, to our knowledge, is the first report of the isolation and analysis of polycystin‐1 at the protein level using mass spectrometry to confirm its predicted structure. The availability of purified polycystin‐1 will allow a new approach to unravelling the complexity of the cell–cell and cell–matrix interactions of this large molecule in normal cells and its perturbation in disease.
A novel serum chitinase that is expressed in bovine liver
Authors: Suzuki Masako , Morimatsu Masami , Yamashita Tetsuro , Iwanaga Toshihiko , Syuto Bunei
Published: October 2002

Chitinases are ubiquitous chitin‐fragmenting hydrolases. They are synthesized by a vast array of organisms, including those not composed of chitin. Here, we describe a novel serum chitinase (chitin‐binding protein b04, CBPb04), which is expressed in bovine liver. Although CBPb04 is secreted as an endocrine chitinase, it shows higher homology with human gastrointestinal tract exocrine chitinase (AMCase) than with macrophage endocrine chitinase (human chitotriosidase). This suggests that cows have a specific defense against chitin‐containing microorganisms. CBPb04 mRNA is expressed in hepatocytes. This is the first report of a hepatogenic mammalian chitinase.
Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5‐diaminofluorescein
Authors: Leikert Jürgen F , Räthel Thomas R , Müller Christian , Vollmar Angelika M , Dirsch Verena M
Published: October 2002

4,5‐Diaminofluorescein (DAF‐2) and its membrane‐permeable derivate DAF‐2 diacetate are fluorescent probes that have been developed to perform real‐time biological detection of nitric oxide (NO). Their use for intracellular imaging, however, has recently been seriously questioned and data using DAF‐2 for extracellular NO detection at low levels, as for example released from endothelial cells, are rare. Here we show that a reliable detection of low levels of NO in biological systems by DAF‐2 is possible (a) by using low DAF‐2 concentrations (0.1 μM) and (b) by subtracting the DAF‐2 auto‐fluorescence from the measured total fluorescence. The described method allows easy real‐time detection of endothelial NO formation.
An amino acid residue whose change by mutation affects drug binding to the HERG channel
Authors: Ishii Kuniaki , Kondo Kazumi , Takahashi Masahiro , Kimura Minako , Endoh Masao
Published: October 2002

We did the experiments to search for amino acids that affect quinidine binding to the HERG channel, and have identified an amino acid whose change by mutation affects the binding of various drugs. The residue is located at position 647 in the S6 and is not involved in the recently identified methanesulfonanilide binding pocket. The homology model of the HERG channel indicated that the residue faces toward the outside of the channel pore. We conclude that the residue at position 647 does not interact directly with drug molecules but plays an important role in keeping the binding site's high affinity for drugs.
Membrane interactions and self‐association of the TatA and TatB components of the twin‐arginine translocation pathway
Authors: De Leeuw Erik , Porcelli Ida , Sargent Frank , Palmer Tracy , Berks Ben C.
Published: October 2002

The Escherichia coli Tat system mediates Sec‐independent export of protein precursors bearing twin‐arginine signal peptides. The essential Tat pathway components TatA, TatB and TatC are shown to be integral membrane proteins. Upon removal of the predicted N‐terminal transmembrane helix TatA becomes a water‐soluble protein. In contrast the homologous TatB protein retains weak peripheral interactions with the cytoplasmic membrane when the analogous helix is deleted. Chemical crosslinking studies indicate that TatA forms at least homotrimers, and TatB minimally homodimers, in the native membrane environment. The presence of such homo‐oligomeric interactions is supported by size exclusion chromatography.
Effects of Fe(III) binding to the nucleotide‐independent site of F1‐ATPase: enzyme thermostability and response to activating anions
Authors: Contessi Stefania , Tanfani Fabio , Scirè Andrea , Mavelli Irene , Lippe Giovanna
Published: October 2002

