bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of interleukin 1-inducible genes and production of interleukin 1 by aging human fibroblasts.
Authors: Kumar S , Millis A J , Baglioni C
Published: May 2006

The interleukin 1 (IL-1)-inducible mRNAs for plasminogen activator inhibitor type 2, manganese superoxide dismutase, and urokinase are overexpressed in old (greater than 70% of life-span completed) but not in young (less than 40% of life-span completed) human foreskin fibroblasts. Furthermore, the activity of this superoxide dismutase is greater in old than in young fibroblasts. IL-1 beta mRNA is detected by Northern blot analysis in old fibroblasts and its expression is further enhanced by a treatment with IL-1 alpha. IL-1 alpha and IL-1 beta mRNAs are detected in old foreskin and lung fibroblasts by a sensitive reverse transcription-PCR assay. IL-1 mRNA is consistently expressed after fibroblasts have completed 85% of their in vitro life-span; an assay with specific antibodies shows that IL-1 alpha is present in these fibroblasts. Prolonged treatment with IL-1 receptor antagonist decreases the levels of IL-1 alpha and of IL-1 alpha and IL-1 beta mRNAs. This observation suggests that IL-1 receptor antagonist inhibits an autocrine loop responsible for IL-1 expression. IL-1 mRNA accumulates in young fibroblasts treated with cycloheximide, suggesting that it is transcribed but unstable in these cells; accumulation of IL-1 mRNA in old fibroblasts may be due at least in part to increased stability. IL-1 alpha stimulates DNA synthesis in young fibroblasts but has progressively less effect as the cells age in culture. These data indicate that IL-1 is "constitutively" produced by aging fibroblasts and that IL-1 induces the expression of specific proteins in these cells. The mechanism for this constitutive production of IL-1 is explored in this paper.
Observing the 1H NMR signal of the myoglobin Val-E11 in myocardium: an index of cellular oxygenation.
Authors: Kreutzer U , Wang D S , Jue T
Published: May 2006

The 1H NMR signal from oxymyoglobin, a low-concentration diamagnetic protein, is visible in myocardial tissue. The methyl group of the Val-E11 resonates in a clear spectral region at -2.76 ppm and responds to dynamic changes in cellular oxygenation. With CO, the signal shifts to -2.4 ppm. The Val-E11 peak assignment and its response to oxygen and CO agree perfectly with previous myoglobin solution studies. Intracellular oxygen level can now be determined in vivo with the signal intensity ratio of oxymyoglobin/deoxymyoglobin, reflected by the Val-E11 and His-F8 peaks in the 1H NMR spectra. Moreover, protein structure-function relationship in vivo can now be probed.
Heat capacity changes and hydrophobic interactions in the binding of FK506 and rapamycin to the FK506 binding protein.
Authors: Connelly P R , Thomson J A
Published: May 2006

Differential interactions among nonpolar moieties at protein/ligand interfaces, and of these nonpolar groups with water, collectively termed hydrophobic interactions, are widely believed to make important energetic contributions to the stability of protein/ligand complexes. Quantitative estimates of hydrophobic interactions, and an evaluation of their structural basis, are essential for obtaining structure-based predictions of the free energies of binding for the purpose of drug design. Two largely nonpolar, immunosuppressive agents, FK506 and rapamycin, each bind with high affinity to a common hydrophobic pocket on a small peptidylproline cis-trans isomerase known as FK506 binding protein (FKBP-12) and inhibit its activity. In an effort to elucidate the structural features of these ligands responsible for the observed energetics, we have undertaken an investigation of the thermodynamics of binding of FK506 and rapamycin to FKBP-12. Enthalpies of binding have been determined by high-precision titration calorimetry over a range of temperature, allowing estimates of heat capacity changes. By analyzing the distribution of changes in solvent-accessible surface area upon binding of FK506 to FKBP-12 from crystallographic data, it is found that 99% of the net surface buried upon binding involves nonpolar groups. This leads to a heat capacity change of FK506 binding, normalized to the amount of nonpolar surface, of -0.40 +/- 0.02 cal.K-1.mol-1.A-2 (1 cal = 4.18 J), a value similar to that obtained for the aqueous dissolution of hydrophobic substances. Our observations are discussed in view of the general nature of hydrophobic interaction processes.
cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase.
Authors: O'Banion M K , Winn V D , Young D A
Published: May 2006

The antiinflammatory glucocorticoids are potent inhibitors of cyclooxygenase, a key regulator of prostaglandin synthesis; yet, the mechanism(s) by which this occurs is not fully understood. We have cloned a 4.1-kilobase (kb) cDNA, distinct from the previously cloned cyclooxygenase (2.8 kb), that confers cyclooxygenase activity to transfected cells. The mRNA for this newly discovered cyclooxygenase is unique for its long 3' untranslated region containing many AUUUA repeats. Levels of the 4.1-kb cyclooxygenase mRNA are rapidly increased by serum or interleukin 1 beta in mouse fibroblasts and human monocytes, respectively, and decreased by glucocorticoids, whereas levels of the 2.8-kb cyclooxygenase mRNA do not change. Similar effects are seen in the presence of cycloheximide where the 4.1-kb, but not the 2.8-kb, mRNA is greatly superinduced. Thus, there are both constitutive (2.8 kb) and regulated (4.1 kb) cyclooxygenase species, the latter most likely being a major mediator of inflammation.
Wind-induced plant motion immediately increases cytosolic calcium.
Authors: Knight M R , Smith S M , Trewavas A J
Published: May 2006

