bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a cDNA encoding the heparin and collagen binding domains of human thrombospondin.
Authors: Dixit V M , Hennessy S W , Grant G A , Rotwein P , Frazier W A
Published: May 2006

Thrombospondin (TSP) cDNA clones were isolated from a human fibroblast cDNA library by screening with degenerate synthetic oligodeoxynucleotides. The probes were designed based on the sequences of peptides isolated from tryptic digests of TSP. The largest clone identified contains an open reading frame that encodes the amino-terminal heparin binding domain of TSP and part of the immediately adjacent collagen binding domain, a region of TSP that includes the site of disulfide crosslinking responsible for trimer formation. In addition to the known amino-terminal sequence of mature TSP (which is identical to that of the heparin binding domain) and that of the collagen binding domain, the open reading frame predicts the amino acid sequences of four tryptic peptides including the one from which the probe sequences were derived. The sequence of the mature protein is preceded by an unusual signal peptide sequence of 18 residues. The heparin binding domain contains 240 amino acids including only one intradomain disulfide bond. In contrast, 80 residues beyond the start of the collagen binding domain, a cluster of 10 cysteine residues occurs within 40 amino acids corresponding to the point of interchain disulfide crosslinking in the TSP trimer. RNA blot analysis indicates a TSP mRNA size of approximately equal to 8 kilobases and Southern blots are consistent with a single gene encoding TSP.
BSC-1 growth inhibitor/type beta transforming growth factor is a strong inhibitor of thymocyte proliferation.
Authors: Ristow H J
Published: May 2006

Growth inhibitor/type beta transforming growth factor purified from BSC-1 cells and human platelets is shown to strongly inhibit the proliferation of Con A-stimulated mouse thymocytes. The inhibition can be achieved with growth inhibitor/type beta transforming growth factor concentrations approximately equal to 1/10th those necessary to inhibit keratinocyte cultures. The inhibitory effect in thymocyte cultures can be reversed by the addition of interleukin 2. These findings suggest that growth inhibitor/type beta transforming growth factor is a naturally occurring immunoregulator.
Human J chain gene: chromosomal localization and associated restriction fragment length polymorphisms.
Authors: Max E E , McBride O W , Morton C C , Robinson M A
Published: May 2006

Gene clones encoding the human J chain, the protein that links immunoglobulin monomers, were recently described. Using probes from J chain clones we have now investigated the chromosomal location of this gene by analysis of somatic cell hybrids and by in situ chromosome hybridization. The gene is located on the long arm of chromosome 4 in band q21, the chromosomal band in which a consistent translocation with chromosome 11 has been observed in some acute leukemias. An additional human sequence that cross-hybridizes with some J chain gene probes is located on a different chromosome. Restriction fragment length polymorphisms deriving from length variations in a tandemly repeated region 5' to the J chain gene were detected; these should facilitate the analysis of genetic linkage between this gene and other markers on chromosome 4.
GH3 pituitary adenoma cells can reverse thymic aging in rats.
Authors: Kelley K W , Brief S , Westly H J , Novakofski J , Bechtel P J , Simon J , Walker E B
Published: May 2006

Thymic size and T-cell function decrease with age, and it has not yet been possible to totally reverse this thymic atrophy and completely restore T-cell-dependent immune functions. In this study, GH3 pituitary adenoma cells, which secrete growth hormone and prolactin, were implanted subcutaneously into 16- and 22-month-old female Wistar-Furth rats and the rats were sacrificed approximately 2 months later. Only thymic remnants were detected in aged, non-implanted rats, but thymus glands were found in both the 18- and the 24-month-old rats that had been implanted with GH3 cells. Thymus glands from the GH3-implanted 18-month-old rats contained distinct cortical thymocytes and medullary epithelial cells. Depending on the concentration of phytohemagglutinin or concanavalin A, T-cell proliferative responses of splenocytes from these implanted rats were 2- to 5-fold greater than those of 18-month-old controls. At the optimal concentration of mitogen, proliferative responses to either lectin could be restored to those levels observed in splenocytes from 3-month-old Wistar-Furth females. Thymus glands from 24-month-old GH3-implanted rats contained more cortical thymocytes and fewer fat vacuoles than controls, but they were not totally reconstituted. No significant lectin-induced T-cell proliferative responses or IL-2 secretion were detected in 24-month-old control rats, but splenocytes from GH3-implanted rats showed augmented T-cell proliferative responses and increased synthesis of IL-2. Fluorescence-activated cell-sorter analysis of thymocytes revealed that 24-month-old rats implanted with GH3 cells had a higher proportion of lymphocytes with the Thy-1.1 and helper-T-cell phenotypes. These data show that it is possible to regenerate normal thymic tissue in situ and reverse the natural loss in cell-mediated immunity that occurs with aging.
Definitive identification of a member of the Epstein-Barr virus nuclear protein 3 family.
Authors: Hennessy K , Wang F , Bushman E W , Kieff E
Published: May 2006

