bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligand-induced stimulation of epidermal growth factor receptor mutants with altered transmembrane regions.
Authors: Kashles O , Szapary D , Bellot F , Ullrich A , Schlessinger J , Schmidt A
Published: May 2006

The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic kinase domain by a single transmembrane (TM) region. To explore the role of the TM region in the process of receptor activation, we have generated EGF-receptor mutants with altered TM regions by utilizing in vitro site-directed mutagenesis. The TM regions of two mutant receptors were either extended (designated i626-3) or shortened (designated d625.3) by three hydrophobic amino acid residues. In the other two mutant receptors, hydrophobic amino acids were substituted by charged residues--i.e., Val-627 was replaced by glutamic acid (designated V627E) or Leu-642 was replaced by an arginine residue (designated L642R). NIH 3T3 cells lacking endogenous EGF receptors were transfected with constructs encoding either wild-type or mutant receptors and shown to express the receptor molecules using 125I-labeled EGF binding and immunoprecipitation experiments. The mutant receptors were expressed on the cell surface as polypeptides of Mr 170,000 exhibiting typical high- and low-affinity binding sites for 125I-labeled EGF. Similar to its effect on wild-type receptors, phorbol 12-myristate 13-acetate abolished the mutant-receptor high-affinity binding sites for EGF. Moreover, EGF was able to stimulate the kinase activities of wild-type and mutant receptors both in vitro and in living cells. The mutant receptors were also able to undergo EGF-induced receptor dimerization as revealed by cross-linking experiments with a bifunctional covalent cross-linking agent. These results are compatible with an intermolecular allosteric oligomerization model for receptor activation rather than with a model based on an intramolecular mechanism for receptor activation.
Detection of two growth hormone receptor mRNAs and primary translation products in the mouse.
Authors: Smith W C , Linzer D I , Talamantes F
Published: May 2006

Two mouse growth hormone-receptor primary translation products of Mr 95,900 and 31,800 were identified from in vitro-translated late pregnant mouse liver mRNA. RNA isolated from mouse liver was translated in a rabbit reticulocyte lysate system containing [35S]methionine, and the growth hormone receptor primary translation products were identified by immunoprecipitation with anti-mouse growth hormone receptor antiserum followed by sodium dodecyl sulfate/PAGE and fluorography. Detectable amounts of the Mr 95,900 and 31,800 proteins were not present in in vitro-translated nonpregnant mouse liver mRNA. This result is consistent with previous observations of the up-regulation of growth hormone receptors in the liver during pregnancy in the mouse. Northern (RNA) blot analysis of mouse liver and adipose tissue RNA with a rabbit growth hormone receptor cDNA probe revealed two hybridizing mRNAs of approximately 3.9 and 1.2 kilobases. These two RNAs were greatly up-regulated in liver, but not in adipose tissue, during pregnancy. The sizes of these mRNAs closely matched predictions of the sizes of the mRNAs coding for the proteins of Mr 95,900 and 31,800 made by in vitro translation of size-fractionated late-pregnant mouse liver poly(A)+RNA. These results suggest a mechanism for the generation of both the heterogeneous forms of the growth hormone receptor identified in mouse liver membrane preparations and the mouse serum growth hormone-binding protein.
Antisense ribosomal protein gene expression specifically disrupts oogenesis in Drosophila melanogaster.
Authors: Qian S , Hongo S , Jacobs-Lorena M
Published: May 2006

To assess the functional importance of ribosomal protein rpA1 gene expression during development of Drosophila melanogaster, we have transformed into the fly's genome an antisense rpA1 gene driven by a heat shock promoter. Antisense rpA1 expression severely disrupted oogenesis and produced a "small egg" female-sterile phenotype. The severities of these defects were proportional to the level of antisense rpA1 expression. Anti-rpA1 expression did not affect larval or pupal development. Quantitative RNA analysis suggested that high anti-rpA1 expression results in a general decrease of mRNA in the ovary.
Interaction of spatially separated protein-DNA complexes for control of gene expression: operator conversions.
Authors: Haber R , Adhya S
Published: May 2006

Two operators, spatially separated from each other and from the promoters, repress the gal operon when bound to Gal repressor. Conversion of either gal operator to a lac operator results in derepression, although both Gal and Lac repressors are present, suggesting that mere occupation of operator sites is not sufficient to cause repression. Conversion of both operators to lac operators restores normal repression in the presence of Lac repressor protein. We propose that normal repression requires interaction between operator-bound like repressor molecules; this generates a DNA loop, which is part of a higher order structure. RNA polymerase and cyclic AMP receptor protein are present in this complex but unable to initiate transcription because of the higher order structure. Such higher order DNA-multiprotein complexes could occur in a variety of genetic regulatory systems that are controlled from distal sites by regulatory proteins.
Activation of human T lymphocytes: differential effects of CD3- and CD8-mediated signals.
Authors: Samstag Y , Emmrich F , Staehelin T
Published: May 2006