Wind is one of the most unusual and more dramatic of the environmental signals to modify plant development. Wind-stimulated crops are also known to experience considerable reductions in growth and subsequent yield. There is at present no experimental data to suggest how wind signals are perceived and transduced by plant cells. We have genetically transformed Nicotiana plumbaginifolia to express aequorin and thus produced luminous plants that directly report cytosolic calcium by emitting blue light. With these plants we have found wind stimulation to cause immediate increases in cytosolic calcium and our evidence, based on the use of specific inhibitors, suggests that this calcium is mobilized from organelle sources. Our data further suggest that wind-induced movement of tissues, by mechanically stimulating and stressing constituent plant cells, is responsible for the immediate elevation of cytosolic calcium; increases occur only when the plant tissue is actually in motion. Repeated wind stimulation renders the cells refractory to further calcium signaling but responsiveness is rapidly recovered when stimulation is subsequently diminished. Our data suggest that mechanoperception in plant cells may possibly be transduced through intracellular calcium. Since mechanoperception and transduction are considered crucial to plant morphogenesis, our observations suggest that calcium could be central in the control and generation of plant form.
Yeast flavohemoglobin is an ancient protein related to globins and a reductase family.
Authors: Zhu H , Riggs A F
Published: May 2006

The hemoglobin of yeast is a two-domain protein with both heme and flavin prosthetic groups. The nucleotide sequences of the cDNA and genomic DNA encoding the protein from Saccharomyces cerevisiae show that introns are absent and that both domains are homologous with a flavoheme protein from Escherichia coli. The heme domains are also homologous with those of O2-binding heme proteins from several other distantly related bacteria, plants, and animals; all appear to be members of the same globin superfamily. Although the homologous hemoglobin of the bacterium Vitreoscilla sp. is a single-domain protein, several bacteria have related O2-binding heme proteins whose second domains have different structures and enzymatic activities: dihydropteridine reductase (E. coli), cytochrome c reductase (Alcaligenes eutrophus), and kinase in the O2 sensor of Rhizobium meliloti. This indicates that one evolutionary pathway of hemoglobin is that of a multipurpose domain attached to a variety of unrelated proteins to form molecules with different functions. The flavin domain of yeast hemoglobin is homologous with members of a flavoprotein family that includes ferredoxin reductase, nitric oxide synthase, and cytochrome P-450 reductase. The correspondence of yeast and E. coli flavohemoglobins indicates that the two-domain protein has been conserved intact for as long as 1.8 billion years, the estimated time of divergence of prokaryotes and eukaryotes provided that cross-species gene transfer has not occurred.
Monaural interaction of excitation and inhibition in the medial superior olive of the mustached bat: an adaptation for biosonar.
Authors: Grothe B , Vater M , Casseday J H , Covey E
Published: May 2006

In most mammals, the superior olive is the first stage for binaural interaction. Neurons in the medial superior olive (MSO) receive excitatory input from both ears and are sensitive to interaural time or phase differences of low-frequency sounds. The mustached bat (Pteronotus parnellii parnellii), a small echolocating species with high-frequency hearing, probably does not use interaural time or phase differences as cues for sound localization. Although the mustached bat has a large MSO, there is some evidence that it is functionally different from the MSO in nonecholocating mammals. Most MSO neurons in the mustached bat are monaural, excited by a contralateral sound. Their responses are phasic and correlated with either the onset or the offset of a sound. As a first step in determining the origin of these phasic monaural responses, we traced the connections of the MSO by using both retrograde and anterograde transport methods. Excitatory inputs to the MSO originate from spherical cells in the anteroventral cochlear nucleus, almost exclusively from the contralateral side. Glycinergic inhibitory input is relayed from the contralateral cochlear nucleus through the medial nucleus of the trapezoid body. To investigate the interactions of the contralateral excitatory and inhibitory inputs at the level of the MSO cell, we recorded sound-evoked responses and applied glycine or its antagonist by using microiontophoresis. The results show that the phasic response to a contralateral sound is created by interaction of a sustained excitatory input with a sustained inhibitory input, also from the contralateral ear. Whether the response is to the onset or offset of a sound is determined by the relative timing between the excitatory and inhibitory inputs. Thus, in MSO of the mustached bat, the ipsilateral excitatory pathway from the cochlear nucleus seen in animals with low-frequency hearing is virtually absent, and the MSO is adapted for timing analysis by using input from only the contralateral ear.
Overlap of the p53-responsive element and cAMP-responsive element in the enhancer of human T-cell leukemia virus type I.
Authors: Aoyama N , Nagase T , Sawazaki T , Mizuguchi G , Nakagoshi H , Fujisawa J I , Yoshida M , Ishii S
Published: May 2006