Some Epstein-Barr virus (EBV) immune human antisera are known to react with a 142-kDa protein, EBV-encoded nuclear antigen 3 (EBNA3), which, like EBNA1 and EBNA2, is likely to be involved in the establishment of latent infection or growth transformation. We have now constructed gene fusions between Escherichia coli lacZ and an EBV DNA open reading frame (BERF1; BamHI E fragment rightward open reading frame 1), which is transcribed into an mRNA in latently infected cells. Purified hybrid protein from one of these constructs, chosen because of its reactivity with EBNA3-positive human antisera, was used to affinity purify the specific antibody from human antiserum. This specific antibody was used to prove that EBNA3 is encoded, at least in part, by BERF1, and that EBNA3 is in the nucleus of each latently infected cell. In rodent cells, BERF1 encodes a 120- to 130-kDa protein, which translocates to the nucleus and is recognized by EBNA3-positive human antisera. Two other proteins similar in size to EBNA3 are detected in latently infected cells by EBV immune human antisera. Two EBV open reading frames related to BERF1 may encode these proteins.
Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines.
Authors: Modrow S , Wolf H
Published: May 2006

Two related but differentially expressed potential membrane proteins of Epstein-Barr virus are encoded by the same reading frame in the EcoRI D het region of the viral genome. Potential antigenic sites in the amino acid sequence of these proteins were selected by computer-aided prediction of the secondary structure and two oligopeptides corresponding to regions located in different parts of the proteins were synthesized chemically. Rabbit antisera to these peptides were used for immunoprecipitation of the native viral proteins from Epstein-Barr virus-positive cell lines from various sources. Both predicted membrane proteins could be precipitated from cell lines that had been transformed in vitro with EBV or from cell lines derived from infectious mononucleosis patients. In cell lines established from Burkitt lymphoma, only the smaller polypeptide, which lacks 138 amino acids from the amino terminus, could be identified. Using the synthetic peptides as antigens in ELISA, we detected elevated antibody titers in sera from patients with infectious mononucleosis and nasopharyngeal carcinoma.
Inhibition of platelet function with synthetic peptides designed to be high-affinity antagonists of fibrinogen binding to platelets.
Authors: Ruggeri Z M , Houghten R A , Russell S R , Zimmerman T S
Published: May 2006

We have constructed synthetic peptides modeled on the sequences of (i) Arg-Gly-Asp, present in fibrinogen, fibronectin, and von Willebrand factor, and of (ii) the fibrinogen gamma chain (gamma 400-411) His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val. The concentration of each peptide that inhibits 50% of 125I-labeled fibrinogen binding to thrombin-stimulated platelets (IC50) was then determined. The IC50 for (gamma 400-411) was 48-180 microM at a fibrinogen concentration of 60 micrograms/ml. A substitution of arginine for alanine at position 9 decreased the IC50 to 14.5 microM. Arginine substitutions for all other residues on the amino-terminal side of the peptide Arg9-Gly-Asp-Val resulted in an IC50 of 0.4-0.8 microM, and the IC50 of the peptide Arg13-Gly-Asp-Val was 0.2-0.3 microM. This contrasts with an IC50 of 200 microM for Arg5-Gly-Asp-Val-Arg4 and an IC50 greater than 1 mM for the peptide Arg12. The inhibitory effect resulted primarily in a decreased affinity of fibrinogen binding to platelets, although the number of available binding sites had also decreased. Binding was completely inhibited. At concentrations between 10 and 18 microM, Arg9-Gly-Asp-Val blocked all ADP-induced aggregation in citrated platelet-rich plasma. The peptide Tyr-His-His-Lys-Arg-Lys-Arg-Lys-Gln-Arg-Gly-Asp-Val was labeled with 125I to quantitate its binding to thrombin-stimulated platelets; at saturation, 59,990 molecules were bound per cell (Kd = 3.8 X 10(-7) M). These modified synthetic peptides bind to platelets with the same affinity as does intact fibrinogen and inhibit platelet function. The increased affinity of these modified peptides is greater than 20-fold that of peptides comprised of only native sequences and is a prerequisite for the potential antithrombotic use of these agents.
Regulation of fungal cell wall growth: a guanine nucleotide-binding, proteinaceous component required for activity of (1----3)-beta-D-glucan synthase.
Authors: Kang M S , Cabib E
Published: May 2006

By treatment with detergent and NaCl, particulate (1----3)-beta-D-glucan synthase (EC 2.4.1.34) from Hansenula anomala or Neurospora crassa was dissociated into a "soluble fraction" and a "membrane fraction." Each fraction alone was almost inactive, but enzymatic activity could be reconstituted by mixing the two fractions and adding GTP or one of its analogs. Based on their lability to heat and to incubation with trypsin, the activity in both fractions is proteinaceous. The active component in the soluble fraction appears to bind guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), since it was specifically protected by this nucleotide against heat inactivation and against inactivation in the presence of EDTA. Furthermore, precipitation of the soluble component with ammonium sulfate in the presence of GTP[gamma S] gave rise to a fraction that was highly active in the absence of added nucleotide, indicating either tight binding or covalent interaction between GTP[gamma S] and the soluble component. The membrane fraction probably contains the catalytic moiety, because it was partially protected against heat inactivation by the substrate, UDP-glucose. Soluble fractions that stimulated membrane fractions from H. anomala and N. crassa were obtained from several other fungi, including Saccharomyces cerevisiae. We propose that the soluble fraction contains a GTP-binding protein that modulates the biosynthesis of (1----3)-beta-D-glucan of fungal cell walls and probably has a major role in the regulation of cell wall morphogenesis.
Amino acid sequence of a prothoracicotropic hormone of the silkworm
Bombyx mori
Authors: Nagasawa Hiromichi , Kataoka Hiroshi , Isogai Akira , Tamura Saburo , Suzuki Akinori , Mizoguchi Akira , Fujiwara Yuko , Suzuki Atsushi , Takahashi Susumu Y. , Ishizaki Hironori
Published: May 2006

