bluuebunny/crossref_metadata_2025 huggingface dataset:
Colony-stimulating factor 1-mediated regulation of a chimeric c-fms/v-fms receptor containing the v-fms-encoded tyrosine kinase domain.
Authors: Roussel M F , Downing J R , Ashmun R A , Rettenmier C W , Sherr C J
Published: May 2006

A chimeric gene specifying the 308 N-terminal amino acids of the extracellular ligand binding domain of the human c-fms protooncogene joined to the remainder of the feline v-fms oncogene product encodes a functional colony-stimulating factor 1 (CSF-1) receptor. When expressed in mouse NIH 3T3 fibroblasts, the chimeric gene product was rapidly transported to the cell surface, was autophosphorylated on tyrosine only in response to human recombinant CSF-1, underwent ligand-induced but not phorbol ester-induced down-modulation, and stimulated CSF-1-dependent cell proliferation. By contrast, the C-terminally truncated glycoprotein encoded by the v-fms oncogene is partially inhibited in its transport to the plasma membrane, is constitutively phosphorylated on tyrosine, and is relatively refractory to both ligand-induced and phorbol ester-induced down-modulation. Although the v-fms oncogene can transform cells in the absence of CSF-1, its tyrosine kinase activity and turnover can be appropriately regulated by the human c-fms-encoded ligand binding domain. The results confirm that C-terminal truncation of the c-fms gene is insufficient to activate its transforming potential and suggest that an additional mutation in its distal extracellular domain is required for oncogenic activation.
Autocrine growth induced by the insulin-related factor in the insulin-independent teratoma cell line 1246-3A.
Authors: Yamada Y , Serrero G
Published: May 2006

An insulin-independent teratoma-derived cell line, called 1246-3A, has been isolated from the adipogenic cell line 1246, which stringently requires insulin for proliferation. The 1246-3A cell line, which can proliferate in the absence of exogenous insulin, produces in its conditioned medium a growth factor similar to pancreatic insulin by its biological and immunological properties. This factor, called "insulin-related factor" (IRF), was purified and iodinated to study its binding to cell surface receptors. 125I-labeled IRF binding to intact 1246-3A cells is lower than to 1246 cells. Cell surface binding can be restored by culturing the 1246-3A cells in the presence of an anti-porcine insulin monoclonal antibody or by acid prewash of the cells prior to performing the binding. Scatchard analysis of binding indicates that IRF secreted by the 1246-3A cells partially occupies high-affinity binding sites on the producer cells. Moreover, insulin monoclonal antibody inhibits the proliferation of the IRF-producing 1246-3A cells, suggesting that these cells are dependent on the secreted IRF for growth in culture. We conclude that the insulin-related factor secreted by the insulin-independent 1246-3A cells stimulates their proliferation in an autocrine fashion.
Comparative mapping of DNA markers from the familial Alzheimer disease and Down syndrome regions of human chromosome 21 to mouse chromosomes 16 and 17.
Authors: Cheng S V , Nadeau J H , Tanzi R E , Watkins P C , Jagadesh J , Taylor B A , Haines J L , Sacchi N , Gusella J F
Published: May 2006

Mouse trisomy 16 has been proposed as an animal model of Down syndrome (DS), since this chromosome contains homologues of several loci from the q22 band of human chromosome 21. The recent mapping of the defect causing familial Alzheimer disease (FAD) and the locus encoding the Alzheimer amyloid beta precursor protein (APP) to human chromosome 21 has prompted a more detailed examination of the extent of conservation of this linkage group between the two species. Using anonymous DNA probes and cloned genes from human chromosome 21 in a combination of recombinant inbred and interspecific mouse backcross analyses, we have established that the linkage group shared by mouse chromosome 16 includes not only the critical DS region of human chromosome 21 but also the APP gene and FAD-linked markers. Extending from the anonymous DNA locus D21S52 to ETS2, the linkage map of six loci spans 39% recombination in man but only 6.4% recombination in the mouse. A break in synteny occurs distal to ETS2, with the homologue of the human marker D21S56 mapping to mouse chromosome 17. Conservation of the linkage relationships of markers in the FAD region suggests that the murine homologue of the FAD locus probably maps to chromosome 16 and that detailed comparison of the corresponding region in both species could facilitate identification of the primary defect in this disorder. The break in synteny between the terminal portion of human chromosome 21 and mouse chromosome 16 indicates, however, that mouse trisomy 16 may not represent a complete model of DS.
Binding site for human immunodeficiency virus coat protein gp120 is located in the NH2-terminal region of T4 (CD4) and requires the intact variable-region-like domain.
Authors: Richardson N E , Brown N R , Hussey R E , Vaid A , Matthews T J , Bolognesi D P , Reinherz E L
Published: May 2006

The external segment of the T4 (CD4) glycoprotein functions as the T-cell surface receptor for human immunodeficiency virus by binding the major viral coat protein (gp120) with relatively high affinity. To more precisely define the region of T4 involved in gp120 interaction, we used purified, soluble forms of T4 anchor-minus polypeptides (produced in a baculovirus system) in conjunction with proteolytic fragmentation, microsequencing, and a specific T4-gp120 binding assay. The results indicate that the NH2-terminal region of T4 including the immunoglobulin variable-region-like domain is required for gp120 interaction. In contrast, the COOH-terminal half of the molecule, containing the two potential N-linked glycosylation sites, is not necessary. Furthermore, reduction of intrachain disulfide bonds in the T4 molecular abrogates gp120 binding, thereby strongly implying that the binding site for gp120 is dependent on the stabilized domain structure of the active binding region.
Anemia lessens and its prevention with recombinant human erythropoietin worsens glomerular injury and hypertension in rats with reduced renal mass.
Authors: Garcia D L , Anderson S , Rennke H G , Brenner B M
Published: May 2006

