bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of activating stimulus on functional phenotype: interleukin 2 mRNA accumulation differentially induced by ionophore and receptor ligands in subsets of murine T cells.
Authors: McGuire K L , Yang J A , Rothenberg E V
Published: May 2006

We have investigated the linkage between CD4/CD8 phenotype and programming for specific responses in primary T-cell populations. In situ hybridization has been used to determine the frequency of cells competent to express the interleukin 2 (IL-2) gene after short-term stimulation with various polyclonal activators. The effects of the T-cell receptor ligands Con A and anti-CD3 monoclonal antibody were compared with those of a calcium ionophore that bypasses membrane receptors altogether. Induction with a calcium ionophore and phorbol ester revealed that potential IL-2 producers not only constitute greater than 85% of the cells with a CD4+ "helper/inducer" phenotype but also constitute over half of the cells with a CD8+ "killer/suppressor" phenotype. There is no defect in the ability of these CD8+ cells to accumulate IL-2 transcripts under these conditions. By contrast, in response to phorbol ester and either Con A or anti-CD3, the CD8+ cells show an abortive IL-2 production response with rapid disappearance of IL-2 mRNA. This results in substantially lower yields of IL-2 per cell than is made by CD4+ cells in response to the same stimuli. The extent to which these populations appear to have diverged in function thus depends on the stimulus used to trigger the response. The results suggest that differences in signal transduction or posttranscriptional regulatory mechanisms, rather than effector gene inducibility per se, may initially underlie the commitment of CD4+ and CD8+ cells to distinct functional roles.
95- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor.
Authors: Nygren A , Bergman T , Matthews T , Jörnvall H , Wigzell H
Published: May 2006

125I-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extent with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25-kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing 125I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule.
Expression of preproenkephalin mRNA by cultured astrocytes and neurons.
Authors: Vilijn M H , Vaysse P J , Zukin R S , Kessler J A
Published: May 2006

Expression of preproenkephalin mRNA by developing glia and neurons was examined in cultures of embryonic and neonatal rat brain. Cultured glia from specific regions of embryonic day 17 and neonatal day 1 rat brain were identified as astrocytes on the basis of both morphology and expression of immunoreactivity for glial fibrillary acidic protein. The level of preproenkephalin mRNA in cultured neonatal hypothalamic astrocytes was comparable to levels present in cultured embryonic striatal and hypothalamic neurons. Levels of the mRNA were significantly higher in astrocytes derived from neonatal hypothalamus compared to astrocytes derived from other areas of the brain. Thus, there is heterogeneity among astrocytes with respect to preproenkephalin expression. Levels of preproenkephalin mRNA in cultured neonatal striatal astrocytes were only one-third as high as levels in embryonic striatal astrocytes; this observation suggests that glial expression of the gene may be down-regulated during development. Although cultured hypothalamic neurons contained substantial levels of prodynorphin mRNA, levels of this mRNA were not detectable in cultured astrocytes from any brain region or in cultured striatal neurons. Thus, glia do not express all opioid peptide genes during development. These observations suggest that expression of the preproenkephalin gene by astrocytes may play a role in development of the brain.
The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhancer.
Authors: Fink J S , Verhave M , Kasper S , Tsukada T , Mandel G , Goodman R H
Published: May 2006

cAMP-regulated transcription of the human vasoactive intestinal peptide gene is dependent upon a 17-base-pair DNA element located 70 base pairs upstream from the transcriptional initiation site. This element is similar to sequences in other genes known to be regulated by cAMP and to sequences in several viral enhancers. We have demonstrated that the vasoactive intestinal peptide regulatory element is an enhancer that depends upon the integrity of two CGTCA sequence motifs for biological activity. Mutations in either of the CGTCA motifs diminish the ability of the element to respond to cAMP. Enhancers containing the CGTCA motif from the somatostatin and adenovirus genes compete for binding of nuclear proteins from C6 glioma and PC12 cells to the vasoactive intestinal peptide enhancer, suggesting that CGTCA-containing enhancers interact with similar transacting factors.
Covalent modification of the beta-1,4-N-acetylmuramoylhydrolase of Streptococcus faecium with 5-mercaptouridine monophosphate.
Authors: Dolinger D L , Schramm V L , Shockman G D
Published: May 2006

Purified beta-1,4-N-acetylmuramoylhydrolase (muramidase-1; EC 3.2.1.17) of Streptococcus faecium ATCC 9790 has been shown to be covalently substituted with approximately 12 mol equivalents of monomeric 5-mercaptouridine monophosphate. All 12 residues are present on the proteolytically processed 87-kDa active form of the enzyme. A peptide fragment containing 5-mercaptouridine, tyrosine, alanine, glycine, and leucine was isolated consistent with an O-phosphate linkage of the nucleotide to tyrosine.
Cultured mammalian cells attach to the invasin protein of Yersinia pseudotuberculosis.
Authors: Isberg R R , Leong J M
Published: May 2006