Mitochondrial F1‐ATPase was induced in different conformations by binding of specific ligands, such as nucleotides. Then, Fourier transform infrared spectroscopy (FT‐IR) and kinetic analyses were run to evaluate the structural and functional effects of Fe(III) binding to the nucleotide‐independent site. Binding of one equivalent of Fe(III) induced a localised stabilising effect on the F1‐ATPase structure destabilised by a high concentration of NaCl, through rearrangements of the ionic network essential for the maintenance of enzyme tertiary and/or quaternary structure. Concomitantly, a lower response of ATPase activity to activating anions was observed. Both FT‐IR and kinetic data were in accordance with the hypothesis of the Fe(III) site location near one of the catalytic sites, i.e. at the α/β subunit interface.
Molecular interactions between poly(ADP‐ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding
Authors: Bauer Pal I. , Chen Hui-Je , Kenesi Erzsebet , Kenessey Istvan , Buki Kalman G. , Kirsten Eva , Hakam Alaeddin , Hwang Jaulang I. , Kun Ernest
Published: October 2002

The molecular interactions of poly(ADP‐ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the ‘global’ Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP‐ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the ‘global’ Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I–Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S‐transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the ‘C clamp’ model derived from X‐ray crystallography.
A possible regulatory role for the metal‐binding domain of CadA, the Listeria monocytogenes Cd2+‐ATPase
Authors: Bal Nathalie , Mintz Elisabeth , Guillain Florent , Catty Patrice
Published: October 2002

Using the baculovirus/Sf9 expression system, we produced CadA and ΔMBD, a metal‐binding domain, truncated CadA. Both proteins had the expected properties of P‐type ATPases: ATP‐induced Cd2+ accumulation, Cd2+‐sensitive ATP and Pi phosphorylation and ATPase activity. ΔMBD displayed lower initial transport velocity as well as lower maximal ATPase activity than CadA. MBD truncation flattened the Cd2+ dependence of the ATPase activity and increased apparent Cd2+ affinity, suggesting a positive cooperativity between MBD and membranous transport sites. We propose that occupancy of MBD by Cd2+ modulates CadA activity.
TAK1 activation of the mouse JunB promoter is mediated through a CCAAT box and NF‐Y
Authors: Eggen B.J.L. , Benus G.F.J.D. , Folkertsma S. , Jonk L.J. , Kruijer W.
Published: October 2002

The JunB gene is activated by many stimuli including transforming growth factor β (TGFβ) family members and interleukin‐6 (IL‐6). Here the effect of TGFβ activated kinase 1 (TAK1), a mitogen activated protein kinase kinase kinase (MAPKKK) implicated in TGFβ, bone morphogenetic protein (BMP) and interleukin‐1 (IL‐1) signaling, on JunB promoter activity was investigated. Promoter analysis led to the identification of a CCAAT motif in the JunB gene, essential for activation by TAK1. Transfer of this CCAAT element to a heterologous minimal promoter conferred TAK1‐responsiveness. The CCAAT‐binding transcription factor, nuclear factor Y (NF‐Y), activated the JunB promoter and a dominant negative NF‐YA construct inhibited TAK1 activation of JunB. Our results demonstrate that JunB gene activation by TAK1 is mediated by the CCAAT‐binding factor NF‐Y.
Palmitoylethanolamide enhances anandamide stimulation of human vanilloid VR1 receptors
Authors: De Petrocellis Luciano , Davis John B , Di Marzo Vincenzo
Published: October 2002