We have determined the complete amino acid sequence of 4K-PTTH-II, one of three forms of the
M
r
4400 prothoracicotropic hormone of the silkworm
Bombyx mori
, active to brainless pupae of
Samia cynthia ricini
. Like vertebrate insulin, it consists of two nonidentical peptide chains (A and B chains). The A chain consists of 20 amino acid residues. The B chain is a mixture of four microheterogeneous peptides, two of which consist of 28 residues, and the other two, of 26 residues. 4K-PTTH-II has considerable sequence homology (40%) with human insulin, and it resembles porcine relaxin both in the carboxyl-terminal cysteine residue of the A chain and in the amino-terminal pyroglutamic acid residue of the B chain. The identical distribution of the six cysteine residues also indicates that 4K-PTTH-II belongs to the insulin family.
Identification of a factor in HeLa cells specific for an upstream transcriptional control sequence of an EIA-inducible adenovirus promoter and its relative abundance in infected and uninfected cells.
Authors: SivaRaman L , Subramanian S , Thimmappaya B
Published: May 2006

Utilizing the gel electrophoresis/DNA binding assay, a factor specific for the upstream transcriptional control sequence of the EIA-inducible adenovirus EIIA-early promoter has been detected in HeLa cell nuclear extract. Analysis of linker-scanning mutants of the promoter by DNA binding assays and methylation-interference experiments show that the factor binds to the 17-nucleotide sequence 5' TGGAGATGACGTAGTTT 3' located between positions -66 and -82 upstream from the cap site. This sequence has been shown to be essential for transcription of this promoter. The EIIA-early-promoter specific factor was found to be present at comparable levels in uninfected HeLa cells and in cells infected with either wild-type adenovirus or the EIA-deletion mutant dl312 under conditions in which the EIA proteins are induced to high levels [7 or 20 hr after infection in the presence of arabinonucleoside (cytosine arabinoside)]. Based on the quantitation in DNA binding assays, it appears that the mechanism of EIA-activated transcription of the EIIA-early promoter does not involve a net change in the amounts of this factor.
Differentiation-inducing factor purified from conditioned medium of mitogen-treated spleen cell cultures stimulates bone resorption.
Authors: Abe E , Tanaka H , Ishimi Y , Miyaura C , Hayashi T , Nagasawa H , Tomida M , Yamaguchi Y , Hozumi M , Suda T
Published: May 2006

Spleen cells treated with mitogens produce a potent bone-resorbing factor called osteoclast-activating factor (OAF). To examine the relationship between the bone-resorbing factor and other protein factors produced by spleen cells, the colony-stimulating factor (CSF), the differentiation-inducing factor (DIF), the macrophage fusion factor (MFF), and the macrophage growth factor (MGF) were purified from 2.68 liters of conditioned medium of mouse spleen cell cultures treated with concanavalin A. Purification was performed successively by DEAE-cellulose, Blue Sepharose, and Sephadex G-150 column chromatography and high-pressure liquid chromatography (HPLC). The DIF was successfully separated from CSF and MGF on HPLC. CSF coincided with MGF on HPLC, but MFF disappeared before application to HPLC. Only the DIF exhibited bone-resorbing activity, whereas CSF and MGF did not. The DIFs purified from L929 cells and Ehrlich ascites tumors similarly exhibited bone-resorbing activity. The DIFs purified from spleen cells and Ehrlich ascites tumor cells exhibited neither interleukin 1 (IL-1) activity nor tumor necrosis factor (TNF) activity, though the unfractionated conditioned medium from spleen cells did exhibit them. In the light of recent reports that IL-1 beta and TNF also stimulate bone resorption, the term OAF should refer to a generic activity rather than a single factor.
Receptor-mediated folate accumulation is regulated by the cellular folate content.
Authors: Kamen B A , Capdevila A
Published: May 2006

Cultured monkey kidney cells are shown to possess a cell-surface receptor that binds 5-methyltetrahydro[3',5',7,9-3H4]folic acid (5-methyltetrahydro[3H]folic acid) with high affinity (Kd = 3 nM at 4 degrees C). The cell-surface binding capacity for [3H]folic acid or [3H]methotrexate is equivalent to that for 5-methyltetrahydro[3H]folate, but the Kd values are 0.4 and 20 nM, respectively. These nonlabeled folates also inhibit cellular binding and accumulation of 5-methyltetrahydro[3H]folate, whereas specific moieties of folic acid (i.e., p-aminobenzoylglutamic acid, pteroic acid, and glutamic acid) had no effect. Surface-bound folate(s) can be released by incubation of the cells at pH 3.5. At 37 degrees C the bound material is slowly transferred into cells, where it becomes resistant to acid release. Within 4 hr the cells internalize about 3-4 times as much folate as is bound to the surface in the steady state. The amount of receptor activity increases markedly when cells are depleted of folate through growth in folate-depleted medium. Binding of folate was inhibited by an antibody raised against a soluble plasma folate-binding protein, suggesting that the cell-surface receptor and the circulating folate-binding protein are immunologically related. These data indicate that cells possess a high-affinity, high-specificity folate receptor whose expression is regulated by the folate content of the cell. They also suggest that a small molecule such as folate can enter cells by receptor-mediated endocytosis.
Homologous genes encode two distinct histidine-rich proteins in a cloned isolate of Plasmodium falciparum.
Authors: Wellems T E , Howard R J
Published: May 2006

