bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of the structure of the RGD‐containing loop in the disintegrins echistatin and eristostatin for recognition of αIIbβ3 and αvβ3 integrins
Authors: McLane Mary Ann , Vijay-Kumar Senadhi , Marcinkiewicz Cezary , Calvete Juan J. , Niewiarowski Stefan
Published: April 2003

Echistatin and eristostatin are structurally homologous disintegrins which exhibit significant functional differences in interaction with various integrins. We hypothesized that this may reflect differences in the sequences of their RGD loops: 20CKRARGDDMDDYC32 and 23CRVARGDWNDDYC35, respectively. Mapping of eristostatin peptides obtained by proteolytic digestion suggested that it has the same alignment of SS bridges as echistatin. Synthetic echistatin D27W resembled eristostatin since it had increased platelet aggregation inhibitory activity, increased potency to block fibrinogen binding to αIIbβ3, and decreased potency to block vitronectin binding to αvβ3 as compared to wild‐type echistatin. Since eristostatin and echistatin have a similar pattern of disulfide bridges, we constructed molecular models of eristostatin based on echistatin NMR coordinates. The RGD loops of eristostatin and echistatin D27W were wider than echistatin's due to the placement of tryptophan (rather than aspartic acid) immediately after the RGD sequence. We propose a hypothesis that the width and shape of the RGD loop are important ligand structural features that affect fitting of ligand to the binding pocket of αIIbβ3 and αvβ3.
Dependence of critical micelle concentration of a zwitterionic detergent on ionic strength: implications in receptor solubilization
Authors: Chattopadhyay Amitabha , Harikumar K.G.
Published: April 2003

The zwitterionic detergent, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS), is mild, non‐denaturing, and extensively used for solubilizing membrane proteins and receptors. We report here that the critical micelle concentration (CMC) of CHAPS is dependent on the concentration of NaCl in the solution. Thus, the CMC of CHAPS decreases from 6.41 mM in absence of any salt to 4.10 mM in presence of 1.5 M NaCl. The logarithm of the CMC values appear to have a linear relationship with the salt concentration. Such changes in CMC with ionic strength have important implications in solubilization of membrane‐bound neuronal receptors. This is shown by optimal solubilization of the serotonin receptor type 1A (5‐HT1A) from bovine brain hippocampal crude (native) membrane by CHAPS at premicellar concentration under high salt conditions.
Isolation and characterization of a cDNA encoding a sulfate transporter from Arabidopsis thaliana
Authors: Takahashi Hideki , Sasakura Noriko , Noji Masaaki , Saito Kazuki
Published: April 2003

A cDNA encoding a sulfate transporter was isolated from Arabidopsis thaliana. The isolated clone contained an open reading frame encoding a polypeptide of 658 amino acids, exhibiting the highest similarity (62%) with the sequence of the low‐affinity sulfate transporter of a tropical legume Stylosanthes hamata. Northern blot analysis indicated the constitutive accumulation of a 2.6 kb length transcript in leaves and roots of seedlings. We also propose that A. thaliana contains three sulfate transporter genes which are expressed as 3.0, 2.7 and 2.6 kb length transcripts, respectively, in an organ‐specific manner.
Elevated cytosolic free Ca2+ concentrations and massive Ca2+ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca2+‐ATPase
Authors: Halachmi D. , Eilam Y.
Published: April 2003

The Ca2+‐ATPase homolog of Saccharomyces cerevisiae, PMR1, cloned by Rudolph et al. (Cell 58 (1989) 133–145) is required for normal Golgi functions. We have investigated the role of Pmr1‐protein in maintaining homeostasis of cytosolic free Ca2+ concentration ([Ca2+]i). It was found that exposure to moderately high Ca2+ concentrations led to elevated levels of [Ca2+]i in cells of pmr1 null mutant, in comparison with cells of pmr2 isogenic mutant (defective in cell‐membrane Na+‐ATPase) and of an isogenic wild type. In addition, we showed that PMR1 deletion causes massive accumulation of Ca2+ in the vacuoles and affects the rates of Ca2+ influx and efflux.
CPP32/Yama/apopain cleaves the catalytic component of DNA‐dependent protein kinase in the holoenzyme
Authors: Teraoka Hirobumi , Yumoto Yoshiko , Watanabe Fumiaki , Tsukada Kinji , Suwa Akira , Enari Masato , Nagata Shigekazu
Published: April 2003

