bluuebunny/crossref_metadata_2025 huggingface dataset:
SARPs: A family of secreted apoptosis-related proteins
Authors: Melkonyan Hovsep S. , Chang Wei C. , Shapiro John P. , Mahadevappa Mamatha , Fitzpatrick Paul A. , Kiefer Michael C. , Tomei L. David , Umansky Samuil R.
Published: July 2002

Quiescent mouse embryonic C3H/10T½ cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T½ cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T½ cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated
sarp1
(secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T½ cells. Hybridization studies with
sarp1
revealed two additional family members. Cloning and sequencing of
sarp2
and
sarp3
revealed 38% and 40% sequence identity to
sarp1
, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with
sarp1
or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with
sarp2
displayed increased sensitivity to different proapoptotic stimuli. Expression of
sarp
family members is tissue specific.
sarp
mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of β-catenin, suggesting that SARPs interfere with the Wnt–frizzled proteins signaling pathway.
Transcriptional repression at a distance through exclusion of activator binding
in vivo
Authors: Shimizu Mitsuhiro , Li Weishi , Shindo Heisaburo , Mitchell Aaron P.
Published: July 2002

The yeast repressor Rme1p acts from distant binding sites to block transcription of the chromosomal
IME1
gene. Rme1p can also repress the heterologous
CYC1
promoter when Rme1p binding sites are placed 250–300 bp upstream of
CYC1
transcriptional activator binding sites (UAS1 and UAS2). Here,
in vivo
footprinting studies indicate that Rme1p acts over this distance by preventing the binding of the
CYC1
transcriptional activators to UAS1 and UAS2. Inhibition of activator binding by Rme1p has the same genetic requirements as repression: both depend upon sequences flanking the Rme1p binding sites and upon Rgr1p and Sin4p, two subunits of the RNA polymerase II-associated Mediator complex that are required for normal nucleosome density. Thus Rme1p may alter chromatin to prevent binding of transcriptional activators to distant DNA sequences.
A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro
Authors: Baker Elizabeth K. , Tozer Eileen Collins , Pfaff Martin , Shattil Sanford J. , Loftus Joseph C. , Ginsberg Mark H.
Published: July 2002

Glanzmann thrombasthenia, an inherited bleeding disorder, can be caused by a defect or deficiency in platelet integrin αIIbβ3(GPIIb-IIIa). Studies of thrombasthenia variants have facilitated identification of sites involved in the functions of αIIbβ3and other integrins. Such sites include those that bind ligand and those that participate in the “activation” of αIIbβ3required for high affinity binding of ligands such as fibrinogen or PAC1, a monoclonal antibody. Here we describe the isolation of such variants, createdin vitrowith Chinese hamster ovary cells that express an activated form of αIIbβ3. These cells were exposed to a mutagen, ethyl methane sulfonate, and variants that lost the capacity to bind PAC1 were isolated by fluorescence-activated cell sorting. These variants were grouped into three phenotypic classes. One comprised integrin mutations that disrupt ligand binding function; a second comprised mutations that interfere with the capacity of cells to activate the integrin. Most of these activation-defective mutations were in the integrin cytoplasmic domain, but surprisingly, several were caused by mutations affecting three closely spaced residues in the β3extracellular domain. A third class of mutants exhibited a defect in integrin activation not ascribable to changes in the integrin sequence. Thus, these may represent mutated signaling molecules required for integrin activation. This unbiased genetic approach provides new insights into the structural basis of integrin function and may assist in identifying the cellular events that regulate integrin function.
Physical interaction between retinoic acid receptor and the oncoprotein Myb inhibits retinoic acid-dependent transactivation
Authors: Pfitzner Edith , Kirfel Jutta , Becker Peter , Rolke Andreas , Schüle Roland
Published: July 2002

The c
-myb
protooncogene is predominantly expressed in hematopoietic cells and plays a vital role in hematopoiesis. Retinoic acid (RA) is able to induce differentiation of several hematopoietic cells. This differentiation is linked to decreased c
-myb
expression, suggesting that retinoid receptors (RAR/RXR) may down-regulate c-
myb
gene expression. Furthermore, recent data indicate that RAR inhibits the function of the Myb protein itself. In addition, the Myb-Ets oncogenic fusion protein has been shown to inhibit transcriptional activation by RAR and thyroid hormone receptor. Myb-Ets also antagonizes the biological response of erythrocytic progenitor cells to RA and thyroid hormone. This prompted us to investigate a possible cross talk between RAR and Myb. Here, we demonstrate that RA inhibits the expression of the endogenous Myb target gene
tom-1
. Conversely, Myb functions as a potent inhibitor of RA-induced biological responses. Functional analysis of Myb mutants in transfection studies revealed that the Myb DNA-binding domain (DBD) is necessary for repression whereas the transactivation domain is dispensable. Furthermore, we show that v-Myb and RAR interact
in vitro
and
in vivo
. This interaction requires the DBD of RAR. In contrast, glutathione
S
-transferase-pulldown assays with v-Myb mutants indicate that the DBD and the C terminus of Myb directly interact with RAR. Our results suggest that the physical interaction between Myb and RAR may play a role in the regulation of hematopoietic gene expression.
Structure and function of the
Bacillus
hybrid enzyme GluXyn-1: Native-like jellyroll fold preserved after insertion of autonomous globular domain
Authors: Aÿ Jacqueline , Götz Frank , Borriss Rainer , Heinemann Udo
Published: July 2002