Two genes encoding distinct histidine-rich proteins in a Plasmodium falciparum clone exhibit high levels of homology, suggesting they have originated by duplication and divergence from a common ancestral sequence. Both genes have a similar interrupted structure and an exon that encodes closely related tandem repeats of very high histidine and alanine content. The most common repeat encoded by one gene, the hexapeptide Ala-His-His-Ala-Ala-Asp, differs in the sixth position from the most common repeat encoded by the other gene, the hexapeptide Ala-His-His-Ala-Ala-Asn. The divergence of the repeat domains is greater than that of the flanking regions, which exhibit 85-90% homology, including untranslated sequences. This suggests the tandem repeats are relatively labile elements within the genome that may provide the parasite with a means of rapid evolutionary change.
Characterization of major peptides in Creutzfeldt-Jakob disease and scrapie.
Authors: Sklaviadis T , Manuelidis L , Manuelidis E E
Published: May 2006

In Creutzfeldt-Jakob disease three major peptides cosediment with the infectious agent. These distinct peptides are not present in identical fractions from uninfected brain, and bind to polyclonal antibodies raised against "prion protein" purified by protease treatment. Three similar distinct peptides are also found in scrapie-infected brain fractions purified without the use of proteases. To clarify the relationships between these distinct peptides and prion protein, peptides were analyzed on immunoblots after cleavage with various glycosidases. There are two different 34-kDa peptides. One binds to ricin and cannot be detected by nonequilibrium pH gradient electrophoresis, presumably due to its highly acidic or basic pI. A second basic 34-kDa glycopeptide (Gp34) contains multiple terminal sialic acid residues responsible for charge heterogeneity (pI values, 7.2-7.8) and is reduced to a single spot with a pI value of 7.8 after neuraminidase treatment. After (but not before) neuraminidase treatment, secondary D-galactose-like sugars are detectable on Gp34, and a small number of N-acetylglucosamine residues probably represent the third sugar residue in an N-linked chain. When virtually all sugar residues are removed with endoglycosidase H the molecular weight of Gp34 is reduced by only approximately equal to 2 kDa. The residual peptide strongly binds antibody. A third acidic 24- to 26-kDa species (p26) also binds polyclonal antibodies but, in contrast to Gp34, was unaffected by any glycosidase treatment. Protease-treated peptides showed a very broad array of pI spots, consistent with a heterogeneous protein origin. None of the nonproteolyzed peptides show a clear relationship to prion protein. The number of sugar residues on Gp34 is not consistent with those estimated for prion protein. Although p26 could be the source of the "prion sequence," p26 does not appear to be glycosylated. Regardless, it is likely that all the major peptides described thus far are accumulated or modified normal gene products and are not integral components of the infectious agent.
Aggressive behavior induces release of nerve growth factor from mouse salivary gland into the bloodstream.
Authors: Aloe L , Alleva E , Böhm A , Levi-Montalcini R
Published: May 2006

Intraspecific fighting induced by 6-8 weeks of social isolation results in massive release of nerve growth factor (NGF) into the bloodstream of adult male mice. The amount of circulating NGF is highly correlated with the number of fighting episodes. Biological, radioimmunological, immunohistochemical, and ultrastructural studies show that NGF is discharged from the salivary gland into the blood within minutes after fighting and reaches the highest level 3-4 hr later. Adrenergic innervation of the salivary gland or adrenalectomy does not abolish the NGF release. Corticotropic hormones do not induce NGF increase in the blood. Daily administrations of highly purified NGF (3 micrograms per g of body weight) result in a considerable increase in the volume of adrenal glands. These findings are unequivocable evidence for a physiological role of the mouse salivary glands as a major source of blood NGF.
Cellular localization of proenkephalin mRNA in rat brain: gene expression in the caudate-putamen and cerebellar cortex.
Authors: Shivers B D , Harlan R E , Romano G J , Howells R D , Pfaff D W
Published: May 2006

The cellular locations of proenkephalin mRNA have been determined for the caudate-putamen and cerebellar cortex of the rat brain by in situ hybridization. In the caudate-putamen, more than half of the neurons express the proenkephalin gene. Morphologically, they are medium-sized cells that may represent projection neurons. In the cerebellar cortex, proenkephalin mRNA is present in a subpopulation of neurons in the granule layer that appear to be Golgi cells--i.e., inhibitory interneurons. The presence of [Met]enkephalin, a pentapeptide derived from proenkephalin, in these two brain areas is consistent with a synthetic role for this mRNA and implicates proenkephalin gene expression in the control of motor function.
Steady-state level and turnover rate of the tripeptide Tyr-Gly-Gly as indexes of striatal enkephalin release in vivo and their reduction during pentobarbital anesthesia.
Authors: Llorens-Cortes C , Gros C , Schwartz J C
Published: May 2006