T cells are activated physiologically by triggering the T-cell receptor-CD3 complex. There is evidence that invariant accessory molecules on the T-cell membrane (CD8 and CD4) are involved in the major histocompatibility complex-restricted recognition process. Moreover, binding and crosslinking of these accessory molecules to the T-cell receptor-CD3 complex exerts a positive synergistic signal, as has been shown by stimulation with crosslinked antibodies. Here we demonstrate that stimulation mediated by immobilized anti-CD3/CD8 antibodies differs from stimulation mediated solely by anti-CD3. Whereas interleukin 2 receptor expression and interferon gamma production are seen to a similar extent in both cases, a second signal provided by the additional involvement of CD8 seems to be essential for interleukin 2 production and full interleukin 2 responsiveness in CD8+ T cells. This second signal is much more sensitive to inhibition by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C and cGMP/cAMP-dependent kinases. Our results also show that substantial modulation of the T-cell receptor complex and most likely CD3 phosphorylation are not essential for initiating the activation of resting T cells. Instead, we found a 22- to 24-kDa phosphoprotein whose strong phosphorylation correlated reliably with T-cell activation.
Human T-cell antigen receptor (TCR) delta-chain locus and elements responsible for its deletion are within the TCR alpha-chain locus.
Authors: Hockett R D , de Villartay J P , Pollock K , Poplack D G , Cohen D I , Korsmeyer S J
Published: May 2006

Individual T cells express the CD3 molecule in association with alternative gamma delta or alpha beta heterodimeric T-cell receptors (TCRs). T-cell precursors and occasional gamma delta-expressing T cells in humans possess an unexpected 2.0-kilobase (kb) mRNA in which a tandemly repeated motif, TEA (T early alpha), has been spliced to the constant (C alpha) region. Long-range pulsed-field gel mapping as well as molecular cloning showed that TEA is located immediately 5' to the most upstream joining (J alpha) segment of the TCR alpha-chain locus. The TCR delta-chain locus is immediately 5' to TEA, and diversity (D delta) gene segments, J delta, C delta, and TEA are linked within 35 kb. The human TCR delta locus conserves a 12/23-base-pair (bp) spacer paradigm in which J delta possesses a 12-bp and V delta a 23-bp spacer, while the D delta segments have a 12 bp-D delta-23 bp spacer motif. Considerable TCR delta diversity can be generated despite the predominant use of one V delta and one J delta segment. Two D delta segments, D delta 1 and D delta 2, are 9 and 13 bp long, are frequently recombined as D delta 1-D delta 2, and reveal exonucleolytic trimming with extensive N-segment addition. A gamma delta clonal T cell possessed an effective VDDJ delta rearrangement and an intermediate DDJ delta rearrangement, arguing that the TCR delta locus displays allelic exclusion. Specific rearranging elements that delete the delta locus, delta Rec and psi J alpha, were mapped and found to separate the delta locus from the alpha locus. The delta locus including D delta 1-D delta 2-J delta 1-C delta-TEA was deleted in mature, alpha beta-expressing T cells, whereas V delta 1 was frequently retained. The location of the delta locus within the alpha locus may necessitate an exclusive choice between delta or alpha expression.
Anti-human immunodeficiency virus type 1 antibody complexes on platelets of seropositive thrombocytopenic homosexuals and narcotic addicts.
Authors: Karpatkin S , Nardi M , Lennette E T , Byrne B , Poiesz B
Published: May 2006

Patients with human immunodeficiency virus type 1 (HIV-1) infection develop an immunologic thrombocytopenic purpura associated with markedly elevated platelet IgG, IgM, and C3C4 as well as serum immune complexes determined by the polyethylene glycol (PEG) method. Analysis of their serum PEG-precipitable immune complexes as well as platelet acid eluates revealed the presence of anti-HIV-1 antibody existing as a complex that eluted in the void volume of a Sephadex G-200 gel-filtration column. The complex binds to washed normal platelets, whereas affinity-purified anti-HIV-1 (gp120) antibody does not. HIV-1 antigen or proviral DNA was not detectable in the immune complexes or platelet extracts. However, anti-antibodies directed against anti-HIV-1 antibody were detectable in the immune complexes as well as platelet eluates. Approximately 50% of eluted platelet IgG contained anti-HIV-1 antibody. Thus the markedly elevated platelet immunoglobulin is partly due to the presence of anti-HIV-1 antibody complexes. This may be responsible for the enhanced platelet clearance and thrombocytopenia in patients with acquired immunodeficiency syndrome-related immunologic thrombocytopenia.
Nerve growth factor increases mRNA levels for the prion protein and the beta-amyloid protein precursor in developing hamster brain.
Authors: Mobley W C , Neve R L , Prusiner S B , McKinley M P
Published: May 2006

Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the beta protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the beta-protein precursor (beta-PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and beta-PP mRNAs. The increases in PrP and beta-PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and beta-PP mRNA levels. Increased PrP and beta-PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and beta-PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.
Regulatory role of galanin in control of hypothalamic-anterior pituitary function.
Authors: Ottlecz A , Snyder G D , McCann S M
Published: May 2006

