bluuebunny/crossref_metadata_2025 huggingface dataset:
Removal of Infected Canine Cemented Total Hip Prostheses Using a Femoral Window Technique
Authors: Dyce Jonathan , Olmstead Marvin L.
Published: November 2002

Objective—To evaluate a femoral window technique for retrieval of cemented total hip prostheses.

Study Design—Retrospective clinical study.

Animals—Twelve dogs with infection of a cemented modular total hip prosthesis.

Methods—Implant removal was performed by an extended craniolateral approach to the hip and proximal femur without trochanteric osteotomy. The femoral cement mantle was fragmented and removed with simple orthopedic instrumentation by a lateral femoral window that was repaired using cerclage wires. Surgical technique, intraoperative and postoperative complications, bacterial culture results, histopathologic findings, and completeness of cement removal were recorded. Follow‐up radiographs were taken 5 to 9 weeks postoperatively. Long‐term follow‐up information was obtained by client questionnaire. Functional outcome was assessed by scoring ability to stand, sit, walk, run, play, climb stairs, and get into a car.

Results—Prosthesis retrieval was performed 2 to 41 months after implantation (median, 14 months). Complete removal of femoral cement was achieved in 10 dogs. A nondisplaced femoral fissure, extending proximally from the window, was an intraoperative complication in 2 dogs.Staphylococcusspp was most commonly isolated (6 dogs) from interfacial membrane samples. Systemic antibiotic therapy, dependent on susceptibility testing, was administered for 3 to 10 weeks postoperatively. There was radiographic evidence of osteotomy healing at 5‐ to 9‐week reassessment. Recurrence of osteomyelitis was not observed. Long‐term functional outcome was considered mildly abnormal.

Conclusions—The lateral window was an effective technique for retrieval of retained femoral cement.

Clinical Relevance—Removal of an infected prosthesis using this technique generally resulted in a clinical outcome comparable to that withab initiofemoral head and neck excision.
Membrane Potential and Bicarbonate Secretion in Isolated Interlobular Ducts from Guinea-pig Pancreas
Authors: Ishiguro H. , Steward M.C. , Sohma Y. , Kubota T. , Kitagawa M. , Kondo T. , Case R.M. , Hayakawa T. , Naruse S.
Published: October 2002

The interlobular duct cells of the guinea-pig pancreas secrete HCO3− across their luminal membrane into a HCO3−-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO3− transport cannot be achieved by luminal Cl−/HCO3− exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO3− efflux, we have measured the intracellular potential (Vm) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl−-25 mM HCO3− solution, a condition similar to the basal state, the resting potential was approximately −60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (∼5 mV) due to activation of electrogenic Na+-HCO3− cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately −50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO3− concentration to 125 mM caused a hyperpolarization (∼10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl− efflux across the luminal membrane is minimized by the depletion of intracellular Cl− and offset by the hyperpolarizing effects of Na+-HCO3− cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO3− that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO3− to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO3−.
Control of Outer Vestibule Dynamics and Current Magnitude in the Kv2.1 Potassium Channel
Authors: Andalib Payam , Wood Michael J. , Korn Stephen J.
Published: October 2002

In Kv2.1 potassium channels, changes in external [K+] modulate current magnitude as a result of a K+-dependent interconversion between two outer vestibule conformations. Previous evidence indicated that outer vestibule conformation (and thus current magnitude) is regulated by the occupancy of a selectivity filter binding site by K+. In this paper, we used the change in current magnitude as an assay to study how the interconversion between outer vestibule conformations is controlled. With 100 mM internal K+, rapid elevation of external [K+] from 0 to 10 mM while channels were activated produced no change in current magnitude (outer vestibule conformation did not change). When channels were subsequently closed and reopened in the presence of elevated [K+], current magnitude was increased (outer vestibule conformation had changed). When channels were activated in the presence of low internal [K+], or when K+ flow into conducting channels was transiently interrupted by an internal channel blocker, increasing external [K+] during activation did increase current magnitude (channel conformation did change). These data indicate that, when channels are in the activated state under physiological conditions, the outer vestibule conformation remains fixed despite changes in external [K+]. In contrast, when channel occupancy is lowered, (by channel closing, an internal blocker or low internal [K+]), the outer vestibule can interconvert between the two conformations. We discuss evidence that the ability of the outer vestibule conformation to change is regulated by the occupancy of a nonselectivity filter site by K+. Independent of the outer vestibule-based potentiation mechanism, Kv2.1 was remarkably insensitive to K+-dependent processes that influence current magnitude (current magnitude changed by <7% at membrane potentials between −20 and 30 mV). Replacement of two outer vestibule lysines in Kv2.1 by smaller neutral amino acids made current magnitude dramatically more sensitive to the reduction in K+ driving force (current magnitude changed by as much as 40%). When combined, these outer vestibule properties (fixed conformation during activation and the presence of lysines) all but prevent variation in Kv2.1 current magnitude when [K+] changes during activation. Moreover, the insensitivity of Kv2.1 current magnitude to changes in K+ driving force promotes a more uniform modulation of current over a wide range of membrane potentials by the K+-dependent regulation of outer vestibule conformation.
Regulation of AE2-mediated Cl− Transport by Intracellular or by Extracellular pH Requires Highly Conserved Amino Acid Residues of the AE2 NH2-terminal Cytoplasmic Domain
Authors: Stewart A.K. , Chernova M.N. , Shmukler B.E. , Wilhelm S. , Alper S.L.
Published: October 2002

