bluuebunny/crossref_metadata_2025 huggingface dataset:
A mathematical model of the cytosolic-free calcium response in endothelial cells to fluid shear stress
Authors: Wiesner Theodore F. , Berk Bradford C. , Nerem Robert M.
Published: July 2002

Important among the responses of endothelial cells to flow stimuli
are cytosolic-free calcium transients. These transients are mediated by
several factors, including blood-borne agonists, extracellular calcium,
and fluid-imposed shear forces. A mathematical model has been developed
describing the recognition and transduction of shear stress to the
second messenger cytosolic calcium. Shear stress modulates the calcium
response via at least two modalities. First, mass transfer of agonist
to the cell surface is enhanced by perfusion and is thus related to
shear stress. Second, the permeability of the cell membrane to
extracellular calcium increases upon exposure to shear stress. A mass
balance for agonist in the perfusate is coupled to a previously
published calcium dynamics model. Computations indicate a flow region
where the transient moves from transport limited to kinetically
limited. Parametric studies indicate distinct contributions to the time
course by each step in the process. These steps include the time to
develop the concentration boundary layer of agonist, receptor
activation, and the mobilization of calcium from intracellular stores.
Exogenous calcium is presumed to enter the cell via shear stress-gated
ion channels. The model predicts a sigmoidal dependence of calcium
influx upon shear stress. The peak value of the transient is determined
largely by the agonist pathway, whereas the plateau level is governed
by calcium influx. The model predicts the modulation of the calcium
transient in the physiologically relevant range of flow and the
associated shear stress. This implies that hemodynamics is important in
regulating endothelial biology.
Antigen receptor engagement delivers a stop signal to migrating T lymphocytes
Authors: Dustin Michael L. , Bromley Shannon K. , Kan Zhengyan , Peterson Daniel A. , Unanue Emil R.
Published: July 2002

We investigated the role of the T cell antigen receptor (TcR) in control of T cell migration in anin vitrosystem. We used T cells from transgenic mice bearing a TcR for the lysozyme peptide 48–62 bound to I-Ak(3A9). T cells from the 3A9 TcR transgenic mice crawled on purified intercellular adhesion molecule-1 substrates, but strikingly, stopped upon interaction with the physiological ligand, i.e., the mouse I-Akwith covalently attached hen egg white lysozyme peptide residues 48–62 complex. TcR-triggered stopping was reversible by treatment with adhesion-strengthening phorbol esters. The microtubule organizing center of stopped cells was positioned adjacent to the site of stable cell anchorage. Direct conversion of lymphocyte function associated-1 to the high-affinity conformation with antibodies also stopped T cells in a similar manner to antigen. Thus, physiological TcR engagement triggers a stop signal through lymphocyte function associated-1. We propose that the stop signal is an early and essential event in T cell activation that also will play an important role in control of T cell migration.
Shortage of mitogen-activated protein kinase is responsible for resistance to AP-1 transactivation and transformation in mouse JB6 cells
Authors: Huang Chuanshu , Ma Wei-Ya , Young Matthew R. , Colburn Nancy , Dong Zigang
Published: July 2002

The JB6 mouse epidermal cell system, which includes tumor promotion-sensitive (P
+
) and tumor promotion-resistant (P
−
) cells, is a well-established and extensively used cell culture model for studying the mechanism of late-stage tumor promotion. Tumor promoters, such as 12-
O
-tetradecanoylphorbol 13-acetate (TPA) or epidermal growth factor (EGF), induce high levels of activator protein 1 (AP-1) activity and large, tumorigenic, anchorage-independent colonies in soft agar at a high frequency in JB6 P
+
cells, but not in JB6 P
−
cells. We report here a molecular explanation for the defect in the AP-1 activation and promotion-resistant phenotype of P
−
cells. We demonstrate that the lack of AP-1 activation and cell transformation responses to TPA and EGF in P
−
cells appears attributable to the low level of mitogen-activated protein kinase (MAPK) (extracellular signal-regulated protein kinase, Erk) in these cells. TPA and EGF induce transactivation of AP-1 activity in P
+
cells but not in P
−
cells. Nonphosphorylated forms and TPA- or EGF-induced phosphorylated forms of Erks (Erk1 and Erk2) in P
−
cells were much lower than those in P
+
cells. Stable transfection of wild-type MAPK (Erk2) into P
−
cells restored its response to TPA and EGF for both AP-1 activation and cell transformation. These results suggest that the shortage of MAPK (Erk1 and Erk2) appears to be an important contributor to the tumor promotion-resistant phenotype in JB6 cells.
Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library
Authors: Chen Yao-Tseng , Güre Ali O. , Tsang Solam , Stockert Elisabeth , Jäger Elke , Knuth Alexander , Old Lloyd J.
Published: July 2002