DNA‐dependent protein kinase (DNA‐PK) is composed of a 460‐kDa catalytic component (p460) and a DNA‐binding component Ku protein. Immunoblot analysis after treatment of Jurkat cells with anti‐Fas antibody demonstrated the cleavage of p460 concomitantly with an increase in CPP32/Yama/apopain activity. Recombinant CPP32/Yama/apopain specifically cleaved p460 in the DNA‐PK preparation that had been purified from Raji cells into 230‐ and 160‐kDa polypeptides, the latter of which was detected in anti‐Fas‐treated Jurkat cells. The regulatory component Ku protein was not significantly affected by CPP32/Yama/apopain. DNA‐PK activity was decreased with the disappearance of p460 in the incubation of DNA‐PK with CPP32/Yama/apopain. These results suggest that the catalytic component of DNA‐PK is one of the target proteins for CPP32/Yama/apopain in Fas‐mediated apoptosis.
Attenuated function of a variant form of the helix‐loop‐helix protein, Id‐3, generated by an alternative splicing mechanism
Authors: Deed Richard W. , Jasiok Michelle , Norton John D.
Published: April 2003

The Id family of helix‐loop‐helix proteins function as negative regulators of DNA binding, basic helix‐loop‐helix proteins in the regulation of cell growth and differentiation. We report here on the identification of a 17 kDa variant of the 14 kDa Id‐3 protein termed Id‐3L (long version) which possesses a unique 60 amino acid carboxy‐terminus generated by readthrough of a ‘coding intron’ and alternative splicing. Northern analysis revealed expression of a minor 1.1 kb Id‐3L transcript together with the predominant 0.95 kd Id‐3 transcript in the majority of adult human tissues analysed. The variant Id‐3L protein is functionally distinguishable from conventional Id‐3 since in in vitro DNA mobility shift assays, it was greatly impaired in its ability to abrogate binding of the basic helix‐loop‐helix protein, E47, to an E box recognition sequence.
Dominant role of mitochondria in protection against a delayed neuronal Ca2+ overload induced by endogenous excitatory amino acids following a glutamate pulse
Authors: Khodorov B. , Pinelis V. , Storozhevykh T. , Vergun O. , Vinskaya N.
Published: April 2003

The objective of this study was to evaluate the contribution of mitochondria to the clearance of Ca2+ loads induced by glutamate or 25 mM K+ pulses. The mitochondrial Ca2+ uptake was suppressed by application of 0.5 μM antimycin A or 3–5 mM NaCN in combination with 2.5 μg/ml oligomycin. In most cells such treatments both in the presence and in the absence of external Na+ failed to abolish the early fast phase of [Ca2+]
i
recovery following a 1‐min 100 μM glutamate pulse. However, the late slow phase of [Ca2+]
i
recovery in the presence of mitochondrial poisons was transformed into a delayed [Ca2+]
i
elevation culminating in the neuronal Ca2+ overload. Suppression of the Na+/Ca2+ exchange caused by glutamate‐induced [Na+]
i
elevation promoted the development of delayed Ca2+ increase. Under identical conditions, the high [Ca2+]
i
transient induced by 25 mM K+ was never accompanied by a delayed Ca2+ elevation. The glutamate‐induced delayed Ca2+ increase could be readily abolished by the removal of external Ca2+ or by application in the post‐glutamate period of the antagonist of NMDA receptors, 100–200 μM AP‐5. The results obtained suggest that mitochondria play a dominant role in the protection against the neuronal Ca2+ overload induced by endogenous excitatory amino acids released in response to a short‐term glutamate challenge.
Pyruvate inhibition of pyruvate dehydrogenase kinase
Authors: Priestman David A. , Orfali Karen A. , Sugden Mary C.
Published: April 2003

Both prolonged starvation and hyperthyroidism evoke stable increases in cardiac pyruvate dehydrogenase kinase (PDHK) activity. Pyruvate inhibits PDHK in rat heart mitochondria with activation of PDHC. The sensitivity of PDHK to inhibition by pyruvate declines after prolonged starvation. In the present study, pyruvate concentrations giving 50% active complex (PDHa) in mitochondria from fed, control and fed, hyperthyroid rats were 0.3 and 0.8 mM, respectively, compared with 1.0 and 2.8 mM, respectively in mitochondria from 24‐h‐starved and 48‐h‐starved rats. The results demonstrate that altered pyruvate sensitivity is not of necessity linked with altered PDHK activity. PDHK activities in mitochondria prepared from cardiac myocytes from fed rats were increased after culture for 24 h with dibutyryl cyclic AMP (50 μM) plus n‐octanoate (1 mM), with a concomitant decline in sensitivity of PDHK to pyruvate inhibition, suggesting that changes in sensitivity of PDHK to pyruvate inhibition in vivo may be secondary to increased fatty acid supply and cyclic AMP concentrations.
Gene expression of Alzheimer‐associated presenilin‐2 in the frontal cortex of Alzheimer and aged control brain
Authors: Deng Gangmin , Su Joseph H. , Cotman Carl W.
Published: April 2003