We reported recently that regulation by intracellular pH (pHi) of the murine Cl−/HCO3− exchanger AE2 requires amino acid residues 310–347 of the polypeptide's NH2-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl− efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pHo) with unclamped or clamped pHi, or during variation of pHi at constant pHo. Wild-type AE2–mediated 36Cl− efflux was profoundly inhibited by acid pHo, with a value of pHo(50) = 6.87 ± 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312–347 identified the greatest acid shift in pHo(50) value, ∼0.8 pH units in the mutant (A)6342–347, but only a modest acid-shift in the mutant (A)6336–341. Two of the six (A)6 mutants retained normal pHi sensitivity of 36Cl− efflux, whereas the (A)6 mutants 318–323, 336–341, and 342–347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336–347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pHo and to pHi were found independently and in concert. The E346A mutation acid-shifted the pHo(50) value to the same extent whether pHi was unclamped or held constant during variation of pHo. Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pHo as well as pHi.
Transport Function of the Renal Type IIa Na+/Pi Cotransporter Is Codetermined by Residues in Two Opposing Linker Regions
Authors: Köhler Katja , Forster Ian C. , Stange Gerti , Biber Jürg , Murer Heini
Published: October 2002

Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/Pi cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region. The effect of cysteine modification by different MTS reagents on two electrogenic transport modes (leak and cotransport) was investigated. MTSEA (2-aminoethyl MTS hydrobromide) and MTSES (MTS ethylsulfonate) led to full inhibition of cotransport and increased the leak, whereas incubation in MTSET (2-[trimethylammonium]ethyl MTS bromide) inhibited only cotransport. The behavior of other double mutants with a cysteine retained at one site and hydrophobic or hydrophilic residues substituted at the other site, indicated that most likely only Cys-460 was modifiable, but the residue at Ala-203 was critical for conferring the leak and cotransport mode behavior. Substrate interaction with the double mutant was unaffected by MTS exposure as the apparent Pi and Na+ affinities for Pi-induced currents and respective activation functions were unchanged after cysteine modification. This suggested that the modified site did not interfere with substrate recognition/binding, but prevents translocation of the fully loaded carrier. The time-dependency of cotransport loss and leak growth during modification of the double cysteine mutant was reciprocal, which suggested that the modified site is a kinetic codeterminant of both transport modes. The behavior is consistent with a kinetic model for NaPi-IIa that predicts mutual exclusiveness of both transport modes. Together, these findings suggest that parts of the opposing linker regions are associated with the NaPi-IIa transport pathway.
Ser165 in the Second Transmembrane Region of the Kir2.1 Channel Determines its Susceptibility to Blockade by Intracellular Mg2+
Authors: Fujiwara Yuichiro , Kubo Yoshihiro
Published: October 2002

The strong inward rectification of Kir2.1 currents is reportedly due to blockade of the outward current by cytoplasmic magnesium (Mg2+i) and polyamines, and is known to be determined in part by three negatively charged amino acid residues: Asp172, Glu224, and Glu299 (D172, E224, E299). Our aim was to identify additional sites contributing to the inward rectification of Kir2.1 currents. To accomplish this, we introduced into wild-type Kir2.1 and its D172N and D172N & E224G & E299S mutants various point mutations selected on the basis of a comparison of the sequences of Kir2.1 and the weak rectifier sWIRK. By analyzing macroscopic currents recorded from Xenopus oocytes using two-electrode voltage clamp, we determined that S165L mutation decreases inward rectification, especially with the triple mutant. The susceptibility to blockade by intracellular blockers was examined using HEK293 transfectants and the inside-out patch clamp configuration. The sensitivity to spermine was significantly diminished in the D172N and triple mutant, but not the S165L mutant. Both the S165L and D172N mutants were less susceptible to blockade by Mg2+i than the wild-type channel, and the susceptibility was still lower in the D172N & S165L double mutant. These results suggest that S165 is situated deeper into the pore from inside than D172, where it is accessible to Mg2+i but not to spermine. The single channel conductance of the D172N mutant was similar to that of the wild-type Kir2.1, whereas the conductance of the S165L mutant was significantly lower. Permeation by extracellular Rb+ (Rb+o) was dramatically increased by S165L mutation, but was increased only slightly by D172N mutation. By contrast, the Rb+/K+ permeability ratio was increased equally by D172N and S165L mutation. We therefore propose that S165 forms the narrowest part of the Kir2.1 pore, where both extracellular and intracellular blockers plug the permeation pathway.
Hydrolyzable ATP and PIP2 Modulate the Small-conductance K+ Channel in Apical Membranes of Rat Cortical-Collecting Duct (CCD)
Authors: Lu Ming , Hebert Steven C. , Giebisch Gerhard
Published: October 2002