The 1,3–1,4-β-glucanase from
Bacillus macerans
(wtGLU) and the 1,4-β-xylanase from
Bacillus subtilis
(wtXYN) are both single-domain jellyroll proteins catalyzing similar enzymatic reactions. In the fusion protein GluXyn-1, the two proteins are joined by insertion of the entire XYN domain into a surface loop of cpMAC-57, a circularly permuted variant of wtGLU. GluXyn-1 was generated by protein engineering methods, produced in
Escherichia coli
and shown to fold spontaneously and have both enzymatic activities at wild-type level. The crystal structure of GluXyn-1 was determined at 2.1 Å resolution and refined to
R
= 17.7% and
R
(free) = 22.4%. It shows nearly ideal, native-like folding of both protein domains and a small, but significant hinge bending between the domains. The active sites are independent and accessible explaining the observed enzymatic activity. Because in GluXyn-1 the complete XYN domain is inserted into the compact folding unit of GLU, the wild-type-like activity and tertiary structure of the latter proves that the folding process of GLU does not depend on intramolecular interactions that are short-ranged in the sequence. Insertion fusions of the GluXyn-1 type may prove to be an easy route toward more stable bifunctional proteins in which the two parts are more closely associated than in linear end-to-end protein fusions.
The “Spot 14” gene resides on the telomeric end of the 11q13 amplicon and is expressed in lipogenic breast cancers: Implications for control of tumor metabolism
Authors: Moncur Joel T. , Park Jonathan P. , Memoli Vincent A. , Mohandas T. K. , Kinlaw William B.
Published: July 2002

Enhanced long chain fatty acid synthesis may occur in breast cancer, where it is necessary for tumor growth and predicts a poor prognosis. “Spot 14” (S14) is a carbohydrate- and thyroid hormone-inducible nuclear protein specific to liver, adipose, and lactating mammary tissues that functions to activate genes encoding the enzymes of fatty acid synthesis. Amplification of chromosome region 11q13, where the S14 gene (
THRSP
) resides, also predicts a poor prognosis in breast tumors. We localized the S14 gene between markers D11S906 and D11S937, at the telomeric end of the amplified region at 11q13, and found that it was amplified and expressed in breast cancer-derived cell lines. Moreover, concordant expression of S14 and a key lipogenic enzyme (acetyl-CoA carboxylase) in a panel of primary breast cancer specimens strongly supported a role for S14 as a determinant of tumor lipid metabolism. S14 expression provides a pathophysiological link between two prognostic indicators in breast cancer: enhanced lipogenesis and 11q13 amplification.
Cocaine alters the accessibility of endogenous cysteines in putative extracellular and intracellular loops of the human dopamine transporter
Authors: Ferrer Jasmine V. , Javitch Jonathan A.
Published: July 2002

Cocaine and other psychostimulants act by blocking the dopamine transporter. Binding of the cocaine analog, [
3
H]2-β-carbomethoxy-3-β-(4-fluorophenyl) tropane (CFT) to the dopamine transporter is sensitive to polar sulfhydryl-specific derivatives of methanethiosulfonate (MTS). These reagents preferentially react with water-accessible, reduced cysteines. The human dopamine transporter has 13 cysteines. Their topology is not completely determined. We sought to identify those cysteine residues the modification of which affects CFT binding and to determine the topology of these reactive cysteines. We mutated each of the cysteines, one at a time and in various combinations, to residues that preserved binding and transport, and we tested the sensitivity of each of the mutant transporters to the reagents. One construct, X5C, had five mutated cysteines (C90A, C135A, C306A, C319F, and C342A). Using a membrane preparation in which both extracellular and intracellular cysteines could be accessible, we found that CFT binding in X5C, as compared with wild-type transporter, was two orders of magnitude less sensitive to MTS ethylammonium (MTSEA). The wild-type cysteines were substituted back into X5C, one at a time, and these constructs were tested in cells and in membranes. Cys-90 and Cys-306 appear to be extracellular, and Cys-135 and Cys-342 appear to be intracellular. Each of these residues is predicted to be in extramembranous loops. The binding of cocaine increases the rate of reaction of MTSEA and MTS ethyltrimethylammonium with the extracellular Cys-90 and therefore acts by inducing a conformational change. Cocaine decreases the rate of reaction of MTSEA with Cys-135 and Cys-342, acting either directly or indirectly on these intracellular residues.
The disassembly and reassembly of functional centrosomes
in vitro
Authors: Schnackenberg Bradley J. , Khodjakov Alexey , Rieder Conly L. , Palazzo Robert E.
Published: July 2002