Chronic renal disease is frequently characterized by anemia, which may modify systemic and renal hemodynamics. In adult Munich-Wistar rats, the mild anemia (hematocrit, approximately equal to 42 vol/dl) that accompanies five-sixths nephrectomy was either made more severe (approximately equal to 30 vol/dl) by feeding a low iron diet or prevented (approximately equal to 50 vol/dl) by administration of recombinant human erythropoietin (r-HuEpo). In functional studies performed 4 weeks after renal ablation, untreated rats exhibited mild anemia with systemic hypertension and elevation of the single nephron glomerular filtration rate due to glomerular capillary hyperperfusion and hypertension. Preventing anemia with r-HuEpo worsened systemic and glomerular hypertension, effects largely obviated by induction of more marked anemia with the low iron diet. Untreated rats followed for 6 weeks postablation exhibited progressive proteinuria and sclerosis involving 12% of glomeruli, contrasted with 33% in rats given r-HuEpo. Even after 12 weeks, sclerosis involved only 6% of glomeruli in rats with more severe anemia but progressed to 30% in untreated rats. Thus, anemia limits systemic and glomerular hypertension and glomerular injury, whereas its prevention by r-HuEpo severely accelerates hemodynamically mediated glomerular injury in this model. These results suggest that anemia is a hemodynamically favorable adaptation to chronic renal disease and that its overly vigorous correction may have adverse renal hemodynamic and structural consequences.
Kinesin ATPase: rate-limiting ADP release.
Authors: Hackney D D
Published: May 2006

The ATPase rate of kinesin isolated from bovine brain by the method of S.A. Kuznetsov and V.I. Gelfand [(1986) Proc. Natl. Acad. Sci. USA 83, 8530-8534)] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from approximately equal to 0.009 sec-1 to 9 sec-1). The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (Ki less than 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by [14C]ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.
Ca2+/calmodulin-dependent protein kinase II: identification of threonine-286 as the autophosphorylation site in the alpha subunit associated with the generation of Ca2+-independent activity.
Authors: Thiel G , Czernik A J , Gorelick F , Nairn A C , Greengard P
Published: May 2006

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II converts the enzyme to a Ca2+-independent form. The time course for this conversion correlates with the autophosphorylation of a threonine residue located within a thermolytic phosphopeptide common to the alpha and beta/beta' subunits. In the present study, this site was identified in the alpha subunit. After autophosphorylation under conditions that produced near-maximal Ca2+-independent activity, the alpha and beta/beta' subunits were separated by NaDodSO4/PAGE, and the alpha subunit was cleaved with cyanogen bromide. The major phosphopeptide (CB-1), containing phosphothreonine as the only radiolabeled amino acid, was purified by reverse-phase high performance liquid chromatography and subjected to automated gas-phase Edman degradation. The sequence obtained, Xaa-Arg-Gln-Glu-Thr-Val-Asp-Xaa-Leu-Lys-Lys-Phe-Asn-Ala-Arg-Arg-Lys-Leu, represented the NH2-terminal 18 residues (residues 282-299) of a 26-amino acid cyanogen bromide peptide predicted from the deduced primary structure of the alpha subunit and contained a consensus sequence for Ca2+/calmodulin-dependent kinase II phosphorylation that included Thr-286. The sequences obtained for two phosphopeptides derived from secondary chymotryptic digestion of CB-1 confirmed that Thr-286 was the phosphorylated residue.
Purification and biochemical properties of a human monocyte-derived growth factor.
Authors: Singh J P , Bonin P D
Published: May 2006

A monocyte-derived growth factor (MDGF) that stimulates proliferation of fibroblast and smooth muscle cells was purified from mitogen-stimulated human peripheral blood lymphocyte conditioned medium by using anion-exchange, Bio-Sil TSK-250 HPLC gel-permeation chromatography, and NaDodSO4/PAGE. Purified MDGF exhibited acidic charge characteristics (pI 5.0) and migrated with an apparent Mr of 40,000 +/- 2000 in molecular sizing HPLC columns. Elution from NaDodSO4/polyacrylamide gels showed that the growth-promoting activity was associated with three or four protein bands. The highest molecular weight species representing the most intense silver-stained band corresponded to 42,000; the lowest molecular weight species was 33,000. MDGF activity was stable to treatment with acid (pH 2.0) or base (pH 10.0) and heating (100 degrees C, 5 min) but was inactivated upon reduction with 2-mercaptoethanol. The acidic MDGF did not effectively compete with platelet-derived growth factor (PDGF) for receptor binding and was not inhibited by PDGF antibodies. Previous studies have suggested that fibroblast growth-stimulating activity of macrophages is largely due to their secretion of interleukin 1 and a PDGF-like molecule. Our purification and biochemical characterization studies reveal the occurrence of multiple forms of fibroblast growth-stimulating activity in human monocyte conditioned medium. The MDGF activity characterized here appears to be structurally and functionally distinct from the previously described fibroblast growth-promoting activities including interleukin 1, basic fibroblast growth factor, and PDGF.
Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7.
Authors: Shoyab M , McDonald V L , Bradley J G , Todaro G J
Published: May 2006