The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATPγS and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.
Tracking Voltage-dependent Conformational Changes in Skeletal Muscle Sodium Channel during Activation
Authors: Chanda Baron , Bezanilla Francisco
Published: October 2002

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.
Probing an Open CFTR Pore with Organic Anion Blockers
Authors: Zhou Zhen , Hu Shenghui , Hwang Tzyh-Chang
Published: October 2002

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl− current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose–response relationships of both blockers follow a simple Michaelis-Menten function with Kd values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl− increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl− binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl− concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl− and isethionate compete for a binding site in the pore. The voltage dependence and external Cl− concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.
Structural and Functional Role of the Extracellular S5-P Linker in the HERG Potassium Channel
Authors: Liu Jie , Zhang Mei , Jiang Min , Tseng Gea-Ny
Published: October 2002

C-type inactivation in the HERG channel is unique among voltage-gated K channels in having extremely fast kinetics and strong voltage sensitivity. This suggests that HERG may have a unique outer mouth structure (where conformational changes underlie C-type inactivation), and/or a unique communication between the outer mouth and the voltage sensor. We use cysteine-scanning mutagenesis and thiol-modifying reagents to probe the structural and functional role of the S5-P (residues 571–613) and P-S6 (residues 631–638) linkers of HERG that line the outer vestibule of the channel. Disulfide formation involving introduced cysteine side chains or modification of side chain properties at “high-impact” positions produces a common mutant phenotype: disruption of C-type inactivation, reduction of K+ selectivity, and hyperpolarizing shift in the voltage-dependence of activation. In particular, we identify 15 consecutive positions in the middle of the S5-P linker (583–597) where side chain modification has marked impact on channel function. Analysis of the degrees of mutation-induced perturbation in channel function along 583–597 reveals an α-helical periodicity. Furthermore, the effects of MTS modification suggest that the NH2-terminal of this segment (position 584) may be very close to the pore entrance. We propose a structural model for the outer vestibule of the HERG channel, in which the 583–597 segment forms an α-helix. With the NH2 terminus of this helix sitting at the edge of the pore entrance, the length of the helix (∼20 Å) allows its other end to reach and interact with the voltage-sensing domain. Therefore, the “583–597 helix” in the S5-P linker of the HERG channel serves as a bridge of communication between the outer mouth and the voltage sensor, that may make important contribution to the unique C-type inactivation phenotype.
Coupling between Voltage Sensors and Activation Gate in Voltage-gated K+ Channels
Authors: Lu Zhe , Klem Angela M. , Ramu Yajamana
Published: October 2002

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1–S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5–S6, equivalent to M1–M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4–S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.
Essential role of calcium in vascular endothelial growth factor A‐induced signaling: mechanism of the antiangiogenic effect of carboxyamidotriazole
Authors: Faehling Martin , Kroll Jens , FöHr Karl J. , Fellbrich Guido , Mayr Ulrike , Trischler Gerlinde , Waltenberger Johannes
Published: October 2002

Vascular endothelial growth factor‐α (VEGF‐A) plays a major role in tumor angiogenesis and raises the concentration of intracellular free calcium ([Ca2+]i). Carboxyamidotriazole (CAI), an inhibitor of calcium influx and of angiogenesis, is under investigation as a tumoristatic agent. We studied the effect of CAI and the role of [Ca2+]i in VEGF‐α signaling in human endothelial cells. VEGF‐α induced a biphasic [Ca2+]i signal. VEGF‐α increased the level of intracellular inositol 1,4,5‐trisphosphate (IP3), which suggests that VEGF‐A releases Ca2+ from IP3‐sensitive stores and induces store‐operated calcium influx. Reduction of either extracellular or intracellular free Ca2+ inhibited VEGF‐A‐induced proliferation. CAI inhibited IP3 formation, both phases of the calcium signal, nitric oxide (NO) release, and proliferation induced by VEGF‐A. CAI prevented neither activation of VEGF receptor‐2 (VEGFR‐2) (KDR/Flk‐1), phospholipase C‐γ, or mitogen‐activated protein kinase (MAP kinase) nor translocation of nuclear factor of activated T cells (NFAT). We conclude that calcium signaling is necessary for VEGF‐A‐induced proliferation. MAP kinase activation occurs independently of [Ca2+]i but is not sufficient to induce proliferation in the absence of calcium signaling. Inhibition of the VEGF‐Ainduced [Ca2+]i signal and proliferation by CAI can be explained by inhibition of IP3 formation and may contribute to the antiangiogenic action of CAI. Calcium‐dependent NO formation may represent a link between calcium signaling and proliferation.
High glucose‐induced oxidative stress and mitochondrial dysfunction in neurons
Authors: Russell James W. , Golovoy David , Vincent Andrea M. , Mahendru Pia , Olzmann James A. , Mentzer Alice , Feldman Eva L.
Published: October 2002