The role of the neuropeptide galanin in the regulation of anterior pituitary function was studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Galanin (50-200 ng; 15-60 pmol) injected into the third cerebral ventricle of rats produced highly significant, dose-related increases of plasma growth hormone (GH) concentrations, whereas galanin increased prolactin (PRL) and decreased thyroid-stimulating hormone (TSH) levels only at the highest dose (60 pmol) tested. Intravenous galanin failed to alter PRL and TSH levels in these rats. In contrast with the results with intraventricular injection of the peptide, intravenous injection of 30 or 300 pmol of galanin produced small, brief, dose-related increases in plasma GH. The response to the 300-pmol dose was less than that induced by a factor-of-20-lower intraventricular dose, which establishes a central action of galanin. Galanin in concentrations ranging from 1 nM to 1 microM failed to alter significantly GH, PRL, or TSH release from dispersed anterior pituitary cells. It also failed to alter GH secretion in response to 100 nM GH-releasing hormone; however, at this dose galanin did potentiate the effect of 100 nM TSH-releasing hormone on TSH and PRL release. Thus, the effects of third-ventricular injection of the peptide are mediated by the hypothalamus. To determine the physiological significance of galanin in control of pituitary hormone release, highly specific antiserum against galanin was injected intraventricularly. Third-ventricular injection of 3 microliter of galanin antiserum resulted in a dramatic decrease in plasma GH values as compared with those of normal rabbit serum-injected controls within 15 min, which persisted until the end of the experiment (5 hr postinjection). Galanin antiserum did not decrease plasma PRL or TSH levels at any time period after its third-ventricular injection; however, a transient increase of plasma TSH levels occurred after 30 and 60 min in comparison with TSH levels in normal rabbit serum-injected controls. Since there was no effect of the antiserum on plasma PRL and only a transient elevation in TSH, galanin may not be physiologically significant enough during resting conditions to alter PRL and TSH release in the male rat. The results of the experiments with galanin antiserum indicate that endogenous galanin has a tonic action within the hypothalamus to stimulate GH release. The rapidity of onset of the effects of galanin and the antiserum directed against it suggest that it acts to stimulate release of GH-releasing hormone from periventricular structures, which then stimulates the release of GH.
Use of antiserum to neurotensin reveals a physiological role for the peptide in rat prolactin release.
Authors: Vijayan E , Carraway R , Leeman S E , McCann S M
Published: May 2006

Previous studies have indicated that the brain peptide neurotensin can stimulate prolactin release by direct action on the pituitary gland, whereas its action within the hypothalamus is inhibitory. The inhibitory action is mediated by the release of dopamine into the hypophyseal portal veins, which deliver the neurotransmitter to the anterior pituitary gland to inhibit prolactin release. Our experiments were done to evaluate the physiologic significance of these neurotensin actions by injecting the globulin fraction of highly specific neurotensin antiserum either intravenously or intraventricularly. Injection into the third ventricle of either 1 or 3 microliter of neurotensin antiserum significantly increased plasma prolactin concentrations in (i) ovariectomized and (ii) ovariectomized estrogen- and progesterone-primed rats within 1 hr of injection. The response was more pronounced in the ovariectomized than in the ovariectomized estrogen- and progesterone-treated animals and was dose related. Intraventricular injection of these doses of neurotensin antiserum also evoked elevations in plasma prolactin in intact males, which were significant but smaller in magnitude than those seen in female rats. To evaluate the effect of the antiserum on the pituitary directly, the antiserum was injected intravenously at a dose of 40 microliter, which was sufficient to block the blood pressure-lowering effect of neurotensin. After the intravenous injection of antiserum, a highly significant suppression of plasma prolactin occurred, detectable when first measured at 1 hr after injection in both ovariectomized and ovariectomized estrogen- and progesterone-treated animals; however, the intravenous injection of antiserum had no significant effect on the prolactin release in males. These data indicate the physiological significance of the hypothalamic inhibitory actions of neurotensin on prolactin release, which are probably mediated by its stimulation of dopamine release that in turn, inhibits prolactin secretion by the lactotropes. The direct stimulatory effect of the peptide on prolactin release after its presumed release into portal vessels also appears to be physiologically significant in female but not in male rats.
Simultaneous transfection of COS-1 cells with mitochondrial and microsomal steroid hydroxylases: incorporation of a steroidogenic pathway into nonsteroidogenic cells.
Authors: Zuber M X , Mason J I , Simpson E R , Waterman M R
Published: May 2006

Transfected, nonsteroidogenic COS-1 cells derived from monkey kidney are found to be capable of supporting the initial and rate-limiting step common to all steroidogenic pathways, the side-chain cleavage of cholesterol to produce pregnenolone. Endogenous COS-1 kidney cell renodoxin reductase and renodoxin are able to sustain low levels of this activity catalyzed by bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) whose synthesis is directed by a transfected plasmid containing P450scc cDNA. Double transfection with both P450scc and adrenodoxin plasmids leads to greater pregnenolone production and indicates that adrenodoxin plays a role as a substrate for this reaction or that bovine adrenodoxin serves as a better electron donor than the endogenous iron-sulfur protein renodoxin. Also it is found that both the bovine adrenodoxin and P450scc precursor proteins are proteolytically processed upon their uptake by COS-1 cell mitochondria to forms having the same electrophoretic mobility as mature bovine adrenodoxin and P450scc. Following triple transfection of COS-1 cells with P450scc, adrenodoxin, and 17 alpha-hydroxylase cytochrome P450 plasmids, pregnenolone produced in mitochondria by the side-chain cleavage reaction can be further metabolized in the endoplasmic reticulum to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Although this functional steroidogenic pathway can be incorporated into this nonsteroidogenic cell type, it is found to be nonresponsive to cAMP, a potent activator of steroid hormone biosynthesis in adrenal cortex, testis, and ovary. Thus the cellular mechanisms necessary to support both microsomal and mitochondrial steroid hydroxylase activities appear not to be tissue specific, whereas the acute cAMP-dependent regulation of steroidogenesis is not present in transformed kidney (COS-1) cells.
Structure of a pre-mRNA branch point/3' splice site region.
Authors: Hall K B , Green M R , Redfield A G
Published: May 2006