The wild-type p53 protein suppresses transformation, but certain missense mutants of p53 can transform cells. Although the wild-type p53 protein contains a transcriptional activation domain, no p53-responsive element has been identified. Here, we identified the p53-responsive element within the Tax-responsive element [21-base-pair (bp) enhancer] of human T-cell leukemia virus type I. Mutation analysis of the 21-bp enhancer indicated that the 16-bp sequence containing the cAMP-responsive element and its surrounding sequence was responsible for p53-induced transactivation. This 16-bp sequence was demonstrated to bind specifically to wild-type human p53 protein in vitro. Using a series of deletion mutants of p53, we showed that almost the entire region of p53 is needed for the transactivating capacity. Furthermore, the transforming mutants of p53 were unable to act as transcriptional activators. The p53-responsive element identified here should be useful to analyze the mechanism by which p53 regulates expression of a set of genes with a negative effect on cellular growth.
Cytokinetic failure and asynchronous nuclear division in BHK cells overexpressing a truncated protein-tyrosine-phosphatase.
Authors: Cool D E , Andreassen P R , Tonks N K , Krebs E G , Fischer E H , Margolis R L
Published: May 2006

Previous work has shown that a T-cell protein-tyrosine-phosphatase truncated in its carboxyl-terminal domain (delta C11.PTP) has full enzymatic activity but no longer localizes in the particulate fraction of the cell. Two baby hamster kidney (BHK) cell lines overexpressing the truncated protein are markedly multinucleate, a state likely caused by a failure in cytokinesis. Nuclei within syncytial cells overexpressing delta C11.PTP display a remarkable asynchronous entry into mitosis. The effects require tyrosine phosphatase activity because expression of an inactive form of the truncated enzyme yields cells indistinguishable from the parental cell line. Redistribution of the enzyme from the particulate to the soluble fraction is apparently important to these observed effects because cells overexpressing the full-length, wild-type enzyme are morphologically similar to controls. Further, when these cells contain more than one nucleus, their syncytial nuclei undergo mitosis synchronously.
Molecular cloning of a serotonin receptor from human brain (5HT1E): a fifth 5HT1-like subtype.
Authors: McAllister G , Charlesworth A , Snodin C , Beer M S , Noble A J , Middlemiss D N , Iversen L L , Whiting P
Published: May 2006

Degenerate primers, suitable for polymerase chain reaction studies and based on the conserved structure of G protein-coupled receptors, were used to isolate cDNA clones encoding putative G protein-coupled receptors from a human hippocampal cDNA library. One clone isolated by this approach (AC1) encoded a putative receptor with 39% amino acid sequence identity to the serotonin 5HT1A receptor and 47% identity to the 5HT1D receptor. When expressed transiently in the human embryonic kidney cell line 293, AC1 cDNA-encoded receptor displayed high affinity (Kd = 15 nM) and saturability for [3H]serotonin, suggesting that AC1 encodes a 5HT1-like receptor. However, 5-carboxamidotryptamine demonstrated low affinity (pKi = 5.15) compared with serotonin (pKi = 8.14), consistent with the observed binding of the putative 5HT1E receptor. The excellent correlation observed between the pharmacology of the expressed receptor encoded by AC1 and the human brain 5HT1E binding site confirms that AC1 encodes a 5HT1E receptor and establishes a fifth 5HT1-like receptor subtype.
[Val12] HRAS downregulates GLUT2 in beta cells of transgenic mice without affecting glucose homeostasis.
Authors: Tal M , Wu Y J , Leiser M , Surana M , Lodish H , Fleischer N , Weir G , Efrat S
Published: May 2006

Glucose-induced insulin release from pancreatic beta cells depends on the beta-cell metabolism of glucose, which generates intracellular signals for secretion. The beta-cell glucose transporter isotype GLUT2 and the glucose phosphorylating enzyme glucokinase have both been implicated in coupling insulin secretion to extracellular glucose levels. Here we present evidence that a pronounced decrease in beta-cell GLUT2 has no immediate effect on glucose homeostasis. Analysis of transgenic mice overexpressing human [Val12]HRAS oncoprotein under control of the insulin promoter reveals a great reduction in plasma-membrane GLUT2 levels. These mice are nonetheless able to maintain normal fed and fasting plasma glucose and insulin levels for a period of several months. Insulin secretion studied in isolated islets and the perfused pancreas is characterized by a normal incremental response to increasing glucose concentrations. Glucose metabolism, as measured by glucose phosphorylation and oxidation in isolated islets, shows a normal dose dependence on extracellular glucose concentrations. These findings suggest that normal GLUT2 expression in beta cells is not essential for glucose sensing. The transgenic mice provide an experimental system for studying the role of glucose phosphorylation in regulation of insulin release in the absence of GLUT2.
Regulation of immediate early gene expression and AP-1 binding in the rat nucleus accumbens by chronic cocaine.
Authors: Hope B , Kosofsky B , Hyman S E , Nestler E J
Published: May 2006