Cancer/testis (CT) antigens—immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types—are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5′ end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.
Farnesyltransferase inhibitors induce cytochrome
c
release and caspase 3 activation preferentially in transformed cells
Authors: Suzuki Nobutaka , Urano Jun , Tamanoi Fuyuhiko
Published: July 2002

Farnesyltransferase inhibitors (FTIs) represent a new class of anticancer drugs that show promise in blocking the growth of tumors. Here, we report that FTIs are capable of inducing apoptosis of transformed but not untransformed cells. Treatment of v-K-
ras
-transformed normal rat kidney (KNRK) cells with FTIs leads to the induction of apoptotic cell morphology, chromatin condensation and DNA fragmentation. In addition, fluorescence-activated cell sorter analysis of FTI-treated KNRK cells shows a sub-G
1
apoptotic peak (chromosome content of <2 N). This FTI-induced apoptosis is evident only when the cells are grown in low serum conditions (0.1% fetal calf serum) and is observed selectively with transformed KNRK cells and not with untransformed NRK cells. Further analysis of the mechanism underlying this apoptosis has shown that FTI treatment of KNRK cells results in the activation of caspase 3 but not caspase 1. Moreover, the addition of Z-DEVD-fmk, an agent that interferes with caspase 3 activity, can inhibit FTI-induced apoptosis in a dose-dependent manner. Introduction of the
CASP-3
gene into MCF7 cells, which lack caspase 3 activity, results in a significant increase of FTI-induced apoptosis. Furthermore, FTI induces the release of cytochrome
c
into the cytosol. This release is an important feature of caspase 3-mediated apoptosis. These results suggest that FTIs induce apoptosis through the release of cytochrome
c
from the mitochondria resulting in caspase 3 activation.
Identification and characterization of a cell membrane nucleic acid channel
Authors: Hanss Basil , Leal-Pinto Edgar , Bruggeman Leslie A. , Copeland Terry D. , Klotman Paul E.
Published: July 2002

We have identified a 45-kDa protein purified from rat renal brush border membrane that binds short single-stranded nucleic acid sequences. This activity was purified, reconstituted in proteoliposomes, and then fused with model planar lipid bilayers. In voltage-clamp experiments, the reconstituted 45-kDa protein functioned as a gated channel that allows the passage of nucleic acids. Channel activity was observed immediately after addition of oligonucleotide. Channel activity was not observed in the absence of purified protein or of oligonucleotide or when protein was heat-inactivated prior to forming proteoliposomes. In the presence of symmetrical buffered solution and oligonucleotide, current passed linearly over the range of holding potentials tested. Conductance was 10.4 ± 0.4 picosiemens (pS) and reversal potential was 0.2 ± 1.7 mV. There was no difference in channel conductance or reversal potential between phosphodiester and phosphorothioate oligonucleotides. Ion-substitution experiments documented a shift in reversal potential only when a concentration gradient for oligonucleotide was established, indicating that movement of oligonucleotide alone was responsible for current. Movement of oligonucleotide across the bilayer was confirmed by using32P-labeled oligonucleotides. Channel open probability decreased significantly in the presence of heparan sulfate. These studies provide evidence for a cell surface channel that conducts nucleic acids.
MP2C, a plant protein phosphatase 2C, functions as a negative regulator of mitogen-activated protein kinase pathways in yeast and plants
Authors: Meskiene Irute , Bögre Laszlo , Glaser Walter , Balog Judit , Brandstötter Markus , Zwerger Karin , Ammerer Gustav , Hirt Heribert
Published: July 2002

By interference of the yeast pheromone mitogen-activated protein kinase (MAPK) pathway with an alfalfa cDNA expression library, we have isolated the
MP2C
gene encoding a functional protein phosphatase type 2C. Epistasis analysis in yeast indicated that the molecular target of the MP2C phosphatase is Ste11, a MAPK kinase kinase that is a central regulator of the pheromone and osmosensing pathways. In plants, MP2C functions as a negative regulator of the stress-activated MAPK (SAMK) pathway that is activated by cold, drought, touch, and wounding. Although activation of the SAMK pathway occurs by a posttranslational mechanism,
de novo
transcription and translation of protein factor(s) are necessary for its inactivation. MP2C is likely to be this or one of these factors, because wound-induced activation of SAMK is followed by
MP2C
gene expression and recombinant glutathione
S
-transferase–MP2C is able to inactivate extracts containing wound-induced SAMK. Wound-induced
MP2C
expression is a transient event and correlates with the refractory period, i.e., the time when restimulation of the SAMK pathway is not possible by a second stimulation. These data suggest that MP2C is part of a negative feedback mechanism that is responsible for resetting the SAMK cascade in plants.
Peroxisomal
d
-hydroxyacyl-CoA dehydrogenase deficiency: Resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency
Authors: van Grunsven Elisabeth G. , van Berkel Emanuel , Ijlst Lodewijk , Vreken Peter , de Klerk Johannis B. C. , Adamski Jerzy , Lemonde Hugh , Clayton Peter T. , Cuebas Dean A. , Wanders Ronald J. A.
Published: July 2002