We have analyzed the solution structure of RNA containing the branch point/3' splice site region of the first intervening sequence (IVS1) of human beta-globin pre-mRNA by nuclease mapping and NMR. Nuclease mapping indicates that there are two distinct structural domains: one contains the branch point region, and the other includes the 3' splice site and second exon sequences. In the branch point domain, the adenosine at which the RNA branch forms appears to be in the loop of a stem/loop structure. The branch point structure does not appear to interact with other parts of the RNA, since its unique nuclease digestion pattern is conserved among transcripts containing the entire intron or only the branch point region. This is confirmed by a comparison of the NMR spectra of two RNA transcripts; a distinct set of resonances appears in the spectra of the RNA containing only branch sequences or including 3' splice site/exon 2 sequences. NMR studies further show that the 3' splice site/exon 2 domain has a lower melting temperature than the branch point domain, suggesting that the two regions are distinct dynamically as well as structurally. Nuclease mapping studies of adenovirus major late IVS1 indicate that this RNA has structural features in common with the human beta-globin transcript.
Positive charges at the NH2 terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide.
Authors: Szczesna-Skorupa E , Browne N , Mead D , Kemper B
Published: May 2006

The NH2-terminal sequences of cytochromes P-450 resemble signal peptides, but these sequences are not cleaved during the insertion of these integral membrane proteins into the microsomes. To examine whether these putative signal peptides are functionally equivalent to signal peptides of secretory proteins, cDNA coding for a fusion protein was produced, in which the signal peptide for preproparathyroid hormone was replaced with the putative signal peptide of cytochrome P450IIC2. The translational product of RNA synthesized in vitro from the cDNA was neither processed nor translocated by chicken oviduct microsomal membranes in a reticulocyte cell-free system but was resistant to extraction from the membranes by alkaline solutions. In addition, the translation of the hybrid RNA was arrested by signal recognition particle. Unlike most signal peptides, the cytochrome P450IIC2 NH2-terminal sequence does not contain basic amino acids preceding the hydrophobic core. Introduction by oligonucleotide-directed mutagenesis of lysine and arginine at the NH2 terminus resulted in a fusion protein that was partially processed by the microsomal membranes, with translocation across the membrane of both the processed and unprocessed proteins. The positive charges convert the cytochrome P450IIC2 NH2 terminus from a combination membrane insertion-halt transfer signal to a more classical secretory membrane-insertion signal, possibly by altering the orientation of the signal peptide in the membrane.
The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5' stem-loop of U1 RNA and interacts with Sm domain proteins.
Authors: Patton J R , Pederson T
Published: May 2006

The U1 small nuclear ribonucleoprotein (snRNP) particle, a cofactor in mRNA splicing, contains nine proteins, six of which are also present in other U snRNPs and three of which are specific to the U1 snRNP. Here we have used a reconstituted human U1 snRNP together with snRNP monoclonal antibodies to define the RNA binding sites of one of the U1 snRNP-specific proteins. When Sm monoclonal antibody (specific for the B', B, and D proteins of U snRNPs) was bound to U1 snRNPs prior to micrococcal nuclease digestion, the same approximately equal to 24 nucleotide fragment of U1 RNA (corresponding to nucleotides 120-143 and termed the "Sm domain") was protected as when no antibody was bound prior to digestion. In contrast, when RNP monoclonal antibody, which reacts with the U1 snRNP-specific Mr 70,000 protein, was bound, additional U1 RNA regions were protected against nuclease digestion. This phenomenon, which we term "antibody-mediated nuclease protection," was exploited to map the position of the Mr 70,000 protein to stem-loop I of U1 RNA. However, there were also sites of Mr 70,000 protein interaction with more 3'-ward regions of U1 RNA, particularly the Sm domain. This indicates that in the three-dimensional structure of the U1 snRNP, the RNP and Sm antigens are in contact with each other. The proximity of the Mr 70,000 protein's RNA binding site (stem-loop I) to the functionally important 5' end of U1 RNA suggests that this protein may be involved in the recognition of, or stabilization of base pairing with, pre-mRNA 5' splice sites.
Antimicrobial activity of synthetic magainin peptides and several analogues.
Authors: Zasloff M , Martin B , Chen H C
Published: May 2006

We have previously reported the isolation of two broad-spectrum antimicrobial peptides ("magainins") from the skin of the African clawed frog Xenopus laevis. These natural peptides are active against many species of bacteria and fungi and also induce osmotic lysis of protozoa. In this report we demonstrate that synthetic magainin peptides appear to be indistinguishable from the natural products with respect to chromatographic properties and biological activity. These studies demonstrate conclusively that the magainin peptides are potent antimicrobial substances.
Selective induction of brain type II Na+ channels by nerve growth factor.
Authors: Mandel G , Cooperman S S , Maue R A , Goodman R H , Brehm P
Published: May 2006