Chronic treatment of rats with cocaine leads to long-term biochemical changes in the nucleus accumbens (NAc), a brain region implicated in mediating the reinforcing effects of cocaine and other drugs of abuse. Immediate early genes (IEGs) and their protein products appear to play an important role in transducing extracellular stimuli into altered patterns of cellular gene expression and, therefore, into long-term changes in cellular functioning. We therefore examined changes in the mRNA levels for the IEGs c-fos, c-jun, fosB, junB, and zif268 in the NAc of rats treated acutely and chronically with cocaine. A single cocaine injection increased the mRNA levels of all of the IEGs examined. Following chronic cocaine treatment, however, IEG expression had returned to control levels and was not significantly increased following a further acute challenge with cocaine, suggesting desensitization in the ability of cocaine to induce these IEGs. Similarly, levels of Fos-like immunoreactivity, which are increased in the NAc by acute cocaine, were reduced to control levels in chronic cocaine-treated rats. Fos, Jun, and a number of related proteins activate or repress transcription of genes by binding to DNA response elements called AP-1 sites. As would be expected from the RNA data and immunohistochemistry, acute cocaine administration increased AP-1 binding activity in the NAc, an effect that reverted completely to control levels within 8-12 hr. In contrast, AP-1 binding activity in the NAc of animals treated chronically with cocaine remained elevated at acute levels 18 hr after the last chronic injection, a time at which c-fos and c-jun mRNA levels and Fos-like immunoreactivity had returned to control values. An additional acute cocaine challenge did not further increase AP-1 binding. The data suggest that chronic cocaine treatment leads to a persistent increase in AP-1 binding activity, which may be involved in some of the physiological and behavioral aspects of cocaine addiction.
On the definition of the concepts thinking, consciousness, and conscience.
Authors: Monin A S
Published: May 2006

A complex system (CS) is defined as a set of elements, with connections between them, singled out of the environment, capable of getting information from the environment, capable of making decisions (i.e., of choosing between alternatives), and having purposefulness (i.e., an urge towards preferable states or other goals). Thinking is a process that takes place (or which can take place) in some of the CS and consists of (i) receiving information from the environment (and from itself), (ii) memorizing the information, (iii) the subconscious, and (iv) consciousness. Life is a process that takes place in some CS and consists of functions i and ii, as well as (v) reproduction with passing of hereditary information to progeny, and (vi) oriented energy and matter exchange with the environment sufficient for the maintenance of all life processes. Memory is a complex of processes of placing information in memory banks, keeping it there, and producing it according to prescriptions available in the system or to inquiries arising in it. Consciousness is a process of realization by the thinking CS of some set of algorithms consisting of the comparison of its knowledge, intentions, decisions, and actions with reality--i.e., with accumulated and continuously received internal and external information. Conscience is a realization of an algorithm of good and evil pattern recognition.
Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun.
Authors: Chevray P M , Nathans D
Published: May 2006

To identify proteins that interact with Jun or Fos we have used the protein interaction cloning system developed by S. Fields and O.-K. Song [(1989) Nature (London) 340, 245-246] to clone mammalian cDNAs encoding polypeptides that interact with the dimerization and DNA-binding motif (bZIP; basic domain leucine zipper motif) of Jun. For this purpose, yeast cells lacking GAL4 activity but expressing a GAL4 DNA-binding domain-Jun bZIP fusion protein were transformed with a mouse embryo cDNA plasmid library in which the cDNA was joined to a gene segment encoding the GAL4 transcriptional activation domain. Several transformants exhibiting GAL4 activity were identified and shown to harbor plasmids encoding polypeptides predicted to form coiled-coil structures with Jun and/or Fos. One of these is a bZIP protein of the ATF/CREB protein family--probably the murine homolog of TAXREB67. Two others encode polypeptides with predicted potential to form coiled-coil structures, and seven other isolates encode segments of alpha- or beta-tropomyosin, classical coiled-coil proteins. The tropomyosin polypeptides were found to interact in the yeast assay system with the bZIP region of Jun but not with the bZIP region of Fos. Our results illustrate the range of protein interaction cloning for discovering proteins that bind to a given target polypeptide.
Molecular cloning of a Drosophila diacylglycerol kinase gene that is expressed in the nervous system and muscle.
Authors: Masai I , Hosoya T , Kojima S , Hotta Y
Published: May 2006

We have isolated a Drosophila melanogaster diacylglycerol kinase (DGK, EC 2.7.1.107) homologue by using a porcine DGK cDNA probe and we have characterized its structure and expression. The DGK cDNA has a single open reading frame that encodes 791 amino acids. The Drosophila and porcine DGKs share a similar carboxyl-terminal region, a putative catalytic domain, which is divided into two separate domains in Drosophila. The DGK gene was mapped to the cytogenetic position 43F1, and its DGK mRNA is abundant both in embryo and in adult fly. By in situ hybridization to sections of adult flies, we demonstrated that the mRNA is present predominantly in the nervous system and muscles, including compound eyes, brain cortex, fibrillar muscle, and tubular muscle. In a 10- to 11-hr embryo, the DGK gene is expressed abundantly in a limited number of cells in the procephalic region and in the ventral nerve cord. The pattern of temporal and spatial expression suggests that the DGK protein has an important function in the adult nervous system and muscle and during the development of the embryonic nervous system.
Fragmentation of proteins in the 13- to 29-kDa mass range observed by 252Cf-plasma desorption mass spectrometry.
Authors: Bunk D M , Macfarlane R D
Published: May 2006