A glycoprotein, termed amphiregulin (AR), inhibits growth of several human carcinoma cells in culture and stimulates proliferation of human fibroblasts and certain other tumor cells. It has been purified to apparent homogeneity from serum-free conditioned medium of MCF-7 human breast carcinoma cells that had been treated with phorbol 12-myristate 13-acetate. AR is a single-chain extremely hydrophilic glycoprotein containing cysteines in disulfide linkage(s) that are essential for biological activity; it is stable between pH 2 and pH 12 and after heating for 30 min at 56 degrees C but unstable at 100 degrees C. The apparent molecular weights of AR and N-Glycanase-treated AR are 14,000 and 15,000, respectively, as assessed by gel chromatography, and approximately 22,500 and approximately 14,000, respectively, as determined by polyacrylamide gel electrophoresis. Treatment of AR with N-Glycanase, O-Glycanase, or neuraminidase does not affect its activity. The pI of AR is approximately 7.8. The amino-terminal amino acid sequence of AR has been determined, and no significant sequence homology between AR and other proteins was found. The molecule thus appears to be a distinct growth regulatory protein.
Photolabile chelators for the rapid photorelease of divalent cations.
Authors: Kaplan J H , Ellis-Davies G C
Published: May 2006

The properties of a recently synthesized photolabile chelator for divalent cations are described, the affinity of which for Ca2+ changes by some 5 orders of magnitude on illumination. The compound 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis[(oxycarbonyl)me thyl]-1,2-ethanediamine (DM-nitrophen) binds Ca2+ (Kd approximately 5.0 x 10(-9) M) and Mg2+ (Kd approximately 2.5 x 10(-6) M) with relatively high affinities. On exposure of the DM-nitrophen-Ca2+ complex to UV light in the 350-nm range, the chelator is cleaved yielding iminodiacetic products with a much lower affinity for Ca (Kd approximately 3 x 10(-3) M) and the free [Ca2+] increases. The quantum yield for Ca2+ release is 0.18. In experiments with chemically skinned skeletal muscle fibers, a fully relaxed fiber equilibrated with DM-nitrophen-Ca2+ complex produced maximal contraction after a single flash from a frequency-doubled ruby laser (347 nm). Half-maximal tension was achieved in approximately 40 ms, some 5 times faster than that obtained after a rapid solution change from a Ca2+-free to a Ca2+-containing solution. In experiments with resealed human erythrocyte ghosts, irradiation of ghosts containing the DM-nitrophen-Ca2+ complex activates a Ca2+-dependent K+ efflux pathway, which is not observed in the absence of illumination. DM-nitrophen is sufficiently stable and photolabile to be used as a caged Ca (or caged Mg) for the rapid photoinitiation of divalent cation-dependent processes over a wide concentration range with a significant increase in temporal resolution over conventional mixing methods.
Translational control of anionic trypsinogen and amylase synthesis in rat pancreas in response to caerulein stimulation.
Authors: Steinhilber W , Poensgen J , Rausch U , Kern H F , Scheele G A
Published: May 2006

Infusion of rats with optimal doses of caerulein for up to 24 hr resulted in divergent changes in protein synthesis in the exocrine pancreas: a 3-fold increase in synthesis of anionic trypsinogen and a 75% decrease in synthesis of amylase. Lipase synthesis showed no change. Rates of total protein synthesis increased 2-fold, while DNA, RNA, and poly(A)+ mRNA concentrations were unchanged during hormonal stimulation. mRNA concentrations for anionic trypsinogen, lipase, and amylase were determined by dot blot hybridization analysis with cDNA and cRNA probes. Despite 12-fold changes in the ratio of synthesis of anionic trypsinogen to amylase at 24 hr of caerulein stimulation, changes in levels of mRNA encoding these two proteins were not observed. The slight decreases observed in amylase mRNA concentrations were found in both hormone and saline-infused animals. In vitro pulse-chase experiments after 12 hr of saline or caerulein infusion indicated that differential turnover of anionic trypsinogen and amylase did not occur during hormone stimulation. These data demonstrate that the differential regulation observed in protein synthesis that results from a single period of hormone stimulation is mediated by differential regulation of mRNA translation. The high degree of conservation observed in the 5' terminal sequences of both amylase and anionic trypsinogen mRNAs between mouse, rat, and dog suggests that sequence-specific mechanisms and secondary structure may play a role in the translational control of these two mRNAs.
Activation of the human "beta 2-interferon/hepatocyte-stimulating factor/interleukin 6" promoter by cytokines, viruses, and second messenger agonists.
Authors: Ray A , Tatter S B , May L T , Sehgal P B
Published: May 2006