Tyr-Gly-Gly (YGG) was recently shown to be an extraneuronal metabolite of opioid peptides derived from proenkephalin A, formed in brain by the action of "enkephalinase" (membrane metalloendopeptidase, EC 3.4.24.11) and degraded by aminopeptidases. The dynamic state of YGG in mouse striatum was studied by evaluating the changes in its level elicited by inhibitors of these peptidases. Inhibition of YGG synthesis by Thiorphan or acetorphan reduced YGG levels with a t1/2 (mean +/- SEM) of 12 +/- 2 min, indicating an apparent turnover rate (mean +/- SEM) of 18 +/- 2 pmol/mg of protein per hr. An apparent turnover rate of 18 +/- 2 pmol/mg of protein per hr was derived from the rate of YGG accumulation elicited by the aminopeptidase inhibitor bestatin. In addition, accumulation of Tyr-Gly-Gly-Phe-Met (YGGFM) in an extrasynaptosomal fraction after blockade of its degradation by Thiorphan and bestatin occurred at a rate of 18 +/- 3 pmol/mg of protein per hr, which is likely to reflect the rate of enkephalin release in vivo. Hence, the three series of data suggest that striatal enkephalins rapidly turn over--e.g., with a t1/2 in the 1-hr range. Pentobarbital anesthesia reduced by about 60% the rate of YGG accumulation elicited by bestatin and the extrasynaptosomal YGGFM accumulation elicited by Thiorphan and bestatin. This suggests that the activity of striatal enkephalin neurons is depressed during anesthesia. Pentobarbital (and chloral hydrate) did not affect the steady-state level of YGGFM but rapidly reduced that of YGG. Hence, the steady-state levels of YGG seem a reliable index of changes in enkephalin release, and measuring levels of characteristic fragments might therefore provide a general means of evaluating neuropeptide release in vivo.
The pseudopterosins: anti-inflammatory and analgesic natural products from the sea whip Pseudopterogorgia elisabethae.
Authors: Look S A , Fenical W , Jacobs R S , Clardy J
Published: May 2006

The Caribbean sea whip Pseudopterogorgia elisabethae (Octocorallia, Cnidaria) has been found to contain the pseudopterosins, a newly described class of natural products, which have been characterized as diterpene-pentoseglycosides. The pseudopterosins possess anti-inflammatory and analgesic properties that exceed, in our assays, the potencies of existing drugs such as indomethacin. As anti-inflammatory agents, the pseudopterosins appear to modify the arachidonic acid cascade by an as yet undefined mechanism of pharmacological action.
Peroxidases depolymerize lignin in organic media but not in water
Authors: Dordick Jonathan S. , Marletta Michael A. , Klibanov Alexander M.
Published: May 2006

Horseradish peroxidase and milk lactoperoxidase, while unable to degrade either synthetic or natural lignins in aqueous solutions, vigorously depolymerize polyconiferyl alcohol, milled wood lignin, and kraft pine lignin in dioxane, dimethylformamide, or methyl formate containing 5% aqueous buffer (10 mM acetate, pH 5). Horseradish peroxidase, solubilized in organic media by chemical modification, can also degrade lignin in native lignocellulose (wheat straw).
Regulatory mechanisms for induction of synthesis of repair enzymes in response to alkylating agents: ada protein acts as a transcriptional regulator.
Authors: Nakabeppu Y , Sekiguchi M
Published: May 2006

Expression of the ada and alkA genes, both of which are involved in the adaptive response of Escherichia coli to alkylating agents, is positively controlled by Ada protein, the product of the ada gene. Large amounts of ada- and alkA-specific RNA were formed in cells treated with a methylating agent, whereas little such RNA was produced in untreated cells. The in vivo transcription-initiation sites for the two genes were determined by primer-extension cDNA synthesis. In an in vitro reconstituted system, both ada and alkA transcripts were formed in an Ada protein-dependent manner. However, responses of the two transcription reactions to methylating agents differed; ada transcription was stimulated by methylnitrosourea, while alkA transcription was suppressed. We prepared a methylated form of Ada protein by an in vitro reaction and compared the activity with that of the normal, unmethylated form. The methylated form was more effective in promoting ada transcription than was the unmethylated form, but the effects of both forms were much the same with regard to alkA transcription. Based on these findings, we propose a model for the molecular mechanism of adaptive response.
p21 ras proteins and guanine nucleotides modulate the phosphorylation of 36- and 17-kilodalton mitochondria-associated proteins.
Authors: Backer J M , Weinstein I B
Published: May 2006

We have found that, when isolated rat liver mitochondria are incubated with [gamma-32P]ATP, there is phosphorylation of 36- and 17-kDa proteins. These proteins together with their protein kinase(s) are released as a complex by incubation of the isolated rat liver mitochondria at 20 degrees C for 30 min with 10 mM glucose 6-phosphate, 0.5 mM inositol phosphate, or 0.01 mM inositol triphosphate. Phosphorylation of the 36- and 17-kDa proteins in this soluble protein fraction is modulated by p21 proteins encoded by ras oncogenes and synthesized in Escherichia coli via recombinant DNA methods. A normal p21 ras protein stimulates phosphorylation of the 36-kDa protein and inhibits phosphorylation of the 17-kDa protein, whereas two transforming p21 ras proteins inhibit phosphorylation of both the 36- and 17-kDa proteins. Although GDP and 5'-guanylyl imidodiphosphate also influence the phosphorylation of these proteins, we present evidence that the effects of p21 ras protein are not simply due to their bound GDP. This novel system may be useful for further studies on the biochemical functions of the p21 ras proteins.
Intracellular pools of transferrin receptors result from constitutive internalization of unoccupied receptors.
Authors: Ajioka R S , Kaplan J
Published: May 2006