The current study examines the association between glucose induction of reactive oxygen species (ROS), mitochondrial (Mt) depolarization, and programmed cell death in primary neurons. In primary dorsal root ganglion (DRG) neurons, 45 mM glucose rapidly induces a peak rise in ROS Corresponding to a 50% increase in mean Mt size at 6 h (P<0.001). This is coupled with loss of regulation of the Mt membrane potential (Mt membrane hyperpolarization, followed by depolarization, MMD), partial depletion of ATP, and activation of caspase‐3 and ‐9. Glucose‐induced activation of ROS, MMD, and caspase‐3 and ‐9 activation is inhibited by myxothiazole and thenoyltrifluoroacetone (P<0.001), which inhibit specific components of the Mt electron transfer chain. Similarly, MMD and caspase‐3 activation are inhibited by 100 fM bongkrekic acid (an inhibitor of the adenosine nucleotide translocase ANT). These results indicate that mild increases in glucose induce ROS and Mt swelling that precedes neuronal apoptosis. Glucotoxicity is blocked by inhibiting ROS induction, MMD, or caspase cleavage by specific inhibitors of electron transfer, or by stabilizing the ANT.—Russell, J. W., Golovoy, D., Vincent, A. M., Mahendru, P., Olzmann, J. A., Mentzer, A., Feldman, E. L. High glucose‐induced oxidative stress and mitochondrial dysfunction in neurons. FASEB J. 16, 1738–1748 (2002)
A fluorescent Tie1 reporter allows monitoring of vascular development and endothelial cell isolation from transgenic mouse embryos
Authors: Iljin Kristiina , Petrova Tatiana V. , Veikkola Tanja , Kumar Vijay , Poutanen Matti , Alitalo Kari
Published: October 2002

Tie1 is an endothelial receptor tyrosine kinase essential for development and maintenance of the vascular system. Here we report generation of transgenic mice expressing enhanced green fluorescent protein (EGFP) or a chimeric protein consisting of a Zeosin resistance marker and EGFP under the control of mouse Tie1 promoter. Intravital monitoring of fluorescence showed that the EGFP reporter recapitulates the Tie1 expression pattern in the developing vasculature, and flow cytometry using EGFP allowed the isolation of essentially pure Tie1‐expressing endothelial cells from transgenic mouse embryos. However, EGFP and LacZ transgenic markers were strongly down‐regulated in the adult vasculature; unlike the Tie1‐LacZ knock‐in locus, the promoter was not reactivated during tumor neovascularization, indicating the presence of additional regulatory elements in the Tie1 locus. Starting at midgestation, Tie1 promoter activity became stronger in the arterial than in the venous endothelium; in adult mice, promoter activity was observed in arterioles, capillaries, and lymphatic vessels, indicating a significant degree of specificity in different types of endothelial cells. Our results establish Tie1‐Z/EGFP transgenic mice as a useful model to study embryonic vascular development and a convenient source for the isolation of primary endothelial cells.—Iljin, K., Petrova, T., Veikkola, T., Kumar, V., Poutanen, M., Alitalo, K. A fluorescent Tie1 reporter allows monitoring of vascular development and endothelial cell isolation from transgenic mouse embryos. FASEB J. 16, 1764–1774 (2002)
A concerted, rational design of type 1 17β‐hydroxysteroid dehydrogenase inhibitors: estradiol‐adenosine hybrids with high affinity
Authors: Qiu Wei , Campbell Robert L. , Gangloff Anne , Dupuis Philippe , Boivin Roch P. , Tremblay Martin R. , Poirier Donald , Lin Sheng‐Xiang
Published: October 2002