Results are presented on the observation of sequence-specific fragmentation of proteins in the 13- to 29-kDa mass range (ribonuclease A, papain, and proteinase K) by 252Cf-plasma desorption mass spectrometry. For these proteins, extensive N-terminal an, cn + 2, and dn fragment ions are observed with mass accuracies approaching 200 ppm for the most prominent fragment ions. The fragmentation pattern of these proteins shows the same fundamental behavior (arginine-directed, charge-remote fragmentation) that is observed in the fragmentation of small peptides, indicating that similar processes are occurring when large proteins and small peptides fragment in plasma desorption mass spectrometry.
Interaction between G-protein beta and gamma subunit types is selective.
Authors: Pronin A N , Gautam N
Published: May 2006

Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association.
Antiproliferative prostaglandins activate heat shock transcription factor.
Authors: Amici C , Sistonen L , Santoro M G , Morimoto R I
Published: May 2006

Treatment of human K562 erythroleukemia cells with the antiproliferative prostaglandin A1 results in the elevated transcription of two heat shock genes, HSP70 and HSP90. Parallel with increased heat shock gene transcription is the activation of heat shock transcription factor. Heat shock transcription factor levels are induced within 60 min after prostaglandin A1 addition to levels similar to that achieved during heat shock. The requirement for protein synthesis for prostaglandin A1 activation of heat shock transcription factor suggests that effects on nascent protein synthesis may be involved in the signaling mechanism. Although it is unclear whether the activation of a heat shock response by prostaglandins is relevant to the biochemical properties of these natural substances, cells pretreated with prostaglandin A1 are protected against a subsequent heat shock, indicative of a thermotolerant state.
Targeted oncogene activation by site-specific recombination in transgenic mice.
Authors: Lakso M , Sauer B , Mosinger B , Lee E J , Manning R W , Yu S H , Mulder K L , Westphal H
Published: May 2006

An efficient and accurate method for controlled in vivo transgene modulation by site-directed recombination is described. Seven transgenic mouse founder lines were produced carrying the murine lens-specific alpha A-crystallin promoter and the simian virus 40 large tumor-antigen gene sequence, separated by a 1.3-kilobase-pair Stop sequence that contains elements preventing expression of the large tumor-antigen gene and Cre recombinase recognition sites. Progeny from two of these lines were mated with transgenic mice expressing the Cre recombinase under control of either the murine alpha A-crystallin promoter or the human cytomegalovirus promoter. All double-transgenic offspring developed lens tumors. Subsequent analysis confirmed that tumor formation resulted from large tumor-antigen activation via site-specific, Cre-mediated deletion of Stop sequences.
Identification of a structural domain that distinguishes the actions of the type 1 and 2 isoforms of transforming growth factor beta on endothelial cells.
Authors: Qian S W , Burmester J K , Merwin J R , Madri J A , Sporn M B , Roberts A B
Published: May 2006

A chimeric transforming growth factor beta (TGF-beta) molecule has been synthesized to map the amino acids responsible for the substantially greater activity of TGF-beta 1 than TGF-beta 2 on growth and migration of endothelial cells. This chimera consists of a dimer of a monomeric unit composed of amino acids 1-39 of TGF-beta 2, 40-82 of TGF-beta 1, and 83-112 of TGF-beta 2. Structural identity of the purified recombinant protein has been confirmed by immunoblotting and NH2-terminal sequencing. The biological potency of the TGF-beta 2-1-2 chimera was equal to that of TGF-beta 1 in inhibition of growth of both fetal bovine heart endothelial cells and rat epididymal fat pad microvascular endothelial cells. Similarly, the TGF-beta 2-1-2 chimera was nearly equivalent to TGF-beta 1 and at least 10-fold more active than TGF-beta 2 in inhibiting migration of bovine aortic endothelial cells. These results identify the sequence between amino acids 40-82 as an important region within TGF-beta that functions to specify a TGF-beta 1- or TGF-beta 2-like activity.
Development of [125I]RB104, a potent inhibitor of neutral endopeptidase 24.11, and its use in detecting nanogram quantities of the enzyme by "inhibitor gel electrophoresis".
Authors: Fournié-Zaluski M C , Soleilhac J M , Turcaud S , Laï-Kuen R , Crine P , Beaumont A , Roques B P
Published: May 2006