Peroxisomes play an essential role in a number of different metabolic pathways, including the β-oxidation of a distinct set of fatty acids and fatty acid derivatives. The importance of the peroxisomal β-oxidation system in humans is made apparent by the existence of a group of inherited diseases in which peroxisomal β-oxidation is impaired. This includes X-linked adrenoleukodystrophy and other disorders with a defined defect. On the other hand, many patients have been described with a defect in peroxisomal β-oxidation of unknown etiology. Resolution of the defects in these patients requires the elucidation of the enzymatic organization of the peroxisomal β-oxidation system. Importantly, a new peroxisomal β-oxidation enzyme was recently described called
d
-bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity primarily reacting with α-methyl fatty acids like pristanic acid and di- and trihydroxycholestanoic acid. In this patient we describe the first case of
d
-bifunctional protein deficiency as resolved by enzyme activity measurements and mutation analysis. The mutation found (Gly
16
Ser) is in the dehydrogenase coding part of the gene in an important loop of the Rossman fold forming the NAD
+
-binding site. The results show that the newly identified
d
-bifunctional protein plays an essential role in the peroxisomal β-oxidation pathway that cannot be compensated for by the
l
-specific bifunctional protein.
Transmembrane heme delivery systems
Authors: Goldman Barry S. , Beck David L. , Monika Elizabeth M. , Kranz Robert G.
Published: July 2002

Heme proteins play pivotal roles in a wealth of biological processes. Despite this, the molecular mechanisms by which heme traverses bilayer membranes for use in biosynthetic reactions are unknown. The biosynthesis of
c
-type cytochromes requires that heme is transported to the bacterial periplasm or mitochondrial intermembrane space where it is covalently ligated to two reduced cysteinyl residues of the apocytochrome. Results herein suggest that a family of integral membrane proteins in prokaryotes, protozoans, and plants act as transmembrane heme delivery systems for the biogenesis of
c
-type cytochromes. The complete topology of a representative from each of the three subfamilies was experimentally determined. Key histidinyl residues and a conserved tryptophan-rich region (designated the WWD domain) are positioned at the site of cytochrome
c
assembly for all three subfamilies. These histidinyl residues were shown to be essential for function in one of the subfamilies, an ABC transporter encoded by
helABCD
. We believe that a directed heme delivery pathway is vital for the synthesis of cytochromes
c
, whereby heme iron is protected from oxidation via ligation to histidinyl residues within the delivery proteins.
Generation of CD8 suppressor factor and β chemokines, induced by xenogeneic immunization, in the prevention of simian immunodeficiency virus infection in macaques
Authors: Wang Yufei , Tao Louisa , Mitchell Elaine , Bogers Willy M. J. M. , Doyle Carl , Bravery Christopher A. , Bergmeier Lesley A. , Kelly Charles G. , Heeney Jonathan L. , Lehner Thomas
Published: July 2002

Previous xenogeneic immunization experiments in rhesus macaques with simian immunodeficiency virus (SIV) grown in human CD4
+
T cells consistently elicited protection from challenge with live SIV. However, the mechanism of protection has not been established. We present evidence that xenogeneic immunization induced significant CD8 suppressor factor, RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP) 1α, and MIP-1β (
P
< 0.001 -
P
< 0.02). The concentrations of these increased significantly in protected as compared with infected macaques (
P
< 0.001). Xenogeneic stimulation
in vitro
also up-regulated CD8 suppressor factors (SF;
P
< 0.001) and the β chemokines which were neutralized by antibodies to the 3 β chemokines. Recombinant human RANTES, MIP-1α and MIP-1β which bind to simian CCR5, suppressed SIV replication in a dose-dependent manner, with RANTES being more effective than the other two chemokines. The results suggest that immunization with SIV grown in human CD4
+
T cells induces CD8-suppressor factor, RANTES, MIP-1α and MIP-1β which may block CCR5 receptors and prevent the virus from binding and fusion to CD4
+
cells.
Polyglutamine-mediated dysfunction and apoptotic death of a
Caenorhabditis elegans
sensory neuron
Authors: Faber Peter W. , Alter Janet R. , MacDonald Marcy E. , Hart Anne C.
Published: July 2002

