bluuebunny/crossref_metadata_2025 huggingface dataset:
Neural control of the endocrine rat heart.
Authors: Jiao J H , Baertschi A J
Published: May 2006

Although atrial stretch is the accepted stimulus for atrial natriuretic factor (ANF), in vivo studies suggest a stretch-independent, neurally induced ANF release mechanism. Thus the hypothesis that cardiac nerves can stimulate ANF secretion was tested in the Langendorff-perfused, paced rat heart. Venom from the scorpion Centruroides sculpturatus was used to activate neuronal sodium channels, veratridine was added to activate sodium channels (predominantly in myocytes), and electrical stimulation was applied to the right atrial appendage. The efficacy of nerve stimulation was verified by measurements of increased neuropeptide Y in the effluent. ANF levels in the effluent increased by 120% over baseline with 0.5 microM scorpion venom and by 88% with 0.5 microM veratridine (P < 0.01). Cardiac mechanics did not explain the large, concentration-dependent ANF response to the scorpion venom, since changes in the left ventricular developed pressure were small, opposite to those induced by veratridine, and unaffected by sympathectomy or adrenergic receptor blockade. Prior chemical sympathectomy and adrenergic receptor blockade almost abolished the ANF response to scorpion venom but hardly affected the ANF response to veratridine. Addition of 1 microM tetrodotoxin abolished all ANF responses. Electrical stimulation of the atrial appendage increased the ANF secretion by 60.2% (P < 0.02), in conjunction with neuropeptide Y, whereas control stimulations were ineffective. These studies unequivocally demonstrate that stimulation of cardiac sympathetic nerves potently stimulates ANF secretion.
Profound decrement of mesolimbic dopaminergic neuronal activity during ethanol withdrawal syndrome in rats: electrophysiological and biochemical evidence.
Authors: Diana M , Pistis M , Carboni S , Gessa G L , Rossetti Z L
Published: May 2006

Activity of the mesolimbic dopaminergic system was investigated in rats withdrawn from chronic ethanol administration by single-cell extracellular recordings from dopaminergic neurons of the ventrotegmental area, coupled with antidromic identification from the nucleus accumbens, and by microdialysis-technique experiments in the nucleus accumbens. Spontaneous firing rates, spikes per burst, and absolute burst firing but not the number of spontaneously active neurons were found drastically reduced; whereas absolute and relative refractory periods increased in rats withdrawn from chronic ethanol treatment as compared with chronic saline-treated controls. Consistently, dopamine outflow in the nucleus accumbens and its acid metabolites were reduced after abruptly stopping chronic ethanol administration. All these changes, as well as ethanol-withdrawal behavioral signs, were reversed by ethanol administration. This reversal suggests that the abrupt cessation of chronic ethanol administration plays a causal role in the reduction of mesolimbic dopaminergic activity seen in the ethanol-withdrawal syndrome. Results indicate that during the ethanol-withdrawal syndrome the mesolimbic dopaminergic system is tonically reduced in activity, as indexed by electrophysiological and biochemical criteria. Considering the role of the mesolimbic dopaminergic system in the reinforcing properties of ethanol, the depressed activity of this system during the ethanol-withdrawal syndrome may be relevant to the dysphoric state associated with ethanol withdrawal in humans.
Reaction sequence and molecular mass of a Cl(-)-translocating P-type ATPase.
Authors: Gerencser G A , Zelezna B
Published: May 2006

The basolateral membranes of Aplysia californica foregut absorptive cells contain both Cl(-)-stimulated ATPase and ATP-dependent Cl- transport activities, and each was inhibited by orthovanadate. Both of these orthovanadate-sensitive activities were reconstituted into proteoliposomes. The reaction sequence kinetics were determined by [gamma-32P]ATP-induced phosphorylation of the reconstituted Cl- pump. Rapid phosphorylation and dephosphorylation kinetics of acyl phosphate bonding were confirmed by destabilization of the phosphoprotein by either hydroxylamine or high pH. Mg2+ caused phosphorylation of the enzyme; Cl- caused dephosphorylation. Orthovanadate almost completely inhibited the Mg(2+)-driven phosphorylation reaction. The molecular mass of the catalytic unit (subunit) of the enzyme appeared to be 110 kDa, which is in agreement with molecular masses of all other catalytic units (subunits) of P-type ATPases.
Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.
Authors: Miller A M , Savinelli E A , Couture S M , Hannigan G M , Han Z , Selden R F , Treco D A
Published: May 2006

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases.
Evidence that the 60-kDa protein of 17S U2 small nuclear ribonucleoprotein is immunologically and functionally related to the yeast PRP9 splicing factor and is required for the efficient formation of prespliceosomes.
Authors: Behrens S E , Galisson F , Legrain P , Lührmann R
Published: May 2006