Neutral endopeptidase 24.11, also known as the common acute lymphoblastic leukemia antigen, is a zinc metallopeptidase involved in the inactivation of biologically active peptides, such as the enkephalins and atrial natriuretic peptide. The highly potent radiolabeled inhibitor 2-((3-[125I]iodo-4-hydroxy)phenylmethyl)-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)propyl]amino-4-oxobutanoic acid ([125I]RB104; Ki = 30 pM) has been developed for the enzyme. [125I]RB104 is highly specific, its Ki for another widely distributed zinc peptidase, angiotensin-converting enzyme, being 15 microM. In binding studies using rat brain slices, [125I]RB104 was shown to have a high affinity (Kd = 300 +/- 20 pM) and high specific binding at the Kd concentration (90%). With rat brain homogenates the Kd of [125I]RB104 was 26.8 +/- 0.9 pM, close to the kinetically derived Kd, 7.0 +/- 0.8 pM. Using the inhibitor, we have developed a simple, rapid, and quantitative technique to detect low nanogram quantities of the endopeptidase directly from tissue extracts after SDS/PAGE. The method has been used to show the presence of low quantities of the enzyme in rabbit bone marrow. Apart from its sensitivity, "inhibitor gel electrophoresis" using [125I]RB104 has the advantage over immunohistochemical methods of being able to label the enzyme in all tissues and species. It will therefore be of great value in determining the exact role of this important regulatory peptidase in a number of biological systems. Moreover, this one-step characterization of neutral endopeptidase 24.11 could be extended to other zinc metallopeptidases such as angiotensin-converting enzyme or collagenases, and inhibitors with affinities as high as RB104 could open the way to visualization of zinc metallopeptidases in different tissues by electron microscopy.
Induced polypeptides associated with filarial worm refractoriness in Aedes aegypti.
Authors: Wattam A R , Christensen B M
Published: May 2006

Brugia malayi and Wuchereria bancrofti are mosquito-borne parasitic nematodes responsible for lymphatic filariasis in approximately 90 million people. The genetic control of the susceptibility of Aedes aegypti mosquitoes to B. malayi was well defined 30 years ago, but no data have since been provided regarding the gene products responsible for susceptibility or refractoriness or both. We addressed this problem by assessing polypeptide synthesis in thoracic tissue, the developmental site of this parasite, in susceptible and refractory strains of A. aegypti. Polyacrylamide gel electrophoresis of radiolabeled polypeptides synthesized in vivo were compared between (i) established susceptible and refractory strains and (ii) a refractory strain newly isolated from the established susceptible strain. Six polypeptide differences recognized by SDS/PAGE and two-dimensional gel electrophoresis were seen only in the refractory strains after they took a blood meal. A seventh polypeptide was present in those refractory mosquitoes that had ingested sucrose but increased in intensity after blood-feeding. The presence of parasites in the blood meal was not necessary to stimulate the synthesis of these polypeptides. These refractory strain-associated molecules may mediate genetically determined variation in susceptibility.
Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P.
Authors: Barik S , Banerjee A K
Published: May 2006

We have previously shown that phosphorylation of vesicular stomatitis virus (VSV) phosphoprotein P by cellular protein kinase activity is an essential prerequisite for its transcriptional function. We have now purified this protein kinase by monitoring its ability to phosphorylate bacterially expressed, unphosphorylated P protein. Biochemical studies showed that the kinase is indistinguishable from casein kinase II, a ubiquitous cyclic AMP-independent protein kinase present in a wide variety of eukaryotic cells and tissues. Functional VSV transcription could be reconstituted with viral L protein, N-RNA template, and P protein phosphorylated by either purified cellular protein kinase or purified casein kinase II. The unusual role of casein kinase II in the transcription process of a nonsegmented negative-strand RNA virus would have important implications in host-virus interactions and antiviral therapy.
Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.
Authors: Mancini G M , Beerens C E , Galjaard H , Verheijen F W
Published: May 2006

The lysosomal carrier for the acidic monosaccharides sialic acid and glucuronic acid was solubilized from rat liver lysosomal membranes and reconstituted into phospholipid vesicles. Membrane proteins were extracted from lysosomal membranes with Triton X-100. Upon removal of the detergent by absorption on Amberlite XAD-2 beads, the solubilized proteins were incorporated in egg yolk phospholipids. The reconstituted proteoliposomes show proton-driven carrier-mediated uptake of acidic monosaccharides. The reconstituted carrier was compared in several characteristics with the transporter as present in the native lysosomal membrane. Transporter substrate affinity (Kt for glucuronic acid = 0.4 mM) and specificity for acidic monosaccharides are completely retained. The proteoliposomes also demonstrate trans-stimulation properties with both substrates sialic acid and glucuronic acid. The transporter is inhibited, both in its native and in the reconstituted state, by the sulfhydryl-modifying agents p-chloromercuribenzoic acid, N-ethylmaleimide, and phenyl isothiocyanate. In native membrane vesicles, arginine and histidine modifiers phenylglyoxal and diethyl pyrocarbonate inactivated transport in a substrate-protectable manner. In reconstituted proteoliposomes, similar inhibition was observed. However, protection by substrates was achieved only after treatment with phenylglyoxal. These data suggest that arginine or histidine residues or both are present at or near the substrate binding site of the carrier. Possibly, other essential histidines become exposed in the reconstituted state. The successful functional reconstitution of the lysosomal sialic acid carrier represents an important step towards its purification and its detailed molecular characterization.
Crystal structure determination at 2.3-A resolution of human transthyretin-3',5'-dibromo-2',4,4',6-tetrahydroxyaurone complex.
Authors: Ciszak E , Cody V , Luft J R
Published: May 2006