Animal cells contain a single centrosome that nucleates and organizes a polarized array of microtubules which functions in many cellular processes. In most cells the centrosome is composed of two centrioles surrounded by an ill-defined “cloud” of pericentriolar material. Recently, γ-tubulin-containing 25-nm diameter ring structures have been identified as likely microtubule nucleation sites within the pericentriolar material of isolated centrosomes. Here we demonstrate that when
Spisula
centrosomes are extracted with 1.0 M KI they lose their microtubule nucleation potential and appear by three-dimensional electron microscopy as a complex lattice, built from 12- to 15-nm thick elementary fiber(s), that lack centrioles and 25-nm rings. Importantly, when these remnants are incubated in extracts prepared from
Spisula
oocytes they recover their 25-nm rings, γ-tubulin, and microtubule nucleation potential. This recovery process occurs in the absence of microtubules, divalent cations, and nucleotides. Thus, in animals the centrosome is structurally organized around a KI-insoluble filament-based “centromatrix” that serves as a scaffold to which those proteins required for microtubule nucleation bind, either directly or indirectly, in a divalent cation and nucleotide independent manner.
Clustering of non-major histocompatibility complex susceptibility candidate loci in human autoimmune diseases
Authors: Becker Kevin G. , Simon Richard M. , Bailey-Wilson Joan E. , Freidlin Boris , Biddison William E. , McFarland Henry F. , Trent Jeffrey M.
Published: July 2002

Human autoimmune diseases are thought to develop through a complex combination of genetic and environmental factors. Genome-wide linkage searches of autoimmune and inflammatory/immune disorders have identified a large number of non-major histocompatibility complex loci that collectively contribute to disease susceptibility. A comparison was made of the linkage results from 23 published autoimmune or immune-mediated disease genome-wide scans. Human diseases included multiple sclerosis, Crohn’s disease, familial psoriasis, asthma, and type-I diabetes (IDDM). Experimental animal disease studies included murine experimental autoimmune encephalomyelitis, rat inflammatory arthritis, rat and murine IDDM, histamine sensitization, immunity to exogenous antigens, and murine lupus (systemic lupus erythematosus; SLE). A majority (≈65%) of the human positive linkages map nonrandomly into 18 distinct clusters. Overlapping of susceptibility loci occurs between different human immune diseases and by comparing conserved regions with experimental autoimmune/immune disease models. This nonrandom clustering supports a hypothesis that, in some cases, clinically distinct autoimmune diseases may be controlled by a common set of susceptibility genes.
Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia
Authors: Harrison Jeffrey K. , Jiang Yan , Chen Shizong , Xia Yiyang , Maciejewski Dominique , McNamara Robert K. , Streit Wolfgang J. , Salafranca Mina N. , Adhikari Soumya , Thompson Darren A. , Botti Paolo , Bacon Kevin B. , Feng Lili
Published: July 2002

A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuron-expressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.
Analysis of the regulatory phosphorylation site in
Acanthamoeba
myosin IC by using site-directed mutagenesis
Authors: Wang Zhen-Yuan , Wang Fei , Sellers James R. , Korn Edward D. , Hammer John A.
Published: July 2002

The actin-activated ATPase activity of
Acanthamoeba
myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from
Acanthamoeba
as determined by (
i
) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (
ii
) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (
iii
) its
K
m
for ATP, and (
iv
) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same
K
m
for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of
Acanthamoeba
myosin IC
in vitro
and indicate that the length of the side chain that delivers this charge is important.
Lamellar lipoproteins uniquely contribute to hyperlipidemia in mice doubly deficient in apolipoprotein E and hepatic lipase
Authors: Bergeron Nathalie , Kotite Leila , Verges Marcel , Blanche Patricia , Hamilton Robert L. , Krauss Ronald M. , Bensadoun Andre , Havel Richard J.
Published: July 2002