Cells derived from a rat pheochromocytoma (PC12 cells) can generate an action potential only upon treatment with nerve growth factor. Using electrophysiological methods, we found that the appearance of action potentials in nerve growth factor-treated PC12 cells can be explained by an increase in the density of Na+ channels. The functional properties of Na+ channels in PC12 cells are similar to those described for peripheral nerves but appear to be different from Na+ channels synthesized in Xenopus oocytes injected with brain type II Na+ -channel mRNA. To determine if PC12 cells express the brain type II Na+ -channel gene, we performed RNase-protection analyses using probes that can distinguish between the brain type I and type II Na+ -channel mRNAs. The results from these studies indicate that undifferentiated PC12 cells express the type II but not the type I Na+ -channel gene. Treatment with nerve growth factor increases expression of the type II Na+ -channel gene but has no effect on type I gene expression. Our findings suggest that Na+ -channel excitability in PC12 cells is due to the specific induction of the brain type II gene by nerve growth factor.
Isolation and expression of rat 1,25-dihydroxyvitamin D3 receptor cDNA.
Authors: Burmester J K , Maeda N , DeLuca H F
Published: May 2006

The cDNA for the 1,25-dihydroxyvitamin D3 receptor has been isolated by screening a rat kidney lambda gt11 expression library with a mixture of three previously identified anti-receptor monoclonal antibodies. Protein produced from this cDNA reacts with each of the three monoclonal antibodies but does not react with an antibody specific for the porcine receptor or an irrelevant antibody. In addition, the expressed protein specifically binds 1,25-dihydroxyvitamin D3 but does not bind estradiol, progesterone, testosterone, cortisol, or aldosterone. The nucleotide sequence for this cDNA has been determined and used to deduce the amino acid sequence of the protein. This amino acid sequence contains a region that matches perfectly the partial amino acid sequence determined for the receptor isolated from porcine intestine. This amino acid sequence also closely matches the reported amino acid sequence of the putative DNA-binding finger of the avian 1,25-dihydroxyvitamin D3 receptor.
Intrinsic membrane glycoproteins with cytosol-oriented sugars in the endoplasmic reticulum.
Authors: Abeijon C , Hirschberg C B
Published: May 2006

We have examined the topography of N-acetylglucosamine-terminating glycoproteins in membranes from rat liver smooth and rough endoplasmic reticulum (SER and RER). It was found that some of these glycoproteins are intrinsic membrane proteins with their sugars facing the cytosolic rather than the luminal side. This conclusion was reached by using vesicles from the SER and RER that were sealed and of the same topographical orientation as in vivo. These vesicles were incubated with UDP-[14C]galactose (which does not enter the vesicles) and saturating amounts of soluble galactosyltransferase from milk, an enzyme that does not penetrate the lumen of the vesicles and that specifically adds galactose to terminal N-acetylglucosamine in a beta 1-4 linkage. Radioactive galactose was mainly transferred to SER proteins of apparent molecular mass 56 and 110 kDa and to a lesser extent to RER and SER proteins of apparent molecular mass 46 and 72 kDa. These proteins are intrinsic membrane proteins, based on the inability of sodium carbonate at pH 11.5 to remove them from the membranes. Studies with peptide N-glycosidase F and chemical beta-elimination showed that the 56-kDa protein of the SER vesicles contained terminal N-acetylglucosamine in an O-linkage to the protein. The above results suggest that some sugars of glycoproteins in the endoplasmic reticulum may attain their final orientation in the membrane by mechanisms yet to be determined.
Chimeric proteins define variable and essential regions of Ha-ras-encoded protein.
Authors: Lowe D G , Ricketts M , Levinson A D , Goeddel D V
Published: May 2006

The biological role of amino acid differences between the human 21-kDa Ha-ras protein (p21) and the human 23-kDa R-ras protein (p23) was investigated by engineering mutant Ha-ras p21 molecules containing divergent amino acid sequences from R-ras p23. Variant amino acids from R-ras p23 regions 1-30, 52-57, 67-78, 1-30 and 67-78 together, and 112-124 were substituted for the corresponding Ha-ras p21 amino acid regions 1-4, 26-31, 41-52, 1-4 and 41-52 together, and 86-98, respectively. Rat fibroblasts transfected with genes encoding these position-12 valine-substituted chimeric Ha-ras proteins displayed the same properties of morphological transformation and anchorage-independent growth as Ha-ras T24 oncogene-transformed fibroblasts. However, substitution of variant amino acids from the 80 C-terminal residues (amino acids 138-218) of R-ras p23 for the corresponding p21 amino acids (residues 112-189) inactivated the transforming activity of position-12 valine-substituted p21. The converse substitution of Ha-ras p21 C-terminal residues into R-ras p23 did not result in transformation by position-38 valine-substituted p23. These data are discussed in terms of the structure of ras proteins and the nature of interactions determining the specificity of effector function.
Tissue-specific expression of the rat galanin gene.
Authors: Kaplan L M , Spindel E R , Isselbacher K J , Chin W W
Published: May 2006