The presenilin‐2 (PS2) gene expression pattern in Alzheimer's disease (AD) and control brains was examined using nonradioactive in situ hybridization. Message for PS2 was primarily detectable in neurons, particularly in somal cytoplasm. Intense staining signal was most commonly found in large pyramidal neurons, whereas moderate or faint staining was usually present in smaller neurons. The pattern of PS2 gene expression exhibited a laminar distribution profile in the frontal cortex. A small subset of tangle‐bearing neurons exhibited PS2 hybridization signal in AD. PS2 mRNA expression appeared correlated to a high degree with lipofuscin autofluorescence in a large subset of neurons.
Bell pepper fruit fatty acid hydroperoxide lyase is a cytochrome P450 (CYP74B)
Authors: Matsui Kenji , Shibutani Mizuyoshi , Hase Toshiharu , Kajiwara Tadahiko
Published: April 2003

Fatty acid hydroperoxide lyases cleave a C−C bond adjacent to a hydroperoxide group in lipoxygenase derived lipid hydroperoxides to form short‐chain aldehydes and oxo‐acids. Previously, we showed that fatty acid hydroperoxide lyase from bell pepper fruits is a heme protein whose spectrophotometric properties greatly resemble a cytochrome P450. In order to ascertain the relationship of it to the P450 gene family, we have cloned cDNA encoding fatty acid hydroperoxide lyase from immature bell pepper fruits. The cDNA encodes 480 amino acids, and shares homology with P450s mostly at the C terminus. The heme binding cysteine is recognizable at position 441. The most closely related P450 is allene oxide synthase (CYP74A), with which it has 40% identity. It qualifies the lyase as a member of a new P450 subfamily, CYP74B. From this finding, the enzyme is thought to be a novel member of P450 specialized for the metabolism of lipid peroxides.
The liver‐specific promoter of the human insulin‐like growth factor‐II gene contains two negative regulatory elements
Authors: Rodenburg Richard J.T. , Krijger Jorrit-Jan T. , Holthuizen P.Elly , Sussenbach John S.
Published: April 2003

The adult liver‐specific IGF‐II promoter P1 is activated by CCAAT/Enhancer Binding Proteins α and β. Here we present evidence that promoter P1, in addition to positively regulating elements, contains two elements of 67 nucleotides that form an inverted repeat (IR) and suppress P1 activity. The two IR elements are specifically bound by a protein (inverted repeat binding factor, IRBF). The amounts of IRBF in various cell lines correlate with the levels of suppression of P1 activity, suggesting that this factor is responsible for the suppression of P1 mediated by the IR elements.
Plasmalogen and anionic phospholipid dependence of the cardiac sarcolemmal sodium‐calcium exchanger
Authors: Ford David A. , Hale Calvin C.
Published: April 2003

Although plasmalogens are the predominant phospholipids of cardiac sarcolemma, their physiological role has not been forthcoming. Since the cardiac sarcolemmal sodium‐calcium exchanger has been proposed to be regulated by anionic phospholipids, the roles of plasmalogens and anionic phospholipids as regulators of the sodium‐calcium exchanger were explored. Reconstituted sodium‐calcium exchange activity in plasmalogen‐containing proteoliposomes was 10‐fold higher than that in control proteoliposomes comprised of only diacyl phospholipids. Additionally, exchange activity in plasmalogen‐containing proteoliposomes was regulated by anionic phospholipids. Thus, plasmalogens provide a critical lipid environment in which anionic phospholipids serve as boundary lipids for the regulation of the trans‐sarcolemmal sodium‐calcium exchanger.
Regulation of the katG‐dpsA operon and the importance of KatG in survival of Burkholderia pseudomallei exposed to oxidative stress
Authors: Loprasert Suvit , Whangsuk Wirongrong , Sallabhan Ratiboot , Mongkolsuk Skorn
Published: April 2003

Homologues of the catalase‐peroxidase gene katG and the gene for the non‐specific DNA binding protein dpsA were identified downstream of oxyR in Burkholderia pseudomallei. Northern experiments revealed that both katG and dpsA are co‐transcribed during oxidative stress. Under conditions where the katG promoter is not highly induced, dpsA is transcribed from a second promoter located within the katG‐dpsA intergenic region. A katG insertion mutant was found to be hypersensitive to various oxidants. Analysis of katG expression in the oxyR mutant indicates that OxyR is a dual function regulator that represses the expression of katG during normal growth and activates katG during exposure to oxidative stress. Both reduced and oxidized OxyR were shown to bind to the katG promoter.
A PLCγ2‐independent platelet collagen aggregation requiring functional association of GPVI and integrin α2β1
Authors: Mangin P , Nonne C , Eckly A , Ohlmann P , Freund M , Nieswandt B , Cazenave J-P , Gachet C , Lanza F
Published: April 2003

