bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of the human parathyroid hormone-like peptide gene.
Authors: Mangin M , Ikeda K , Dreyer B E , Broadus A E
Published: May 2006

A parathyroid hormone-like peptide (PTH-LP) has recently been identified in human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The peptide appears to be encoded by a single-copy gene that gives rise to multiple mRNAs that are heterogeneous at both their 5' and their 3' ends. Alternative RNA splicing is responsible for the 3' heterogeneity and results in mRNAs encoding three different peptides, each with a unique C terminus. We have isolated and characterized the human PTHLP gene. The gene is a complex transcriptional unit spanning more than 12 kilobases of DNA and containing six exons. Two 5' exons encode distinct 5' untranslated regions and are separated by a putative promoter element, indicating that the gene either has two promoter or is alternatively spliced from a single promoter upstream of the first exon. The middle portion of the PTHLP gene, comprising exons 2-4, has an organizational pattern of introns and exons identical to that of the parathyroid hormone gene, consistent with a common ancestral origin of these two genes. Exon 4 of the PTHLP gene encodes the region common to all three peptides and the C terminus of the shortest peptide, and exons 5 and 6 encode the unique C termini of the other two peptides. Northern analysis of mRNAs from four human tumors of different histological types reveals the preferential use of 3' splicing patterns by individual tumors.
Molecular cloning of feline immunodeficiency virus.
Authors: Olmsted R A , Barnes A K , Yamamoto J K , Hirsch V M , Purcell R H , Johnson P R
Published: May 2006

Feline immunodeficiency virus (FIV) is a T-lymphotropic retrovirus associated with immunodeficiency and opportunistic infections in cats. The discovery of FIV provides an opportunity for the development of a small animal model for AIDS. To initiate the molecular and biological characterization of FIV, cDNA clones were synthesized and used to isolate a proviral clone of FIV. Molecular cross-hybridization analysis of FIV with five lentiviruses revealed that nucleotide-sequence similarities exist between FIV and these lentiviruses in the gag-pol genes. However, nucleotide-sequence similarities were not seen upon comparison of the FIV long terminal repeat sequence with known viral sequences. Common antigenic determinants appeared to be shared by FIV, caprine arthritis encephalitis virus, and visna virus as shown by serological cross-reactivity of rabbit antibodies to caprine arthritis encephalitis virus and visna virus with the putative FIV core protein p28. These studies demonstrated that FIV is a member of the lentivirus subfamily and is distantly related to the AIDS lentiviruses of primates. Importantly, progeny virions of our molecular clone were infectious for experimentally inoculated cats. The availability of an infectious molecular clone will make possible a detailed dissection of the molecular pathogenesis of FIV, which may facilitate the development of vaccine and therapeutic strategies for AIDS.
A glucose transport protein expressed predominately in insulin-responsive tissues.
Authors: Charron M J , Brosius F C , Alper S L , Lodish H F
Published: May 2006

Using low-stringency hybridization to the rat brain glucose transporter (GT), a 2489-base-pair cDNA clone was isolated from a rat soleus lambda gt10 cDNA library. It encodes a 509-amino acid protein whose sequence and predicted membrane structure is very similar to those of the rat brain and liver GTs. The muscle GT-like protein is 65% identical in amino acid sequence to the rat brain GT and 52% identical to the rat liver GT; the major differences are in the NH2- and COOH-terminal hydrophilic segments. This GT-like mRNA is expressed predominately in tissues where glucose transport is sensitive to insulin, including striated muscle, cardiac muscle, and adipose tissue; low-level expression is also detected in smooth muscle and kidney mRNA. This GT-like cDNA is the fourth member of the mammalian GT-related gene family identified to date. We propose that it encodes an insulin-sensitive GT.
Reconstitution of purified brown adipose tissue mitochondria uncoupling protein: demonstration of separate identity of nucleotide binding and proton translocation sites by chemical probes.
Authors: Katiyar S S , Shrago E
Published: May 2006

The reaction of two arginine-modifying reagents, phenylglyoxal and 2,3-butanedione, with a highly purified uncoupling protein of brown adipose tissue mitochondria decreased the GDP binding to the uncoupling protein. This inhibition was irreversible and dependent on time and concentration of the reagent. Complete inhibition of GDP binding by both reagents establishes that arginine is one of the critical amino acid residues involved in the binding of GDP. Reconstitution of the uncoupling protein (both unmodified and modified) into phospholipid vesicles by our procedure showed no effect of phenylglyoxal modification on the H+ conductance, thus demonstrating that the proton translocation site is structurally different and distinct from the GDP binding site.
Plasmin-mediated fibrinolysis by variant recombinant tissue plasminogen activators.
Authors: Urano S , Metzger A R , Castellino F J
Published: May 2006