In HeLa cells the majority of transferrin (Tf) receptors are found within the endocytic apparatus, with only 20% of receptors exposed at the cell surface. Receptor distribution is unaltered by the presence or absence of Tf. The mechanism responsible for the cellular distribution of receptors was explored by selectively inactivating receptors within the endocytic apparatus. This was accomplished by employing Tf-horseradish peroxidase conjugates. Peroxidase-catalyzed oxidation of diaminobenzidine within an endosome destroys Tf receptor activity. Using such conjugates, we have demonstrated that the majority of internal Tf receptors could be inactivated when less than 6.0% of receptors were occupied by the conjugate at steady state. This result indicates that occupied and unoccupied receptors are in the same compartment. Furthermore, horseradish peroxidase that was internalized by fluid-phase pinocytosis inactivated intracellular Tf receptors in the absence of Tf; this indicates that the presence of internal receptors is ligand independent. Following exposure of cells to the conjugate, receptor inactivation was proportional to the percentage of the endocytic cycle traversed by the conjugate--that is, the rate of ligand accumulation was the same as the rate of endosomal Tf receptor inactivation. When the Tf-horseradish peroxidase conjugate and 125I-labeled Tf were internalized simultaneously, both ligands were found in the same compartment. However, if the two ligands were administered as separate pulses and the period between pulses was as short as 1 min, the ligands remained separate within the cell. Together these results demonstrate that the intracellular pool of Tf receptors reflects the constitutive internalization of unoccupied Tf receptors, which, once internalized, remain segregated.
cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins.
Authors: Fisher D Z , Chaudhary N , Blobel G
Published: May 2006

The amino acid sequences deduced from cDNA clones of human lamin A and lamin C show identity between these two lamins except for an extra 9.0-kDa carboxyl-terminal tail that is present only in lamin A. Both lamins A and C contain an alpha-helical domain of approximately 360 residues that shows striking homology to a corresponding alpha-helical rod domain that is the structural hallmark of all intermediate filament proteins. However, the lamin alpha-helical domain is 14% larger than that of the intermediate filament proteins. In addition to the extensive homology to intermediate filament proteins as reported [McKeon, F., Kirschner, M. & Caput, D. (1986) Nature (London) 319, 463-468], a different 82-amino acid residue stretch at the carboxyl terminus of lamin A has been deduced and verified by amino acid sequencing. This region contains sequence homology to amino- and carboxylterminal domains of type I and type II epidermal keratins. Implications of the presence of these and other domains in lamins A and C for the assembly of the nuclear lamina are discussed.
Molecular cloning of cDNAs encoding rat and human medium-chain acyl-CoA dehydrogenase and assignment of the gene to human chromosome 1.
Authors: Matsubara Y , Kraus J P , Yang-Feng T L , Francke U , Rosenberg L E , Tanaka K
Published: May 2006

Rat liver mRNA encoding the precursor of medium-chain acyl-CoA dehydrogenase was purified to near homogeneity by polysome immunoadsorption using a polyclonal, monospecific antibody. A single-stranded, 32P-labeled cDNA probe was synthesized using the enriched mRNA as template and was used to screen directly 15,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone [600 base pairs (bp)] was positively identified by hybrid-selected translation combined with mitochondrial processing of translated products. Using the isolated rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Three overlapping clones (1100 bp, 500 bp, and 400 bp) were isolated and positively identified by hybrid-selected translation. The largest human cDNA clone was subcloned into the transcription vector pGEM-2, which contains a bacteriophage T7 RNA polymerase promoter. In vitro transcription of this recombinant, followed by in vitro translation, showed that the cDNA clone coded for approximately 80% of the medium-chain acyl-CoA dehydrogenase protein. The sizes of rat and human mRNAs encoding the precursor of medium-chain acyl-CoA dehydrogenase were 2.2 and 2.4 kilobases long, respectively, as determined by blot hybridization analysis of electrophoretically fractionated poly(A)+ RNA. Southern blot analysis of DNAs from human-rodent somatic cell hybrids with an isolated human cDNA assigned the gene coding for this enzyme to the short arm of chromosome 1, band p31. The chromosomal assignment was confirmed by in situ hybridization of the probe to human metaphase cells. Direct screening of cDNA libraries using a highly enriched mRNA to generate a probe, as demonstrated in this study, may provide the most rapid and convenient approach to cDNA cloning of low-abundance mRNAs.
Multiple calcium channels mediate neurotransmitter release from peripheral neurons.
Authors: Perney T M , Hirning L D , Leeman S E , Miller R J
Published: May 2006

We examined the effects of dihydropyridine drugs on evoked neurotransmitter release from cultured neonatal rat sensory and sympathetic neurons. Depolarization with K+-rich solutions increased the release of substance P from cultured sensory neurons. This release was enhanced by BAY K8644 and (+)-202791 and was blocked by a variety of other dihydropyridines including (-)-202791, by Co2+, or in Ca2+-free solutions. K+-rich solutions also stimulated the release of [3H]norepinephrine from cultured sympathetic neurons. This release was also completely blocked by Co2+ or in Ca2+-free solution. In contrast to the situation in sensory neurons, however, the evoked release of [3H]norepinephrine was completely resistant to the blocking effects of dihydropyridine such as nimodipine. However, BAY K8644 was able to enhance the evoked release of [3H]norepinephrine, and this enhancement was blocked by nimodipine. These results are discussed in relation to the possible participation of multiple types of calcium channels in the release of neurotransmitters.
Extra-atrial expression of the gene for atrial natriuretic factor.
Authors: Gardner D G , Deschepper C F , Ganong W F , Hane S , Fiddes J , Baxter J D , Lewicki J
Published: May 2006