The effect of expressing human huntingtin fragments containing polyglutamine (polyQ) tracts of varying lengths was assessed in
Caenorhabditis elegans
ASH sensory neurons in young and old animals. Expression of a huntingtin fragment containing a polyQ tract of 150 residues (Htn-Q150) led to progressive ASH neurodegeneration but did not cause cell death. Progressive cell death and enhanced neurodegeneration were observed in ASH neurons that coexpressed Htn-Q150 and a subthreshold dose of a toxic OSM-10∷green fluorescent protein (OSM-10∷GFP) fusion protein. Htn-Q150 huntingtin protein fragments formed protein aggregates in ASH neurons, and the number of ASH neurons containing aggregates increased as animals aged. ASH neuronal cell death required
ced-3
caspase function, indicating that the observed cell death is apoptotic. Of interest,
ced-3
played a critical role in Htn-Q150-mediated neurodegeneration but not in OSM10∷GFP-mediated ASH neurodegeneration.
ced-3
function was important but not essential for the formation of protein aggregates. Finally, behavioral assays indicated that ASH neurons, coexpressing Htn-Q150 and OSM10∷GFP, were functionally impaired at 3 days before the detection of neurodegeneration, cell death, and protein aggregates.
Specific, rapid synthesis of Phe-RNA by RNA
Authors: Illangasekare Mali , Yarus Michael
Published: July 2002

RNA 77, derived by selection amplification, accelerates its own conversion to Phe-RNA (relative to randomized RNA) more than 6 × 10
7
-fold, by using amino acid adenylates as substrate. A modified assay system allows measurement of slow rates of aa-RNA formation, which for disfavored amino acid substrates can be more than 10
4
-fold slower than phenylalanine. Thus unlike previously characterized self-aminoacylators, RNA 77 catalysis is highly amino acid selective. Remarkably, both rates of aminoacyl transfer and amino acid specificities are greater for RNA 77 than measured for protein PheRS. These data experimentally support the possible existence of an ancestral amino acid-specific translation system relying entirely on RNA catalysis. RNA 77 itself embodies a possible transitional evolutionary state, in which side-chain-specific aa-RNA formation and anticodon-codon pairing were invested in the same molecule.
Plasmodium falciparum
subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1–42 maturase
Authors: Barale Jean-Christophe , Blisnick Thierry , Fujioka Hisashi , Alzari Pedro M. , Aikawa Masamishi , Braun-Breton Catherine , Langsley Gordon
Published: July 2002

The process of human erythrocyte invasion by
Plasmodium falciparum
parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a
P. falciparum
subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases.
Pfsub2
is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2’s subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.
Identification of SH2-Bβ as a potent cytoplasmic activator of the tyrosine kinase Janus kinase 2
Authors: Rui Liangyou , Carter-Su Christin
Published: July 2002

Janus kinases (JAKs) are cytoplasmic tyrosine kinases critical for signaling by growth hormone (GH) and many other ligands that bind to members of the cytokine receptor superfamily. SH2-Bβ was previously identified as a JAK2-interacting protein that is tyrosyl phosphorylated in response to GH and other cytokines that activate JAK2. In this study, we examined whether SH2-Bβ alters the activity of JAK2. SH2-Bβ, when coexpressed with JAK2, significantly increased the tyrosyl phosphorylation of JAK2 and multiple other cellular proteins and stimulated the kinase activity of JAK2 by ≈20-fold. Coexpression of SH2-Bβ with JAK2 dramatically increased tyrosyl phosphorylation of signal transducer and activator of transcription (Stat)5B and Stat3, physiological substrates of JAK2. SH2-Bβ(R555E) with a defective Src homology 2 domain was unable to stimulate JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B and Stat3. More importantly, SH2-Bβ enhanced GH-induced tyrosyl phosphorylation of endogenous JAK2 and ligand-induced tyrosyl phosphorylation of Stat5B by endogenous JAK2. In contrast, SH2-Bβ did not potentiate the activation of other tyrosine kinases including the receptors for platelet-derived growth factor, epidermal growth factor, or nerve growth factor (TrkA), tyrosine kinases that also bind SH2-Bβ. These data demonstrate that SH2-Bβ is a potent cytoplasmic activator of JAK2 and is thereby expected to be an important cellular regulator of signaling by GH and other hormones and cytokines that activate JAK2.
Crystal structure of the α appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly
Authors: Traub Linton M. , Downs Maureen A. , Westrich Jennifer L. , Fremont Daved H.
Published: July 2002