The hallmark of "beta 2-interferon (IFN-beta 2)/hepatocyte-stimulating factor/interleukin 6" gene expression is its inducibility in different types of human cells (fibroblasts, monocytes, epithelial cells, and endothelial cells) by different stimuli, which include cytokines such as tumor necrosis factor, interleukin 1 (IL-1) and platelet-derived growth factor, different viruses, and bacterial products such as endotoxin. The activation by cytokines, viruses, and second messenger agonists of the IFN-beta 2 promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was studied after transfection into HeLa cells. A chimeric gene containing IFN-beta 2 DNA from -1180 to +13 linked to the CAT gene was inducible approximately 10-fold by phorbol 12-myristate 13-acetate (PMA), followed, in decreasing order, by pseudorabies and Sendai viruses (7- to 11-fold each); serum (6- to 9-fold); the cytokines tumor necrosis factor, IL-1, and epidermal growth factor (3- to 5-fold each); the cAMP agonists BrcAMP and forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (2- to 6-fold each); poly(I).poly(C) (2- to 4-fold); 1,2-diacylglycerol and the calcium ionophore A23187 (1.5- to 2-fold each). Bacterial endotoxin did not activate this IFN-beta 2/CAT fusion gene in HeLa cells. Deletion of the 5' boundary of the IFN-beta 2 DNA from -1180 to -596 in the fusion gene preserved its activation by IL-1, tumor necrosis factor, epidermal growth factor, serum, pseudorabies, and Sendai viruses and by PMA, Br-cAMP, and forskolin; deletion to -225 led to a small reduction (by a factor of 1.5-2) in the responsiveness to serum, PMA, and Sendai virus but not to the other inducers; a further deletion to -112 greatly reduced all responsiveness. Thus, the region between -225 and -113 in IFN-beta 2, which contains DNA motifs similar to the regulatory elements in the human c-fos gene, appears to contain the major cis-acting regulatory elements responsible for the activation of the IFN-beta 2 promoter by several different cytokines, viruses, and second messenger agonists.
Cultured lymphocytes from alcoholic subjects have altered cAMP signal transduction.
Authors: Nagy L E , Diamond I , Gordon A
Published: May 2006

Previous work has shown that freshly isolated lymphocytes from alcoholic subjects show significantly reduced basal and adenosine receptor-stimulated cAMP levels. This decrease could be due to ethanol-induced cellular adaptation or to a genetic difference in the regulation of cAMP signal transduction. Therefore, we cultured human lymphocytes in defined medium without ethanol for 7-8 days and then examined differences in receptor-dependent cAMP accumulation between lymphocytes from alcoholic and nonalcoholic subjects. After four to six generations in culture without ethanol, lymphocytes from alcoholic subjects have significantly higher cAMP levels than do cells from nonalcoholic subjects. Thus, a difference in cAMP signal transduction is demonstrable in cells from alcoholic subjects grown without ethanol. We also found that cultured lymphocytes from both alcoholic and nonalcoholic subjects show a decrease in receptor-stimulated cAMP levels after exposure to 200 mM ethanol for 48 hr. To determine whether alcoholic subjects have increased sensitivity to ethanol, lymphocytes were exposed to only 100 mM ethanol for 24 hr. Under these conditions, receptor-dependent cAMP levels did not change in cells from nonalcoholic subjects. However, lymphocytes from alcoholic subjects showed a 39% decrease (P less than 0.003) in adenosine receptor-stimulated cAMP levels. Taken together, the results show that (i) chronic ethanol treatment in culture reproduces the suppression of cAMP levels found in circulating lymphocytes from alcoholic subjects and (ii) despite four to six cell divisions in culture without ethanol, lymphocytes from alcoholic subjects exhibit significantly increased adenosine receptor-dependent cAMP levels and increased sensitivity to chronic exposure to ethanol. These findings suggest that the suppression of cAMP levels observed in freshly isolated lymphocytes from alcoholic subjects results from both a direct effect of chronic exposure to ethanol and a genetic difference leading to altered cAMP signal transduction.
A sensitive method for the identification of uncharacterized viruses related to known virus groups: hepadnavirus model system.
Authors: Mack D H , Sninsky J J
Published: May 2006

Amino acid sequence similarity of the reverse transcriptases encoded by retroviruses and hepadnaviruses was first reported by Toh, H., Hayashida, H. & Miyata, T. (1983) Nature (London) 305, 827-829. The regions of similarity extend over a small number of amino acids and require the introduction of gaps through the open reading frame. By using an octapeptide region as the sole criterion for "taxonomic" classification, we have grouped the oncoviruses into two distinct categories and the lentiviruses and hepadnaviruses into two additional groupings. This classification suggests that murine and feline leukemia viruses may be more closely related to the viruses that are associated with leukemia in primates and cattle than had been appreciated. We have exploited a portion of this region because of the minimal translational codon degeneracy of the conserved residues. Unique oligonucleotides from this region have been designed and used in the primer-directed in vitro DNA amplification of the hepadnaviruses as a model system. In addition, mixtures of oligonucleotides with various sequences but of the same length were demonstrated to be efficient primers. The amplification procedure enabled dramatic increases in sensitivity and coincident detection of mammalian and avian genomes. This approach will be a valuable tool to detect and characterize members of viral groups. In addition, since short stretches of similarity have been frequently identified in related but distinct genes, such an approach could prove a valuable asset to molecular studies in general.
Role of the ribosome in suppressing transcriptional termination at the pyrBI attenuator of Escherichia coli K-12.
Authors: Roland K L , Liu C G , Turnbough C L
Published: May 2006

Pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12 occurs primarily by an attenuation control mechanism. Previous studies have suggested a model for attenuation control in which low intracellular levels of UTP cause close coupling of transcription and translation within the pyrBI leader region. This close coupling apparently prevents transcriptional termination at an attenuator (a rho-independent transcriptional terminator) located 23 base pairs before the pyrBI structural genes within an open reading frame for a 44-amino acid leader polypeptide. Presumably, a ribosome involved in the synthesis of the leader polypeptide disrupts or precludes the formation of the attenuator-encoded RNA hairpin, which is required for transcriptional termination. In this study, we examined the role of the ribosome in inhibiting transcriptional termination at the pyrBI attenuator. Using oligonucleotide-directed mutagenesis, we systematically introduced termination codons into the reading frame for the leader polypeptide to determine the distance a ribosome must translate to suppress transcriptional termination. These mutations were incorporated individually into a pyrB::lacZ gene fusion, which was then introduced into the E. coli chromosome. The resulting fusion strains were used to measure the effect of each mutation on pyrB::lacZ expression. The results show that a ribosome must translate to within 14-16 nucleotides of the attenuator-encoded RNA hairpin to inhibit transcriptional termination efficiently, which indicates a direct interaction between the ribosome and the termination hairpin sequence as proposed in the present model. Additional results indicate that factors not included in the present model for attenuation control contribute to the expression and regulation of the pyrBI operon.
Two transcripts encode rat cytochrome b5 reductase.
Authors: Pietrini G , Carrera P , Borgese N
Published: May 2006

A cDNA expression library in lambda gt11 was screened with affinity-purified polyclonal anti-rat cytochrome b5 reductase antibodies. One positive clone out of 450,000 clones was isolated and found to be incomplete. This clone was used to rescreen the library, and a second, overlapping clone that contained the entire coding sequence was isolated. RNA gel blots showed that the two overlapping clones contained approximately 90% of the reductase mRNA sequence. Sequencing data showed (i) that rat reductase has a 93% sequence similarity with bovine and human reductase and (ii) that reductase is not synthesized as a high molecular weight precursor. Results of Southern blot analysis were consistent with the hypothesis that a single gene codes for the soluble and membrane-bound (microsomal and mitochondrial) forms of the reductase, present in erythrocytes and liver, respectively. The cloned cDNA was used to study reductase transcripts in liver and reticulocytes. Two antisense RNA probes that together covered the entire coding region and part of the noncoding region of reductase mRNA were used in RNase A protection experiments. These probes detected only one transcript in liver, suggesting that endoplasmic reticulum and mitochondrial reductase are translated from the same mRNA. In contrast, two transcripts were detected in reticulocytes, one of which mismatched the liver probe approximately 30 nucleotides downstream from the initiation codon. Since the soluble and membrane form of the reductase are known to differ at the N terminus, we suggest that this second transcript encodes soluble reductase.
Carboxyl-terminal mutants of the large tumor antigen of simian virus 40: a role for the early protein late in the lytic cycle.
Authors: Khalili K , Brady J , Pipas J M , Spence S L , Sadofsky M , Khoury G
Published: May 2006

Simian virus 40 (SV40) mutants dl1066 and dl1140 contain deletions within the region encoding the carboxyl terminus of the large tumor (T) antigen. Although these mutations have little effect on the efficiency of viral DNA replication, they decrease the yield of infectious virus particles by 3-4 orders of magnitude [Pipas, J. (1985) J. Virol. 54, 569-575]. Here we show that the level of late RNA is lower by a factor of 5-15 in CV-1P monkey cells infected with these mutants compared to cells infected with wild-type SV40. Consistent with this decrease in RNA, synthesis of late viral structural proteins VP1 and VP3 decreases by a factor of 5-15. In contrast, the synthesis of SV40 agnoprotein decreases by a factor greater than 100. Intercistronic complementation of these mutants with pm1493 and dl121, two SV40 mutants that are defective in agnoprotein but encode wild-type T antigen, results in an increased synthesis of agnoprotein in the infected cells. These results suggest that the carboxyl-terminal portion of T antigen participates in the posttranscriptional regulation of agnoprotein.
Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein.
Authors: Lamond A I , Konarska M M , Grabowski P J , Sharp P A
Published: May 2006

Splicing complexes that form a rabbit beta-globin precursor mRNA (pre-mRNA) have been analyzed for their small nuclear RNA (snRNA) content by both affinity chromatography and specific probe hybridization of replicas of native electrophoretic gels. A pathway of spliceosome assembly was deduced that has at least three stages. (i) U2 small nuclear ribonucleoprotein (snRNP) alone binds to sequences of mRNA upstream of the 3' splice site. (ii) U4, U5, and U6 snRNPs bind, apparently simultaneously. (iii) U4 snRNP is released to generate a spliceosome that contains U2, U5, and U6 snRNPs together with the RNA intermediates in splicing. U1 snRNP was not detected in association with any of these complexes. A parallel analysis of the spliceosome found with an adenovirus precursor mRNA substrate yielded an identical snRNP composition with one additional, unidentified RNA species, called X. This latter RNA species was not detected in the spliceosome bound to the beta-globin substrate.
Transformation of yeast with synthetic oligonucleotides.
Authors: Moerschell R P , Tsunasawa S , Sherman F
Published: May 2006

Genomic DNA of the yeast, Saccharomyces cerevisiae, can be conveniently and specifically altered by transforming spheroplasts or lithium acetate-treated cells directly with synthetic oligonucleotides. Altered forms of iso-1-cytochrome c were generated by transforming a cyc1 mutant with oligonucleotides and selecting for at least partially functional revertants; the oligonucleotides contained a sequence that corrected the cyc1 mutation and produced additional alterations at nearby sites. Transformation has been accomplished with oligonucleotides as short as 20 nucleotides and with amounts as low as 100 micrograms. This method of site-directed mutagenesis in vivo has been used to produce alterations in the NH2-terminal region of iso-1-cytochrome c in which the NH2-terminal methionine is excised and the penultimate residue is acetylated.
3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency.
Authors: Dayton E T , Pharr P , Ogawa M , Serafin W E , Austen K F , Levi-Schaffer F , Stevens R L
Published: May 2006