The crystal structure of the complex of 3',5'-dibromo-2',4,4',6-tetrahydroxyaurone, a flavone derivative, with human transthyretin (TTR), a serum thyroid hormone transport protein, has been determined and refined to R = 17.9% for data to 2.3-A resolution and provides a detailed description of a protein-bound flavonoid structure. This bromoaurone is a potent competitor for thyroid hormone binding to TTR, a 54,980-dalton alpha 4 tetrameric protein of 222 molecular symmetry, as well as an inhibitor of iodothyronine deiodinase. Crystals of the TTR-bromoaurone complex are isomorphous to those of native TTR. Interpretation of difference Fourier electron density maps revealed two binding modes for the bromoaurone in each of the two independent binding sites of the TTR tetramer: deep in the channel near Ser-117 (mode I) and near the channel entrance (mode II). None of the binding modes can be fully occupied because of overlap between binding positions. A statistical disorder for bromoaurone binding was also applied, as it binds along the twofold crystallographic axis and does not possess such symmetry. The binding of mode I and that of mode II were refined at half occupancy, resulting in two molecules per tetramer. The bromoaurone binds in a nonplanar antiskewed conformation. The molecular pattern for TTR binding consists of halogen groups able to anchor between beta-sheets to form both hydrophobic and hydrophilic contacts. Comparison of structural data for bromoaurone- and thyroxine-TTR complexes indicates that bromoaurone binding mode I is 3 A deeper in the channel and binding mode II is 4 A further from the channel center than thyroxine. The bromoaurone binding observed in this TTR complex differs significantly from that based upon computer modeling studies.
A hybrid plant RNA virus made by transferring the noncapsid movement protein from a rod-shaped to an icosahedral virus is competent for systemic infection.
Authors: De Jong W , Ahlquist P
Published: May 2006

For many plant RNA viruses, multiple viral gene products, including noncapsid movement proteins and capsid proteins, contribute to the spread of infection within plants. The extent to which these factors interact to support infection spread is not known, but, for movement protein mutants of certain viruses, the inability of coinoculated "helper" viruses to complement defective movement has suggested a possible requirement for coadaptation between noncapsid movement proteins and other virus factors. To test directly for required coadaptation, the 3a movement protein gene of cowpea chlorotic mottle virus, an icosahedral bromovirus, was replaced with the nonhomologous 30-kDa movement protein gene of sunn-hemp mosaic virus, a rod-shaped, cowpea-adapted tobamovirus. The resulting hybrid virus is competent for systemic infection of cowpea, with systemic infection dependent upon expression of the 30-kDa gene. In view of the dramatic differences between cowpea chlorotic mottle virus and sunn-hemp mosaic virus in genetic organization and particle morphology, the ability of the hybrid to systemically infect cowpea implies that the tobamovirus 30-kDa movement protein functions independently of sequence-specific interactions with other viral components or sequences. Similarly, the required contribution of bromovirus capsid protein to infection movement appears to be independent of specific interaction with the natural 3a movement protein. In addition to other implications concerning movement protein and coat protein function, the results are consistent with the possibility that two or more distinguishable transfer processes may be involved in crossing different tissue barriers to achieve full systemic spread of infection.
Carotid-aortic and renal baroreceptors mediate the atrial natriuretic peptide release induced by blood volume expansion.
Authors: Antunes-Rodrigues J , Machado B H , Andrade H A , Mauad H , Ramalho M J , Reis L C , Silva-Netto C R , Favaretto A L , Gutkowska J , McCann S M
Published: May 2006

Our previous studies have shown that stimulation of the anteroventral third ventricle (AV3V) region of the brain increases atrial natriuretic peptide (ANP) release, whereas lesions of the AV3V region or median eminence of the tuber cinereum block the release of ANP caused by blood volume expansion. These results suggest that participation of the central nervous system is critical to this response. The role of baroreceptors in the response was evaluated in the current research by studying the response of plasma ANP to blood volume expansion induced by intravenous injection of hypertonic saline solution (0.3 M NaCl, 2 ml/100 g of body weight, over 1 min) in conscious, freely moving male rats. Plasma samples were assayed for ANP by radioimmunoassay. In sham-operated rats, blood volume expansion induced a rapid increase in plasma ANP: the concentration peaked at 5 min and remained elevated at 15 min after saline injection. One week after deafferentation of the carotid-aortic baroreceptors, basal plasma ANP concentrations were highly significantly decreased on comparison with values of sham-operated rats; plasma ANP levels 5 min after blood volume expansion in the deafferented rats were greatly reduced. Unilateral right vagotomy reduced resting levels of plasma ANP but not the response to blood volume expansion; resting concentrations of plasma ANP and responses to expansion were normal in bilaterally vagotomized rats. In rats that had undergone renal deafferentation, resting levels of ANP were normal but the response to blood volume expansion was significantly suppressed. The evidence indicates that afferent impulses via the right vagus nerve may be important under basal conditions, but they are not required for the ANP release induced by blood volume expansion. In contrast, baroreceptor impulses from the carotid-aortic sinus regions and the kidney are important pathways involved in the neuroendocrine control of ANP release. The evidence from these experiments and our previous stimulation and lesion studies indicates that the ANP release in response to volume expansion is mediated by afferent baroreceptor input to the AV3V region, which mediates the increased ANP release via activation of the hypothalamic ANP neuronal system.
The two-step conversion of big endothelin 1 to endothelin 1 and degradation of endothelin 1 by subcellular fractions from human polymorphonuclear leukocytes.
Authors: Kaw S , Hecker M , Vane J R
Published: May 2006