A rapid and quantitative fibrinolytic assay has been used to measure the overall activity of a recombinant tissue plasminogen activator (rTPA) preparation for dissolution of a fibrin clot by its ability to activate [Glu1]plasminogen (containing glutamic acid at position 1) to plasmin. A standard curve constructed for wild-type two-chain rTPA that contains, from the amino terminus, the finger (F)-growth factor (E)-kringle 1 (K1)-kringle 2 (K2)-serine protease (P) domains was used to assess the overall fibrin-dissolving abilities of variant recombinant molecules. Two-chain deletion mutants lacking the E domain, the F-E domains, the F-E-K1 domains, and the K1-K2 domains yielded activities ranging from 22% to 35% of the overall activity of wild-type two-chain rTPA, suggesting that both the K2 and F domains are individually responsible for a portion of the function of the molecule. Comparison of variant molecules containing F-K1-K2-P and F-K2-K2-P domains showed that the latter variant possessed a 4-fold higher activity (1.4-fold greater than that of wild-type two-chain rTPA), indicating that, for the activity measured, the presence of K2 leads to a greater effectiveness than that of K1. A plasmin cleavage-resistant mutant (Arg-275----Ser) has been used to assess possible differences in one- and two-chain rTPA in this overall activity, the former displaying 86% of the activity of the latter, suggesting that such differences are indeed small. Finally, the proper covalent attachment of the light and heavy chains of two-chain rTPA are very important to its overall fibrinolytic activity, since replacement of Cys-264 with glycine and concomitant disruption of one of the covalent attachment sites of the two chains provides a variant of rTPA with less than 2% of the activity of the wild-type two-chain molecule. The effector molecule, epsilon-amino hexanoic acid (epsilon Ahx; epsilon-aminocaproic acid), inhibits the overall fibrinolytic effect of rTPA in this system, with an effective Ki of approximately 1.5 mM. Its efficacy, as measured by the Ki, is independent of the presence of the epsilon Ahx binding regions of plasminogen and rTPA and is similar to the efficacy obtained when urokinase was the activator in place of wild-type two-chain rTPA or when activation of plasminogen was bypassed as a result of provision of preformed plasmin to the assay. The results suggest that in the overall clot lysis system, an important epsilon Ahx binding site may exist on fibrin that inhibits its dissolution by plasmin.
Characterization of the human 5-lipoxygenase gene.
Authors: Funk C D , Hoshiko S , Matsumoto T , Rdmark O , Samuelsson B
Published: May 2006

The gene for human 5-lipoxygenase has been isolated from three different bacteriophage genomic libraries and a genomic cosmid library. The gene spans greater than 82 kilobases and consists of 14 exons. The size range for the exons is 82-613 base pairs, whereas that for the introns is approximately 200 bp to greater than 26 kb. A major site of transcription initiation in leukocytes was mapped to a thymidine residue 65 base pairs upstream of the ATG initiation codon by nuclease S1 protection and primer extension experiments. Other potential minor initiation sites were found. The putative promoter region contains no TATA and CCAAT sequences in the expected positions upstream of the major transcription initiation site but contains multiple GC boxes within a (G + C)-rich region, as does the immediate 5' region of the first intron. Characteristics common to the 5' end of the human 5-lipoxygenase gene and the promoter regions of the housekeeping genes raise important questions concerning the regulation of 5-lipoxygenase gene expression.
Cytosolic factor purified from Escherichia coli is necessary and sufficient for the export of a preprotein and is a homotetramer of SecB.
Authors: Watanabe M , Blobel G
Published: May 2006

We have purified to homogeneity a cytosolic factor from Escherichia coli that is required for the translocation of a preprotein into inverted vesicles of the E. coli plasma membrane. The preprotein used is a precursor of mutant maltose-binding protein. This mutant contains alterations of the carboxyl terminus. Unlike the precursor for wild-type maltose-binding protein, the mutant precursor does not acquire a protease-resistant conformation after synthesis and retains posttranslational translocation competence. The purified cytosolic factor, added posttranslationally, is necessary and sufficient to yield virtually 100% translocation of the mutant precursor into inverted vesicles. The purified factor amounts to 0.08% of the cytosolic proteins and is a 64-kDa tetramer consisting of four identical 16-kDa subunits. Amino-terminal sequence analysis revealed that it is identical to the secB gene product. The purified SecB homotetramer is part of a larger 150-kDa complex that represents the "export" factor activity. During purification, the export factor activity dissociates into a 64-kDa SecB homotetramer and unidentified component(s). For the posttranslational integration of another preprotein, the precursor for the lamB gene product, into inverted vesicles, the 64-kDa SecB homotetramer is also required but additional factor(s) makes integration more efficient.
Identification of amino acid residues essential for the enzymatic activities of pertussis toxin.
Authors: Locht C , Capiau C , Feron C
Published: May 2006