Remnants of triglyceride-rich lipoproteins containing apolipoprotein (apo) B-48 accumulate in apo E-deficient mice, causing pronounced hypercholesterolemia. Mice doubly deficient in apo E and hepatic lipase have more pronounced hypercholesterolemia, even though remnants do not accumulate appreciably in mice deficient in hepatic lipase alone. Here we show that the doubly deficient mice manifest a unique lamellar hyperlipoproteinemia, characterized by vesicular particles 600 Å–1,300 Å in diameter. As seen by negative-staining electron microscopy, these lipoproteins also contain an electron-lucent region adjacent to the vesicle wall, similar to the core of typical lipoproteins. Correlative chemical analysis indicates that the vesicle wall is composed of a 1:1 molar mixture of cholesterol and phospholipids, whereas the electron-lucent region appears to be composed of cholesteryl esters (about 12% of the particle mass). Like the spherical lipoproteins of doubly deficient mice, the vesicular particles contain apo B-48, but they are particularly rich in apo A-IV. We propose that cholesteryl esters are removed from spherical lipoproteins of these mice by scavenger receptor B1, leaving behind polar lipid-rich particles that fuse to form vesicular lipoproteins. Hepatic lipase may prevent such vesicular lipoproteins from accumulating in apo E-deficient mice by hydrolyzing phosphatidyl choline as scavenger receptor B1 removes the cholesteryl esters and by gradual endocytosis of lipoproteins bound to hepatic lipase on the surface of hepatocytes.
Evidence for functional and physical association between
Caenorhabditis elegans
SEL-10, a Cdc4p-related protein, and SEL-12 presenilin
Authors: Wu Guangyu , Hubbard E. Jane Albert , Kitajewski Jan K. , Greenwald Iva
Published: July 2002

Mutations in either of two human presenilin genes (
PS1
and
PS2
) cause Alzheimer’s disease. Here we describe genetic and physical interactions between
Caenorhabditis elegans
SEL-10, a member of the Cdc4p family of proteins, and SEL-12, a
C. elegans
presenilin. We show that loss of
sel-10
activity can suppress the egg-laying defective phenotype associated with reducing
sel-12
activity, and that SEL-10 can physically complex with SEL-12. Proteins of the Cdc4p family have been shown to target proteins for ubiquitin-mediated turnover. The functional and physical interaction between
sel-10
and
sel-12
therefore offers an approach to understanding how presenilin levels are normally regulated.
Nlk is a murine protein kinase related to Erk/MAP kinases and localized in the nucleus
Authors: Brott Barbara K. , Pinsky Benjamin A. , Erikson Raymond L.
Published: July 2002

Extracellular-signal regulated kinases/microtubule-associated protein kinases (Erk/MAPKs) and cyclin-directed kinases (Cdks) are key regulators of many aspects of cell growth and division, as well as apoptosis. We have cloned a kinase,
Nlk
, that is a murine homolog of the
Drosophila nemo
(
nmo
) gene. The
Nlk
amino acid sequence is 54.5% similar and 41.7% identical to murine Erk-2, and 49.6% similar and 38.4% identical to human Cdc2. It possesses an extended amino-terminal domain that is very rich in glutamine, alanine, proline, and histidine. This region bears similarity to repetitive regions found in many transcription factors.
Nlk
is expressed as a 4.0-kb transcript at high levels in adult mouse brain tissue, with low levels in other tissues examined, including lung, where two smaller transcripts of 1.0 and 1.5 kb are expressed as well. A 4.0-kb
Nlk
message is also present during embryogenesis, detectable at day E
10.5
, reaching maximal steady state levels at day E
12.5
, and then decreasing. Nlk transiently expressed in COS7 cells is a 60-kDa kinase detectable by its ability to autophosphorylate. Mutation of the ATP-binding Lys-155 to methionine abolishes its ability to autophosphorylate, as does mutation of a putative activating threonine in kinase domain VIII, to valine, aspartic, or glutamic acid. Subcellular fractionation indicates that 60–70% of Nlk is localized to the nucleus, whereas 30–40% of Nlk is cytoplasmic. Immunofluorescence microscopy confirms that Nlk resides predominantly in the nucleus. Nlk and Nmo may be the first members of a family of kinases with homology to both Erk/MAPKs and Cdks.
A previously unidentified host protein protects retroviral DNA from autointegration
Authors: Lee Myung Soo , Craigie Robert
Published: July 2002

Integration of a DNA copy of the viral genome into a host chromosome is an essential step in the retrovirus life cycle. The machinery that carries out the integration reaction is a nucleoprotein complex derived from the core of the infecting virion. To successfully integrate into host DNA, the viral DNA within this complex must avoid self-destructive integration into itself, a reaction termed autointegration. We have previously shown [Lee, M. S. and Craigie, R. (1994)
Proc. Natl. Acad. Sci. USA
91, 9823–9827] that viral nucleoprotein complexes isolated from Moloney murine leukemia virus-infected cells exhibit a barrier to autointegration. This autointegration barrier could be destroyed by stripping factors from the complexes and subsequently restored by incubation with a host cell extract, but not by incubation with an extract of disrupted virions. We have now used this autointegration barrier reconstitution assay to purify the host factor from uninfected NIH 3T3 fibroblasts. It is a single polypeptide of 89 aa that does not match any previously identified protein. The identity of the protein was confirmed by expressing it in
Escherichia coli
and demonstrating the activity of the heterologously expressed protein in the reconstitution assay.
Centrosome hypertrophy in human breast tumors: Implications for genomic stability and cell polarity
Authors: Lingle Wilma L. , Lutz Ward H. , Ingle James N. , Maihle Nita J. , Salisbury Jeffrey L.
Published: July 2002