AP-2 adaptors regulate clathrin-bud formation at the cell surface by recruiting clathrin trimers to the plasma membrane and by selecting certain membrane proteins for inclusion within the developing clathrin-coat structure. These functions are performed by discrete subunits of the adaptor heterotetramer. The carboxyl-terminal appendage of the AP-2 α subunit appears to regulate the translocation of several endocytic accessory proteins to the bud site. We have determined the crystal structure of the α appendage at 1.4-Å resolution by multiwavelength anomalous diffraction phasing. It is composed of two distinct structural modules, a β-sandwich domain and a mixed α–β platform domain. Structure-based mutagenesis shows that alterations to the molecular surface of a highly conserved region on the platform domain differentially affect associations of the appendage with amphiphysin, eps15, epsin, and AP180, revealing a common protein-binding interface.
Recycling MHC class I molecules and endosomal peptide loading
Authors: Grommé Monique , Uytdehaag Fons G. C. M. , Janssen Hans , Calafat Jero , van Binnendijk Robert S. , Kenter Marcel J. H. , Tulp Abraham , Verwoerd Désirée , Neefjes Jacques
Published: July 2002

MHC class I molecules usually present peptides derived from endogenous antigens that are bound in the endoplasmic reticulum. Loading of exogenous antigens on class I molecules, e.g., in cross-priming, sometimes occurs, but the intracellular location where interaction between the antigenic fragment and class I takes place is unclear. Here we show that measles virus F protein can be presented by class I in transporters associated with antigen processing-independent, NH4Cl-sensitive manner, suggesting that class I molecules are able to interact and bind antigen in acidic compartments, like class II molecules. Studies on intracellular transport of green fluorescent protein-tagged class I molecules in living cells confirmed that a small fraction of class I molecules indeed enters classical MHC class II compartments (MIICs) and is transported in MIICs back to the plasma membrane. Fractionation studies show that class I complexes in MIICs contain peptides. The pH in MIIC (around 5.0) is such that efficient peptide exchange can occur. We thus present evidence for a pathway for class I loading that is shared with class II molecules.
Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target
Authors: Selzer Paul M. , Pingel Sabine , Hsieh Ivy , Ugele Bernhard , Chan Victor J. , Engel Juan C. , Bogyo Matthew , Russell David G. , Sakanari Judy A. , McKerrow James H.
Published: July 2002

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture orin vivo. We now report that specific cysteine protease inhibitors killLeishmaniaparasitesin vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition ofLeishmaniacysteine protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stablein vivoto ameliorate the pathology associated with a mouse model ofLeishmaniainfection.
DNA adenine methylase mutants of
Salmonella typhimurium
show defects in protein secretion, cell invasion, and M cell cytotoxicity
Authors: García-Del Portillo Francisco , Pucciarelli M. Graciela , Casadesús Josep
Published: July 2002

Mutants of
Salmonella typhimurium
lacking DNA adenine methylase are attenuated for virulence in BALB/c mice. LD
50
values of a DNA adenine methylation (Dam)
−
mutant are at least 10
3
- to 10
4
-fold higher than those of the parental strain when administrated by oral or intraperitoneal routes. Dam
−
mutants are unable to proliferate in target organs but persist in low numbers in these locations. Efficient protection to challenge with the virulent parental strain is observed in mice infected with a Dam
−
mutant. Use of the ileal loop assay shows that Dam
−
mutants are less cytotoxic to M cells and fail to invade enterocytes. In the tissue culture model, lack of DNA adenine methylation causes reduced ability to invade nonphagocytic cells. In contrast, no effect is observed either in intracellular proliferation within nonphagocytic cells or in survival within macrophages. The invasion defect of Dam
−
mutants is correlated with a distinct pattern of secreted proteins, which is observed in both PhoP
+
and PhoP
−
backgrounds. Altogether, our observations suggest a multifactorial role of Dam methylation in
Salmonella
virulence.
Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase
Authors: Huang Dongqing , Patrick Gentry , Moffat Jason , Tsai Li-Huei , Andrews Brenda
Published: July 2002