In human embryonic kidney cells over‐expressing the human vanilloid receptor type 1 (VR1), palmitoylethanolamide (PEA, 0.5–10 μM) enhanced the effect of arachidonoylethanolamide (AEA, 50 nM) on the VR1‐mediated increase of the intracellular Ca2+ concentration. PEA (5 μM) decreased the AEA half‐maximal concentration for this effect from 0.44 to 0.22 μM. The PEA effect was not due to inhibition of AEA hydrolysis or adhesion to non‐specific sites, since bovine serum albumin (0.01–0.25%) potently inhibited AEA activity, and PEA also enhanced the effect of low concentrations of the VR1 agonists resiniferatoxin and capsaicin. PEA (5 μM) enhanced the affinity of AEA for VR1 receptors as assessed in specific binding assays. These data suggest that PEA might be an endogenous enhancer of VR1‐mediated AEA actions.
Nucleotide requirements for CDX2 binding to the cis promoter element mediating intestine‐specific expression of guanylyl cyclase C
Authors: Di Guglielmo Matthew D. , Park Jason , Schulz Stephanie , Waldman Scott A.
Published: October 2002

Guanylyl cyclase C (GC‐C), specifically expressed by intestinal epithelial cells, is the receptor for the Escherichia coli heat‐stable enterotoxin that causes diarrhea. Tissue‐specific expression of GC‐C is mediated by the intestinal transcriptional regulator CDX2. This trans‐activating protein regulates intestine‐specific expression by binding to a critical sequence in the proximal promoter of GC‐C. The precise nucleotide elements mediating CDX2 binding to promoter elements remain undefined. Several nuclear proteins form complexes with a DNA probe containing the promoter element of GC‐C mediating CDX2 binding. The present study examined the nucleotide requirements in the consensus binding site and flanking regions in the cis element that mediates specific CDX2 binding to the promoter of GC‐C. These studies identified seven core base pairs in the critical promoter element mediating tissue‐specific expression of GC‐C that are required for CDX2 binding. In addition, base pairs flanking this core sequence contribute to and are required for CDX2 recognition. These studies describe the precise nucleotide sequence within the GC‐C promoter that comprises the CDX2 binding site required for intestine‐specific expression.
Kinetic study of the processing by dipeptidyl‐peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion
Authors: Lambeir Anne-Marie , Durinx Christine , Proost Paul , Van Damme Jo , Scharpé Simon , De Meester Ingrid
Published: October 2002

Dipeptidyl‐peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady‐state kinetics of DPPIV/CD26‐mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase‐activating peptide (PACAP27 and PACAP38), gastrin‐releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C‐terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.
Ht31: the first protein kinase A anchoring protein to integrate protein kinase A and Rho signaling1
Authors: Klussmann Enno , Edemir Bayram , Pepperle Barbara , Tamma Grazia , Henn Volker , Klauschenz Erhard , Hundsrucker Christian , Maric Kenan , Rosenthal Walter
Published: October 2002

In an attempt to isolate protein kinase A anchoring proteins (AKAPs) involved in vasopressin‐mediated water reabsorbtion, the complete sequence of the human AKAP Ht31 was determined and a partial cDNA of its rat orthologue (Rt31) was cloned. The Ht31 cDNA includes the estrogen receptor cofactor Brx and the RhoA GDP/GTP exchange factor proto‐lymphoid blast crisis (Lbc) sequences. The Ht31 gene was assigned to chromosome 15 (region q24–q25). It encodes Ht31 and the smaller splice variants Brx and proto‐Lbc. A protein of the predicted size of Ht31 (309 kDa) was detected in human mammary carcinoma and HeLa cells. Anti‐Ht31/Rt31 antibodies immunoprecipitated RhoA from primary cultured rat renal inner medullary collecting duct cells, indicating an interaction between the AKAP and RhoA in vivo. These results suggest that Ht31/Rt31 represent a new type of AKAP, containing both an anchoring and a catalytic domain, which appears to be capable of modulating the activity of an interacting partner. Ht31/Rt31 have the potential to integrate Rho and protein kinase A signaling pathways, and thus, are prime candidates to regulate vasopressin‐mediated water reabsorbtion.
Molecular cloning of the cDNA coding for Xenopus laevis prion protein
Authors: Strumbo Bice , Ronchi Severino , Bolis Liana C. , Simonic Tatjana
Published: October 2002