The centrosome plays an important role in maintenance of cell polarity and in progression through the cell cycle by determining the number, polarity, and organization of interphase and mitotic microtubules. By examining a set of 35 high grade human breast tumors, we show that centrosomes of adenocarcinoma cells generally display abnormal structure, aberrant protein phosphorylation, and increased microtubule nucleating capacity in comparison to centrosomes of normal breast epithelial and stromal tissues. These structural and functional centrosome defects have important implications for understanding the mechanisms by which genomic instability and loss of cell polarity develop in solid tumors.
Increased skin tumorigenesis in mice lacking pi class glutathione
S
-transferases
Authors: Henderson Colin J. , Smith Austin G. , Ure Jan , Brown Ken , Bacon E. Jane , Wolf C. Roland
Published: July 2002

The activity of chemical carcinogens is a complex balance between metabolic activation by cytochrome P450 monooxygenases and detoxification by enzymes such as glutathione
S
-transferase (GST). Regulation of these proteins may have profound effects on carcinogenic activity, although it has proved impossible to ascribe the observed effects to the activity of a single protein. GstP appears to play a very important role in carcinogenesis, although the precise nature of its involvement is unclear. We have deleted the murine
GstP
gene cluster and established the effects on skin tumorigenesis induced by the polycyclic aromatic hydrocarbon 7,12-dimethylbenz anthracene and the tumor promoting agent 12-
O
-tetradecanoylphorbol-13-acetate. After 20 weeks, a highly significant increase in the number of papillomas was found in the
GstP1/P2
null mice [
GstP1/P2
(−/−)
mice, 179 papillomas, mean 9.94 per animal vs.
GstP1/P2
(+/+)
mice, 55 papillomas, mean 2.89 per animal, (
P
< 0.001)]. This difference in tumor incidence provides direct evidence that a single gene involved in drug metabolism can have a profound effect on tumorigenicity, and demonstrates that GstP may be an important determinant in cancer susceptibility, particularly in diseases where exposure to polycyclic aromatic hydrocarbons is involved, for instance in cigarette smoke-induced lung cancer.
Phosphorylation of a twitchin-related protein controls catch and calcium sensitivity of force production in invertebrate smooth muscle
Authors: Siegman Marion J. , Funabara Daisuke , Kinoshita Shigeharu , Watabe Shugo , Hartshorne David J. , Butler Thomas M.
Published: July 2002

“Catch” is a condition of prolonged, high-force maintenance at resting intracellular Ca
2+
concentration ([Ca
2+
]) and very low energy usage, occurring in invertebrate smooth muscles, including the anterior byssus retractor muscle (ABRM) of
Mytilus edulis
. Relaxation from catch is rapid on serotonergic nerve stimulation in intact muscles and application of cAMP in permeabilized muscles. This release of catch occurs by protein kinase A-mediated phosphorylation of a high (≈600 kDa) molecular mass protein, the regulator of catch. Here, we identify the catch-regulating protein as a homologue of the mini-titin, twitchin, based on (
i
) a partial cDNA of the purified isolated protein showing 77% amino acid sequence identity to the kinase domain of
Aplysia californica
twitchin; (
ii
) a polyclonal antibody to a synthetic peptide in this sequence reacting with the phosphorylated catch-regulating protein band from permeabilized ABRM; and (
iii
) the similarity of the amino acid composition and molecular weight of the protein to twitchin. In permeabilized ABRM, at all but maximum [Ca
2+
], phosphorylation of twitchin results in a decreased calcium sensitivity of force production (half-maximum at 2.5 vs. 1.3 μM calcium). At a given submaximal force, with equal numbers of force generators, twitchin phosphorylation increased unloaded shortening velocity ≈2-fold. These data suggest that aspects of the catch state exist not only at resting [Ca
2+
], but also at higher submaximal [Ca
2+
]. The mechanism that gives rise to force maintenance in catch probably operates together, to some extent, with that of cycling myosin crossbridges.
Acute cholesterol depletion inhibits clathrin-coated pit budding
Authors: Subtil Agathe , Gaidarov Ibragim , Kobylarz Keith , Lampson Michael A. , Keen James H. , McGraw Timothy E.
Published: July 2002