Small nuclear ribonucleoprotein (snRNP) U2 functions in the splicing of mRNA by recognizing the branch site of unspliced mRNA. The binding of U2 snRNP and other components to pre-mRNA leads to the formation of a stable prespliceosome. In HeLa nuclear extracts, U2 snRNP exists either as a 17S form (under low salt conditions) or a 12S form (at higher salt concentrations). We have recently shown that the purified 17S U2 snRNP contains nine proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa in addition to the common snRNP proteins and the U2 proteins A' and B" that are found in the 12S U2 snRNP form. By using antibodies against the PRP9 protein from Saccharomyces cerevisiae (a protein required for the addition of U2 to prespliceosomes in yeast), we have shown that the 60-kDa protein specific to human U2 snRNP particles is structurally related to the yeast PRP9 protein. Interestingly, anti-PRP9 antibodies strongly inhibit prespliceosome formation in HeLa nuclear splicing extracts, resulting in a complete inhibition of the mRNA splicing reaction in vitro. This indicates that the U2 60-kDa protein may also be functionally related to its yeast counterpart PRP9. Most importantly, the addition of purified 17S U2 snRNPs, but not of 12S U2 snRNPs, to HeLa splicing extracts in which the endogeneous U2 snRNPs have been functionally neutralized with anti-PRP9 antibodies fully restores the mRNA-splicing activity of the extracts. These data suggest further that the 17S form is the functionally active form of U2 snRNP in the spliceosome.
Design of peptide enzymes (pepzymes): surface-simulation synthetic peptides that mimic the chymotrypsin and trypsin active sites exhibit the activity and specificity of the respective enzyme.
Authors: Atassi M Z , Manshouri T
Published: May 2006

Two 29-residue peptides were prepared, one of which (ChPepz) was designed by surface-simulation synthesis to mimic the active site of alpha-chymotrypsin, and the other (TrPepz), which contained four substitutions relative to ChPepz, was fashioned after the active site of trypsin. Each peptide was cyclized by a disulfide bond. The ChPepz monomer effected hydrolysis of the ester group in N-benzoyl-L-tyrosine ethyl ester, an alpha-chymotrypsin substrate, with Km and kcat values that were comparable to those of alpha-chymotrypsin. ChPepz was completely inactivated by diisopropyl fluorophosphate (DIFP), L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK), or reduction of the disulfide bond. It had no catalytic activity on N-tosyl-L-arginine methyl ester, a trypsin substrate. On the other hand, TrPepz, which had no effect on N-benzoyl-L-tyrosine ethyl ester, hydrolyzed N-tosyl-L-arginine methyl ester with a Km value that was essentially identical to that of trypsin, but its kcat value was almost half that of trypsin. TrPepz was fully inactivated by reduction of the disulfide bond, by DIFP, or by phenylmethylsulfonyl fluoride but not by TPCK. It was also completely inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, and human alpha 1-antitrypsin. ChPepz and TrPepz hydrolyzed proteins (myoglobin and casein) to give panels of peptides that were similar to those of the same protein obtained with the respective enzyme. However, TrPepz was more efficient than trypsin at hydrolyzing the C bonds of two or more consecutive lysine and/or arginine residues. Like its esterase activity, the proteolytic activity of ChPepz was inhibited by either DIFP or TPCK whereas that of TrPepz was inhibited by either DIFP or phenylmethylsulfonyl fluoride but not by TPCK. Finally, ChPepz and TrPepz were each more active at low temperature than the respective enzyme. This ability to construct fully functional peptide enzymes (pepzymes) of chosen specificities should find many practical applications.
The role of active-site aromatic and polar residues in catalysis and substrate discrimination by xylose isomerase.
Authors: Meng M , Bagdasarian M , Zeikus J G
Published: May 2006

The functions of individual amino acid residues in the active site of Thermoanaerobacterium thermosulfurigenes D-xylose ketol-isomerase (EC 5.3.1.5) were studied by site-directed substitution. The role of aromatic residues in the active-site pocket was not limited to the creation of a hydrophobic environment. For example, Trp-188 provided for substrate binding and Trp-139 allowed for the discrimination between D-xylose and D-glucose. Substrate discrimination was accomplished by steric hindrance caused by the side chain of Trp-139 toward the larger glucose molecule. Preference of the enzyme for the alpha-anomer of glucose depended on the His-101/Asp-104 pair. Wide differences observed in the catalytic constant (kcat) for alpha- versus beta-glucose in the wild-type enzyme and the fact that only the kcat for alpha-glucose was changed in the His-101-->Asn mutants strongly suggest that the substrate molecule entering the hydride-shift step is still in the cyclic form. On the basis of these results a revised hypothesis for the catalytic mechanism of D-xylose isomerase has been proposed that involves His-101, Asp-104, and Asp-339 functioning as a catalytic triad.
Genomic organization of the melanoma-associated glycoprotein MUC18: implications for the evolution of the immunoglobulin domains.
Authors: Sers C , Kirsch K , Rothbächer U , Riethmüller G , Johnson J P
Published: May 2006