The expression of invasin, the product of the Yersinia pseudotuberculosis inv gene, allows enteric bacteria to enter cultured mammalian cells. The ability of invasin to bind animal cells and the potential significance of this interaction in the entry process were investigated. It was found that HEp-2 cells could attach to surfaces coated with bacterial membranes containing invasin. By fractionating bacterial membrane proteins on NaDodSO4/polyacrylamide gels and transferring the protein to filters, we demonstrated that the cell-binding component of the membranes comigrated with invasin. Mutations that changed the electrophoretic mobility of the protein also caused a corresponding shift in the migration of the cell-binding activity, showing that the comigrating protein was indeed invasin. Monoclonal antibodies directed against invasin that blocked invasin-HEp-2 cell interaction also inhibited bacteria from penetrating HEp-2 cells, indicating that interaction of this protein with animal cells is critical for cellular penetration.
Automatic microinjection system facilitates detection of growth inhibitory mRNA.
Authors: Pepperkok R , Zanetti M , King R , Delia D , Ansorge W , Philipson L , Schneider C
Published: May 2006

Naturally quiescent human lymphocytes, consisting predominantly of T cells, contain mRNA(s) that can inhibit DNA synthesis when injected into either human diploid fibroblasts (IMR-90) or transformed recipient cells (HeLa). By using an automated capillary microinjection system and a fluorescent coinjection marker (fluorescein isothiocyanate-dextran), individually injected cells can be retrieved and analyzed for DNA synthesis. mRNA isolated from resting T cells is able to block the cells from entering the S phase. The block is reversible and leads to a delay in DNA synthesis. The inhibitory effect is not observed if the injected mRNA is isolated from growth-activated T cells. The disappearance of the inhibition coincides with the approach of the G1/S boundary in both the donor T cells and the recipient human fibroblasts. The mRNA of resting T cells was size-fractionated and the peak inhibitory activity was recovered in a fraction approximately equal to 1.5 kilobases long.
Patterns of mRNA expression during early cell growth differ in kidney epithelial cells destined to undergo compensatory hypertrophy versus regenerative hyperplasia.
Authors: Norman J T , Bohman R E , Fischmann G , Bowen J W , McDonough A , Slamon D , Fine L G
Published: May 2006

An increase in cell size and protein content is characteristic of cells undergoing hypertrophy and of replicating cells prior to DNA synthesis. Cell enlargement in the two situations could be regulated by similar early events with an interruption of the cell cycle occurring in hypertrophy, or the two processes could be uncoupled. In vivo models were used to compare hypertrophy induced by unilateral nephrectomy and hyperplasia induced by folic acid injection in rabbit renal cortical cells. Within 48 hr, cell volume increased in both groups but the number of cells in the cell cycle and DNA synthesis was increased only after folic acid. Patterns of mRNA expression of the following three groups of cell cycle-dependent genes were analyzed: (i) protooncogenes (c-fos, c-myc, and c-Ha-ras), (ii) structural protein genes (vimentin and beta-actin), and (iii) transport protein genes (Na+, K+-ATPase, ADP-ATP translocase, and calcyclin). mRNAs for all genes, except calcyclin and c-Ha-ras, were detected in controls. Folic acid generally induced rapid, transient increases in mRNA levels, but after unilateral nephrectomy, expression of most mRNAs showed a gradual, progressive increase. These data indicate that gene expression in the early stages of cell enlargement differs in cells destined to undergo proliferation vs. hypertrophy. The term "sustained message amplification" is proposed to describe the hypertrophied cell.
Antisense Myc sequences induce differentiation of F9 cells.
Authors: Griep A E , Westphal H
Published: May 2006

Down-regulation of Myc expression is the earliest documented change in gene expression in retinoic acid-induced differentiation of murine F9 teratocarcinoma cells. F9 cells transfected with plasmids expressing antisense Myc sequences under control of the simian virus 40 (SV40) early promoter exhibit a decrease in Myc protein. The result of this decrease is the spontaneous differentiation into cells that resemble retinoic acid-treated F9 cells as judged by plasminogen activator assays. In contrast, when F9 cells are transfected with a plasmid expressing Myc under control of the SV40 early promoter, resulting cell clones are resistant to differentiation by retinoic acid as shown by the lack of induction of plasminogen activator. These results suggest that down-regulation of Myc is sufficient and necessary for F9 cell differentiation.
Bacteriophage Mu sites required for transposition immunity.
Authors: Darzins A , Kent N E , Buckwalter M S , Casadaban M J
Published: May 2006

Plasmids with bacteriophage Mu sequences receive additional Mu insertions 20-700 times less frequently than plasmids without Mu sequences. The Mu sites required for this transposition immunity were mapped near each end, either of which was sufficient. The left site was between 127 and 203 base pairs from the left end, and the right site was between 22 and 93 base pairs from the right end. These sequences include the innermost but not the outermost of the three binding sites for the Mu A transposition protein at each end of Mu. Transposition immunity was cis-acting and independent of its location on a target plasmid. An additional copy of an immunity site reduced transposition a factor of 10 further. Transposition immunity was seen both during full phage lytic growth, with all the bacteriophage Mu genes, and during normal cellular growth, with a mini-Mu element containing only the Mu c and ner regulatory and A and B transposition genes.
Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go.
Authors: Ueda H , Harada H , Nozaki M , Katada T , Ui M , Satoh M , Takagi H
Published: May 2006