Many biologically important macromolecules are internalized into cells by clathrin-coated pit endocytosis. The mechanism of clathrin-coated pit budding has been investigated intensively, and considerable progress has been made in characterizing the proteins involved in internalization. Membrane lipid composition and the lateral organization of lipids and proteins within membranes are believed to play an important role in the regulation of membrane-trafficking processes. Here we report that membrane cholesterol plays a critical role in clathrin-coated pit internalization. We show that acute cholesterol depletion, using β-methyl-cyclodextrin, specifically reduces the rate of internalization of transferrin receptor by more than 85%, without affecting intracellular receptor trafficking back to the cell surface. The effect on endocytosis is attributable to a failure of coated pits to detach from the plasma membrane, as visualized by using a green fluorescent protein–clathrin conjugate in living cells. Ultrastructural studies indicate that acute cholesterol depletion causes accumulation of flat-coated membranes and a corresponding decrease in deep-coated pits, consistent with the possibility that flat clathrin lattices are direct precursors of indented pits and endocytic vesicles in intact cells. We conclude that clathrin is unable to induce curvature in the membrane depleted of cholesterol.
p53
gene mutations are not required for early dissemination of cancer cells
Authors: Offner Sonja , Schmaus Werner , Witter Klaus , Baretton Gustavo B. , Schlimok Günter , Passlick Bernward , Riethmüller Gert , Pantel Klaus
Published: July 2002

The p53 protein is involved in several central cellular processes, including gene transcription, DNA repair, cell cycling, genomic stability, chromosomal segregation, senescence, and apoptosis.
p53
mutations frequently result in an immunocytochemically detectable accumulation of the p53 protein in tumor cells. To evaluate whether
p53
gene mutations are required for the onset of hematogeneous tumor cell dissemination, we compared the p53 status of primary and micrometastatic tumor cells. Disseminated carcinoma cells could be detected in bone marrow aspirates obtained from 46 (40%) of 114 patients with various types of epithelial tumors without overt skeleton metastases. There was no correlation between the detection of p53 protein in primary lung carcinomas and the presence of tumor cells in bone marrow. Further analyses revealed that the disseminated carcinoma cells rarely accumulate mutated p53 protein and that 10 cell lines derived thereof did not harbor
p53
mutations even in the presence of such mutations in the autologous primary tumors. These observations indicate that tumor cells can leave the primary tumor before mutations of the
p53
gene occur and that these mutations are not essential for such early hematogeneous dissemination of cancer cells. Thus, the value of mutated p53 as a target for diagnosis and treatment of micrometastatic disease in cancer patients is questionable.
A “midinfrared” scenario for cuprate superconductivity
Authors: Leggett A. J.
Published: July 2002

I conjecture that the mechanism of superconductivity in the
cuprates is a saving, due to the improved screening resulting from
Cooper pair formation, of the part of the Coulomb energy associated
with long wavelengths and midinfrared frequencies. This scenario is
shown to provide a plausible explanation of the trend of transition
temperature with layering structure in the Ca-spaced compounds and to
predict a spectacularly large decrease in the electron-energy-loss
spectroscopy cross-section in the midinfrared region on transition to
the superconducting state, as well as less spectacular but still
surprisingly large changes in the optical behavior. Existing
experimental results appear to be consistent with this picture.
A model for the mechanism of strand passage by DNA gyrase
Authors: Kampranis Sotirios C. , Bates Andrew D. , Maxwell Anthony
Published: July 2002

The mechanism of type II DNA topoisomerases involves the formation of an enzyme-operated gate in one double-stranded DNA segment and the passage of another segment through this gate. DNA gyrase is the only type II topoisomerase able to introduce negative supercoils into DNA, a feature that requires the enzyme to dictate the directionality of strand passage. Although it is known that this is a consequence of the characteristic wrapping of DNA by gyrase, the detailed mechanism by which the transported DNA segment is captured and directed through the DNA gate is largely unknown. We have addressed this mechanism by probing the topology of the bound DNA segment at distinct steps of the catalytic cycle. We propose a model in which gyrase captures a contiguous DNA segment with high probability, irrespective of the superhelical density of the DNA substrate, setting up an equilibrium of the transported segment across the DNA gate. The overall efficiency of strand passage is determined by the position of this equilibrium, which depends on the superhelical density of the DNA substrate. This mechanism is concerted, in that capture of the transported segment by the ATP-operated clamp induces opening of the DNA gate, which in turn stimulates ATP hydrolysis.
An enhancer element located downstream of the major glutamate dehydrogenase gene of
Bacillus subtilis
Authors: Belitsky Boris R. , Sonenshein Abraham L.
Published: July 2002