The cell surface glycoprotein MUC18, a member of the immunoglobulin superfamily and homologous to several cell adhesion molecules, is associated with tumor progression and the development of metastasis in human malignant melanoma. Immunohistochemical and Northern blot analysis revealed that expression of the antigen is restricted to advanced primary and metastatic melanomas and to cell lines of the neuroectodermal lineage. The genomic sequence encoding the cell surface antigen spans approximately 14 kb and consists of 16 exons. The organization of the gene, which is related to that of the neural cell adhesion molecule N-CAM, shows a structure where each immunoglobulin-related domain is encoded by more than one exon. Sequencing of the putative MUC18 promoter region revealed a G + C-rich promoter lacking conventional TATA and CAAT boxes. Several motifs for binding of transcription factor Sp1 are present in the regulatory region, and only a single transcription start site within a presumed initiator sequence was identified. Sequence elements which might confer melanocyte-specific expression were not detected. Instead, recognition sequences for the transcription factors CREB, AP-2, and c-Myb, as well as CArG-box motifs, were observed. These elements may contribute to the differential regulation of the MUC18 gene in normal and malignant tissues and suggest a role for this putative adhesion molecule in neural crest cells during embryonic development.
Cysteine-102 is positioned in the metal binding activation site of the Corynebacterium diphtheriae regulatory element DtxR.
Authors: Tao X , Murphy J R
Published: May 2006

DNA sequence analysis of dtxR has shown that the M(r) 25,316 regulatory protein contains a single cysteine residue at position 102. DtxR readily forms inactive disulfide-linked dimers. We have used saturation site-directed mutagenesis of the cysteine codon (TGC) at position 102 in order to determine the role of this residue in metal ion binding. We show that the insertion of amino acids other than cysteine or aspartic acid into this position abolishes DtxR function both in vitro and in recombinant Escherichia coli DH5 alpha:lambda RS45toxPO/lacZ. Only those mutant alleles in which the TGC codon for Cys-102 was replaced by either TGT (Cys) or GCA (Asp) were found to direct the expression of active forms of DtxR that regulate the expression of beta-galactosidase from the toxPO/lacZ transcriptional fusion.
Tissue culture-induced DNA methylation variation in maize.
Authors: Kaeppler S M , Phillips R L
Published: May 2006

Twenty-one progeny lines derived from tissue cultures of two embryo sources of maize inbred strain A188 were examined for DNA methylation changes. Total DNA was cut with the isoschizomers Hpa II and Msp I and probed with 18 single-copy Pst I genomic clones and two cDNA clones. Eight of these probes could detect both increases and decreases in methylation. With these probes 39% of the families were found to contain an altered methylation pattern. All changes represented a decrease in methylation. The other 12 probes could detect only increases in methylation; no methylation variation was seen with these probes. Fifteen percent of the methylation changes were homozygous in the original regenerated plant. Changes were stably inherited upon two generations of self-pollination. No sequence variation was observed in Msp I-digested DNA from the same 21 progeny lines. Certain probes detected methylation changes much more often than others. Our study provides evidence that demethylation occurs at a high frequency and could be an important cause of tissue culture-induced variation. Occurrence of the frequent homozygous alterations in original regenerated plants implies a non-random mutational mechanism.
v-jun cooperates with v-erbB to transform the thrombocytic/megakaryocytic lineage.
Authors: Garcia M , Samarut J
Published: May 2006

The transforming properties of v-jun, the viral counterpart of the transcription factor AP1, were investigated in avian hematopoietic cells. Two retroviruses, called JB and JBN, expressing both v-jun and v-erbB, were constructed using an avian erythroblastosis-based vector. We show that the cooperative action of both oncogenes allowed the virus to efficiently transform bone marrow cells. No such transformation was obtained with either oncogene alone. JB-transformed bone marrow cells expressed GATA-1, TAL-1, and histone H5, suggesting that they belong to the erythrocytic/thrombocytic lineage. (Thrombocytes are the avian homologues of mammal megakaryocytes.) Moreover, after induction with phorbol 12-myristate 13-acetate JB-transformed bone marrow cells began to differentiate and synthesized high levels of platelet glycoproteins, indicating that they were of thrombocytic origin. These results were confirmed by c-ets1 analysis since this transcription factor, specifically found in cells with megakaryocytic but not erythrocytic features, was clearly detected in these cells.
Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system.
Authors: Roselli-Rehfuss L , Mountjoy K G , Robbins L S , Mortrud M T , Low M J , Tatro J B , Entwistle M L , Simerly R B , Cone R D
Published: May 2006

Corticotropin (ACTH) and melanotropin (MSH) peptides (melanocortins) are produced not only in the pituitary but also in the brain, with highest concentrations in the arcuate nucleus of the hypothalamus and the commisural nucleus of the solitary tract. We have identified a receptor for MSH and ACTH peptides that is specifically expressed in regions of the hypothalamus and limbic system. This melanocortin receptor (MC3-R) is found in neurons of the arcuate nucleus known to express proopiomelanocortin (POMC) and in a subset of the nuclei to which these neurons send projections. The MC3-R is 43% identical to the MSH receptor present in melanocytes and is strongly coupled to adenylyl cyclase. Unlike the MSH or ACTH receptors, MC3-R is potently activated by gamma-MSH peptides, POMC products that were named for their amino acid homology with alpha- and beta-MSH, but lack melanotropic activity. The primary biological role of the gamma-MSH peptides is not yet understood. The location and properties of this receptor provide a pharmacological basis for the action of POMC peptides produced in the brain and possibly a specific physiological role for gamma-MSH.
Altered expression of the cyclin D1 and retinoblastoma genes in human esophageal cancer.
Authors: Jiang W , Zhang Y J , Kahn S M , Hollstein M C , Santella R M , Lu S H , Harris C C , Montesano R , Weinstein I B
Published: May 2006