The metabolism of big endothelin 1 (bET) and endothelin 1 (ET-1) by subcellular fractions from human polymorphonuclear leukocytes (PMNs) was investigated by bioassay and reversed-phase high-performance liquid chromatography. More than 80% of endothelin-converting activity was recovered from the cytosolic fraction, which in addition to ET-1 generated other peptides from bET. The processing of bET to all its metabolites including ET-1 was prevented by the serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the elastase inhibitor ONO-5046 (100 microM) but not by phenylmethylsulfonyl fluoride (PMSF; 143 microM), another serine protease inhibitor. Paradoxically, human leukocyte elastase, despite generating a bET fragmentation pattern similar to that of PMN cytosol, produced very little ET-1. However, subsequent treatment of the elastase-derived metabolites of bET with PMN cytosol in the presence of ONO-5046 dramatically increased the amount of ET-1 formed. The generation of ET-1 following this intervention was inhibited by DCI. The PMN membrane preparation degraded ET-1 to a major metabolite, similar to that produced from ET-1 by elastase, and several minor products, paralleled by a loss of its smooth muscle contracting activity. The degradation of ET-1 by PMN microsomes was prevented by DCI, PMSF, or ONO-5046. Our results suggest that an elastase-initiated serine protease cascade is responsible for the sequential conversion of bET to ET-1 by the PMN cytosol. Elastase also partly accounts for the ET-metabolizing properties of PMN microsomes.
Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.
Authors: McKnight R A , Shamay A , Sankaran L , Wall R J , Hennighausen L
Published: May 2006

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. We have tested whether MAR sequences from the chicken lysozyme locus, the so-called A-elements, can confer position-independent regulation to a whey acidic protein (WAP) transgene in mammary tissue of mice. In the absence of MARs, expression of WAP transgenes was observed in 50% of the lines, and regulation during pregnancy, during lactation, and upon hormonal induction did not mimic that of the endogenous WAP gene and varied with the integration site. In contrast, all 11 lines in which WAP transgenes were juxtaposed to MAR elements showed expression. Accurate position-independent hormonal and developmental regulation was seen in four out of the five lines analyzed. These results indicate that MARs can establish independent genetic domains in transgenic mice.
At least five related, but distinct, hepatitis C viral genotypes exist.
Authors: Cha T A , Beall E , Irvine B , Kolberg J , Chien D , Kuo G , Urdea M S
Published: May 2006

Hepatitis C virus, the major causative agent of blood-borne non-A, non-B hepatitis in the world, has been the subject of considerable nucleic acid sequence analysis. Although all reported hepatitis C sequences from the United States have been represented by the prototype hepatitis C virus type 1 sequence, two groups of variant sequences have been reported in Japan. However, we have noted five distinct, but related, genotypes (I-V) throughout the world, based on detailed sequence determination and analysis of the first 1700 nucleotides and part of the nonstructural region 5 at the C terminus of the open reading frame. The nucleotide sequence for a large number of hepatitis C virus isolates spanning six continents was obtained by direct sequence analysis of PCR products after reverse transcription. Genotype was classified by using several distinct sequence motifs. We observed that most genotypes coexist in several geographic regions, including the United States, Japan, Germany, and Italy. So far, genotype V has been found only in South Africa. Interestingly, each distinct genotype seems to be maintained throughout the genome in the segments studied. These genotype distinctions should be considered when designing specific diagnostic tests, developing potential vaccines, and studying viral transmission.
Carboxyl-terminal deletion and point mutations decrease the transforming potential of the activated rat neu oncogene product.
Authors: Mikami Y , Davis J G , Dobashi K , Dougall W C , Myers J N , Brown V I , Greene M I
Published: May 2006

The rat neu oncogene encodes a constitutively activated growth factor receptor/transmembrane tyrosine kinase, p185Tneu, that is structurally similar to yet distinct from the epidermal growth factor receptor. To explore the role of the carboxyl-terminal region and of putative autophosphorylation sites in regulating the activity of the rat p185Tneu (T, transforming) protein, we used site-directed mutagenesis to generate a p185Tneu mutant in which a putative tyrosine autophosphorylation site (residue 1253) at the extreme carboxyl terminus was replaced by a phenylalanine residue and a mutant in which the carboxyl-terminal 122 amino acids were deleted. These proteins were expressed in NIH 3T3 cells at comparable levels and exhibited similar autophosphorylation activity, exogenous substrate phosphorylation ability, oligomerization levels, and responsiveness to a partially purified neu-activating factor. However, the mutant p185Tneu proteins displayed a decreased transforming capacity both in vitro and in vivo. This analysis demonstrated that the carboxyl-terminal domain and at least one putative tyrosine autophosphorylation site of p185Tneu play a role in positively regulating the cell growth-regulating properties of the neu protein.