Reconstitution of purified mu opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. mu opioid receptors were purified by 6-succinylmorphine AF-AminoTOYOPEARL 650M affinity chromatography and by PBE isoelectric chromatography. The purified mu opioid receptor (pI 5.6) migrated as a single Mr 58,000 polypeptide by NaDodSO4/PAGE, a value identical to that obtained by affinity cross-linking purified mu receptors. When purified mu receptors were reconstituted with purified Gi, the G protein that mediates the inhibition of adenylate cyclase, the displacement of [3H]naloxone (a mu opioid antagonist) binding by [D-Ala2,MePhe4,Gly-ol5]enkephalin (a mu opioid agonist) was increased 215-fold; this increase was abolished by adding 100 microM (guanosine 5'-[gamma-thio]triphosphate. Similar increases in agonist displacement of [3H]naloxone binding (33-fold) and its abolition by guanosine 5'-[gamma-thio]triphosphate were observed with Go, the G protein of unknown function, but not with the v-Ki-ras protein p21. In reconstituted preparations with Gi or Go, neither [D-Pen2,D-Pen5]enkephalin (a delta opioid agonist; where Pen is penicillamine) nor U-69,593 (a kappa opioid agonist) showed displacement of the [3H]naloxone binding. In addition, the mu agonist stimulated both [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding (in exchange for GDP) and the low-Km GTPase in such reconstituted preparations, with Gi and Go but not with the v-Ki-ras protein p21, in a naloxone-reversible manner. The stoichiometry was such that the stimulation of 1 mol of mu receptor led to the binding of [3H]guanosine 5'-[beta,gamma-imido]triphosphate to 2.5 mol of Gi or to 1.37 mol of Go. These results suggest that the purified mu opioid receptor is functionally coupled to Gi and Go in the reconstituted phospholipid vesicles.
Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver.
Authors: Pohlentz G , Klein D , Schwarzmann G , Schmitz D , Sandhoff K
Published: May 2006

Competition experiments using lactosylceramide, ganglioside GM3 and ganglioside GD3 as substrates, as well as mutual inhibitors for ganglioside N-acetylgalactosaminyltransferase, in Golgi vesicles derived from rat liver suggested that N-acetylgalactosamine transfer to these three respective compounds, leading to gangliosides GA2, GM2, and GD2, respectively, is catalyzed by one enzyme. Analogous studies with gangliosides GA1, GM1, and GD1b as glycolipid acceptors in sialyltransferase assays indicated GM1b, GD1a, and GT1b synthases to be identical. These results are incorporated into a model for ganglioside biosynthesis and its regulation.
Conserved cysteine and histidine residues of the avian myeloblastosis virus nucleocapsid protein are essential for viral replication but are not "zinc-binding fingers".
Authors: Jentoft J E , Smith L M , Fu X D , Johnson M , Leis J
Published: May 2006

The nucleocapsid protein from the Rous sarcoma virus has two regions of sequence with the motif Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Cys. All retrovirus nucleocapsid proteins contain one or two of these motifs, and they represent the only conserved sequences among these proteins. Sequence analysis of nucleocapsid from avian myeloblastosis virus shows that it also contains two Cys-His sequences and, in fact, differs from the Rous sarcoma nucleocapsid protein only in three residues near the carboxyl terminus. The hypothesized role of the conserved cysteines and histidines as zinc ligands was tested experimentally. No tightly bound metal ions were detected for avian myeloblastosis nucleocapsid protein, and the molar amount of zinc in virions was less by a factor of 50 than that of the nucleocapsid protein. Added Zn2+ did not significantly affect nucleocapsid binding to poly(ethenoadenylic acid) or its secondary structure, as determined from circular dichroism. Nevertheless, the conserved cysteine and histidine residues of the Rous sarcoma (Prague-C strain) nucleocapsid protein are essential for fully functional virus, as shown by the fact that single-site substitutions of five of the six conserved cysteines and either of the two histidine residues blocked viral replication.
Chemical synthesis and enzymatic activity of a 99-residue peptide with a sequence proposed for the human immunodeficiency virus protease.
Authors: Nutt R F , Brady S F , Darke P L , Ciccarone T M , Colton C D , Nutt E M , Rodkey J A , Bennett C D , Waxman L H , Sigal I S
Published: May 2006

Retroviral proteins, including those from the human immunodeficiency virus (HIV), are synthesized as polyprotein precursors that require proteolytic cleavage to yield the mature viral proteins. A 99-residue polypeptide, encoded by the 5' end of the pol gene, has been proposed as the processing protease of HIV. The chemical synthesis of the 99-residue peptide was carried out by the solid-phase method, and the isolated product was found to exhibit specific proteolytic activity upon folding under reducing conditions. Upon size-exclusion chromatography, enzymatic activity was eluted at a point consistent with a dimeric molecular size. Specificity was demonstrated by the cleavage of the natural substrate HIV gag p55 into gag p24 and gag p17, as well as cleavage of small peptide substrates representing processing sites of HIV fusion proteins. The proteolytic action of the synthetic product could be inhibited by pepstatin, an aspartic protease inhibitor.
Molecular cloning and expression of the cDNA for the hamster alpha 1-adrenergic receptor.
Authors: Cotecchia S , Schwinn D A , Randall R R , Lefkowitz R J , Caron M G , Kobilka B K
Published: May 2006