We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. We also examined 36 of these 50 carcinomas for expression of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene.
Interpretation of two compact planetary nebulae, IC 4997 and NGC 6572, with aid of theoretical models.
Authors: Hyung S , Aller L H
Published: May 2006

Observations of two dense compact planetary nebulae secured with the Hamilton Echelle spectrograph at Lick Observatory combined with previously published UV spectra secured with the International Ultraviolet Explorer enable us to probe the electron densities and temperatures (plasma diagnostics) and ionic concentrations in these objects. The diagnostic diagrams show that no homogenous model will work for these nebulae. NGC 6572 may consist of an inner torordal ring of density 25,000 atoms/cm3 and an outer conical shell of density 10,000 atoms/cm3. The simplest model of IC 4997 suggests a thick inner shell with a density of about 107 atoms/cm3 and an outer envelope of density 10,000 atoms/cm3. The abundances of all elements heavier than He appear to be less than the solar values in NGC 6572, whereas He, C, N, and O may be more abundant in IC 4997 than in the sun. IC 4997 presents puzzling problems.
Paradoxical structure and function in a mutant human insulin associated with diabetes mellitus.
Authors: Hua Q X , Shoelson S E , Inouye K , Weiss M A
Published: May 2006

The solution structure of a diabetes-associated mutant human insulin (insulin Los Angeles; PheB24-->Ser) was determined by 13C-edited NMR spectroscopy and distance-geometry/simulated annealing calculations. Among vertebrate insulins PheB24 is invariant, and in crystal structures the aromatic ring appears to anchor the putative receptor-binding surface through long-range packing interactions in the hydrophobic core. B24 substitutions are of particular interest in relation to the mechanism of receptor binding. In one analogue ([GlyB24]insulin), partial unfolding of the B chain has been observed with paradoxical retention of near-native bioactivity. The present study of [SerB24]insulin extends this observation: relative to [GlyB24]insulin, near-native structure is restored despite significant loss of function. To our knowledge, our results provide the first structural study of a diabetes-associated mutant insulin and support the hypothesis that insulin undergoes a change in conformation on receptor binding.
Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration.
Authors: Biro S , Fu Y M , Yu Z X , Epstein S E
Published: May 2006

Smooth muscle cell (SMC) proliferation and migration play pivotal roles in restenosis following angioplasty. c-myc is an immediate early response gene induced by various mitogens, and several lines of evidence derived from experiments using transformed or hematopoietic cell lines, or transgenic mice, suggest its protein product plays a role in numerous signaling transduction pathways, including those modulating cell division. We therefore reasoned that a strategy employing oligodeoxynucleotides (ODNs) complementary to c-myc mRNA (antisense ODNs) might be potent inhibitors of SMC proliferation and, perhaps, of SMC migration. To evaluate this concept, we tested several antisense ODNs targeted to c-myc mRNA (15- or 18-mer ODNs complementary to different c-myc mRNA sequences) by introducing them individually into the medium of cultured rat aortic SMCs. Phosphoroamidate-modified ODNs were employed to retard degradation. Antisense ODNs inhibited, in a concentration-dependent manner, SMC proliferation and SMC migration. Maximal inhibitory effect was 50% for proliferation and > 90% for migration. These effects were associated with decreased SMC expression of c-myc-encoded protein by Western immunoblotting and immunocytochemical staining. ODNs with the same nucleotides but a scrambled sequence caused no effect. These results indicate that the c-myc gene product is involved in the signal transduction pathways mediating SMC proliferation and migration in the in vitro model we employed. The results also suggest a potential role of antisense strategies designed to inhibit c-myc expression for the prevention of coronary restenosis.
Cell-type-specific expression of rat steroid 5 alpha-reductase isozymes.
Authors: Berman D M , Russell D W
Published: May 2006

The enzyme steroid 5 alpha-reductase (EC 1.3.99.5) is a component of an intercellular signaling pathway that determines cell fate in the primordium of the mammalian reproductive tract. During male phenotypic sexual differentiation, the dihydrotestosterone product of this enzyme binds to the androgen receptor and initiates development of the external genitalia and prostate. Genes encoding two isozymes of steroid 5 alpha-reductase with different biochemical properties and tissue distributions have recently been isolated. In the current study, we utilize in situ hybridization analysis to determine cell-type-specific expression patterns of the 5 alpha-reductase isozyme mRNAs in two androgen target tissues (regenerating ventral prostate and epididymis) and a peripheral tissue (liver). In regenerating ventral prostate, the type 1 mRNA is expressed in basal epithelial cells whereas expression of the type 2 mRNA is largely confined to stromal cells. These results were confirmed by immunohistochemical analysis and are consistent with distinct roles played by the isozymes in the prostate. In the epididymis, both 5 alpha-reductase isozyme mRNAs are expressed in epithelial cells. Only the type 1 mRNA is present in the liver. This mRNA is distributed in a striking spatial gradient extending from hepatocytes surrounding the portal triad (high expression) to those surrounding the central vein (low to absent expression). These findings demonstrate cell-type-specific expression of the steroid 5 alpha-reductase isozymes and underscore their distinct and overlapping functions in androgen physiology.
Poincaré resonances and the limits of trajectory dynamics.
Authors: Petrosky T , Prigogine I
Published: May 2006