The role of the phospholipase C (PLC)γ2 isotype in platelet activation was evaluated by studying PLCγ2 −/− mice. These mice have a prolonged bleeding time but their platelets respond normally to non‐collagenous agonists. PLCγ2‐null platelets show residual aggregation response to collagen fibres (6% versus 74% for wild‐type) with minimal granule secretion and no shape change. A delayed shape change is observed at later aggregation times. Specific activation by glycoprotein (GP)VI agonists (convulxin, collagen‐related peptide and GPVI crosslinking) is, however, abolished. Antibodies against integrin α2β1 and GPVI each inhibit the residual collagen response, implying a role of α2β1 in platelet activation and a functional association with GPVI. These responses are also prevented by blocking integrin αIIbβ3 and phosphoinositide 3‐kinase, whereas aspirin treatment and ADP receptor blockade only inhibit shape change. These results provide evidence for a PLCγ2‐independent collagen activation pathway requiring cooperation between GPVI and α2β1 leading to αIIbβ3‐dependent aggregation and shape change by released ADP and thromboxane A2.
Redifferentiation of dedifferentiated bovine chondrocytes when cultured in vitro in a PLGA–collagen hybrid mesh
Authors: Chen Guoping , Sato Takashi , Ushida Takashi , Hirochika Rei , Tateishi Tetsuya
Published: April 2003

Bovine articular chondrocytes dedifferentiated and lost their ability to express articular cartilage‐specific extracellular matrices such as type II collagen and aggrecan when cultured in a culture flask during in vitro multiplication. A poly(DL‐lactic‐co‐glycolic acid) (PLGA)–collagen hybrid mesh was prepared and used to redifferentiate the dedifferentiated cells. The two passaged dedifferentiated chondrocytes were seeded in a PLGA–collagen hybrid mesh and cultured in vitro in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells adhered to the hybrid mesh, distributed evenly, and proliferated to fill the spaces in the scaffold. The gene expression of type I collagen, type II collagen, and aggrecan was analyzed after the cells were cultured in the hybrid mesh for 2–12 weeks. The expression of the gene encoding type I collagen was downregulated, whereas those of type II collagen and aggrecan were upregulated. Histological examination by hematoxylin–eosin and safranin O/fast green staining indicates that the cells regained their original round morphology. In addition, a homogeneous distribution of articular cartilage extracellular matrices was detected around the cells. These results suggest redifferentiation of the differentiated chondrocytes in the hybrid mesh. The hybrid mesh, which facilitated the redifferentiation of the dedifferentiated multiplied chondrocytes, would be an effective scaffold for the assembly of cells to regenerate three‐dimensional cartilaginous tissue.
Solution structure and p43 binding of the p38 leucine zipper motif: coiled‐coil interactions mediate the association between p38 and p43
Authors: Ahn Hee-Chul , Kim Sunghoon , Lee Bong-Jin
Published: April 2003

p38, which has been suggested to be a scaffold protein for the assembly of a macromolecular tRNA synthetase complex, contains a leucine zipper‐like motif. To understand the importance of the leucine zipper‐like motif of p38 (p38LZ) in macromolecular assembly, the p38LZ solution structure was investigated by circular dichroism and nuclear magnetic resonance spectroscopy. The solution structure of p38LZ showed an amphipathic α‐helical structure and characteristics similar to a coiled‐coil motif. The protein–protein interaction mediated by p38LZ was examined by an in vitro binding assay. The p43 protein, another non‐synthetase component of the complex, could bind to p38LZ via its N‐terminal domain, which is also predicted to have a potential coiled‐coil motif. Thus, we propose that the p38–p43 complex would be formed by coiled‐coil interactions, and the formation of the binary complex would facilitate the macromolecular assembly of aminoacyl‐tRNA synthetases.
Understanding the mechanism of proton movement linked to oxygen reduction in cytochrome c oxidase: lessons from other proteins
Authors: Mills Denise A , Ferguson-Miller Shelagh
Published: April 2003

Cytochrome c oxidase is a large intrinsic membrane protein designed to use the energy of electron transfer and oxygen reduction to pump protons across a membrane. The molecular mechanism of the energy conversion process is not understood. Other proteins with simpler, better resolved structures have been more completely defined and offer insight into possible mechanisms of proton transfer in cytochrome c oxidase. Important concepts that are illustrated by these model systems include the ideas of conformational change both close to and at a distance from the triggering event, and the formation of a transitory water‐linked proton pathway during a catalytic cycle. Evidence for the applicability of these concepts to cytochrome c oxidase is discussed.
ATP synthesis driven by proton transport in F1F0‐ATP synthase
Authors: Weber Joachim , Senior Alan E
Published: April 2003