Atrial natriuretic factor (ANF) is a group of peptides, originally isolated from the cardiac atria, that have a number of important effects on blood pressure, renal function, and salt balance. In the current study, expression of the ANF gene in certain extra-atrial tissues of the rat has been examined by radioimmunoassay of extracted ANF protein and by blot-hybridization, nuclease S1 analysis, and primer-extension analysis of the ANF mRNA. ANF peptides and mRNA were detected in cardiac ventricles, lung, and pituitary gland at levels generally less than or equal to 1% those of cardiac atria. The ANF transcripts in extra-atrial tissue appear to be very similar to those synthesized in the atria. They are polyadenylylated, are equivalent in overall length (950-1050 nucleotides), and have identical 5' termini. A secondary transcription start site mapping approximately 80 base pairs upstream from the primary start site is employed in atria and to a lesser extent in other tissues. The ANF transcript is present throughout the cardiac ventricles from apex to base and in the septum as well as the ventricular free walls. The transcript is more prevalent in the left ventricle and interventricular septum than in the right ventricle. Immunocytochemistry using various anti-rat ANF antibodies localized ANF immunoreactivity to the atrial myocytes; the ventricular myocytes, particularly along the endothelial surface of the ventricular chamber; perialveolar cells in the lung; and the gonadotropin-producing cells of the pituitary. The data indicate that the capacity for ANF gene expression extends beyond atrial tissue, albeit at much reduced levels, and may suggest alternative, perhaps paraendocrine, functions for the peptide in these tissues.
Structure and function of a chloroplast DNA replication origin of
Chlamydomonas reinhardtii
Authors: Wu Madeline , Lou J. K. , Chang D. Y. , Chang C. H. , Nie Z. Q.
Published: May 2006

Chloroplast DNA replication in
Chlamydomonas reinhardtii
is initiated by the formation of a displacement loop (D-loop) at a specific site. One D-loop site with its flanking sequence was cloned in recombinant plasmids SC3-1 and R-13. The sequence of the chloroplast DNA insert in SC3-1, which includes the 0.42-kilobase (kb) D-loop region, as well as 0.2 kb to the 5′ end and 0.43 kb to the 3′ end of the D-loop region, was determined. The sequence is A+T-rich and contains four large stem-loop stuctures. An open reading frame potentially coding for a polypeptide of 136 amino acids was detected in the D-loop region. One stem-loop structure and two back-to-back prokaryotic-type promoters were mapped within the open reading frame. The 5.5-kb
Eco
RI fragment cloned in R-13 contains the 1.05-kb SC3-1 insert and its flanking regions. A yeast autonomously replicating (ARS) sequence and an ARC sequence, which promotes autonomous replication in
Chlamydomonas
, have been mapped within the flanking regions [Vallet, J.-M. & Rochaix, J.-D. (1985)
Curr. Genet.
9, 321-324]. Both R-13 and SC3-1 were active as templates in a crude algal preparation that supports DNA synthesis. In this
in vitro
system, chloroplast DNA synthesis initiated near the D-loop site.
Secretion and processing of insulin precursors in yeast.
Authors: Thim L , Hansen M T , Norris K , Hoegh I , Boel E , Forstrom J , Ammerer G , Fiil N P
Published: May 2006

A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor alpha 1 leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.
Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata.
Authors: Kranz R G , Haselkorn R
Published: May 2006

A Rhodopseudomonas capsulata nifH::lacZ gene fusion was used to isolate constitutive mutants of R. capsulata, unable to repress nif gene transcription anaerobically with every fixed-nitrogen source tested. When these nifc strains were grown aerobically, nif gene transcription was repressed. These results indicate that the regulation of nif gene transcription by fixed nitrogen is different from the regulation by oxygen. Under anaerobic conditions, nif gene transcription in both R. capsulata and Klebsiella pneumoniae is specifically prevented by inhibitors of DNA gyrase [DNA topoisomerase type II (ATP-hydrolyzing), EC 5.99.1.3]. A recent study has shown that anaerobically grown Salmonella typhimurium have high DNA gyrase activity, whereas aerobically grown cells have high DNA topoisomerase type I (EC 5.99.1.2) activity and DNA that is more relaxed [Yamamoto, N. & Droffner, M. L. (1985) Proc. Natl. Acad. Sci. USA 82, 2077-2081]. In view of these results, we suggest that the control of nif gene transcription in response to oxygen is determined by the action of DNA gyrase and DNA topoisomerase I. Thus, although nitrogen control of nif gene expression requires the products of regulatory genes for which constitutive mutations can be isolated, oxygen appears instead to prevent the adoption of a DNA conformation necessary, directly or indirectly, for nif gene transcription.
Cloning and nitrate induction of nitrate reductase mRNA
Authors: Cheng Chi-Lien , Dewdney Julia , Kleinhofs Andris , Goodman Howard M.
Published: May 2006

Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate
in vivo
labeled protein and
in vitro
translation products, demonstrating that nitrate induction increases nitrate reductase protein and translatable mRNA. A partial cDNA clone for barley nitrate reductase has been isolated and identified by hybrid-selected translation. RNA blot-hybridization analysis shows that nitrate induction also causes a marked increase in the steady-state level of nitrate reductase mRNA.
The C-C (6-4) UV photoproduct is mutagenic in Escherichia coli.
Authors: Glickman B W , Schaaper R M , Haseltine W A , Dunn R L , Brash D E
Published: May 2006