In previous papers we have shown that the elimination of the resonance divergences in large Poincare systems leads to complex irreducible spectral representations for the Liouville-von Neumann operator. Complex means that time symmetry is broken and irreducibility means that this representation is implementable only by statistical ensembles and not by trajectories. We consider in this paper classical potential scattering. Our theory applies to persistent scattering. Numerical simulations show quantitative agreement with our predictions.
Estrogen receptor and progesterone receptor genes are expressed differentially in mouse embryos during preimplantation development.
Authors: Hou Q , Gorski J
Published: May 2006

Estrogen and progesterone play an important role in the development and implantation of preimplantation embryos. However, it is controversial whether these hormones act directly on the embryos. The effects of these hormones depend on the existence of their specific receptors. To determine whether estrogen receptor (ER) and progesterone receptor genes are expressed in mouse preimplantation embryos, we examined RNA from embryos at different stages of preimplantation development by reverse transcription-polymerase chain reaction techniques. ER mRNA was found in oocytes and fertilized eggs. The message level began to decline at the two-cell stage and reached its lowest level at the five- to eight-cell stage. ER mRNA was not detectable at the morula stage but reappeared at the blastocyst stage. Progesterone receptor mRNA was not detectable until the blastocyst stage. The embryonic expression of ER and progesterone receptor genes in the blastocyst suggests a possible functional requirement for ER and progesterone receptor at this stage of development. These results provide a basis for determining the direct role of estrogen and progesterone in preimplantation embryos.
Leucine aminopeptidase: an inducible component of the defense response in Lycopersicon esculentum (tomato).
Authors: Pautot V , Holzer F M , Reisch B , Walling L L
Published: May 2006

A leucine aminopeptidase (EC 3.4.11.1) cDNA clone (DR57) that was induced in response to Pseudomonas syringae pv. tomato (P.s. tomato) infection was isolated using a subtractive hybridization-enriched cDNA probe. Genomic DNA blot analysis showed that the tomato genome had two leucine aminopeptidase genes. The levels of DR57 mRNAs after P.s. tomato infection and mechanical wounding were determined in two inbred tomato lines that exhibit susceptibility and resistance to P.s. tomato. DR57 mRNAs were detected 12 hours after infection and 4 hours after wounding. Furthermore, DR57 mRNAs were systemically induced in response to wounding. DR57 mRNAs were induced in leaves after Spodoptera littoralis feeding but were not detected in detached leaf controls. Possible roles for the DR57 leucine aminopeptidase in the defense reactions are discussed.
17 beta-estradiol mimics ligand activity of the c-erbB2 protooncogene product.
Authors: Matsuda S , Kadowaki Y , Ichino M , Akiyama T , Toyoshima K , Yamamoto T
Published: May 2006

We report here the physical and functional interaction of estrogen with the ErbB2 protein p185c-erbB2. The ErbB2 protein immunoprecipitated from estrogen-treated [10(-8) to 10(-6) M 17 beta-estradiol (E2)] RC cells showed higher autophosphorylation activity than that from untreated cells. Likewise autophosphorylation activity of ErbB2 protein from untreated cells was stimulated in vitro by E2. In addition, E2 treatment induced down-regulation of ErbB2 protein from the detergent-soluble fraction of the RC cells within 15 min. E2 also induced morphological transformation of the RC cells but not of the parental NIH 3T3 cells, which express little c-erbB2 under the same experimental conditions. This morphological transformation of RC cells was reversed by tamoxifen. However, E2 treatment did not induce anchorage-independent growth of RC cells. Scatchard analysis revealed E2 binding to the ErbB2 protein on RC cells; the Kd value was 2.7 nM. E2 did not bind appreciably to the parental NIH 3T3 cells or cells expressing an ErbB2 protein lacking most of its extracellular domain. These data suggest that estrogen plays an important role in ErbB2-mediated signaling.
Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP.
Authors: D'Mello S R , Galli C , Ciotti T , Calissano P
Published: May 2006

High levels of extracellular K+ ensure proper development and prolong survival of cerebellar granule neurons in culture. We find that when switched from a culture medium containing high K+ (25 mM) to one containing a low but more physiological K+ concentration (5 mM), differentiated granule neurons degenerate and die. Death induced by low K+ is due to apoptosis (programmed cell death), a form of cell death observed extensively in the developing nervous system and believed to be necessary for proper neurogenesis. The death process is accompanied by cleavage of genomic DNA into internucleosome-sized fragments, a hallmark of apoptosis. Inhibitors of transcription and translation suppress apoptosis induced by low K+, suggesting the necessity for newly synthesized gene products for activation of the process. Death can be prevented by insulin-like growth factor I but not by several other growth/neurotrophic factors. cAMP but not the protein kinase C activator phorbol 12-myristate 13-acetate can also support survival in low K+. In view of the large numbers of granule neurons that can be homogeneously cultured, our results offer the prospect of an excellent model system to study the mechanisms underlying apoptosis in the central nervous system and the suppression of this process by survival factors such as insulin-like growth factor I.
cDNAs encoding the large subunit of human replication factor C.
Authors: Bunz F , Kobayashi R , Stillman B
Published: May 2006