The cDNA for the Syrian hamster alpha 1-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the receptor protein purified from DDT1MF-2 smooth muscle cells. The deduced amino acid sequence encodes a 515-residue polypeptide that shows the most sequence identity with the other adrenergic receptors and the putative protein product of the related clone G-21. Similarities with the muscarinic cholinergic receptors are also evident. Expression studies in COS-7 cells confirm that we have cloned the alpha 1-adrenergic receptor that couples to inositol phospholipid metabolism.
Proteins from eight eukaryotic cytochrome P-450 families share a segmented region of sequence similarity.
Authors: Kalb V F , Loper J C
Published: May 2006

Proteins from eight eukaryotic families in the cytochrome P-450 superfamily share one region of sequence similarity. This region begins 275-310 amino acids from the amino terminus of each P-450, continues for approximately 170 residues, and ends 35-50 amino acids before the carboxyl terminus. The region can be divided into four domains of sequence similarity, each possessing its own pattern of invariant, conserved, and variable amino acids. The four domains are 56, 20, 59, and 28 residues long and are connected by three shorter segments of limited sequence similarity. The number of residues in these short segments varies with the P-450 protein but ranges from 0 to 20 residues. Consensus sequences based on these similarities can be used to determine whether the sequence of an unidentified peptide resembles that expected for a P-450. Sequence similarities between proteins sometimes reflect constraints imposed by the requirements of a common function. The fourth domain of the P-450s, for example, contains an invariant cysteine that provides the axial thiolate ligand to the heme iron. Other relationships between the four domains and P-450 function can be examined by in vitro mutagenic procedures that alter the conserved amino acids or modify the distance between domains.
Coordinate regulation of two genes encoding gluconeogenic enzymes by the trans-dominant locus Tse-1.
Authors: Lem J , Chin A C , Thayer M J , Leach R J , Fournier R E
Published: May 2006

Tissue-specific extinguisher-1 (Tse-1) is a mouse genetic locus that can repress liver-specific tyrosine aminotransferase gene expression in trans. To search for other Tse-1-responsive genes, hepatoma microcell hybrids retaining mouse chromosome 11 or human chromosome 17, containing murine Tse-1 and human TSE1, respectively, were screened for expression of liver-specific mRNAs. While most liver gene activity was unaffected in such hybrids, phosphoenolpyruvate carboxykinase and tyrosine aminotransferase gene expression was coordinately repressed in these clones. Extinction of both genes was apparently mediated by a single genetic locus that resides on human chromosome 17.
Identification and characterization of a mitogen-activated S6 kinase.
Authors: Jenö P , Ballou L M , Novak-Hofer I , Thomas G
Published: May 2006

Treatment of Swiss mouse 3T3 cells with epidermal growth factor, orthovanadate, or serum results in the activation of a kinase that phosphorylates protein S6 of the 40S ribosomal subunit in vitro. This kinase is eluted as a single peak of activity from either a Mono Q anion-exchange column at 0.34 M NaCl or a Mono S cation-exchange column at 0.20 M NaCl. Treatment of the peak fraction from the Mono S column with phosphatase 2A completely abolishes the activity of the enzyme. The kinase appears to be distinct from protein kinase C, cAMP-dependent protein kinase, and two protease-activated kinases, PAK II and H4P. The kinase has been purified to apparent homogeneity and migrates as a single band at Mr 70,000 in NaDodSO4/polyacrylamide gels. The kinase exhibits the ability to autophosphorylate, and this activity directly parallels S6 phosphorylation activity on the final step of purification. In vitro, the kinase incorporates up to 5 mol of phosphate into S6, and the tryptic phosphopeptide maps obtained are equivalent to those from S6 phosphorylated in vivo. Most important, treatment of the purified kinase with phosphatase 2A results in complete inactivation of the enzyme, arguing that the activity of the kinase is directly controlled by phosphorylation.
Colonization of the Krakatau Islands by vertebrates: equilibrium, succession, and possible delayed extinction.
Authors: Thornton I W , Zann R A , Rawlinson P A , Tidemann C R , Adikerana A S , Widjoya A H
Published: May 2006