Mammalian Cdk5 is a member of the cyclin-dependent kinase family that is activated by a neuron-specific regulator, p35, to regulate neuronal migration and neurite outgrowth. p35/Cdk5 kinase colocalizes with and regulates the activity of the Pak1 kinase in neuronal growth cones and likely impacts on actin cytoskeletal dynamics through Pak1. Here, we describe a functional homologue of Cdk5 in budding yeast, Pho85. Like Cdk5, Pho85 has been implicated in actin cytoskeleton regulation through phosphorylation of an actin-regulatory protein. Overexpression of
CDK5
in yeast cells complemented most phenotypes associated with
pho85Δ
, including defects in the repression of acid phosphatase expression, sensitivity to salt, and a G
1
progression defect. Consistent with the functional complementation, Cdk5 associated with and was activated by the Pho85 cyclins Pho80 and Pcl2 in yeast cells. In a reciprocal series of experiments, we found that Pho85 associated with the Cdk5 activators p35 and p25 to form an active kinase complex in mammalian and insect cells, supporting our hypothesis that Pho85 and Cdk5 are functionally related. Our results suggest the existence of a functionally conserved pathway involving Cdks and actin-regulatory proteins that promotes reorganization of the actin cytoskeleton in response to regulatory signals.
Crystal structure of the Sec18p N-terminal domain
Authors: Babor S. Mariana , Fass Deborah
Published: July 2002

Yeast Sec18p and its mammalian orthologue
N-
ethylmaleimide-sensitive fusion protein (NSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SNAPs), Sec18p/NSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SNAP receptors). During the ATP hydrolysis cycle, the Sec18p/NSF homohexamer undergoes a large-scale conformational change involving repositioning of the most N terminal of the three domains of each protomer, a domain that is required for SNAP-mediated interaction with SNAP receptors. Whether an internal conformational change in the N-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed. We have determined the structure of the N-terminal domain from Sec18p by x-ray crystallography. The Sec18p N-terminal domain consists of two β-sheet-rich subdomains connected by a short linker. A conserved basic cleft opposite the linker may constitute a SNAP-binding site. Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement. However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled.
The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices
Authors: Zhang Xue-Nong , Zhu Jingya , Spudich John L.
Published: July 2002

Chimeras of the
Halobacterium salinarum
transducers HtrI and HtrII were constructed to study the structural determinants for their specific interaction with the phototaxis receptors sensory rhodopsins I and II (SRI and SRII), respectively. Interaction of receptors and transducers was assessed by two criteria: phototaxis responses by the cells and transducer-modulation of receptor photochemical reaction kinetics in membranes. Coexpression of HtrI with SRII or HtrII with SRI did not result in interaction by either criterion. Each receptor was coexpressed with chimeric transducers in which various domains of the two transducers were interchanged. The results show that the presence of the two transmembrane helices of HtrI in a chimera is necessary and sufficient for functional transducer complexation with SRI, i.e., for wild-type SRI photoreactions and attractant and 2-photon repellent phototaxis responses. Additionally, a previously demonstrated chaperone-like facilitation of SRI folding or stability by HtrI was shown to depend only on the two transmembrane helices of HtrI in chimeric transducers. Similarly, the two transmembrane helices of HtrII specify interaction with the repellent receptor SRII according to motility analysis and laser-flash spectroscopy. The results support a model in which the membrane domains of the receptor/transducer complexes, consisting of the seven helices of the receptor interacting with the four-helix bundle of the transducer dimer, produce SRI- and SRII-specific signals to the flagellar motor by means of interchangeable cytoplasmic domains.
Terreic acid, a quinone epoxide inhibitor of Bruton’s tyrosine kinase
Authors: Kawakami Yuko , Hartman Stephen E. , Kinoshita Eiji , Suzuki Hidefumi , Kitaura Jiro , Yao Libo , Inagaki Naoki , Franco Alessandra , Hata Daisuke , Maeda-Yamamoto Mari , Fukamachi Hiromi , Nagai Hiroichi , Kawakami Toshiaki
Published: July 2002

Bruton’s tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development.Btkmutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using anin vitroassay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects ofbtkmutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors.
Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene
Authors: Zhang Zhongjian , Kundu Gopal C. , Panda Dibyendu , Mandal Asim K. , Mantile-Selvaggi Giuditta , Peri Alessandro , Yuan Chiun-Jye , Mukherjee Anil B.
Published: July 2002

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2–13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (
n
= 25) remained apparently healthy even after 1½ years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.
Ectopic expression of prion protein (PrP) in T lymphocytes or hepatocytes of PrP knockout mice is insufficient to sustain prion replication
Authors: Raeber Alex J. , Sailer Andreas , Hegyi Ivan , Klein Michael A. , Rülicke Thomas , Fischer Marek , Brandner Sebastian , Aguzzi Adriano , Weissmann Charles
Published: July 2002