Replication factor C (RFC) is a multisubunit, DNA polymerase accessory protein required for the coordinated synthesis of both DNA strands during simian virus 40 DNA replication in vitro. Previous studies have shown that RFC is a DNA-dependent ATPase that binds in a structure-specific manner to the 3' end of a primer hybridized to a template DNA, an activity thought intrinsic to the 140-kDa component of this multisubunit complex. Here, the isolation and analysis of cDNAs encoding this subunit is described. Analysis of the full-length coding sequence revealed an open reading frame of 3.4 kb, encoding an 1148-amino acid protein with a predicted molecular mass of 130 kDa. A putative ATP-binding motif was observed that is similar to a motif in several of the smaller subunits of RFC and in functionally homologous replication factors of bacterial and viral origin. A "DEAD" box is also conserved among these proteins. The predicted protein shows significant identity with a DNA-binding protein of murine origin (B. Luckow, P. Lichter, and G. Schütz, personal communication). Regions of similarity were also seen between the amino acid sequences of the 140-kDa subunit of RFC, poly(ADP-ribose) polymerase, and bacterial DNA ligases--possibly representing a conserved structural feature of these proteins that bind similar DNA substrates.
Activated human B lymphocytes express three CTLA-4 counterreceptors that costimulate T-cell activation.
Authors: Boussiotis V A , Freeman G J , Gribben J G , Daley J , Gray G , Nadler L M
Published: May 2006

Signaling via the T-cell receptor complex is necessary but not sufficient to induce antigen-specific T lymphocytes to expand clonally. To proliferate, T cells must receive one or more costimulatory signals provided by antigen presenting cells (APCs). One such critical costimulatory signal is delivered by the CD28/CTLA-4 counterreceptor, B7, expressed on APCs. B7 costimulation induces CD28 signaling, resulting in interleukin 2 (IL-2) secretion, and T-cell proliferation. Conversely, T-cell receptor signaling in the absence of B7 costimulation results in induction of antigen-specific tolerance. Here, we show that activated human B lymphocytes express two additional CTLA-4 counterreceptors also capable of providing T-cell costimulation. At 24 hr postactivation, B cells express a CTLA-4 counterreceptor not recognized by anti-B7 or -BB-1 monoclonal antibodies (mAbs), which induces detectable IL-2 secretion and T-cell proliferation. At 48 and 72 hr postactivation, B cells express both B7 and a third CTLA-4 counterreceptor identified by the anti-BB-1 mAb. BB-1 appears to be a molecule distinct from B7 by its expression on B7- cells and its capacity to induce T cells to proliferate without significant accumulation of IL-2. As observed for B7, costimulatory signals mediated by these alternative CTLA-4/CD28 counterreceptors are likely to be essential for generation of an immune response and their absence may result in antigen-specific tolerance. We propose the following terminology for these CTLA-4 counterreceptors: (i) B7, B7-1; (ii) early CTLA-4 binding counterreceptor, B7-2; and (iii) BB-1, B7-3.
Geometric derivation of the chronometric redshift.
Authors: Segal I E
Published: May 2006

The chronometric redshift-distance relation z = tan 2(1/2rho), where rho is the distance in radians in the Einstein metric, is derived by an elementary geometric analysis comparable to that in traditional analysis of the expanding universe model. The differential dTt of Einstein time evolution Tt through time t, as applied to the local Minkowski coordinates x, takes the form sec2(1/2t). At the point of observation t = rho, implying that for a sufficiently localized source, observed wave lengths are a factor of sec2(1/2rho) greater than the corresponding emitted wave lengths.
Role of intermolecular/intrastructural B- and T-cell determinants in the diversification of autoantibodies to ribonucleoprotein particles.
Authors: Fatenejad S , Mamula M J , Craft J
Published: May 2006

The U1 small nuclear ribonucleoprotein (sn-RNP) particle, which consists of the U1 small RNA and multiple polypeptides, is a central target of the autoimmune response in systemic lupus erythematosus. Autoantibodies to the individual proteins of the U1 snRNP typically co-occur in patients with systemic lupus erythematosus, an observation reconciled by postulating that the intact RNA-protein complex serves as the autoimmunogen and that snRNP-specific autoreactive T cells are necessary for autoantibody production. In this study, we demonstrated that normal mice did not develop antibody responses following immunization with purified self (murine) snRNPs. However, when such mice were coimmunized with self snRNPs in conjunction with the human (foreign) U1 snRNP A protein, they developed autoantibodies directed against individual proteins of the U1 snRNP, in addition to anti-A antibodies; we have previously shown that such mice develop snRNP-specific, autoreactive T cells. Intact snRNPs as a co-immunogen were a prerequisite for antibody expansion, since this response was abrogated by disruption of snRNP particles with pancreatic RNase prior to immunization. These findings indicate that autoreactive helper T cells can drive autoantibody production to the individual proteins of snRNP particles and that such autoantibody responses may require the presence of intact snRNP particles that possess intrastructural B-cell and helper-T-cell determinants. These results also suggest that induction of an immune response to one component of an autoantigenic snRNP complex, possibly through priming with molecular mimics, can induce the diversification of autoantibodies that is characteristic of that found in patients with systemic lupus erythematosus.
Characterization of downstream elements in a Raf-1 pathway.
Authors: Liaw G J , Steingrimsson E , Pignoni F , Courey A J , Lengyel J A
Published: May 2006