Isolation and characterization of the cDNA coding for the 216‐residue Xenopus laevis prion protein is reported. Existence of this protein in amphibians was suggested by an EST fragment (accession number BG813008), while a conclusive demonstration is presented here. This protein exhibits a higher identity level to avian and turtle prion (more than 44%) than to mammalian prion (about 28%). Although most of the structural motifs common to known prion proteins are conserved in X. laevis, the lack of repeats represents a substantial difference. Other features worth noting are the presence of not perfectly conserved hydrophobic stretch, which is considered the prion signature, as well as the complete absence of histidine residues.
Rubiscolin, a δ selective opioid peptide derived from plant Rubisco
Authors: Yang Shuzhang , Yunden Jinsmaa , Sonoda Soushi , Doyama Naomi , Lipkowski Andrzej W , Kawamura Yukio , Yoshikawa Masaaki
Published: October 2002

We found that the sequences YPLDL and YPLDLF in the large subunit of spinach D‐ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) met the structure YP‐aliphatic amino acid which might have opioid activity. We then synthesized these peptides to test their opioid activity. The IC50 of these peptides in mouse vas deferens assay were 51.0 μM and 24.4 μM, respectively, and those in δ receptor binding assay using [3H]deltorphin II as radioligand were 2.09 μM and 0.93 μM, respectively. Both peptides were selective for δ receptor. We named them rubiscolin‐5 and ‐6, respectively. Rubiscolin‐5 and ‐6 have antinociceptive activity in mice after i.c.v. or oral administration. The enzymatic conditions to release rubiscolin were investigated using both spinach Rubisco and synthetic fragment peptides. This is the first example of bioactive peptides derived from plant Rubisco.
Ferredoxins of the third kind
Authors: Meyer Jacques
Published: October 2002

Most low‐potential ferredoxins (Fds) are of the well‐known [2Fe–2S] plant or [4Fe–4S] bacterial type. Yet, an additional class of [2Fe–2S] Fds has been recognized on the basis of sequence and spectroscopic idiosyncrasies. A recent crystal structure has confirmed the uniqueness of this third kind of Fd, and shown that these proteins display an unexpected structural similarity to thioredoxin. The properties of these thioredoxin‐like [2Fe–2S] Fds are summarized, and hypotheses concerning their function are discussed.
Characterization of acute somatosensory pain transmission in P/Q‐type Ca2+ channel mutant mice, leaner
Authors: Ogasawara Miki , Kurihara Takashi , Hu Qiuping , Tanabe Tsutomu
Published: October 2002

To study the role of the Cav2.1/α1A (P/Q‐type) Ca2+ channel in somatosensory pain processing, behavioral and electrophysiological studies were conducted using the leaner (tgla/tgla
) mouse. Behavioral analyses in tgla/tgla
revealed reduced responses to mechanical stimuli, and enhanced responses to heat stimuli. Electrophysiological analyses showed that tgla/tgla
had a significantly reduced ability to evoke dorsal root potentials, suggesting a functional deficit in the spinal dorsal horn local circuitry responsible for presynaptic inhibition of primary sensory fibers. These results suggest the critical importance of the P/Q‐type channel in modulation of acute somatosensory pain transmission in spinal cord.
Histamine deficiency induces tissue‐specific down‐regulation of histamine H2 receptor expression in histidine decarboxylase knockout mice
Authors: Fitzsimons Carlos P. , Lazar-Molnar Eszter , Tomoskozi Zsuzsa , Buzás Edit , Rivera Elena S. , Falus A.
Published: October 2002