Mutation induced by ultraviolet light is predominantly targeted by UV photoproducts. Two primary candidates for the premutagenic lesion are the cyclobutane pyrimidine dimer and the less frequent (by a factor of 10) pyrimidine-pyrimidone (6-4) photoproduct. Methylation of the 3'-cytosine in the sequence 5' CCAGG 3' reduces the yield of (6-4) lesions, but not of cyclobutane dimers, at these sites. By taking advantage of mutants deficient in cytosine methylation, we show here that at the three sites in the lacI gene of Escherichia coli having this sequence, the specific increase in the formation of the (6-4) photoproducts is accompanied by a concomitant increase in mutation. At each site, a G X C to A X T transition results in an amber mutation. In the unmethylated state, these sites become among the most frequent nonsense mutations recovered. We conclude that the (6-4) photoproduct constitutes a major premutagenic lesion in E. coli.
Frameshift mutations produced by proflavin in bacteriophage T4: specificity within a hotspot.
Authors: Ripley L S , Clark A
Published: May 2006

Frameshift mutations were induced by proflavin in the rIIB gene of bacteriophage T4. rIIB DNA from each of 48 independent frameshifts was inserted into M13mp8 and sequenced. Two-thirds of the frameshifts (33/48) lie contiguous to one another in 10 base pairs of the rIIB sequence. This hotspot differs markedly from previously characterized mutagen-induced frameshift hotspots. Distinctive features of the hotspot include the absence of locally repetitive sequences, particularly G X C runs, and the fact that many different sequence changes are induced within the hotspot sequence at appreciable frequencies. Among the 33 mutants at the hotspot, 8 distinguishable DNA sequence changes were seen. All of the mutations were deletions of a single base or duplications of one or more bases. Duplications were more frequent than deletions. The patterns of the base sequence changes suggest that two specific phosphodiester bonds within the hotspot sequence are sites at which proflavin-induced mutation is initiated.
The murine Fc receptor for immunoglobulin: purification, partial amino acid sequence, and isolation of cDNA clones.
Authors: Hibbs M L , Walker I D , Kirszbaum L , Pietersz G A , Deacon N J , Chambers G W , McKenzie I F , Hogarth P M
Published: May 2006

The murine Fc receptor for IgG (Fc gamma R) was purified to homogeneity by immunoaffinity chromatography from detergent lysates of the macrophage cell line J774. Microsequencing of intact protein yielded a single amino-terminal sequence, which was confirmed and extended to 20 residues by the isolation of an overlapping peptide. The isolation of additional proteolytic fragments obtained by using Staphylococcus aureus V8 protease, cyanogen bromide, and lysine C proteinase, facilitated sequence analysis of a total of 119 amino acid residues. Codon usage charts were used to construct oligonucleotide probes based on the amino acid sequences of three nonoverlapping peptides. These probes were used to screen a cDNA library derived from the WEHI-3B myelomonocytic cell line, and a single cDNA clone (pFc24) to which all three probes hybridized was isolated. This clone, containing a 1.02-kilobase cDNA insert, has been characterized by restriction mapping and partial DNA sequencing, and it has been shown to encode the Fc gamma R. The sequence at the 5' end of the clone contained the coding information for the amino-terminal sequence of the Fc gamma R as well as a putative 13-amino acid signal sequence. The 3' end of the clone encoded a peptide identified in purified receptor preparations. Thus, the presence of coding information at the 5' and 3' ends of this clone suggests that full-length Fc receptor cDNA spans greater than 1 kilobase.
Phospholipase treatment of accessory cells that have been exposed to antigen selectively inhibits antigen-specific Ia-restricted, but not allospecific, stimulation of T lymphocytes.
Authors: Falo L D , Benacerraf B , Rock K L
Published: May 2006

The corecognition of antigen and class II major histocompatibility complex (MHC) molecules (Ia molecules) by the T-cell receptor is a cell surface event. Before antigen is recognized, it must be taken up, processed, and displayed on the surface of an Ia-bearing accessory cell (antigen-presenting cell, APC). The exact nature of antigen processing and the subsequent associations of antigen with the APC plasma membrane, Ia molecules, and/or the T-cell receptor are not well defined. To further analyze these events, we have characterized the processing and presentation of the soluble polypeptide antigen bovine insulin. We found that this antigen requires APC-dependent processing, as evidenced by the inability of metabolically inactivated APCs to present native antigen to antigen plus Ia-specific T-T hybridomas. The ability of the same APCs to present antigen after uptake and processing showed that this antigen subsequently becomes stably associated with the APC plasma membrane. To characterize the basis for this association, we analyzed its sensitivity to enzymatic digestion. APCs exposed to antigen, treated with phospholipase A2, and then immediately fixed lost the ability to stimulate bovine insulin plus I-Ad-specific hybridomas. In contrast, the ability of these same APCs to stimulate I-Ad allospecific hybridomas was unaffected. This effect of phospholipase is not mimicked by the broadly active protease Pronase, nor is there evidence for contaminating proteases in the phospholipase preparation. These results suggest that one consequence of antigen processing may be an antigen-lipid association that contributes to the anchoring of antigen to the APC membrane. The implications of this model are discussed.