As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated inter-leukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. We now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content approximately equal to 50-fold and their carboxypeptidase A content approximately equal to 100-fold, and augment approximately equal to 45-fold their biosynthesis of proteoglycans bearing 35S-labeled heparin relative to 35S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.
Metastability transfer spectroscopy with two like ions in the same trap
Authors: Dehmelt Hans , Nagourney Warren
Published: May 2006

High-resolution optical spectroscopy of an individual trapped ion is hampered by lack of sharp lasers. This suggests the use of a second metastable excited ion as an ultrasharp light source. To this end, laser-cool two barium ions to an equilibrium distance of ≈8 μm on the
z
(symmetry) axis of the trap and, in this (earth)(Ba
+
)
2
-molecule, visually or photoelectrically identify them as A and B by their location. Briefly turn on a 455-nm spectral lamp until one of the ions, say the A ion, is pumped into the metastable
D
5/2
level and turns invisible. Focus on the visible, spatially well-resolved B ion and turn off the blue and red illumination lasers for ≈15 s. Then turn them back on again and check on whether the excitation by chance has been transferred to the B ion and is now in the
D
5/2
level and dark while the A ion is bright. The cross section for absorption of the λ(
D
5/2
→
S
1/2
) ≡ λ
0
= 1.76 μm radiation by a stationary ion can be >λ
0
2
/2π. Thus, by pushing the two ions together to ≈λ
0
/4 by turning on a much stronger trapping field during the excitation exchange period, one might be able to detect excitation transfer in >10% of the attempts. The ions are tuned relative to each other by a 0- to 10-mV/cm variable dc field in the
z
direction, which displaces them axially and causes them to see different rf fields, which Stark-shifts their frequencies. In this way, a resonant transfer response as sharp as twice the natural width of the
D
5/2
level, 11 mHz or a
Q
≈ 0.4 × 10
17
, might be demonstrated.
Embryonic chicken cornea and cartilage synthesize type IX collagen molecules with different amino-terminal domains.
Authors: Svoboda K K , Nishimura I , Sugrue S P , Ninomiya Y , Olsen B R
Published: May 2006

We have analyzed embryonic chicken cornea for the presence of type IX collagen mRNA and protein. Using RNA transfer blot analysis, we demonstrate that alpha 1(IX) and alpha 2(IX) mRNAs are expressed by corneal epithelial cells at the time that the primary stromal components are synthesized. The levels of the mRNAs decrease with increasing developmental age and are barely detectable at day 11 of development. In contrast, type IX collagen protein is detectable by immunofluorescence at days 5 and 6 and undetectable by day 8. Using probes specific for alpha 1(IX) and alpha 2(IX) mRNAs, we demonstrate that the size of alpha 2(IX) mRNA is the same in cornea as in chondrocytes, the major source of type IX collagen. However, the alpha 1(IX) mRNA is about 700 nucleotides shorter in the cornea than in cartilage because the corneal form of the mRNA does not contain the 5' region that encodes the non-triple-helical amino-terminal globular domain of cartilage type IX collagen. Therefore, corneal type IX collagen must lack this domain. This structural modulation of an extracellular matrix protein is likely to contribute to the functional differences between cartilage matrix and the early corneal stroma, both of which are rich in type II collagen.
Rate constancy of globin gene evolution in placental mammals.
Authors: Easteal S
Published: May 2006

The molecular clock hypothesis is investigated by comparison of the rates of nucleotide substitution in globin genes of mice, cows and goats, humans, and rabbits, using the relative rate test. These comparisons are based on a branching order of genes and species established by cladistic analysis of nucleotide sequences. The species branching order is shown to be mouse, cow/goat, human, and rabbit. Relative rate tests involving paralogous and orthologous genes provide no evidence of heterogeneity, among species, in the rate of evolution of the genes. This result is discrepant with the conclusions of most other recent, similar studies. By comparison with previous studies, the present study is based on a sound phylogeny and involves a larger sample of species, genes, and genic regions. The result provides strong support for the neutral theory of molecular evolution and demonstrates that molecular evolutionary rate does not depend on generation time.
Convulsant-induced increase in transcription factor messenger RNAs in rat brain.
Authors: Saffen D W , Cole A J , Worley P F , Christy B A , Ryder K , Baraban J M
Published: May 2006

Administration of the convulsants pentylenetetrazole (Metrazole) or picrotoxin to rats caused a dramatic increase in mRNAs of four putative transcription factor genes, zif/268, c-jun, jun-B, and c-fos, in neurons of the hippocampus and dentate gyrus, as well as other areas of the cerebral cortex, including pyriform cortex and cingulate cortex. The increase in these mRNAs was rapid and transient: amounts peaked within 1 hr and returned to baseline within 2 hr. These results extend the observation made by Morgan et al. [Morgan, J. I., Cohen, D. R., Hempstead, J. L. & Curran, T. (1987) Science 237, 192-197] that c-fos mRNA and protein are induced in rat brain after seizures. We hypothesize that the increase of these putative transcription factor mRNAs in the brain is part of a programmed genomic response of neurons to intense stimulation, which is analogous to the genomic response of nonneuronal cells to growth factors.
A Ca2+-linked increase in coupled cAMP synthesis and hydrolysis is an early event in cholinergic and beta-adrenergic stimulation of parotid secretion.
Authors: Deeg M A , Graeff R M , Walseth T F , Goldberg N D
Published: May 2006