Histidine decarboxylase (HDC) is the single enzyme responsible for histamine synthesis. HDC‐deficient mice (HDC−/−) have no histamine in their tissues when kept on a histamine‐free diet. Therefore, the HDC−/− mice provide a suitable model to investigate the involvement of histamine in the regulation of histamine receptor expression. Gene expression of H1 and H2 histamine receptors was studied in several organs of HDC−/− mice and compared to standard (HDC+/+) mice. In many tissues, prolonged absence of histamine induced down‐regulation of the H2 receptor subtype. The expression of the H1 receptor was less sensitive to histamine deficiency. Exogenous histamine present in the diet abolished the differences observed in H2 receptor expression. These results suggest that the expression of mouse H2 receptor is under the control of histamine in a tissue‐specific manner.
A novel dimer of a C‐type lectin‐like heterodimer from the venom of Calloselasma rhodostoma (Malayan pit viper)
Authors: Wang Runhua , Kong Chunguang , Kolatkar Prasanna , Chung Maxey C.M.
Published: October 2002

We have isolated a potent platelet aggregation inducer from the crude venom of Calloselasma rhodostoma (Malayan pit viper), termed rhodoaggretin, with a novel oligomeric structure consisting of a dimer of C‐type lectin‐like heterodimers. On the basis of its native molecular mass of 66 kDa, and a M
r of 30 kDa for its disulfide‐linked αβ‐heterodimer, we propose that rhodoaggretin exists as a (αβ)2 complex in the native state. We postulate that the di‐dimer is stabilized by non‐covalent interactions as well as by an intersubunit disulfide bridge between the two αβ‐heterodimers. This conclusion is based on the following observations: (a) sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of the non‐reduced rhodoaggretin gave a major 28 and a minor 52 kDa band. (b) Prior treatment of rhodoaggretin with a limited amount of 2‐mercaptoethanol (2‐ME; 0.1%) resulted in the complete abolishment of the 52 kDa band in SDS–PAGE. (c) Two‐dimensional SDS–PAGE in the presence of 3% 2‐ME showed that both the 28 and 52 kDa bands gave two bands each with M
rs of 18 (α‐subunit) and 15 (β‐subunit) kDa. (d) Mass spectrometric analyses showed that purified rhodoaggretin had a M
r of 30 155.39±3.25 Da while its s‐pyridylethylated α‐ and β‐subunits had M
rs of 16 535.62±2.98 and 15 209.89±1.61 Da respectively. These molecular weight data suggested the presence of 15 cysteinyl residues in rhodoaggretin as compared to the 14 that are reported for the heterodimeric C‐type lectin‐like proteins. This extra cysteinyl residue is a candidate for the formation of the intersubunit disulfide bond in the (αβ)2 complex. (e) Homology structural modeling studies showed that the extra cysteinyl residue can indeed form a disulfide bond that covalently links the two αβ‐heterodimers as proposed above.
STAM/EAST/Hbp adapter proteins – integrators of signalling pathways
Authors: Lohi Olli , Lehto Veli-Pekka
Published: October 2002

STAM/EAST/Hbp family of proteins consists of eight members well conserved from yeast to mammals. The basic domain architecture is comprised of an N‐terminal Vps27, Hrs and STAM homology domain, a ubiquitin‐interacting motif and a central Src homology‐3 domain. Vertebrate members also carry an immunoreceptor tyrosine‐based activation motif. STAM/EAST/Hbp proteins become tyrosine‐phosphorylated by a variety of cytokines and growth factors. STAM 1 and STAM 2A are involved in cytokine‐mediated signalling for DNA synthesis and c‐myc induction. EAST and STAM 2A/Hbp play a role in receptor‐mediated endo‐ and exocytosis and probably also in the regulation of actin cytoskeleton. Knockout experiments implicate a role for STAM 1 in neural cell survival. A picture is emerging of STAM/EAST/Hbp proteins acting as integrators of thus far mechanistically disparate cellular signalling events.
Functional and molecular characterization of a peptide transporter in the rat PC12 neuroendocrine cell line
Authors: Hussain I , Zanic-Grubisic T , Kudo Y , Boyd C.A.R
Published: October 2002