MacArthur and Wilson [MacArthur, R. H. & Wilson, E. O. (1967) The Theory of Island Biogeography (Princeton Univ. Press, Princeton, NJ)] used the rate of recolonization of the Krakatau Islands (sterilized in the cataclysmic 1883 eruption) by birds and vascular plants to test their equilibrium model of immigration and extinction processes on islands. Working with data only up to 1933, they concluded that the number of resident land bird species had approached equilibrium by 1908-1919, 25-36 years after the eruption, when the number of vascular plant species was still increasing. Recent surveys of vertebrates on the archipelago show continuing colonization, as well as species turnover. Nonmigrant nonmarine birds and reptiles are only now approaching equilibrium species numbers; archipelago acquisition rates for these groups are lower than in the 40 years following the 1883 eruption. Apparent (minimal) annual extinction rates are an order of magnitude lower than those calculated for equilibrium by MacArthur and Wilson. The active volcano Anak Krakatau emerged in 1930 and suffered an eruption in 1952 that destroyed the vegetation. It is still at an early stage of succession and on the threshold of major vegetational change. This island and a physically dynamic, ever-young spit on Sertung I, also held at an early successional stage, may provide (i) ecological refuges for some species whose optimal habitat on the older islands is being extirpated by vegetational succession and (ii) ecological "windows" through which such species may still establish from the mainland, thus postponing their extinction on the archipelago.
Construction of a "mutagenesis cartridge" for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants.
Authors: Kuhn R J , Tada H , Ypma-Wong M F , Dunn J J , Semler B L , Wimmer E
Published: May 2006

By following a strategy of genetic analysis of poliovirus, we have constructed a synthetic "mutagenesis cartridge" spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type 1 (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed us to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. We suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).
Creation of AUG initiation codons by addition of uridines within cytochrome b transcripts of kinetoplastids.
Authors: Feagin J E , Shaw J M , Simpson L , Stuart K
Published: May 2006

The cytochrome b gene of Trypanosoma brucei has an ATG codon near its 5' end but the cytochrome b genes of the related kinetoplastids Leishmania tarentolae and Crithidia fasciculata lack an ATG. Recent results have shown that 34 uridines that are not encoded in the genome are added within the 5' end of T. brucei cytochrome b transcripts during or after transcription. These additions create an AUG in the transcript that is 20 amino acids upstream of the AUG predicted from the genomic sequence. We report here that the cytochrome b transcripts of L. tarentolae and C. fasciculata also contain added uridines within their 5' ends. The additions occur in similar numbers and positions and an in frame AUG is created at a similar site in all three species. These data strongly suggest that the created AUG functions as the initiation codon for cytochrome b in these species. Since some other kinetoplastid mitochondrial genes also lack conventional initiation codons, creation of initiation codons may be an important function of uridine addition.
Exposure to crosslinked IgD induces receptors for IgD on T cells in vivo and in vitro.
Authors: Coico R F , Finkelman F , Swenson C D , Siskind G W , Thorbecke G J
Published: May 2006

IgD is a surface immunoglobulin, which is coexpressed with IgM on greater than 90% of mature B cells, but its levels in serum are extremely low compared to those of IgM. It role as a surface receptor has been reemphasized by our recent findings that IgD receptors are induced on helper T cells by exposure to IgD and that such cells have immunoaugmenting properties. The present study shows that crosslinking of soluble IgD or of monomeric cell surface IgD is required and sufficient for the induction of T cells bearing receptors for IgD, both in vivo and in vitro. Effective IgD crosslinking in this respect can be obtained with antigen or with heterologous and immunogenic as well as nonimmunogenic allotype-specific anti-IgD. These results reinforce the concept that the induction of T cells bearing receptors for IgD is an integral component of the normal immune response.
Synergistic action of glucocorticoid and estradiol responsive elements.
Authors: Ankenbauer W , Strähle U , Schütz G
Published: May 2006

Modulation of gene expression by steroid hormones is mediated by receptor proteins that associate with regulatory elements of responsive genes upon binding the hormone ligand. The finding that two glucocorticoid responsive elements act cooperatively to stimulate transcription of the tyrosine aminotransferase gene prompted us to explore whether synergistic effects also occur when two different steroid hormone receptors are involved. A region of the chicken vitellogenin II gene that displays homologies to glucocorticoid and estradiol responsive elements was tested for its capability to confer estradiol and glucocorticoid inducibility to a heterologous promoter. When positioned immediately upstream of the thymidine kinase gene promoter, this element enhances expression by either steroid. Combination of both hormones results in a synergistic increase of transcription. Mutational analysis shows that sequences that show similarities of glucocorticoid and estradiol responsive elements are absolutely required for hormone induction. Analysis of the dose dependence of induction by both steroids demonstrates that half-maximal activity is observed at lower hormone concentrations when the other steroid is present in saturating amounts, which suggests that the synergistic induction observed with the combination of hormones is based on a functional interaction of the two hormone receptors.
Cloning and nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.
Authors: Niu X D , Browning K S , Behal R H , Reed L J
Published: May 2006