We have isolated and characterized cDNAs encoding rat galanin from a cDNA library prepared from rat hypothalamic tissue. Analysis of these clones reveals that rat galanin is synthesized initially as part of a 124-amino acid precursor that includes a signal peptide, galanin (29 amino acids), and a 60-amino acid galanin mRNA-associated peptide. In the precursor, galanin includes a C-terminal glycine and is flanked on each side by dibasic tryptic cleavage sites. The deduced amino acid sequence of rat galanin is 90% similar to porcine galanin, with all three amino acid differences in the C-terminal heptapeptide. The predicted galanin mRNA-associated peptide includes a 35-amino acid sequence that is 78% similar to the previously reported porcine analogue. This sequence is set off by a single basic tryptic cleavage site and includes a 17-amino acid region that is nearly identical to the porcine counterpart. The high interspecies conservation suggests a biological role for this putative peptide. Blot hybridization analysis using rat genomic DNA is consistent with a single galanin-encoding gene. RNA blot analysis of total RNA prepared from rat tissues reveals a single band of hybridizing mRNA that is approximately 900 nucleotides long. Rat galanin mRNA is located predominantly in the central nervous system and gastrointestinal tract. Highest central nervous system concentrations are found in the hypothalamus, with lower levels in the cortex and brainstem. Gastrointestinal rat galanin mRNA is most abundant in the duodenum, with progressively lower concentrations in the stomach, small intestine, and colon.
A hydroxyproline-rich protein in the soybean cell wall.
Authors: Averyhart-Fullard V , Datta K , Marcus A
Published: May 2006

A cDNA clone that hybridizes to an mRNA (1A10) that accumulates to a substantial level in the axis of the germinating soybean seed was sequenced. The amino acid sequence of the clone indicates an almost perfect repeat of Pro-Pro-Val-Tyr-Lys resulting in a protein containing 40% proline and lacking serine and histidine. On the likelihood that such a protein might be a hydroxyproline-rich cell-wall glycoprotein (HRGP), cell walls of a soybean cell culture were extracted by procedures used to obtain soluble basic cell-wall glycoproteins, and the proteins were fractionated and purified. A 33-kDa protein (and possibly a 28-kDa protein) was obtained that has an amino acid distribution similar to that of the cDNA clone. The protein lacks histidine and serine and contains 20% hydroxyproline and 20% proline. The HRGP is thus distinct both in its amino acid content and in its pentameric repeat of Pro-Pro-Val-Tyr-Lys, with half of the prolines being hydroxylated.
"Calciosome," a cytoplasmic organelle: the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of nonmuscle cells?
Authors: Volpe P , Krause K H , Hashimoto S , Zorzato F , Pozzan T , Meldolesi J , Lew D P
Published: May 2006

Calsequestrin (CS) is the protein responsible for the high-capacity, moderate affinity binding of Ca2+ within the terminal cisternae of the sarcoplasmic reticulum, believed up to now to be specific for striated muscle. The cells of two nonmuscle lines (HL-60 and PC12) and of two rat tissues (liver and pancreas) are shown here to express a protein that resembles CS in many respects (apparent mass and pH-dependent migration in NaDodSO4/PAGE; blue staining with StainsAll dye; Ca2+ binding ability) and is specifically recognized by affinity-purified antibodies against skeletal muscle CS. In these cells, the CS-like protein is shown by immunofluorescence and immunogold procedures to be localized within peculiar, heretofore unrecognized structures distributed throughout the cytoplasm. These structures appear to be discrete organelles, which we propose to be named "calciosomes." By cell fractionation (Percoll gradient and free-flow electrophoresis), the CS-like protein of HL-60 cells is shown to copurify with the markers of the inositol 1,4,5-trisphosphate (Ins-P3)-sensitive Ca2+ store, whereas the markers of other organelles (endoplasmic reticulum, Golgi complex, mitochondria, endosomes) and of the plasma membrane do not. Calciosome might thus be the intracellular target of Ins-P3--i.e., the source of the Ca2+ redistributed to the cytosol following receptor-triggered generation of the messenger.
Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability.
Authors: Penttinen R P , Kobayashi S , Bornstein P
Published: May 2006

Transforming growth factor-beta (TGF-beta) has been shown to stimulate synthesis of extracellular matrix proteins, both in animals and in cell culture. We found that mRNAs for alpha 1(I) collagen, fibronectin, and thrombospondin were markedly increased in TGF-beta-treated 3T3 (mouse) cells. For collagen and fibronectin this increase was 10-to 20-fold, as measured by quantitative blot hybridization analysis. A maximal value was reached at 16-24 hr, with a subsequent gradual decline. Concomitant treatment with cycloheximide prevented the stimulation observed with TGF-beta. Under conditions of confluent growth a clear increase in alpha 1(I) collagen mRNA stability was observed, whereas in subconfluent cells no change in mRNA half-life was found, despite an equally large increase in mRNA levels. We suggest that the mode of action of TGF-beta varies with the target cell and depends on the interplay of a number of complex cellular factors.
Learning from lesions: patterns of tissue inflammation in leprosy.
Authors: Modlin R L , Melancon-Kaplan J , Young S M , Pirmez C , Kino H , Convit J , Rea T H , Bloom B R
Published: May 2006