We have studied functional properties of peptide transport in the pheochromocytoma neuroendocrine cell line from rat. The neutral peptide D‐Phe‐L‐Ala (resistant to hydrolysis) is a good substrate for uptake into these cells. Transport is substantially inhibited by diethylpyrocarbonate pretreatment and is stimulated by external acidification. It is sodium‐independent and, unexpectedly, insensitive to membrane potential. Peptide uptake is inhibited by a wide variety of other di‐ and tripeptides but not by amino acids. The neuropeptide kyotorphin (opioid dipeptide (L‐Tyr‐L‐Arg)) inhibits uptake of labelled peptide and trans‐stimulates efflux showing that it is a transported substrate. These findings are discussed in relation to the molecular basis and physiological role of this transport system.
The effect of molecular confinement on the conformational dynamics of the native and partly folded state of apomyoglobin
Authors: Bismuto Ettore , Irace Gaetano
Published: October 2002

Inclusion in agarose gel significantly affects the conformational dynamics of native and acidic partly folded states of tuna apomyoglobin, a single tryptophan containing protein, as documented by frequency domain fluorometry investigations. The heterogeneity of the tryptophanyl emission decay increases on gel inclusion compared to that observed for free‐in‐solvent protein at both neutral and acidic pH, thus suggesting that the interconversion rate among conformational substates is somewhat reduced. The observation that this effect is much more pronounced for the partly folded state suggests that confined environments such as those existing in the living cells might favor the sequential folding process avoiding that structured intermediates rapidly convert into less structured ones.
Production of β‐defensin‐2 by human colonic epithelial cells induced by Salmonella enteritidis flagella filament structural protein
Authors: Takahashi Akira , Wada Akihiro , Ogushi Ken-ichi , Maeda Kyoko , Kawahara Tsukasa , Mawatari Kazuaki , Kurazono Hisao , Moss Joel , Hirayama Toshiya , Nakaya Yutaka
Published: October 2002

We recently showed that FliC of Salmonella enteritidis increased human β‐defensin‐2 (hBD‐2) expression, and now describe the signaling responsible pathway. FliC increased the intracellular Ca2+ concentration ([Ca2+]in) in Caco‐2 cells. The [Ca2+]in increase induced by FliC was prevented by U73122 and heparin, but not by chelating extracellular Ca2+ or pertussis toxin. The FliC‐induced increase in hBD‐2 promoter activity via nuclear factor κB (NF‐κB) was also inhibited by chelation of intracellular Ca2+ or by U73122. We conclude that FliC increased [Ca2+]in via inositol 1,4,5‐trisphosphate, which was followed by up‐regulating hBD‐2 mRNA expression via an NF‐κB‐dependent pathway.
c‐Jun N‐terminal kinase‐3 (JNK3)/stress‐activated protein kinase‐β (SAPKβ) binds and phosphorylates the neuronal microtubule regulator SCG10
Authors: Neidhart Sybille , Antonsson Bruno , Gilliéron Corine , Vilbois Francis , Grenningloh Gabriele , Arkinstall Steve
Published: October 2002

The neuronal growth‐associated protein SCG10 is enriched in the growth cones of neurons where it destabilizes microtubules and thus contributes to the dynamic assembly and disassembly of microtubules. Since its microtubule‐destabilizing activity is regulated by phosphorylation, SCG10 may link extracellular signals to rearrangements of the neuronal cytoskeleton. To identify signal transduction pathways that may lead to SCG10 phosphorylation, we tested a series of serine–threonine‐directed protein kinases that phosphorylate SCG10 in vitro. We demonstrate that purified SCG10 can be phosphorylated by two subclasses of mitogen‐activated protein (MAP) kinases, c‐Jun N‐terminal/stress‐activated protein kinase (JNK/SAPK) and p38 MAP kinase. Moreover, SCG10 was found to bind tightly and specifically to JNK3/SAPKβ. JNK3/SAPKβ phosphorylation occurs at Ser‐62 and Ser‐73, residues that result in reduced microtubule‐destabilizing activity for SCG10. Endogenous SCG10 also undergoes increased phosphorylation in sympathetic neurons at times of JNK3/SAPKβ activation following deprivation from nerve growth factor. Together these observations indicate that activation of JNK/SAPKs provides a pathway for phosphorylation of SCG10 and control of growth cone microtubule formation following neuronal exposure to cellular stresses.
Spermidine is essential for normal proliferation of trypanosomatid protozoa
Authors: González Nélida S , Huber Alejandra , Algranati Israel D
Published: October 2002