At the poles of the Drosophila embryo, cell fate is established by a pathway that begins with the activation of a membrane-associated tyrosine kinase (the torso gene product); this then leads to activation of a serine/threonine kinase (Drosophila Raf-1). Activated Raf-1 then leads, by an undefined mechanism, to the transcriptional activation of the tailless (tll) gene; the tll gene product, itself a transcription factor, subsequently regulates the expression of an array of target genes. To further define this pathway, we have utilized sequence comparison between Drosophila melanogaster and Drosophila virilis to identify conserved elements in the tll promoter region. As assessed by DNase I footprinting and promoter dissection experiments, two of these elements are potential regulatory targets of Raf-1-activated transcription factors. Sequence comparison also reveals that the unique residues in the DNA-binding domain of the tll protein, the next component in the pathway, are conserved. One of these residues, the alanine after the last cysteine in the first zinc finger, may be responsible for part of the difference between the tll protein DNA binding site and the closely related half-site of the retinoid/estrogen receptors. Consistent with the rapid turnover of the tll protein, it contains a PEST sequence (rich in proline, glutamate and aspartate, serine, and threonine) that is also conserved.
Toward an estimation of binding constants in aqueous solution: studies of associations of vancomycin group antibiotics.
Authors: Williams D H , Searle M S , Mackay J P , Gerhard U , Maplestone R A
Published: May 2006

An approach toward the estimation of binding constants for organic molecules in aqueous solution is presented, based upon a partitioning of the free energy of binding. Consideration is given to polar and hydrophobic contributions and to the entropic cost of rotor restrictions and bimolecular associations. Several parameters (derived from an analysis of entropy changes upon the melting of crystals and from the binding of cell wall peptide analogues to the antibiotic ristocetin A) which may be useful guides to a crude understanding of binding phenomena are presented: (i) amide-amide hydrogen bond strengths of -(1 to 7) +/- 2 kJ.mol-1, (ii) a hydrophobic effect of -0.2 +/- 0.05 kJ.mol-1.A-2 of hydrocarbon removed from exposure to water in the binding process, and (iii) free energy costs for rotor restrictions of 3.5-5.0 kJ.mol-1. The validity of the parameters for hydrogen bond strengths is dependent on the validity of the other two parameters. The phenomenon of entropy/enthalpy compensation is considered, with the conclusion that enthalpic barriers to dissociations will result in larger losses in translational and rotational entropy in the association step. The dimerization of some vancomycin group antibiotics is strongly exothermic (-36 to -51 kJ.mol-1) and is promoted by a factor of 50-100 by a disaccharide attached to ring 4 (in vancomycin and eremomycin) and by a factor of ca. 1000 by an amino-sugar attached to the benzylic position of ring 6 in eremomycin. The dimerization process (which, as required for an exothermic association, appears to be costly in entropy) may be relevant to the mode of action of the antibiotics.
Stimulus-induced association of Ca(2+)-binding proteins with the plasma membrane detected in situ by photolabeling of intact chromaffin and PC12 cells.
Authors: Schwaller B , Calef E , Gitler C , Rosenheck K
Published: May 2006

To investigate the involvement of cytosolic proteins in exocytosis, a system with high temporal and spatial resolution has been developed that allows us to detect the interaction of Ca(2+)- and membrane-binding proteins with the plasma membrane during stimulation of intact chromaffin and PC12 (rat pheochromocytoma) cells. We used 5-iodonaphthalene-1-azide (INA), a hydrophobic label that rapidly partitions into the lipid bilayer of biological membranes. Upon photolysis the label covalently attaches to membrane-embedded domains of proteins. Cells, preincubated with INA in the dark, were stimulated by either 300 microM carbamoylcholine or 60 mM K+ and irradiated (20 s) at various time intervals after stimulation. Subsequently, the cytosolic Ca(2+)- and membrane-binding proteins were isolated in the presence of EGTA (EGTA extract). Of the approximately 40 proteins in the EGTA extract, 15 (15-100 kDa) are labeled in both cell types. Upon stimulation, labeling is increased up to 3-fold in some of the proteins compared to cells labeled under basal conditions. In the absence of external Ca2+, no increase is observed. The rate of label incorporation is similar to the rate of exocytosis in several of these proteins. These results indicate that in the event of triggered exocytosis some of the Ca(2+)-binding proteins interact with the plasma membrane and temporarily embed in the lipid bilayer. Our findings support the hypothesis according to which stimulus-induced alterations in the structure of the Ca(2+)-binding proteins lead to their transient insertion into the membrane and thereby to membrane fusion.
Vitamin D and adaptation to dietary calcium and phosphate deficiencies increase intestinal plasma membrane calcium pump gene expression.
Authors: Cai Q , Chandler J S , Wasserman R H , Kumar R , Penniston J T
Published: May 2006