The clinical forms of leprosy constitute a spectrum that correlates closely with the degree of cell-mediated immunity. Patients with tuberculoid leprosy develop strong cell-mediated responses and have only a few, localized lesions, whereas patients with multibacillary lepromatous leprosy are specifically unresponsive to antigens of Myobacterium leprae. T cells of the CD4+ subset predominate in tuberculoid lesions, whereas CD8+ cells predominate in lepromatous lesions. Monoclonal antibodies that distinguish subpopulations of CD4+ and CD8+ cells were used to analyze the distribution of T cells infiltrating lesions across the disease spectrum. In lepromatous lesions, T cells of T-suppressor phenotype (9.3-) were the predominant CD8+ cells and suppressor/inducer cells (2H4+, Leu-8+) represented half of the CD4+ subset. In tuberculoid lesions, helper T cells (CD4+4B4+) outnumbered suppressor/inducer T cells by 14:1, compared with a ratio of 1.2:1 in peripheral blood. Analysis of the precursor frequency of antigen-reactive T cells permitted us to estimate that there was a 100-fold enrichment of T cells able to proliferate in response to M. leprae antigens in tuberculoid lesions (2/100), when compared with blood from the same patients. The methods used here to characterize the T-lymphocyte subsets and frequency of antigen-reactive T cells in leprosy may be useful in analyzing immunological reactions occurring in lesions of other inflammatory and autoimmune diseases.
Induction of indoleamine 2,3-dioxygenase: a mechanism of the antitumor activity of interferon gamma.
Authors: Ozaki Y , Edelstein M P , Duch D S
Published: May 2006

The antiproliferative effects of interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) were found to be cell-dependent. Among the human cell lines examined, IFN-gamma had a greater antiproliferative effect against cell lines that exhibited induction of indoleamine 2,3-dioxygenase, such as the KB oral carcinoma or WiDr colon adenocarcinoma, than against those that lacked the enzyme activity, such as the SW480 colon adenocarcinoma or NCI-H128 small-cell lung carcinoma. Induction of this dioxygenase showed a clear temporal relationship with increased metabolism of L-tryptophan and the depletion of this amino acid in the culture medium. While 70-80% of L-tryptophan remained in the medium of IFN-alpha- or vehicle-treated cells, virtually all of this amino acid was depleted in the medium of the IFN-gamma-treated group following 2-3 days of culture. Supplementing the growth medium with additional L-tryptophan reversed the antiproliferative effect of IFN-gamma against KB cells in a dose- and time-dependent manner. The antiproliferative effects of IFN-alpha and IFN-gamma on SW480 and NCI-H128 cells, which are independent of the dioxygenase activity, and the inability of added L-tryptophan to reverse the effects of IFN-gamma in WiDr cells suggest multiple mechanisms of action of the IFNs. The data show that the antiproliferative effect of IFN-gamma through induction of indoleamine 2,3-dioxygenase, with a consequent L-tryptophan deprivation, is an effective means of regulating cell growth.
cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor.
Authors: Rydel R E , Greene L A
Published: May 2006

Nerve growth factor (NGF) is a neurotrophic agent for sympathetic and embryonic sensory neurons both in vivo and in vitro. We report here that the membrane-permeant cAMP analogs, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, can replace NGF in promoting long-term survival and neurite outgrowth in cultures of rat neonatal sympathetic and embryonic sensory neurons. N6-substituted analogs, including the more commonly used N6,O2'-dibutyryl-cAMP, are less efficacious. Additivity and switching experiments indicate that the cAMP analogs affect the same neuronal population as that maintained by NGF. However, unlike NGF, the cAMP analogs do not evoke somatic hypertrophy. Moreover, studies with sympathetic neurons reveal that the neurotrophic actions of the cAMP analogs, but not of NGF, are blocked by the axial diastereoisomer of adenosine 3',5'-phosphorothioate, a competitive cAMP antagonist. Thus, the mechanism by which cAMP analogs promote neuronal survival and differentiation appears to involve activation of cAMP-dependent protein kinases, whereas, in contrast, the same effects of NGF neither require nor are mediated by such a pathway. Furthermore, the different efficacies observed with N6- and C8-substituted cAMP analogs suggest that this neurotrophic pathway may involve differential activation of the regulatory subunits of cAMP-dependent protein kinases. The presence of this parallel, cAMP-responsive, neurotrophic pathway in at least two types of NGF-responsive neurons may be developmentally important and has the potential to be exploited for the treatment of injuries or diseases affecting these and possibly other nerve cells.
BamHI E region of the Epstein-Barr virus genome encodes three transformation-associated nuclear proteins.
Authors: Ricksten A , Kallin B , Alexander H , Dillner J , Fåhraeus R , Klein G , Lerner R , Rymo L
Published: May 2006

Recombinant vectors carrying DNA fragments from the BamHI E region of the B95-8 Epstein-Barr virus (EBV) genome were transfected into COS-1 cells, and the transient expression of EBV-encoded nuclear antigens (EBNAs) was analyzed by using polyvalent human antisera and rabbit antibodies to synthetic peptides. Vector DNA containing two rightward open reading frames in the BamHI E fragment, BERF2a and BERF2b, induced the expression of a nuclear antigen identical serologically and with respect to size to the larger of the two polypeptides previously designated as EBNA4 in B95-8 cells. An antigen corresponding to the smaller polypeptide was induced in cells transfected with constructs that contained two neighboring reading frames, BERF3 and BERF4. This antigen also reacted with a rabbit antiserum to the synthetic peptide 203, deduced from BERF4. Thus, the findings show that the two components of the EBNA4 doublet in B95-8 cells are encoded by separate genes. The antigen encoded by BERF2a and/or BERF2b has been designated as EBNA4 and the antigen encoded by BERF3 and/or BERF4 has been designated as EBNA6. Polyvalent human antisera detected EBNA4 and EBNA6 in 9 of 11 lymphoid cell lines carrying independent EBV isolates. In the remaining two lines, either EBNA4 or EBNA6 was not detectable.
Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene.
Authors: Hirakawa T , Ruley H E
Published: May 2006