Trypanosomatid parasites containing a metabolically unstable ornithine decarboxylase (ODC) are naturally resistant to high levels of α‐difluoromethylornithine (DFMO) because this ODC inhibitor, though causing a drastic reduction of intracellular putrescine, elicits only a moderate decrease of the spermidine endogenous pool. In this study we have used a combination of DFMO with cyclohexylamine (CHA; bis‐cyclohexylammonium sulfate), an inhibitor of spermidine synthase, to reach a more complete depletion of spermidine. Under these conditions we have observed the arrest of proliferation not only in trypanosomatids with stable ODC but also in parasites with an enzyme of high turnover rate. In all cases the reinitiation of proliferation occurred only after the addition of exogenous spermidine, and neither putrescine nor spermine were able to induce the same effect.
Paternally inherited transgenes are down‐regulated but retain low activity during early embryogenesis in Arabidopsis
Authors: Baroux Célia , Blanvillain Robert , Gallois Patrick
Published: October 2002

We investigated the timing of transgene activation after fertilisation in Arabidopsis following crosses and using two transgenic promoters (from the AtCYCB1 and AtLTP1 genes). Using both a transactivation system and direct transcriptional fusion to drive β‐glucuronidase reporter expression, reciprocal crosses showed a lack of expression of the paternal components. This is consistent with a lack of paternal genome activity previously reported during early seed development in Arabidopsis [Viella‐Calzada et al. (2000) Nature 404, 91–94]. However, transactivation experiments of the BARNASE gene gave evidence that at least some paternal loci retain transcriptional activity, though at a low level, during early embryogenesis.
Targeting influenza virosomes to ovarian carcinoma cells
Authors: Mastrobattista Enrico , Schoen Pieter , Wilschut Jan , Crommelin Daan J.A , Storm Gert
Published: October 2002

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) have attracted attention as delivery vesicles for cytosolic drug delivery as they possess membrane fusion activity. Here, we show that influenza virosomes can be targeted towards ovarian carcinoma cells (OVCAR‐3) with preservation of fusion activity. This was achieved by incorporating poly(ethylene glycol) (PEG)‐derivatized lipids into the virosome membrane. This PEG layer serves as shield to prevent interaction of HA with ubiquitous sialic acid residues and as spatial anchor for antibody attachment. Coupling of Fab′ fragments of mAb 323/A3 (anti‐epithelial glycoprotein‐2) to the distal ends of PEG lipids resulted in specific binding of virosomes to OVCAR‐3 cells. These antibody‐redirected virosomes fused with membranes of OVCAR‐3 cells in a pH‐dependent fashion.
A novel second myostatin gene is present in teleost fish
Authors: Maccatrozzo Lisa , Bargelloni Luca , Cardazzo Barbara , Rizzo Giorgia , Patarnello Tomaso
Published: October 2002