The cellular form of the Prion protein (PrPC) is necessary for prion replication in mice. To determine whether it is also sufficient, we expressed PrP under the control of various cell- or tissue-specific regulatory elements in PrP knockout mice. The interferon regulatory factor-1 promoter/Eμ enhancer led to high PrP levels in the spleen and low PrP levels in the brain. Following i.p. scrapie inoculation, high prion titers were found in the spleen but not in the brain at 2 weeks and 6 months, showing that the lymphoreticular system by itself is competent to replicate prions. PrP expression directed by theLckpromoter resulted in high PrP levels on T lymphocytes only but, surprisingly, did not allow prion replication in the thymus, spleen, or brain following i.p. inoculation. A third transgenic line, which expressed PrP in the liver under the control of the albumin promoter/enhancer—albeit at low levels—also failed to replicate prions. These results show that expression of PrP alone is not sufficient to sustain prion replication and suggest that additional components are needed.
Tamoxifen inhibits acidification in cells independent of the estrogen receptor
Authors: Altan Nihal , Chen Yu , Schindler Melvin , Simon Sanford M.
Published: July 2002

Tamoxifen has been reported to have numerous physiological effects that are independent of the estrogen receptor, including sensitization of resistant tumor cells to many chemotherapeutic agents. Drug-resistant cells sequester weak base chemotherapeutics in acidic organelles away from their sites of action in the cytosol and nucleus. This work reports that tamoxifen causes redistribution of weak base chemotherapeutics from acidic organelles to the nucleus in drug-resistant cells. Agents that disrupt organelle acidification (e.g., monensin, bafilomycin A
1
) cause a similar redistribution. Measurement of cellular pH in several cell lines reveals that tamoxifen inhibits acidification of endosomes and lysosomes without affecting cytoplasmic pH. Similar to monensin, tamoxifen decreased the rate of vesicular transport though the recycling and secretory pathways. Organellar acidification is required for many cellular functions, and its disruption could account for many of the side effects of tamoxifen.
Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6)
Authors: Dormady Shane P. , Zhang Xin-Min , Basch Ross S.
Published: July 2002

Pluripotential hematopoietic stem cells grow in close
association with bone marrow stromal cells, which play a critical role
in sustaining hematopoiesis in long-term bone marrow cultures. The
mechanisms through which stromal cells act to support pluripotential
hematopoietic stem cells are largely unknown. This study demonstrates
that growth arrest-specific gene-6 (GAS6) plays an important role in
this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky
(Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine
kinase receptors and binds to these receptors via tandem G domains at
its C terminus. After translation, GAS6 moves to the lumen of the
endoplasmic reticulum, where it is extensively γ-carboxylated. The
carboxylation process is vitamin K dependent, and current evidence
suggests that GAS6 must be γ-carboxylated to bind and activate any of
the cognate tyrosine kinase receptors. Here, we show that expression of
GAS6 is highly correlated with the capacity of bone marrow stromal
cells to support hematopoiesis in culture. Nonsupportive stromal cell
lines express little to no GAS6, whereas supportive cell lines express
high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3
fibroblasts is sufficient to render this previously nonsupportive cell
line capable of supporting long-term hematopoietic cultures. 3T3 cells,
genetically engineered to stably express GAS6 (GAS6-3T3), produce a
stromal layer that supports the generation of colony-forming units in
culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically
engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6
does not substitute for coculturing the hematopoietic progenitors with
genetically modified 3T3 cells.
Combinatorial target-guided ligand assembly: Identification of potent subtype-selective c-Src inhibitors
Authors: Maly Dustin J. , Choong Ingrid C. , Ellman Jonathan A.
Published: July 2002

A method for the rapid and efficient identification of ligands to
biological targets is reported. The combinatorial method does not
require structural or mechanistic information and is accomplished in
four straightforward steps. (
i
) A set of potential
binding elements is prepared wherein each molecule incorporates a
common chemical linkage group. (
ii
) The set of potential
binding elements is screened to identify all binding elements that
interact even weakly with the biological target. (
iii
) A
combinatorial library of linked binding elements is prepared whereby
the binding elements are connected by the common chemical linkage
groups through a set of flexible linkers. (
iv
) The
combinatorial library is screened to identify the tightest-binding
ligands. The utility of the method was demonstrated by the
identification of a potent and subtype-selective small molecule
inhibitor of the non-receptor tyrosine kinase c-Src
(IC
50
= 64 nM). Because the method relies on
connecting two distinct binding elements, the relative contributions of
the two binding elements to the potency and selectivity of the
inhibitor were readily determined. This information provides valuable
insight into the molecular basis of inhibition.
Areca nut use following migration and its consequences
Authors: Warnakulasuriya S.
Published: July 2002