A 537-base cDNA encoding a portion of Saccharomyces cerevisiae dihydrolipoamide acetyltransferase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) was isolated from a lambda gt11 yeast cDNA library by immunoscreening. This cDNA was subcloned and used as a probe to screen a lambda gt11 yeast genomic DNA library. Two overlapping clones were used to determine the complete sequence of the acetyltransferase gene. The composite sequence has an open reading frame of 1446 nucleotides encoding a presequence of 28 amino acids and a mature protein of 454 amino acids (Mr = 48,546). The deduced amino acid sequence contains the experimentally determined amino acid sequences of the amino terminus and two internal peptide fragments of the acetyltransferase. Hybridization analysis of yeast genomic DNA showed that the gene has a single copy. A 915-base segment of the acetyltransferase gene hybridized to a yeast mRNA of approximately equal to 1.6 kilobases. Analysis of the deduced amino acid sequence of the dihydrolipoamide acetyltransferase revealed a multidomain structure similar to those reported for the corresponding acetyltransferases from Escherichia coli and rat liver, and extensive sequence similarity among the three enzymes. However, the yeast enzyme contains only one lipoyl domain, in contrast to three lipoyl domains reported for the E. coli enzyme and apparently two for the rat liver enzyme.
Electron transfer in a genetically modified bacterial reaction center containing a heterodimer.
Authors: Kirmaier C , Holten D , Bylina E J , Youvan D C
Published: May 2006

Time-resolved optical measurements encompassing the femtoseconds to seconds time scales have been used to investigate Rhodobacter capsulatus reaction centers (RCs) in which the histidine residue at position 200 on the M polypeptide has been changed to a leucine by site-directed mutagenesis. The HisM200----Leu RC, which has a heterodimer consisting of a bacteriochlorophyll and a bacteriopheophytin, is capable of the primary photochemistry observed in wild-type Rb. capsulatus RCs, but with an overall quantum yield reduced by about half. The lower yield resides in the initial electron transfer reaction and may be associated in part with substantial charge transfer character of the excited heterodimer. These and other comparisons between Rb. capsulatus wild-type and HisM200----Leu RCs have important implications for our understanding of the mechanism of electron transfer in the RC and the efficiency of the charge separation process.
Molecular systematics of higher primates: genealogical relations and classification.
Authors: Miyamoto M M , Koop B F , Slightom J L , Goodman M , Tennant M R
Published: May 2006

We obtained 5' and 3' flanking sequences (5.4 kilobase pairs) from the psi eta-globin gene region of the rhesus macaque (Macaca mulatta) and combined them with available nucleotide data. The completed sequence, representing 10.8 kilobase pairs of contiguous noncoding DNA, was compared to the same orthologous regions available for human (Homo sapiens, as represented by five different alleles), common chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus). The nucleotide sequence for Macaca mulatta provided the outgroup perspective needed to evaluate better the relationships of humans and great apes. Pairwise comparisons and parsimony analysis of these orthologues clearly demonstrated (i) that humans and great apes share a high degree of genetic similarity and (ii) that humans, chimpanzees, and gorillas form a natural monophyletic group. These conclusions strongly favor a genealogical classification for higher primates consisting of a single family (Hominidae) with two subfamilies (Homininae for Homo, Pan, and Gorilla and Ponginae for Pongo).
Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus.
Authors: Gyllensten U B , Erlich H A
Published: May 2006

Single-copy sequences can be enzymatically amplified from genomic DNA by the polymerase chain reaction. By using unequal molar amounts of the two amplification primers, it is possible in a single step to amplify a single-copy gene and produce an excess of single-stranded DNA of a chosen strand for direct sequencing or for use as a hybridization probe. Further, individual alleles in a heterozygote can be sequenced directly by using allele-specific oligonucleotides either in the amplification reaction or as sequencing primers. By using these methods, we have studied the allelic diversity at the HLA-DQA locus and its association with the serologically defined HLA-DR and -DQ types. This analysis has revealed a total of eight alleles and three additional haplotypes. This procedure has wide applications in screening for mutations in human genes and facilitates the linking of enzymatic amplification of genes to automated sequencing.
Divergent disease patterns in granulocyte-macrophage colony-stimulating factor transgenic mice associated with different transgene insertion sites.
Authors: Metcalf D , Moore J G
Published: May 2006

A comparison was made of disease development in two lines of transgenic mice in which the granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene was inserted in different chromosomal locations. Female-line mice (X chromosome insertion) had equivalent elevations of serum GM-CSF levels to those in male-line mice (autosomal insertion) but a shorter survival (median survival, 95 versus 145 days) and a significantly higher incidence of large inflammatory foci in skeletal muscle and gut congestion. Male-line transgenic mice had higher levels of cells in the peritoneal cavity and a higher frequency of spleen enlargement with excess erythropoiesis than female-line mice and uniquely developed fibrotic nodules in the abdominal and pleural cavities. The various diseases in GM-CSF transgenic mice are likely to have been induced by GM-CSF-stimulated products of macrophages, and in the two transgenic lines the macrophages exhibit characteristic differences in morphology and possibly functional activity.
Regulation by cAMP and vasoactive intestinal peptide of phosphorylation of specific proteins in striatal cells in culture.
Authors: Girault J A , Shalaby I A , Rosen N L , Greengard P
Published: May 2006