We report on the isolation and characterisation of the complete cDNA sequence encoding a novel bone morphogenetic protein‐like protein (sbMSTN‐b) in the teleost fish Sparus aurata. The encoded protein is 68% identical to S. aurata MSTN at the amino acid level, and homologues were also found in Umbrina cirrosa and Tetraodon nigroviridis. Phylogenetic analysis suggests that the MSTN‐b gene may be present in most, perhaps all, teleost fish species. RT‐PCR on different tissues/stages indicates that MSTN‐b is expressed almost exclusively in the central nervous system, starting from late larval stages. Quantitative analyses indicate an increase of sbMSTN‐b expression in the brain associated with metamorphosis, at the same time as completion of nervous system differentiation.
Horizontal transfer of a bacterial gene involved in polyglutamate biosynthesis to the plant‐parasitic nematode Meloidogyne artiellia
Authors: Veronico Pasqua , Jones John , Di Vito Mauro , De Giorgi Carla
Published: October 2002

Analysis of a genomic fragment from the plant parasitic nematode Meloidogyne artiellia revealed the presence of a gene which, in bacteria, is involved in the formation of polyglutamate capsule. Searching of various databases, including the Caenorhabditis elegans genome sequence and the large EST datasets from a variety of parasitic nematodes, showed that no similar genes have been identified in other nematodes or in any other eukaryotic organisms. The M. artiellia gene has a typical eukaryotic structure and its mRNA is present in the intestine. The gene is expressed in all life cycle stages tested. These findings demonstrate horizontal gene transfer may be important in catalyzing the diversification of nematode lineages.
Activity of human Δ5 and Δ6 desaturases on multiple n‐3 and n‐6 polyunsaturated fatty acids
Authors: de Antueno Roberto J. , Knickle Leah C. , Smith Heidi , Elliot Michele L. , Allen Stephen J. , Nwaka Solomon , Winther Michael D.
Published: October 2002

Yeast co‐expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n‐3 and n‐6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Δ5 desaturase is active on 20:3n‐6 and 20:4n‐3. Endogenous Δ6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n‐6 or 20:5n‐3 which, through two sequential elongations, produced 24:4n‐6 and 24:5n‐3, respectively. Overall, our data suggest that a single human Δ6 desaturase is active on 18:2n‐6, 18:3n‐3, 24:4n‐6 and 24:5n‐3.
BMP signaling regulates Nkx2‐5 activity during cardiomyogenesis
Authors: Jamali Mina , Karamboulas Christina , Rogerson Parker J , Skerjanc Ilona S
Published: October 2002

Nkx2‐5 regulates the transcription of muscle‐specific genes during cardiomyogenesis. Nkx2‐5 expression can induce cardiomyogenesis in aggregated P19 cells but not in monolayer cultures. In order to investigate the mechanism by which cellular aggregation regulates Nkx2‐5 function, we examined the role of bone morphogenetic protein 4 (BMP4). We showed that the expression of the BMP inhibitor, noggin, was sufficient to inhibit the induction of cardiomyogenesis by Nkx2‐5 during cellular aggregation. Furthermore, soluble BMP4 could activate Nkx2‐5 function in monolayer cultures, resulting in the formation of cardiomyocytes. Therefore, BMP signaling is necessary and sufficient for the regulation of Nkx2‐5 activity during cardiomyogenesis in P19 cells.
Substrate specificity and subsite mobility in T. aurantiacus xylanase 10A
Authors: Lo Leggio Leila , Kalogiannis Stavros , Eckert Kelvin , Teixeira Susana C.M , Bhat Mahalingeswara K , Andrei Carmen , Pickersgill Richard W , Larsen Sine
Published: October 2002

The substrate specificity of Thermoascus aurantiacus xylanase 10A (TAX) has been investigated both biochemically and structurally. High resolution crystallographic analyses at 291 K and 100 K of TAX complexes with xylobiose show that the ligand is in its α anomeric conformation and provide a rationale for specificity on p‐nitrophenyl glycosides at the −1 and −2 subsites. Trp 275, which is disordered in uncomplexed structures, is stabilised by its interaction with xylobiose. Two structural subsets in family 10 are identified, which differ by the presence or absence of a short helical stretch in the eighth βα‐loop of the TIM barrel, the loop bearing Trp 275. This structural difference is discussed in the context of Trp 275 mobility and xylanase function.