The
rocG
gene of
Bacillus subtilis
,
encoding a catabolic glutamate dehydrogenase, is transcribed by SigL
(σ
54
)-containing RNA polymerase and requires for its
expression RocR, a member of the NtrC/NifA family of proteins that
bind to enhancer-like elements, called upstream activating sequences
(UAS). Unlike the case for other σ
54
-dependent genes,
rocG
has no UAS; instead, its expression depends on a
sequence located 1.5 kilobases downstream of the
rocG
promoter, beyond the end of the
rocG
coding region. The
same sequence also serves as the UAS for the downstream
rocABC
operon and can activate
rocG
if moved
upstream of its promoter. Furthermore, the activating sequence can be
moved as far as 15 kilobases downstream of the
rocG
promoter and still retain partial activity.
Suppression of Δ
5
-androstenediol-induced androgen receptor transactivation by selective steroids in human prostate cancer cells
Authors: Chang Hong-Chiang , Miyamoto Hiroshi , Marwah Padma , Lardy Henry , Yeh Shuyuan , Huang Ko-En , Chang Chawnshang
Published: July 2002

Our earlier report suggested that androst-5-ene-3β,7β-diol (Δ
5
-androstenediol or Adiol) is a natural hormone with androgenic activity and that two potent antiandrogens, hydroxyflutamide (Eulexin) and bicalutamide (Casodex), fail to block completely the Adiol-induced androgen receptor (AR) transactivation in prostate cancer cells. Here, we report the development of a reporter assay to screen several selective steroids with anti-Adiol activity. Among 22 derivatives/metabolites of dehydroepiandrosterone, we found 4 steroids [no. 4, 1,3,5(10)-estratriene-17α-ethynyl-3,17β-diol; no. 6, 17α-ethynyl-androstene-diol; no. 8, 3β,17β-dihydroxy-androst-5-ene-16-one; and no. 10, 3β-methylcarbonate-androst-5-ene-7,17-dione] that have no androgenic activity and could also block the Adiol-induced AR transactivation in prostate cancer PC-3 cells. Interestingly, these compounds, in combination with hydroxyflutamide, further suppressed the Adiol-induced AR transactivation. Reporter assays further showed that these four anti-Adiol steroids have relatively lower glucocorticoid, progesterone, and estrogenic activity. Together, these data suggest some selective steroids might have anti-Adiol activity, which may have potential clinical application in the battle against the androgen-dependent prostate cancer growth.
Quinolone signaling in the cell-to-cell communication system of
Pseudomonas
aeruginosa
Authors: Pesci Everett C. , Milbank Jared B. J. , Pearson James P. , McKnight Susan , Kende Andrew S. , Greenberg E. Peter , Iglewski Barbara H.
Published: July 2002

Numerous species of bacteria use an elegant regulatory mechanism known as quorum sensing to control the expression of specific genes in a cell-density dependent manner. In Gram-negative bacteria, quorum sensing systems function through a cell-to-cell signal molecule (autoinducer) that consists of a homoserine lactone with a fatty acid side chain. Such is the case in the opportunistic human pathogen
Pseudomonas aeruginosa
, which contains two quorum sensing systems (
las
and
rhl
) that operate via the autoinducers,
N
-(3-oxododecanoyl)-
l
-homoserine lactone and
N
-butyryl-
l
-homoserine lactone. The study of these signal molecules has shown that they bind to and activate transcriptional activator proteins that specifically induce numerous
P. aeruginosa
virulence genes. We report here that
P. aeruginosa
produces another signal molecule, 2-heptyl-3-hydroxy-4-quinolone, which has been designated as the
Pseudomonas
quinolone signal. It was found that this unique cell-to-cell signal controlled the expression of
lasB
, which encodes for the major virulence factor, LasB elastase. We also show that the synthesis and bioactivity of
Pseudomonas
quinolone signal were mediated by the
P. aeruginosa las
and
rhl
quorum sensing systems, respectively. The demonstration that 2-heptyl-3-hydroxy-4-quinolone can function as an intercellular signal sheds light on the role of secondary metabolites and shows that
P. aeruginosa
cell-to-cell signaling is not restricted to acyl-homoserine lactones.
The unseen color aftereffect of an unseen stimulus: Insight from blindsight into mechanisms of color afterimages
Authors: Barbur John L. , Weiskrantz Lawrence , Harlow J. Alister
Published: July 2002