Human estrogenic 17β‐hydroxysteroid dehydrogenase (17β‐HSD type 1) catalyzes the final step in the synthesis of active estrogens that stimulate the proliferation of breast cancer cells. Based on the initial premise to make use of the binding energies of both the substrate and cofactor sites, and molecular modeling starting from the enzyme structure, several estradiol‐adenosine hybrids were designed and synthesized. Among these hybrids, EM‐1745 with a linker of 8‐CH2 groups is proved to be the best competitive inhibitor with a Ki of 3.0 ± 0.8 nM. The crystal structure of the EM‐1745 enzyme complex at 1.6 Å provides evidence at atomic resolution of strong interactions between both the steroid and cofactor moieties and the enzyme molecule, as illustrated by a σAweighted 2Fo‐Fc electron density map contoured at 3.0 σ. The substrate entry loop is further stabilized in this complex compared with previous complexes of the enzyme. These results confirm our initial strategy of combining studies of structural biology and enzyme mechanism in the inhibitor design, which may be applied to other steroidogenic enzymes involved in human diseases.
RETRACTED: α‐Synuclein regulates neuronal survival via Bcl‐2 family expression and PI3/Akt kinase pathway
Authors: Seo Ji‐Heui , Rah Jong‐Cheol , Choi Se Hoon , Shin Jae Kyung , Min Kyeoungsik , Kim Hye‐Sun , Park Cheol Hyoung , Kim Seonghan , Kim Eun‐Mee , Lee Sang‐Hyoung , Lee Sangho , Won Suh Se , Suh Yoo‐Hun
Published: October 2002

α‐Synuclein (α‐SN) is a ubiquitous protein that is especially abundant in the brain and has been postulated to play a central role in the pathogenesis of Parkinson's disease, Alzheimer's disease, and other neurodegenerative disorders. However, little is known about the neuronal functions of α‐SN and the molecular and cellular mechanisms underlying neuronal loss. Here, we show that α‐SN plays dual roles of neuroprotection and neurotoxicity depending on its concentration or level of expression. At nanomolar concentrations, α‐SN protected neurons against serum deprivation, oxidative stress, and excitotoxicity through the PI3/Akt signaling pathway, and its protective effect was increased by Bcl‐2 overexpression. Conversely, at both low micromolar and overexpressed levels in the cell, α‐SN resulted in cytotoxicity. This might be related to decreased Bcl‐xL expression and increased bax expression, which is subsequently followed by cytochrome c release and caspase activation and also by microglia‐mediated inflammatory responses via the NFκB and mitogen‐activated protein kinase pathways.
Anti‐monocyte chemoattractant protein‐1 gene therapy inhibits restenotic changes (neointimal hyperplasia) after balloon injury in rats and monkeys
Authors: Usui Makoto , Egashira Kensuke , Ohtani Kisho , Kataoka Chu , Ishibashi Minako , Hiasa Ken‐ichi , Katoh Makoto , Zhao Qingwei , Kitamoto Shiro , Takeshita Akira
Published: October 2002

Prevention of restenosis after coronary intervention is a major clinical challenge, which highlights the need of new therapeutic options. Vascular injury may involve inflammatory responses that accelerate the recruitment and activation of monocytes through the activation of chemotactic factors, including monocyte chemoattractant protein‐1 (MCP‐1). However, there is no definitive evidence supporting the role of MCP‐1 in restenosis. We recently devised a new strategy for anti‐MCP‐1 gene therapy by transfecting an N‐terminal deletion mutant of the MCP‐1 gene into skeletal muscles. We demonstrate here that this strategy suppressed monocyte infiltration/activation in the injured site and markedly inhibited restenotic changes (neointimal hyperplasia) after balloon injury of the carotid artery in rats and monkeys. This strategy also suppressed the local production of MCP‐1 and inflammatory cytokines. Therefore, monocyte infiltration and activation mediated by MCP‐1 are essential in the development of restenotic changes after balloon injury. This strategy may be a useful form of gene therapy against human restenosis.
Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c‐erbB‐2 receptor via epidermal growth factor receptor
Authors: Dittmar Thomas , Husemann Anja , Schewe Yvonne , Nofer Jerzy‐Roch , Niggemann Bernd , Zänker Kurt S. , Brandt Burkhard H.
Published: October 2002