Topical questions in ATP synthase research are: (1) how do protons cause subunit rotation and how does rotation generate ATP synthesis from ADP+Pi? (2) How does hydrolysis of ATP generate subunit rotation and how does rotation bring about uphill transport of protons? The finding that ATP synthase is not just an enzyme but rather a unique nanomotor is attracting a diverse group of researchers keen to find answers. Here we review the most recent work on rapidly developing areas within the field and present proposals for enzymatic and mechanoenzymatic mechanisms.
Long term depression of human nociceptive skin senses induced by thin fibre stimulation
Authors: Nilsson Hans‐Jörgen , Psouni Elia , Schouenborg Jens
Published: April 2003

We have recently shown that stimulation, through a multi‐electrode array, of thin nerve fibres close to the dermo‐epidermal junction in the skin, produces powerful inhibition of itch and, to a lesser degree, cutaneous pain in humans. Here, we have studied the induction time and frequency dependency (range 1–10 Hz) of the inhibitory effects of such stimulation on itch, mechanical, and thermal pain, in 20 healthy subjects. Sixteen electrodes applied on the skin were consecutively stimulated using a method termed cutaneous field stimulation (CFS). The results show that different treatment periods with CFS were required for the induction of significant inhibitory effects on different nociceptive qualities: 1st heat pain (1 min), itch (3 min), 2nd heat pain (6 min), pinch evoked pain (8 min). Six to ten minutes stimulation sufficed to induce peak inhibitory effects on all these sensory qualities while longer stimulation (up to 40 min) did not cause significantly stronger inhibition. The effects on itch, 1st and 2nd heat pain lasted over 55 min after termination of CFS. There was no effect on prickle. No significant difference in inhibitory effects of different stimulation frequencies (1, 4 and 10 Hz/electrode) was found. The induction time and effective stimulation frequencies may suggest that the underlying mechanisms are similar to those of long term depression (LTD) previously described in the spinal cord in animal experiments.
Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response
Authors: Churin Yuri , Al-Ghoul Laila , Kepp Oliver , Meyer Thomas F. , Birchmeier Walter , Naumann Michael
Published: April 2003

Infection with the human microbial pathogen Helicobacter pylori is assumed to lead to invasive gastric cancer. We find that H. pylori activates the hepatocyte growth factor/scatter factor receptor c-Met, which is involved in invasive growth of tumor cells. The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response. CagA could represent a bacterial adaptor protein that associates with phospholipase Cγ but not Grb2-associated binder 1 or growth factor receptor–bound protein 2. The H. pylori–induced motogenic response is suppressed and blocked by the inhibition of PLCγ and of MAPK, respectively. Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling. The activation of the motogenic response in H. pylori–infected epithelial cells suggests that CagA could be involved in tumor progression.
The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore–microtubule attachment and in maintaining the spindle assembly checkpoint
Authors: Hauf Silke , Cole Richard W. , LaTerra Sabrina , Zimmer Christine , Schnapp Gisela , Walter Rainer , Heckel Armin , van Meel Jacques , Rieder Conly L. , Peters Jan-Michael
Published: April 2003

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3–5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.
YHR150w and YDR479c encode peroxisomal integral membrane proteins involved in the regulation of peroxisome number, size, and distribution in Saccharomyces cerevisiae
Authors: Vizeacoumar Franco J. , Torres-Guzman Juan C. , Tam Yuen Yi C. , Aitchison John D. , Rachubinski Richard A.
Published: April 2003

The peroxin Pex24p of the yeast Yarrowia lipolytica exhibits high sequence similarity to two hypothetical proteins, Yhr150p and Ydr479p, encoded by the Saccharomyces cerevisiae genome. Like YlPex24p, both Yhr150p and Ydr479p have been shown to be integral to the peroxisomal membrane, but unlike YlPex24p, their levels of synthesis are not increased upon a shift of cells from glucose- to oleic acid–containing medium. Peroxisomes of cells deleted for either or both of the YHR150w and YDR479c genes are increased in number, exhibit extensive clustering, are smaller in area than peroxisomes of wild-type cells, and often exhibit membrane thickening between adjacent peroxisomes in a cluster. Peroxisomes isolated from cells deleted for both genes have a decreased buoyant density compared with peroxisomes isolated from wild-type cells and still exhibit clustering and peroxisomal membrane thickening. Overexpression of the genes PEX25 or VPS1, but not the gene PEX11, restored the wild-type phenotype to cells deleted for one or both of the YHR150w and YDR479c genes. Together, our data suggest a role for Yhr150p and Ydr479p, together with Pex25p and Vps1p, in regulating peroxisome number, size, and distribution in S. cerevisiae. Because of their role in peroxisome dynamics, YHR150w and YDR479c have been designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p.
Shear stress–induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases
Authors: Wojciak-Stothard Beata , Ridley Anne J.
Published: April 2003

Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress–induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.
LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway
Authors: Chen Esther J. , Kaiser Chris A.
Published: April 2003

LST8, a Saccharomyces cerevisiae gene encoding a 34-kD WD-repeat protein, was identified by mutations that caused defects in sorting Gap1p to the plasma membrane. Here, we report that the Gap1p sorting defect in the lst8-1 mutant results from derepression of Rtg1/3p activity and the subsequent accumulation of high levels of intracellular amino acids, which signal Gap1p sorting to the vacuole. To identify the essential function of Lst8p, we isolated lst8 mutants that are temperature-sensitive for growth. These mutants show hypersensitivity to rapamycin and derepressed Gln3p activity like cells with compromised TOR pathway activity. Like tor2 mutants, lst8 mutants also have cell wall integrity defects. Confirming a role for Lst8p in the TOR pathway, we find that Lst8p associates with both Tor1p and Tor2p and is a peripheral membrane protein that localizes to endosomal or Golgi membranes and cofractionates with Tor1p. Further, we show that a sublethal concentration of rapamycin mimics the Gap1p sorting defect of an lst8 mutant. Finally, the different effects of lst8 alleles on the activation of either the Rtg1/3p or Gln3p transcription factors reveal that these two pathways constitute distinct, genetically separable outputs of the Tor–Lst8 regulatory complex.
Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification
Authors: Shirasu-Hiza Mimi , Coughlin Peg , Mitchison Tim
Published: April 2003

Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth–promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.
Active repression by unliganded retinoid receptors in development
Authors: Weston Andrea D. , Blumberg Bruce , Underhill T. Michael
Published: April 2003

The retinoid receptors have major roles throughout development, even in the absence of ligand. Here, we summarize an emerging theme whereby gene repression, mediated by unliganded retinoid receptors, can dictate cell fate. In addition to activating transcription, retinoid receptors actively repress gene transcription by recruiting cofactors that promote chromatin compaction. Two developmental processes for which gene silencing by the retinoid receptors is essential are head formation in Xenopus and skeletal development in the mouse. Inappropriate repression, by oncogenic retinoic acid (RA)**Abbreviations used in this paper: APL, acute promyelocytic leukemia; dnRARα, dominant–negative version of the RARα; E, embryonic age; HDAC, histone deacetylase; LCoR, ligand-dependent corepressor; NCoR, nuclear receptor corepressor; RA, retinoic acid; RAR, RA receptor; RARE, RXR homodimer bound to bipartite response element; RXR, retinoid X receptor; TSA, trichostatin A; CYP26, cytochrome p450, 26; TR, thyroid hormone receptor. receptor (RAR) fusion proteins, blocks myeloid differentiation leading to a rare form of leukemia. Our current understanding of the developmental role of retinoid repression and future perspectives in this field are discussed.
Spatial distribution and specification of mammalian replication origins during G1 phase
Authors: Li Feng , Chen Jianhua , Solessio Eduardo , Gilbert David M.
Published: April 2003

We have examined the distribution of early replicating origins on stretched DNA fibers when nuclei from CHO cells synchronized at different times during G1 phase initiate DNA replication in Xenopus egg extracts. Origins were differentially labeled in vivo versus in vitro to allow a comparison of their relative positions and spacing. With nuclei isolated in the first hour of G1 phase, in vitro origins were distributed throughout a larger number of DNA fibers and did not coincide with in vivo origins. With nuclei isolated 1 h later, a similar total number of in vitro origins were clustered within a smaller number of DNA fibers but still did not coincide with in vivo origins. However, with nuclei isolated later in G1 phase, the positions of many in vitro origins coincided with in vivo origin sites without further change in origin number or density. These results highlight two distinct G1 steps that establish a spatial and temporal program for replication.
Sim4
Authors: Pidoux Alison L. , Richardson William , Allshire Robin C.
Published: April 2003

Fission yeast centromeres are composed of two domains: the central core and the outer repeats. Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins. Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin. Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure. The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region. Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain. Functional Mis6 is required for Sim4 localization at the kinetochore. Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore.
Distinct in vivo roles for double-stranded RNA-binding domains of the Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting
Authors: Doyle Michael , Jantsch Michael F.
Published: April 2003

The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA–binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.
Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow
Authors: Shimonaka Mika , Katagiri Koko , Nakayama Toshinori , Fujita Naoya , Tsuruo Takashi , Yoshie Osamu , Kinashi Tatsuo
Published: April 2003

Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.
FAK induces expression of Prx1 to promote tenascin-C–dependent fibroblast migration
Authors: McKean David M. , Sisbarro Lila , Ilic Dusko , Kaplan-Alburquerque Nihal , Nemenoff Raphael , Weiser-Evans Mary , Kern Michael J. , Jones Peter Lloyd
Published: April 2003