We have studied three low molecular weight phosphoproteins, ARPP-16, ARPP-19, and ARPP-21 (cAMP-regulated phosphoproteins of Mr 16,000, 19,000, and 21,000, respectively) in reaggregate cultures from various regions of fetal mouse brain. ARPP-16 and ARPP-21 were detected only in striatal and cortical cultures. In contrast, ARP-19, which is structurally related to ARPP-16, was also present in reaggregate cultures prepared from thalamus and ventral and dorsal mesencephalon, as well as in monolayer cultures of astroglial cells. In striatal aggregates cultured over a 3-week period, the relative levels of ARPP-16, ARPP-21, and synapsin I/protein IIIa (synaptic vesicle-associated phosphoproteins closely related to each other and treated as a single entity in the present study) increased with time, whereas the level of ARPP-19 decreased. Incubation of striatal aggregates with 8-Br-cAMP, forskolin, or vasoactive intestinal peptide increased the phosphorylation of all these proteins. We conclude that the state of phosphorylation of two proteins enriched in specific neurons (ARPP-16 and ARPP-21) and two more widely distributed proteins (ARPP-19 and synapsin I/protein IIIa) is regulated by cAMP and vasoactive intestinal peptide in striatal cells in culture. These phosphoproteins may therefore play a role in mediating some of the actions of vasoactive intestinal peptide in the caudate-putamen.
A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans.
Authors: Takacs A M , Denker J A , Perrine K G , Maroney P A , Nilsen T W
Published: May 2006

The mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt sequence are transcribed to yield a 109-nt nonpolyadenylated RNA with the 22-nt leader sequence at its 5' end. We suggest that the 22-nt leader is acquired by 63-kDa antigen mRNAs through trans-splicing. These results indicate that trans-splicing is widespread in nematodes and argue for the functional significance of the 22-nt spliced leader exon in nematode mRNA metabolism.
Phosphorylation of yeast phosphatidylserine synthase in vivo and in vitro by cyclic AMP-dependent protein kinase.
Authors: Kinney A J , Carman G M
Published: May 2006

Evidence is presented that demonstrates that phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) from Saccharomyces cerevisiae is phosphorylated in vivo and in vitro by cAMP-dependent protein kinase. Phosphatidylserine synthase activity in cell extracts was reduced in the bcy1 mutant (which has high cAMP-dependent protein kinase activity) and elevated in the cyr1 mutant (which has low cAMP-dependent protein kinase activity) when compared with wild-type cells. The reduced phosphatidylserine synthase activity in the bcy1 mutant correlated with elevated levels of a phosphorylated form of the phosphatidylserine synthase Mr 23,000 subunit. The elevated phosphatidylserine synthase activity in the cyr1 mutant correlated with reduced levels of the phosphorylated form of the enzyme. There was negligible phosphorylation of the phosphatidylserine synthase Mr 23,000 subunit from stationary-phase cells. Pure phosphatidylserine synthase was phosphorylated by the cAMP-dependent protein kinase catalytic subunit, which resulted in a 60-70% reduction in phosphatidylserine synthase activity. The cAMP-dependent protein kinase catalytic subunit catalyzed the incorporation of 0.7 mol of phosphate per mol of phosphatidylserine synthase Mr 23,000 subunit. The specific cAMP-dependent protein kinase inhibitor prevented the phosphorylation of phosphatidylserine synthase and the inhibition of its activity by the catalytic subunit. Analysis of peptides derived from protease-treated labeled phosphatidylserine synthase showed only one labeled peptide. Phospho amino acid analysis of labeled phosphatidylserine synthase showed that the enzyme was phosphorylated at a serine residue.
Interleukin 3 binds to a 140-kDa phosphotyrosine-containing cell surface protein.
Authors: Isfort R J , Stevens D , May W S , Ihle J N
Published: May 2006

Interleukin 3 (IL-3) is a hematopoietic growth factor that supports the proliferation and differentiation of early hematopoietic-lineage progenitors. By using growth factor-dependent cell lines, IL-3 has been shown to induce the appearance of several phosphotyrosine-containing proteins, including a 140-kDa cell surface phosphoprotein. Here we demonstrate that the 140-kDa phosphoprotein binds IL-3 and forms a stable complex with an apparent molecular mass of 170 kDa. The possible relationship of the 140-kDa phosphoprotein to the previously described 65-kDa IL-3-binding protein is discussed.
Extracellular ATP induces a large nonselective conductance in macrophage plasma membranes.
Authors: Buisman H P , Steinberg T H , Fischbarg J , Silverstein S C , Vogelzang S A , Ince C , Ypey D L , Leijh P C
Published: May 2006