The dynamics and compartmental characteristics of cAMP metabolism were examined by 18O labeling of cellular adenine nucleotide alpha phosphoryls in rat parotid gland stimulated to secrete with beta-adrenergic and cholinergic agents. The secretory response occurred in association with a rapidly increased rate of cAMP hydrolysis apparently coordinated with an equivalent increase in the rate of cAMP synthesis, since the cellular concentration of cAMP remained unchanged. The magnitude of this metabolic response was equivalent to the metabolism of 10-75 times the cellular content of cAMP within the first minute of stimulation. This increased metabolic rate occurred only during the early (1-3 min) period of stimulation, in what appeared to be an exclusive cellular compartment distinguished by a unique distribution of 18O among adenine nucleotide alpha phosphoryls. This 18O distribution contrasted with that produced by forskolin, which increased cellular cAMP concentration and elicited only a delayed response missing the early secretory component. The early acceleration of cAMP metabolism appeared linked to a stimulus-induced increase in intracellular Ca2+ concentration, since the Ca2+ ionophore ionomycin produced the same metabolic response in association with secretion. These observations suggest that cAMP metabolism is involved in stimulus-secretion coupling by a Ca2+-linked mechanism different from that in which cAMP plays the role of a second messenger.
Dosage-dependent modifiers of polycomb and antennapedia mutations in Drosophila.
Authors: Kennison J A , Tamkun J W
Published: May 2006

Two genes known to control the determination of segmental identity in Drosophila melanogaster are polycomb and antennapedia. To identify additional genes involved in the determination of segmental identity, we have isolated dominant modifers (both suppressors and enhancers) of polycomb and/or antennapedia mutations. Sixty-four such modifier mutations have been recovered and mapped to 18 complementation groups. All of the mutations identify genes necessary for viability of the zygote. Six of the 18 genes that were identified by mutations that interact with polycomb and/or antennapedia have been previously characterized as homoeotic genes [i.e., Sex combs reduced (Scr), Brista (Ba), trithorax (trx), Polycomb (Pc), Polycomblike (Pcl), and Sex comb on midleg (Scm)]. Mutations in several of the additional loci identified here have also been shown to have homoeotic phenotypes.
Variable region (V delta) gene segment most frequently utilized in adult thymocytes is 3' of the constant (C delta) region.
Authors: Iwashima M , Green A , Davis M M , Chien Y H
Published: May 2006

A variable region (V delta 5) of the T-cell receptor (TcR) delta chain that is preferentially expressed in adult murine thymocytes is located 2.5 kilobases 3' of the constant region (C delta) element. The V delta 5 coding sequence is in a transcriptional orientation opposite the J delta (joining region) and C delta coding elements and rearranges by inversion. The C delta is divided into four exons, three of which encode amino acids of TcR delta polypeptide, and the fourth comprises the entire 3' untranslated region. In this respect, C delta resembles C alpha rather than C beta or C gamma.
Isolation and primary structure of tumor-derived peptides related to human pancreastatin and chromogranin A.
Authors: Schmidt W E , Siegel E G , Kratzin H , Creutzfeldt W
Published: May 2006

Using an antiserum raised against a synthetic C-terminal peptide of porcine pancreastatin, we detected pancreastatin-like immunoreactivity (PLI) in human pancreatic islets, adrenal medulla, and endocrine tumors. From a carcinoid liver metastasis, human PLI was extracted and purified by HPLC. Two C-terminally amidated peptides were isolated and characterized by sequence analysis. The first peptide, hCgA-210-301, consists of 92 amino acid residues with glycinamide as C terminus. It is identical to the cDNA-derived sequence of human chromogranin A, positions 210-301, which is preceded by two basic residues indicating a putative processing site. The C-terminal part, positions 250-301, shows 70% sequence identity to porcine pancreastatin and represents the human pancreastatin-like sequence. The second peptide, hCgA-273-301, represents a C-terminally amidated fragment of the human pancreastatin sequence, generated by an Asp-Pro cleavage at the N terminus. Peptide hCgA-273-301 was synthesized to confirm the structure of the natural peptide. Two other peptides derived from human chromogranin A were isolated and partially characterized. They are generated by proteolytic cleavage after dibasic amino acids Lys-Arg (positions 338-339) and after Trp-376 of the human chromogranin A sequence, respectively. These results indicate that chromogranin A may represent the precursor for pancreastatin-related and possibly other yet-unidentified peptides of unknown physiological function.
Antide and related antagonists of luteinizing hormone release with long action and oral activity.
Authors: Ljungqvist A , Feng D M , Hook W , Shen Z X , Bowers C , Folkers K
Published: May 2006