Induction of tumor cell migration is a key step in invasion and metastasis. Here we report that the epidermal growth factor (EGF)‐induced cell migration of breast cancer cells is attributed to a transient, rather than a sustained, activation of phospholipase C (PLC) ‐γ1 due to c‐erbB‐2 signaling. EGF stimulation of EGF receptor (EGFR) overexpressing cells resulted in long‐term PLC‐γ1 tyrosine phosphorylation and sustained levels of inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG) producing sinusoidal calcium oscillations. In contrast, c‐erbB‐2/EGFR expressing cells displayed baseline transient calcium oscillations after EGF treatment due to short‐term PLC‐γ1 tyrosine phosphorylation and short‐term IP3 and DAG turnover. A third cell line expressing a point‐mutated c‐erbB‐2 receptor that lacks the autophosphorylation Y1248 was generated to investigate whether the different PLC‐γ1 activation was attributed to this structure. Neither PLC‐γ1 tyrosine phosphorylation nor IP3 and DAG turnover and calcium oscillations were observed in this cell line, indicating the modulation of the PLC‐γ1 activation time course by c‐erbB‐2 signaling. Induction of cell migration was solely observable in the c‐erbB‐2‐positive cell line as proved by the mode of actin reorganization and a cell migration assay, using a 3D‐ collagen lattice. In summary, c‐erbB‐2 up‐regulation switches on the cell migration program by modulating the time course of PLC‐γ1 activation.
The antitumoral effect of endostatin and angiostatin is associated with a down‐regulation of vascular endothelial growth factor expression in tumor cells
Authors: Hajitou Amin , Grignet‐Debrus Christine , Devy Laetitia , Berndt Sarah , Blacher Silvia , Deroanne Christophe F. , Bajou Khalid , Fong Timothy , Chiang Yawen , Foidart Jean‐Michel , Noël Agnès
Published: October 2002

Endostatin and angiostatin are known as tumor‐derived angiogenesis inhibitors, but their mechanisms of action are not yet completely defined. We report here that endostatin and angiostatin, delivered by adenoviral vectors, reduced in vitro the neovessel formation in the mouse aortic ring assay by 85 and 40%, respectively. We also demonstrated in vivo that both endostatin and angiostatin inhibited local invasion and tumor vascularization of transplanted murine malignant keratinocytes, and reduced by 50 and 90% the development of highly vascularized murine mammary tumors. This inhibition of tumor growth was associated with a reduction of tumor vascularization. Expression analysis of vascular endothelial growth factor (VEGF) carried out in the mouse aortic ring model revealed a 3‐ to 10‐fold down‐regulation of VEGF mRNA expression in endostatin‐treated rings. A similar down‐regulation of VEGF expression at both mRNA and protein levels was also observed in the two in vivo cancer models after treatment with each angiogenesis inhibitor. This suggests that endostatin and angiostatin effects may be mediated, at least in part, by their ability to down‐regulate VEGF expression within the tumor. This work provides evidence that endostatin and angiostatin act on tumor cells themselves.
Isolation and functional characterization of the actin‐binding region in the tight junction protein ZO‐1
Authors: Fanning Alan S. , Ma Thomas Y. , Anderson James Melvin
Published: October 2002

Zonula occludens (ZO)‐1 is a member of the MAGUK (membrane‐associated guanylate kinase homologs) family of membrane‐associated signaling molecules that binds directly to both cytosolic and transmembrane components of the tight junction and is believed to organize these proteins within the apical junctional complex. It also binds directly to F‐actin, although the functional relevance of this interaction is unknown. To address this issue, we have used VSVG‐ tagged transgenes to dissect ZO‐1 and have identified a 220 amino acid region of ZO‐1 that is necessary for its association with F‐actin in MDCK cell pull‐down assays. A GST fusion expressing this region can bind directly to F‐actin in vitro, whereas a GFP fusion expressing this domain decorates actin stress fibers when expressed in MDCK cells. These results indicate that this actin‐binding region (ABR) is both necessary and sufficient for binding to F‐actin in vitro and in vivo. VSVG‐tagged transgenes that lack the ABR still accumulate at both early and late cell‐cell contacts in MDCK cells, suggesting that the ABR is not required for tight junction localization. However, accumulation of constructs lacking the ABR is markedly reduced at tight junctions in confluent cells, suggesting that the ABR does play an important role in the localization of ZO‐1 at junctions. Furthermore, the ABR is required for localization to a novel actin‐rich pool of ZO‐1 that accumulates in puncta at the free edge of cells before initiation of cell‐cell contact. We conclude that direct interactions between ZO‐1 and F‐actin play a role in several different steps of junction assembly.
Analysis of differential gene expression in stretched podocytes: osteopontin enhances adaptation of podocytes to mechanical stress
Authors: Endlich Nicole , Sunohara Masataka , Nietfeld Wilfried , Wolski Eryk W. , Schiwek Daniel , KräNzlin Bettina , Gretz Norbert , Kriz Wilhelm , Eickhoff Holger , Endlich Karlhans
Published: October 2002