Extracellular ATP in its tetra-anionic form (ATP4-) induces ion fluxes and membrane depolarization in the mouse macrophage-like cell line J774.2 and in resident mouse macrophages. We analyzed the effects of extracellular ATP4- by both patch-clamp and intracellular microelectrode techniques. Whole-cell patch-configuration membrane potential measurements on J774.2 cells revealed that ATP4- -induced depolarization occurred within 40 ms of pulsed application of ATP and was completely reversible. The depolarizations were accompanied by a dramatic increase in membrane conductance and showed no sign of adaptation to ATP over a period of 30 min. At 5 mM total ATP (ATPt) the whole-cell conductance was approximately 10 nS, and an upper limit of 20 pS for a single-channel conductance has been established. The reversal potential associated with the ATP-induced depolarization at asymmetric K+, Na+, Ca2+, and Cl- concentrations across the membrane was 0 mV. In patch-clamped cells depolarization was complete at 20 microM ATP4-, and repolarization from full depolarization occurred in approximately 5 s. In contrast, in intact cells measured by microelectrode impalement, complete depolarization occurred at approximately 2 mM ATP4- and repolarization was much slower (approximately 100 min). These findings indicate that the changes in intracellular ionic composition that occur after ATP treatment affect the rate of cell repolarization. At lower concentrations of ATP, potassium conductances modulated the depolarizing effect of ATP. ATP also depolarized mouse peritoneal macrophages, but a variant cell line (ATPR B2), derived from J774.2 cells by prolonged exposure to ATP, was insensitive to ATP. Our results provide a membrane electrophysiological description and analysis of a large nonselective plasma membrane conductance of macrophages induced by extracellular ATP.
Identification of upstream regulatory elements involved in the developmental expression of the Arabidopsis thaliana cab1 gene.
Authors: Ha S B , An G
Published: May 2006

We studied cis regulatory elements controlling the light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene (cab1) by stably transforming tobacco plants using a tumor-inducing (Ti) plasmid vector system. The results from the 5' and internal deletion analyses indicate that there are at least three cis-acting elements that are involved in the light-dependent developmental expression of cab1 gene. Two such elements are located at the immediate upstream regulatory region and the other element is located at the further upstream region. The 1120-base-pair (bp) DNA fragment containing the immediate and far upstream region can confer light-inducible organ specificity on the truncated nos promoter. However, deletion of the 39-bp DNA fragment at the immediate upstream regulatory region from this hybrid promoter resulted in a nonfunctional promoter, revealing that the 39-bp region is important for the cab promoter specificity. Further analyses of this region suggest that a potential Z-DNA-forming sequence (ATACGTGT) is involved in light-dependent developmental expression of the cab1 gene. Two additional Z-DNA-forming sequences (ACACATAT) that are inverted repeats of this sequence are also found in the upstream region where the additional regulatory elements are expected.
Presence of laminin-binding proteins in trichomonads and their role in adhesion.
Authors: Casta e Silva Filho F , de Souza W , Lopes J D
Published: May 2006

Adhesion is regarded as an important feature in the pathogenesis of various microorganisms. Ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. We report that laminin enhances the adhesion of the parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus to a polystyrene substrate and to the surface of epithelial cells (Madin-Darby canine kidney cell line) in vitro. The enhancement was higher for T. vaginalis than for T. foetus. Addition of anti-laminin antibodies to medium significantly inhibited the adhesion of parasites to polystyrene substrate. Indirect immunofluorescence and transmission electron microscopy of replicas of the parasite's surface labeled with antibody-gold complexes showed laminin-binding sites distributed over the parasite surface. Iodinated P1 fragment of laminin, which retains the laminin-binding site, binds saturably to the parasite surface with a Kd of 19.5 nM, for about 3 X 10(5) binding sites per cell. Immunoblotting analysis of whole parasite extracts showed that a protein of 118 kDa is responsible for laminin binding.
Sodium butyrate increases glucose transporter expression in LLC-PK1 cells.
Authors: Takano M , Rhoads D B , Isselbacher K J
Published: May 2006

The effect of sodium butyrate on the expression of the facilitated glucose transporter (GT) was investigated in the pig kidney cell line LLC-PK1. When cells were treated with butyrate, GT mRNA expression was remarkably enhanced with a maximal effect at 5 mM. Levels of GT mRNA were increased at 1 day after butyrate treatment and continued to increase for at least 4 days; however, acetate and propionate did not affect GT mRNA levels significantly. The induction of GT mRNA by butyrate was accompanied by an increase in GT function. The expression of GT mRNA decreased in HepG2, HT-29, and COS cells by treatment with butyrate for 1 day. Interestingly, glucose deprivation of LLC-PK1 cells reduced the induction of GT mRNA by butyrate, although starvation itself slightly enhanced steady-state GT mRNA levels. Therefore, expression of GT in LLC-PK1 cells is strongly induced by butyrate by a pathway that apparently depends on the presence of glucose in culture medium.
Disruption of Dictyostelium discoideum morphogenesis by overproduction of cAMP phosphodiesterase.
Authors: Faure M , Podgorski G J , Franke J , Kessin R H
Published: May 2006

The development and cellular differentiation of Dictyostelium discoideum are disrupted in transformants secreting high levels of the cyclic nucleotide phosphodiesterase. The aggregation of these cells in the early stage of development proceeds rapidly and without the formation of organized streams. The later stages of development, in which differentiation into stalk and spore cells normally takes place, are completely blocked so that the transformants remain in spherical clusters of undifferentiated cells that do not elaborate the tip structure that regulates morphogenesis. These effects are due to overproduction of extracellular phosphodiesterase and demonstrate the role of cAMP during the aggregation phase of development as well as in the control of differentiation and pattern formation.