We show here that, in the absence of a direct geniculostriate input in human subjects, causing loss of sight in the visual half-field contralateral to the damage, the pupil responds selectively to chromatic modulation toward the long-wavelength (red) region of the spectrum locus even when the stimulus is isoluminant for both rods and cones and entirely restricted to the subjects’ “blind” hemifields. We also show that other colors are less or wholly ineffective. Nevertheless, red afterimages, generated by chromatic modulation toward the green region of the spectrum locus, also cause constrictions of the pupil even when green stimuli are themselves completely ineffective in the blind hemifield. Moreover, human subjects with damage to or loss of V1 are typically completely unaware of the stimulus that generates the aftereffect or of the aftereffect itself, both of which can be seen clearly in normal vision. The results show that pupillary responses can reveal the processing of color afterimages in the absence of primary visual cortex and in the absence of acknowledged awareness. This phenomenon is therefore a striking example of “blindsight” and makes possible the formulation of a model that predicts well the observed properties of color afterimages.
Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from
Parp
-disrupted mouse embryonic stem cells
Authors: Nozaki Tadashige , Masutani Mitsuko , Watanabe Masatoshi , Ochiya Takahiro , Hasegawa Fumio , Nakagama Hitoshi , Suzuki Hiroshi , Sugimura Takashi
Published: July 2002

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (
Parp
+/+
) ES cells and
Parp
+/−
and
Parp
−/−
ES clones generated by disrupting
Parp
exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from
Parp
−/−
clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and
Parp
+/−
clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from
Parp
−/−
clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.
Miriamin, a defensive diterpene from the eggs of a land slug (
Arion
sp.)
Authors: Schroeder Frank C. , Gonzàlez Andrés , Eisner Thomas , Meinwald Jerrold
Published: July 2002

The eggs of the land slug
Arion
sp. contain a diterpene, miriamin, characterized as a polyoxygenated geranylgeraniol derivative. In bioassays with a coccinellid beetle,
Harmonia axyridis
, miriamin was shown to be potently antifeedant, indicating that the compound plays a protective role in nature. It is suggested that mucilaginous soil-inhabiting organisms, given their intense exposure to pathogens and predators, may be a rich source of chemical defensive agents.
Structural distribution of stability in a thermophilic enzyme
Authors: Hollien Julie , Marqusee Susan
Published: July 2002

Stability parameters for individual residues in
Thermus thermophilus
cysteine-free RNase H were determined by native state hydrogen exchange, thus providing a unique comparison of regional thermodynamics between thermophilic and mesophilic homologues. The general distribution of stability in the thermophilic protein is similar to that of its mesophilic homologue, with a proportional increase in stability for almost all residues. As a consequence, the residue-specific stabilities of the two proteins are remarkably similar under conditions where their global stabilities are the same. These results indicate that
T. thermophilus
RNase H is stabilized in a delocalized fashion, preserving a finely tuned balance of stabilizing interactions throughout the structure. Therefore, although protein stability can be altered by single amino acid substitution, evolution for optimal function may require more subtle and delocalized mechanisms.
Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism
Authors: Xie Lai-Hua , Horie Minoru , Takano Makoto
Published: July 2002

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M
1
muscarinic receptors, we activated the ATP-sensitive potassium (K
ATP
) channel with a K
+
channel opener and recorded the whole-cell K
ATP
current. The K
ATP
current was reversibly inhibited by the stimulation of the M
1
receptor, which is linked to phospholipase C (PLC) by the G
q
protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca
2+
with 10 mM 1,2-bis(2-aminophenoxy)ethane-
N
,
N
,
N
′,
N
′-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M
1
-receptor stimulation failed to inhibit the K
ATP
current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP
2
) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP
2
was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5′-[β,γ–imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the K
ATP
channel was significantly higher when the M
1
receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the K
ATP
channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP
2
levels.
Chromosomal instability and cytoskeletal defects in oral cancer cells
Authors: Saunders William S. , Shuster Michele , Huang Xin , Gharaibeh Burhan , Enyenihi Akon H. , Petersen Iver , Gollin Susanne M.
Published: July 2002

Oral squamous cell carcinomas are characterized by complex, often near-triploid karyotypes with structural and numerical variations superimposed on the initial clonal chromosomal alterations. We used immunohistochemistry combined with classical cytogenetic analysis and spectral karyotyping to investigate the chromosomal segregation defects in cultured oral squamous cell carcinoma cells. During division, these cells frequently exhibit lagging chromosomes at both metaphase and anaphase, suggesting defects in the mitotic apparatus or kinetochore. Dicentric anaphase chromatin bridges and structurally altered chromosomes with consistent long arms and variable short arms, as well as the presence of gene amplification, suggested the occurrence of breakage–fusion–bridge cycles. Some anaphase bridges were observed to persist into telophase, resulting in chromosomal exclusion from the reforming nucleus and micronucleus formation. Multipolar spindles were found to various degrees in the oral squamous cell carcinoma lines. In the multipolar spindles, the poles demonstrated different levels of chromosomal capture and alignment, indicating functional differences between the poles. Some spindle poles showed premature splitting of centrosomal material, a precursor to full separation of the microtubule organizing centers. These results indicate that some of the chromosomal instability observed within these cancer cells might be the result of cytoskeletal defects and breakage–fusion–bridge cycles.