Established REF52 cells (rat embryo fibroblasts) completely resist stable transformation by ras oncogenes, and simian virus 40 large tumor (T) antigen collaborates with ras to convert REF52 cells to tumorigenic state. A temperature-sensitive simian virus 40 large T antigen (encoded by tsA58) allowed the T24 Ha-ras oncogene to transform REF52 cells in a temperature-dependent manner. Two thirds of the clones transformed with tsA58 and ras became arrested in G2 or late S phase when shifted to a nonpermissive temperature for T antigen stability. Thus, ras induced growth arrest rather than stable transformation in the absence of a functional collaborating oncogene. These results indicate that collaborating oncogenes can regulate cellular responses to ras and have implications regarding therapeutic strategies to control tumor cells expressing activated ras oncogenes.
Isolation and characterization of the apical surface of polarized Madin-Darby canine kidney epithelial cells.
Authors: Sambuy Y , Rodriguez-Boulan E
Published: May 2006

We have developed a fast and efficient method to isolate the apical surface of Madin-Darby canine kidney epithelial cells. After confluent cell monolayers were coated with alternate layers of cationized colloidal silica and a polyanion, 60% of the apical surface was recovered as large membrane sheets through the application of a polylysine-coated glass surface. Scanning electron microscopy of the cytoplasmic aspect of the apical surface revealed a honeycomb pattern given by the cell borders fractured at or above the level of the tight junctions. By transmission electron microscopy, the apical preparation appeared to be composed of plasma membrane and a thin layer of cytoplasm. Enzyme assays and immunoblots demonstrated a 6- to 7-fold enrichment of an apical marker and a low level of contamination by cytoplasmic and basolateral markers. After removal of cytosolic contaminants and peripheral membrane proteins by alkaline extraction, apical integral membrane proteins were characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). Lectin blotting and [3H]glucosamine labeling identified two major sialoglycoproteins of apparent Mr 200,000 and 100,000. The apical membrane sheets here described provide a useful model for systematic characterization of the molecular components of the membrane, for reconstitution of lipid and protein transport in cell-free systems, and for study of the interactions of submembranous cytoskeletal proteins with the apical plasma membrane domain.
Exon-intron organization and sequence comparison of human and murine T11 (CD2) genes.
Authors: Diamond D J , Clayton L K , Sayre P H , Reinherz E L
Published: May 2006

Genomic DNA clones containing the human and murine genes coding for the 50-kDa T11 (CD2) T-cell surface glycoprotein were characterized. The human T11 gene is approximately equal to 12 kilobases long and comprised of five exons. A leader exon (L) contains the 5'-untranslated region and most of the nucleotides defining the signal peptide [amino acids (aa) -24 to -5]. Two exons encode the extracellular segment; exon Ex1 is 321 base pairs (bp) long and codes for four residues of the leader peptide and aa 1-103 of the mature protein, and exon Ex2 is 231 bp long and encodes aa 104-180. Exon TM is 123 bp long and codes for the single transmembrane region of the molecule (aa 181-221). Exon C is a large 765-bp exon encoding virtually the entire cytoplasmic domain (aa 222-327) and the 3'-untranslated region. The murine T11 gene has a similar organization with exon-intron boundaries essentially identical to the human gene. Substantial conservation of nucleotide sequences between species in both 5'- and 3'-gene flanking regions equivalent to that among homologous exons suggests that murine and human genes may be regulated in a similar fashion. The probable relationship of the individual T11 exons to functional and structural protein domains is discussed.
Antiphagocytic activity of streptococcal M protein: selective binding of complement control protein factor H.
Authors: Horstmann R D , Sievertsen H J , Knobloch J , Fischetti V A
Published: May 2006

Isolated complement components were used to study the regulation of the alternative complement pathway C3 convertase (EC 3.4.21.47), also called C3b,Bb, on M protein-carrying (M+) and M protein-lacking (M-) streptococci. Neither M- nor M+ streptococci directly affected the formation or dissociation of the surface-bound C3b,Bb or the inactivation of surface-bound C3b by factor I. However, the activity of the serum control protein of the alternative complement pathway, factor H, in controlling streptococcus-bound C3b and C3b,Bb was 6-8 times stronger on M+ organisms than on M- organisms. Furthermore, M+ streptococci of different serotypes and purified streptococcal M6 protein were shown to selectively bind factor H, the dissociation constants ranging from 4.5 X 10(-6) M to 6 X 10(-7) M. We conclude that the antiphagocytic activity of streptococcal M protein may be due to complement inhibition mediated by the binding of factor H. Binding of a regulatory protein appears to be a previously unrecognized route by which a pathogen is able to evade alternative pathway activation.