Glomerular hypertension is a major determinant advancing progression to end‐stage renal failure. Podocytes, which are thought to counteract pressure‐mediated capillary expansion, are increasingly challenged in glomerular hypertension. Studies in animal models of glomerular hypertension indicate that glomerulosclerosis develops from adhesions of the glomerular tuft to Bowman's capsule due to progressive podocyte loss. However, the molecular alterations of podocytes in glomerular hypertension are unknown. In this study, we determined the changes in gene expression in podocytes induced by mechanical stress in vitro (cyclic biaxial stretch, 0.5 Hz, 5% linear strain, 3 days) using cDNA arrays (6144 clones). Sixteen differentially regulated genes were identified, suggesting alterations of cell‐matrix interaction, mitochondrial/metabolic function, and protein synthesis/degradation in stretched podocytes. The transcript for the matricellular protein osteopontin (OPN) was most strongly up‐regulated by stretch (approximately threefold). By reverse transcriptase‐polymer chain reaction, up‐regulation of OPN mRNA was also detected in glomeruli of rats treated for 2.5 wk with desoxycorticosterone acetate‐salt, an animal model of glomerular hypertension. In cultured podocytes, OPN coating induced a motile phenotype increasing actin nucleation proteins at cell margins and reducing stress fibers and focal adhesions. Intriguingly, additional OPN coating of collagen IV‐coated membranes accelerated stretch‐induced actin reorganization and markedly diminished podocyte loss at higher strain. This study delineates the molecular response of podocytes to mechanical stress and identifies OPN as a stretch‐adapting molecule in podocytes.
Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide
Authors: Bodennec Jacques , Pelled Dori , Riebeling Christian , Trajkovic Selena , Futerman Anthony H.
Published: October 2002

Glucosylceramide (GlcCer) accumulates in the inherited metabolic disorder, Gaucher disease, because of the defective activity of lysosomal glucocerebrosidase. We previously demonstrated that upon GlcCer accumulation, cultured hippocampal neurons exhibit modified growth patterns, altered endoplasmic reticulum density, and altered calcium release from intracellular stores. We here examined the relationship between GlcCer accumulation and phospholipid synthesis. After treatment of neurons with an active site‐directed inhibitor of glucocerebrosidase, or in neurons obtained from a mouse model of Gaucher disease, [14C]methyl choline incorporation into [14C]phosphatidylcholine ([14C]PC) and [14C]sphingomyelin was elevated, as were [14C]CDPcholine levels, suggesting that CTP:phosphocholine cytidylyltransferase (CCT) is activated. Indeed, CCT activity was elevated in neurons that had accumulated GlcCer. GlcCer, but not galactosylceramide (GalCer), stimulated CCT activity in rat brain homogenates, and significantly higher levels of CCT were membrane associated in cortical homogenates from a mouse model of Gaucher disease compared with wild‐type mice. Because CCT mRNA and protein levels were unaltered in either neurons or brain tissue that had accumulated GlcCer, it appeared likely that GlcCer activates CCT by a post‐translational mechanism. This was verified by examination of the effect of GlcCer on CCT purified about 1200‐fold from rat brain. GlcCer stimulated CCT activity, with stimulation observed at levels as low as 2.5 mol% and with maximal activation reached at 10 mol%. In contrast, GalCer had no effect. Together, these data demonstrate that GlcCer directly activates CCT, which results in elevated PC synthesis, which may account for some of the changes in growth rates observed upon neuronal GlcCer accumulation.
The C‐terminal activation domain of the STAT‐1 transcription enhances ischemia/reperfusion‐induced apoptosis in cardiac myocytes
Authors: Stephanou Anastasis , Scarabelli Tiziano M. , Townsend Paul A. , Bell Robert , Yellon Derek , Knight Richard A. , Latchman David S.
Published: October 2002

We have demonstrated previously that the STAT‐1 transcription factor plays a key role in ischemia/reperfusion (I/R)‐induced apoptosis in cardiac myocytes. In the present study we assessed which region of the STAT‐1 molecule mediates apoptosis in cardiac myocytes. A STAT‐1 construct (amino acid 350–750) lacking the N‐terminus could enhance I/R‐induced apoptosis in cardiac myocytes. However, a STAT‐1 construct, which lacks 60 amino acids at the C‐terminus (amino acid 691–750), was ineffective in promoting I/R‐induced apoptosis in cardiac myocytes. Furthermore, overexpression of a C‐terminal STAT‐1 construct (amino acid 691–750) containing the transcriptional activation domain, but not the DNA binding domain, strongly enhanced I/R‐induced apoptotic cell death. Cardiac myocytes isolated from mice expressing a truncated C‐terminal STAT‐1 were more sensitive to I/R‐induced cell death. Finally, isolated hearts from these animals exposed to I/R injury had larger infarct size and greater number of TUNEL‐positive myocytes than control hearts. These studies demonstrate that the C‐terminal transactivation domain of STAT‐1 is necessary and sufficient for I/R injury‐induced apoptosis in cardiac myocytes.
Inhibition of erythropoietin gene expression signaling involves the transcription factors GATA‐2 and NF‐κB
Authors: La Ferla Katia , Reimann Christian , Jelkmann Wolfgang , Hellwig‐Bürgel Thomas
Published: October 2002