Antide is the decapeptide N-Ac-D-Nal(2),D-Phe(pCl),D-Pal(3),Ser,Lys(Nic),D-Lys(Nic),Leu,Lys(iPr),P ro,D- Ala-NH2 [Nal(2) represents 3-(2-naphthyl)alanine; Phe(p-Cl) represents 3-(4-chlorophenyl)alanine; Pal(3) represents 3-(3-pyridyl)alanine; Lys(Nic) represents N epsilon-nicotinoyllysine; Lys-(iPr) represents N epsilon-isopropyllysine], which is an antagonist of luteinizing hormone-releasing hormone (LHRH), which has high antiovulatory activity, releases negligible histamine, and is scheduled for scale-up, safety testing, and evaluation in the experimental primate and in clinical medicine. Thirty-five more peptides were synthesized, designed on antide with variations in positions 5-8. Of these, N-Ac-D-Nal(2),D-Phe(pCl),D-Pal(3),Ser,Lys(Pic),cis-D- Ala(PzAC),Leu,Lys(iPr),Pro,D-Ala-NH2 [Lys(Pic) represents N epsilon-picoloyllysine; Ala(PzAC) represents 3-(4-pyrazinylcarbonylaminocyclohexyl)alanine] was not only the most potent but also had higher antiovulatory activity than antide--i.e., 73% per 0.25 microgram and 100% per 0.5 microgram vs. 36% per 0.5 microgram and 100% per 1.0 microgram. Antide showed significant (P less than 0.001) duration of action when injected at a dose of 10 micrograms 44 hr before injection of 50 ng of the agonist [D-Qal(3)6]LHRH [Qal(3) represents 3-(3-quinolyl)alanine]. Antide showed oral antiovulatory activity at 600 micrograms (73%) and at 1200 micrograms (100%) with negligible difference between water and corn oil oral formulations.
Lipoxins A4 and B4: comparison of icosanoids having bronchoconstrictor and vasodilator actions but lacking platelet aggregatory activity.
Authors: Lefer A M , Stahl G L , Lefer D J , Brezinski M E , Nicolaou K C , Veale C A , Abe Y , Smith J B
Published: May 2006

Lipoxins A4 (LxA4) and B4 (LxB4), two lipoxygenase-generated icosanoids of arachidonic acid metabolism, were found to have a distinct biological profile. Both LxA4 and LxB4 slowly contracted pulmonary parenchymal strips isolated from guinea pigs, rabbits, and rats in a concentration-dependent manner over the range 0.1-1 microM. This bronchoconstrictor effect was not associated with release of peptide leukotrienes or thromboxane A2, nor was it blocked by lipoxygenase inhibitors or thromboxane receptor antagonists, suggesting it is a direct effect of lipoxins. However, the leukotriene D4 (LTD4) receptor antagonist LY-171883 reduced the LxA4 response, indicating that LTD4 and LxA4 may share the same receptor. LxA4 and LxB4 also exerted an endothelium-dependent vasorelaxation in guinea pig, rat, and, to a lesser extent, rabbit aortic vascular smooth muscle. In contrast to other vasoactive icosanoids, LxA4 and LxB4 failed to aggregate rat, rabbit, or guinea pig platelets or to inhibit ADP-induced aggregation. LxA4 also enhanced the release of liver lysosomal hydrolases in a liver large granule fraction, indicating a lysosomal labilizing action of LxA4. LxA4 and LxB4 share a similar biological profile. It is not clear yet whether the lipoxins could be mediators of circulatory or pulmonary disease states.
High level of accumulation of a mRNA coding for a precursor-like protein in the submaxillary gland of male rats.
Authors: Rosinski-Chupin I , Tronik D , Rougeon F
Published: May 2006

NaDodSO4/PAGE analysis of in vitro translation products of rat submaxillary gland (SMG) mRNAs has revealed an important sexual dimorphism. Moreover, most of the rat male-specific major translation products differ in size from those translated from male mouse SMG mRNAs. To characterize proteins accumulated in the rat SMG under androgen control, a cDNA library was constructed. Here we report the nucleotide sequence of a 0.7-kilobase mRNA that is 1000-3000 times more abundant in male rats than in female rats. The predicted corresponding protein, SMR1, has a molecular weight of 16,000 and contains a signal peptide for secretion and potential signals for glycosylation. An interesting feature of SMR1 is the presence, in a hydrophilic region, of the tetrapeptide Gln-His-Asn-Pro surrounded by two pairs of basic residues that represent potential cleavage sites for maturation enzymes. In rats, the tissue distribution of the SMR1 mRNA is restricted to the SMG and the prostate. Only very low amounts of SMR1 mRNA can be detected in the SMG of male or female mice. Southern blot analysis indicates the presence of three genes in rats but only one in mice. Hypotheses on the physiological role of SMR1-derived peptides in male rats are discussed.
Construction of a genetic map of human chromosome 17 by use of chromosome-mediated gene transfer.
Authors: Xu W M , Gorman P A , Rider S H , Hedge P J , Moore G , Prichard C , Sheer D , Solomon E
Published: May 2006

We used somatic-cell hybrids, containing as their only human genetic contribution part or all of chromosome 17, as donors for chromosome-mediated gene transfer. A total of 54 independent transfectant clones were isolated and analyzed by use of probes or isoenzymes for greater than 20 loci located on chromosome 17. By combining the data from this chromosome-mediated gene transfer transfectant panel, conventional somatic-cell hybrids containing well-defined breaks on chromosome 17, and in situ hybridization, we propose the following order for these loci: pter-(TP53-RNP2-D17S1)-(MYH2-MYH1)-D17Z 1-CRYB1-(ERBA1-GCSF-NGL)-acute promyelocytic leukemia breakpoint-RNU2-HOX2-(NGFR-COLIAI-MPO)-GAA-UM PH-GHC-TK1-GALK-qter. Using chromosome-mediated gene transfer, we have also regionally localized the random probes D17S6 to D17S19 on chromosome 17.