Abstract Areca nut use is widespread in the Oriental countries, affecting approximately 20% of the world's population. The combined use of areca nut and smokeless tobacco (ST) is practiced particularly in the Indo‐Chinese continents. While there is considerable global variation in the use of these products, migrant studies relevant to areca nut use is of considerable interest to epidemiologists in suggesting the extent to which these environment exposures are important in the aetiology of different cancers and other health‐related consequences. Studies on Indian migrants to the Malay peninsula, South and east Africa and various Asian ethnic groups resident in several parts of the United Kingdom have shown that the consumption of areca nut (often mixed with ST) is highly prevalent in these communities. Available data on the prevalence of areca chewing among these migrant populations are reviewed here. The carriage of these risk factors from South Asia to other countries has resulted in excess risk of oral cancer in these new settlements. There is also a high incidence of cardiovascular disease, hypertension and late onset diabetes among Indians living in the United Kingdom and there is new evidence to suggest that the combined roles of areca and ST may be contributory. Because of their enhanced financial situation, substance abuse may increase in their new country of domicile. The two products are psychologically addictive and a dependency syndrome related to their use among Asian immigrants to the United Kingdom has been described recently.
Alcohol usage in sport and exercise
Authors: GUTGESELL MARGARET , CANTERBURY RANDOLPH
Published: July 2002

In this review we consider some of the acute and chronic effects of alcohol on human exercise and sport performance. The 1982 position stand of the American College of Sport Medicine on the use of alcohol in sport emphasized that there was little benefit for an athlete. Subsequent literature continues to demonstrate that there are adverse effects on performance. However, the literature is often confusing and disparate. We will attempt to explain the effects and speculate on the possible mechanisms. We divide the review into acute and chronic metabolic and physiological effects of alcohol on exercise performance, primarily in humans. We also review the epidemiological evidence of the associations between alcohol use and problem alcohol behaviors in various athletic groups. Finally, we review the limited data on the effectiveness of exercise therapy in the treatment of alcohol dependent patients. In spite of scientific evidence that alcohol use is, in general, detrimental (or of no benefit) to sport (exercise) performance, alcohol continues to be used by athletes both on a chronic basis and even immediately prior to sports participation. There is some encouraging but limited evidence that student‐athlete alcohol use is decreasing and exercise can be effective as part of alcohol rehabilitation.
Tomographic Three-dimensional Reconstruction of Insect Flight Muscle Partially Relaxed by AMPPNP and Ethylene Glycol
Authors: Schmitz Holger , Reedy Mary C. , Reedy Michael K. , Tregear Richard T. , Taylor Kenneth A.
Published: July 2002

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5′-[β′γ- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension (“glycol-stiff state”). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of “glycol-stiff” sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90° axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45° angled rigor lead bridges. These 90° “target zone” bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These “nontarget zone” bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90° target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only ∼25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90° target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements.
Calcium Determines the Supramolecular Organization of Fibrillin-rich Microfibrils
Authors: Wess T.J. , Purslow P.P. , Sherratt M.J. , Ashworth J. , Shuttleworth C.A. , Kielty C.M.
Published: July 2002

Microfibrils are ubiquitous fibrillin-rich polymers that are thought to provide long-range elasticity to extracellular matrices, including the zonular filaments of mammalian eyes. X-ray diffraction of hydrated bovine zonular filaments demonstrated meridional diffraction peaks indexing on a fundamental axial periodicity (D) of ∼56 nm. A Ca2+-induced reversible change in the intensities of the meridional Bragg peaks indicated that supramolecular rearrangements occurred in response to altered concentrations of free Ca2+. In the presence of Ca2+, the dominant diffracting subspecies were microfibrils aligned in an axial 0.33-D stagger. The removal of Ca2+ caused an enhanced regularity in molecular spacing of individual microfibrils, and the contribution from microfibrils not involved in staggered arrays became more dominant. Scanning transmission electron microscopy of isolated microfibrils revealed that Ca2+ removal or addition caused significant, reversible changes in microfibril mass distribution and periodicity. These results were consistent with evidence from x-ray diffraction. Simulated meridional x-ray diffraction profiles and analyses of isolated Ca2+-containing, staggered microfibrillar arrays were used to interpret the effects of Ca2+. These observations highlight the importance of Ca2+ to microfibrils and microfibrillar arrays in vivo.