The effect of vitamin D and other variables on the synthesis of the chicken intestinal plasma membrane calcium pump (PMCA) mRNA was assessed. The DNA probe for Northern analysis was obtained by reverse transcription and PCR with intestinal poly(A)+ RNA, using two 20-mer oligonucleotide primers homologous to the 3' coding region of the human teratoma PMCA. An EcoRI restriction fragment of the PCR product was cloned into the pBluescript II KS(-) phagemid vector, and the chimeric plasmid was used to transform Escherichia coli. The amino acid sequence deduced from the nucleotide DNA sequence of the PCR product and the cloned DNA were 96% homologous with the teratoma sequence. Northern blots of intestinal poly(A)+ RNA with 32P-labeled DNA showed the presence of three major species of chicken PMCA mRNAs at about 6.6, 5.4, and 4.5 kb. Northern analysis with the chicken PMCA DNA indicated that repletion of vitamin D-deficient chickens with vitamin D increased PMCA mRNAs in the duodenum, jejunum, ileum, and colon. After injection of 1,25-dihydroxyvitamin D3 intravenously into vitamin D-deficient chickens, duodenal PMCA mRNA tended to increase by 2 hr, reached a maximum at about 16 hr, and returned to baseline levels at 48 hr. Adaptation of chickens to either a calcium- or phosphorus-deficient diet resulted in a 2- to 3-fold increase in duodenal PMCA mRNA. These results indicate that vitamin D and specific variables that affect calcium absorption through the vitamin D-endocrine system increase intestinal PMCA gene expression.
Sequence conversion during postreplicative adenovirus overlap recombination.
Authors: Bennett K L , Pearson G D
Published: May 2006

Sequence conversion efficiently transfers genetic information in high yield during postreplicative adenovirus overlap recombination. This process is intrinsically nonreciprocal, depends on adenovirus-specific strand-displacement replication by both partner molecules, and requires that complementary sequences on displaced strands must exceed a minimal length to form a heteroduplex intermediate.
Crystals of hemagglutinin-neuraminidase of parainfluenza virus contain triple-stranded helices.
Authors: Murti K G , Takimoto T , Laver W G , Portner A
Published: May 2006

When purified dimers of hemagglutinin-neuraminidase molecules released by protease digestion from three strains of human parainfluenza virus 1 were used in crystallization trials, long thin needle crystals formed. Electron microscopic analysis of these needle crystals revealed that they are composed of stacks of triple-stranded helices with each strand of the helix made up of subunits of hemagglutinin-neuraminidase. To our knowledge, this is the first direct demonstration of the assembly of protein subunits into large triple-stranded helices. An understanding of the organization of these triple helices may shed light on the structural properties of the hemagglutinin-neuraminidase molecules that cause them to form these helices.
In vitro selection of optimal DNA substrates for T4 RNA ligase.
Authors: Harada K , Orgel L E
Published: May 2006

We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.
Activation of c-myb is an early bone-marrow event in a murine model for acute promonocytic leukemia.
Authors: Nason-Burchenal K , Wolff L
Published: May 2006

Insertional mutagenesis of c-myb by Moloney murine leukemia virus occurs in 100% of promonocytic leukemias (MMLS) induced by the virus. These leukemias, which resemble acute monocytic leukemia-M5 in humans are induced only in mice undergoing a peritoneal chronic inflammatory response. We have found that two leukemia-specific gag-myb mRNAs in MML provide molecular markers for detection of preleukemic cells in hematopoietic tissue in vivo. The two aberrant RNAs result from splicing of gag to either exon 3 or 4 of c-myb, depending on the site of proviral integration. After reverse transcription-PCR with nested primers and hybridization with specific gag-myb junction probes, one cell, having aberrant c-myb message, could be detected in a minimum of 10(5) liver cells or 10(6) spleen or bone-marrow cells. This approach was used to examine hematopoietic tissues of mice after pristane injection to induce inflammation and virus inoculation. Cells with gag-myb mRNAs could be detected as early as 2 weeks after virus inoculation. In mice receiving both pristane and virus, there was evidence of preleukemic cells in 83% of the mice by 3 weeks after virus infection. Furthermore, 100% of the mice were positive for preleukemic cells by 8 weeks, even though only 50% of mice have been shown to succumb to MML (peak time for disease latency is 12-16 weeks). Cells with these aberrant c-myb messages were initially detected in the bone marrow, but during intermediate stages of disease development these cells disseminated to the spleen, liver, and granuloma. At preleukemic times, from 3 to 8 weeks after virus infection, a lower percentage of mice were positive in the group that did not receive pristane compared with mice in the group receiving pristane. However, at 18 weeks, 100% of the mice in the group receiving virus only had evidence of cells expressing gag-myb RNA in their spleens and/or bone marrow; it is of interest that mice inoculated with virus alone never develop MML. This approach for detecting preleukemic cells will now allow the study of mechanisms by which these preleukemic cells progress to a more transformed state and, perhaps, to a more differentiated state.