The anemia of chronic inflammatory and malignant diseases is partly due to impaired synthesis of the hormone erythropoietin (Epo). The proinflammatory cytokines interleukin‐1 (IL‐1) and tumor necrosis factor α (TNF‐α) suppress in vitro Epo gene expression and Epo protein secretion. However, the molecular mechanisms of this inhibition are poorly understood. The human Epo promoter and the 5' flanking region contain several recognition sequences for transcription factors acting either positively or negatively. Herein, we investigated the roles of the transcription factors GATA‐2 and NF‐κB in the modulation of Epo gene expression by IL‐1β and TNF‐α in the human hepatoma cell line HepG2. Electrophoretic mobility shift assays revealed increased GATA‐2 and NF‐κB DNA binding in cells treated with IL‐1β or TNF‐α. Reporter gene assays with a sequence from the Epo promoter in front of the firefly luciferase gene showed that the cytokines reduced Epo reporter gene activity. Functional inactivation of GATA‐2 and NF‐κB by oligo‐decoy techniques prevented the inhibition of Epo production by IL‐1β and TNF‐α. In HepG2 cells stably transfected with a dominant‐negative form of IκBα, the activation of NF‐κB was inhibited, while Epo mRNA levels and Epo secretion increased. Thus, both GATA‐2 and NF‐κB seem to be involved in the suppression of Epo gene expression by IL1β and TNF‐α in vitro and may be responsible for impaired Epo synthesis in inflammatory diseases in vivo.
Down‐regulation of human CYP3A4 by the inflammatory signal interleukin 6: molecular mechanism and transcription factors involved
Authors: Jover Ramiro , Bort Roque , Gómez‐Lechón Ma. José , Castell José V.
Published: October 2002

The hepatic drug‐metabolizing cytochrome P‐450 (CYP) enzymes are down‐regulated during inflammation. In vitro studies with hepatocytes have shown that the cytokines released during inflammatory responses are largely responsible for this CYP repression. However, the signaling pathways and the cytokine‐activated factors involved remain to be properly identified. Our research has focused on the negative regulation of CYP3A4 (the major drug‐metabolizing human CYP) by interleukin 6 (IL‐6) (the principal regulator of the hepatic acute‐phase response). CYP3A4 down‐regulation by IL‐6 requires activation of the glycoprotein receptor gp130; however, it does not proceed through the JAK/STAT pathway, as demonstrated by the overexpression of a dominant‐negative STAT3 factor by means of an adenoviral vector. The involvement of IL‐6‐activated kinases such as extracellular signal‐regulated kinase ERK1/2 or p38 is also unlikely, as evidenced by the use of specific chemical inhibitors. It is noteworthy that IL‐6 caused a moderated induction in the mRNA of the transcription factor C/EBPβ (CCAAT‐ enhancer binding protein β) and a marked increase in the translation of C/EBPβ‐LIP, a 20‐kDa C/EBPβ isoform lacking a transactivation domain. Adenovirus‐mediated expression of C/EBPβ‐ LIP caused a dose‐dependent repression of CYP3A4 mRNA, whereas overexpression C/EBPα and C/EBPβ‐LAP (35 kDa) caused a significant induction. Our results support the idea that IL‐6 down‐regulates CYP3A4 through translational induction of C/EBPβ‐LIP, which competes with and antagonizes constitutive C/EBP transactivators. From a clinical point of view, these findings could be relevant in the development of therapeutic cytokines with a less repressive effect on hepatic drug‐metabolizing enzymes.
Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter?
Authors: Wang Rui
Published: October 2002

Bearing the public image of a deadly “gas of rotten eggs,” hydrogen sulfide (H2S) can be generated in many types of mammalian cells. Functionally, H2S has been implicated in the induction of hippocampal long‐term potentiation, brain development, and blood pressure regulation. By acting specifically on KATP channels, H2S can hyperpolarize cell membranes, relax smooth muscle cells, or decrease neuronal excitability. The endogenous metabolism and physiological functions of H2S position this gas well in the novel family of endogenous gaseous transmitters, termed “gasotransmitters.” It is hypothesized that H2S is the third endogenous signaling gasotransmitter, besides nitric oxide and carbon monoxide. This positioning of H2S will open an exciting field‐H2S physiology‐encompassing realization of the interaction of H2S and other gasotransmitters, sulfurating modification of proteins, and the functional role of H2Sin multiple systems. It may shed light on the pathogenesis of many diseases related to the abnormal metabolism of H2S. — Wang, R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J. 16, 1792–1798 (2002)