Fibroblast migration depends, in part, on activation of FAK and cellular interactions with tenascin-C (TN-C). Consistent with the idea that FAK regulates TN-C, migration-defective FAK-null cells expressed reduced levels of TN-C. Furthermore, expression of FAK in FAK-null fibroblasts induced TN-C, whereas inhibition of FAK activity in FAK–wild-type cells had the opposite effect. Paired-related homeobox 1 (Prx1) encodes a homeobox transcription factor that induces TN-C by interacting with a binding site within the TN-C promoter, and it also promotes fibroblast migration. Therefore, we hypothesized that FAK regulates TN-C by controlling the DNA-binding activity of Prx1 and/or by inducing Prx1 expression. Prx1–homeodomain binding site complex formation observed with FAK–wild-type fibroblasts failed to occur in FAK-null fibroblasts, yet expression of Prx1 in these cells induced TN-C promoter activity. Thus, FAK is not essential for Prx1 DNA-binding activity. However, activated FAK was essential for Prx1 expression. Functionally, Prx1 expression in FAK-null fibroblasts restored their ability to migrate toward fibronectin, in a manner that depends on TN-C. These results appear to be relevant in vivo because Prx1 and TN-C expression levels were reduced in FAK-null embryos. This paper suggests a model whereby FAK induces Prx1, and subsequently the formation of a TN-C–enriched ECM that contributes to fibroblast migration.
The Voltage-sensitive Release Mechanism of Excitation Contraction Coupling in Rabbit Cardiac Muscle Is Explained by Calcium-induced Calcium Release
Authors: Griffiths H. , MacLeod K.T.
Published: April 2003

The putative voltage-sensitive release mechanism (VSRM) was investigated in rabbit cardiac myocytes at 37°C with high resistance microelectrodes to minimize intracellular dialysis. When the holding potential was adjusted from −40 to −60 mV, the putative VSRM was expected to operate alongside CICR. Under these conditions however, we did not observe a plateau at positive potentials of the cell shortening versus voltage relationship. The threshold for cell shortening changed by −10 mV, but this resulted from a similar change of the threshold for activation of inward current. Cell shortening under conditions where the putative VSRM was expected to operate was blocked in a dose dependent way by nifedipine and CdCl2 and blocked completely by NiCl2. “Tail contractions” persisted in the presence of nifedipine and CdCl2 but were blocked completely by NiCl2. Block of early outward current by 4-aminopyridine and 4-acetoamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS) demonstrated persisting inward current during test depolarizations despite the presence of nifedipine and CdCl2. Inward current did not persist in the presence of NiCl2. A tonic component of cell shortening that was prominent during depolarizations to positive potentials under conditions selective for the putative VSRM was sensitive to rapidly applied changes in superfusate [Na+] and to the outward Na+/Ca2+ exchange current blocking drug KB-R7943. This component of cell shortening was thought to be the result of Na+/Ca2+ exchange–mediated excitation contraction coupling. Cell shortening recorded under conditions selective for the putative VSRM was increased by the enhanced state of phosphorylation induced by isoprenaline (1 μM) and by enhancing sarcoplasmic reticulum Ca2+ content by manipulation of the conditioning steps. Under these conditions, cell shortening at positive test depolarizations was converted from tonic to phasic. We conclude that the putative VSRM is explained by CICR with the Ca2+ “trigger” supplied by unblocked L-type Ca2+ channels and Na+/Ca2+ exchange.
Hydrophobic Coupling of Lipid Bilayer Energetics to Channel Function
Authors: Goforth Robyn L. , Chi Aung K. , Greathouse Denise V. , Providence Lyndon L. , Koeppe Roger E. , Andersen Olaf S.
Published: April 2003

The hydrophobic coupling between membrane-spanning proteins and the lipid bilayer core causes the bilayer thickness to vary locally as proteins and other “defects” are embedded in the bilayer. These bilayer deformations incur an energetic cost that, in principle, could couple membrane proteins to each other, causing them to associate in the plane of the membrane and thereby coupling them functionally. We demonstrate the existence of such bilayer-mediated coupling at the single-molecule level using single-barreled as well as double-barreled gramicidin channels in which two gramicidin subunits are covalently linked by a water-soluble, flexible linker. When a covalently attached pair of gramicidin subunits associates with a second attached pair to form a double-barreled channel, the lifetime of both channels in the assembly increases from hundreds of milliseconds to a hundred seconds—and the conductance of each channel in the side-by-side pair is almost 10% higher than the conductance of the corresponding single-barreled channels. The double-barreled channels are stabilized some 100,000-fold relative to their single-barreled counterparts. This stabilization arises from: first, the local increase in monomer concentration around a single-barreled channel formed by two covalently linked gramicidins, which increases the rate of double-barreled channel formation; and second, from the increased lifetime of the double-barreled channels. The latter result suggests that the two barrels of the construct associate laterally. The underlying cause for this lateral association most likely is the bilayer deformation energy associated with channel formation. More generally, the results suggest that the mechanical properties of the host bilayer may cause the kinetics of membrane protein conformational transitions to depend on the conformational states of the neighboring proteins.
Effects of Heating and Cooling on Nerve Terminal Impulses Recorded from Cold-sensitive Receptors in the Guinea-pig Cornea
Authors: Carr Richard W. , Pianova Svetlana , Fernandez Juana , Fallon James B. , Belmonte Carlos , Brock James A.
Published: April 2003

An in vitro preparation of the guinea-pig cornea was used to study the effects of changing temperature on nerve terminal impulses recorded extracellularly from cold-sensitive receptors. At a stable holding temperature (31–32.5°C), cold receptors had an ongoing periodic discharge of nerve terminal impulses. This activity decreased or ceased with heating and increased with cooling. Reducing the rate of temperature change reduced the respective effects of heating and cooling on nerve terminal impulse frequency. In addition to changes in the frequency of activity, nerve terminal impulse shape also changed with heating and cooling. At the same ambient temperature, nerve terminal impulses were larger in amplitude and faster in time course during heating than those recorded during cooling. The magnitude of these effects of heating and cooling on nerve terminal impulse shape was reduced if the rate of temperature change was slowed. At 29, 31.5, and 35°C, a train of 50 electrical stimuli delivered to the ciliary nerves at 10–40 Hz produced a progressive increase in the amplitude of successive nerve terminal impulses evoked during the train. Therefore, it is unlikely that the reduction in nerve terminal impulse amplitude observed during cooling is due to the activity-dependent changes in the nerve terminal produced by the concomitant increase in impulse frequency. Instead, the differences in nerve terminal impulse shape observed at the same ambient temperature during heating and cooling may reflect changes in the membrane potential of the nerve terminal associated with thermal transduction.
SamCluster: an integrated scheme for automatic discovery of
sample classes using gene expression profile
Authors: Li Wuju , Fan Ming , Xiong Momiao
Published: April 2003

Motivation: Feature (gene) selection can dramatically improve the accuracy of gene expression profile based sample class prediction. Many statistical methods for feature (gene) selection such as stepwise optimization and Monte Carlo simulation have been developed for tissue sample classification. In contrast to class prediction, few statistical and computational methods for feature selection have been applied to clustering algorithms for pattern discovery.

Results: An integrated scheme and corresponding program SamCluster for automatic discovery of sample classes based on gene expression profile is presented in this report. The scheme incorporates the feature selection algorithms based on the calculation of CV (coefficient of variation) and t-test into hierarchical clustering and proceeds as follows. At first, the genes with their CV greater than the pre-specified threshold are selected for cluster analysis, which results in two putative sample classes. Then, significantly differentially expressed genes in the two putative sample classes with p-values ≤ 0.01, 0.05, or 0.1 from t-test are selected for further cluster analysis. The above processes were iterated until the two stable sample classes were found. Finally, the consensus sample classes are constructed from the putative classes that are derived from the different CV thresholds, and the best putative sample classes that have the minimum distance between the consensus classes and the putative classes are identified. To evaluate the performance of the feature selection for cluster analysis, the proposed scheme was applied to four expression datasets COLON, LEUKEMIA72, LEUKEMIA38, and OVARIAN. The results show that there are only 5, 1, 0, and 0 samples that have been misclassified, respectively. We conclude that the proposed scheme, SamCluster, is an efficient method for discovery of sample classes using gene expression profile.

Availability: The related program SamCluster is available upon request or from the web page http://www.sph.uth.tmc.edu:8052/hgc/Downloads.asp

Contact: wujuli@yahoo.com

* To whom correspondence should be addressed.
Attitude and orbit determination for small satellite using magnetometer measurement
Authors: Jianqi Wang , Xibin Cao , Zhaowei Sun
Published: April 2003

The measurement of geomagnetic field can provide a reliable and economical basis for attitude and orbit information of low earth orbiting satellite. Because the earth's magnetic field is a function of position, and its measurement on the orbit are fully observable, orbit estimation can be obtained using extend Kalman filter (EKF) algorithm. With the assistant of angle velocity information from gyro measurement, attitude estimation can also be obtained. At the same time, gyro drift rate estimation is a part of the filter output. Although orbit and attitude determination are independent of each other, the filter can give the orbit and attitude estimation at the same time. The results of the numerical test show that a signal EKF can estimate both orbit and attitude by using magnetometer and gyro measurement only. The accuracy, usually is sufficient for low earth orbiting satellites.