The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid. Mutations at the Glu-106 position do not significantly reduce the enzymatic activities of the S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by a variety of monoclonal antibodies, including neutralizing antibodies. Pertussis toxin containing a deletion or replacement of Trp-26, Glu-129, or both in the S1 subunit should thus be devoid of toxic activities without losing its reactivity with protective antibodies and, therefore, could be safely included in new generation vaccines against whooping cough.
Experimental approaches to hypothetical hormones: detection of a candidate ligand of the neu protooncogene.
Authors: Yarden Y , Weinberg R A
Published: May 2006

There is a growing list of oncogenes encoding transmembrane tyrosine kinases that have structures reminiscent of growth factor receptors. In most cases, the ligands for these putative receptors are unknown. Using the neu oncogene as a model system, we have developed several experimental approaches for the detection of such hypothetical ligands. The following lines of evidence collectively imply that a candidate ligand of the neu-encoded oncoprotein is secreted by ras-transformed fibroblasts: Medium conditioned by ras transformants is able to induce down-modulation of the neu-encoded p185 and to activate its intrinsic tyrosine kinase activity in vitro. In addition, a rapid increase in the phosphorylation in vivo of tyrosine residues of the neu-encoded protein is induced by the conditioned medium. Finally, transfer of the neu gene into hematopoietic cells renders them mitogenically responsive to the conditioned medium. The possibility of indirect activation of the oncoprotein through other known receptors, especially the receptor for the epidermal growth factor, was experimentally excluded.
Heparin suppresses the induction of c-fos and c-myc mRNA in murine fibroblasts by selective inhibition of a protein kinase C-dependent pathway.
Authors: Wright T C , Pukac L A , Castellot J J , Karnovsky M J , Levine R A , Kim-Park H Y , Campisi J
Published: May 2006

Heparin is a complex glycosaminoglycan that inhibits the proliferation of several cell types in culture and in vivo. To begin to define the mechanism(s) by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the expression of the growth factor-inducible protooncogenes c-fos and c-myc. We show that heparin suppressed the induction of c-fos and c-myc mRNA by serum in murine (BALB/c) 3T3 fibroblasts. Using purified mitogens, we further show that suppression was most marked when protooncogene expression was induced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. By contrast, there was little or no suppression when the cells were stimulated by epidermal growth factor, which, in these cells, utilizes a protein kinase C-independent pathway for the induction of gene expression. Heparin also inhibited the change in cell morphology induced by the phorbol ester but had no effect on the morphological change induced by epidermal growth factor and agents that raise intracellular cAMP. Heparin did not inhibit intracellular protein kinase C activity, phorbol ester-induced down-regulation of protein kinase C, or phosphorylation of the 80-kDa intracellular protein kinase C substrate. These results suggest that heparin inhibits a protein kinase C-dependent pathway for cell proliferation and suppresses the induction of c-fos and c-myc mRNA at a site distal to activation of the kinase.
Identification of a human transcription unit affected by the variant chromosomal translocations 2;8 and 8;22 of Burkitt lymphoma.
Authors: Shtivelman E , Henglein B , Groitl P , Lipp M , Bishop J M
Published: May 2006

Chromosomal translocations in Burkitt lymphoma and mouse plasmacytomas typically lie within or near the protooncogene MYC. In some instances, however, these tumors contain variant translocations with breakpoints located more distant from and downstream of MYC, in a domain commonly known as pvt-1. Until now, there has been no evidence that pvt-1 marks the location of a functional gene. Here we report the identification of a large transcriptional unit in human DNA that includes pvt-1. We have designated this unit as PVT. PVT begins 57 kilobase pairs downstream of MYC and occupies a minimum of 200 kilobase pairs of DNA. Some of the translocations that occur downstream of MYC in Burkitt lymphoma transect PVT; others lie between the two genes. None of the translocations we have studied appear to enhance transcription from an intact allele of PVT (indeed, they may inactivate that transcription), but some are associated with the production of abundant and anomalous 0.8- to 1.0-kilobase RNAs that contain the 5' exon of PVT and sequences transcribed from the constant region of an immunoglobulin gene (the reciprocal participant in the translocation). Identification of PVT should facilitate the exploration of how translocations downstream of MYC and insertions of retroviral DNA in the vicinity of pvt-1 might contribute to tumorigenesis.
Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor.
Authors: Schols D , Baba M , Pauwels R , Desmyter J , De Clercq E
Published: May 2006

The triphenylmethane derivative aurintricarboxylic acid (ATA), but not aurin, selectively prevented the binding of OKT4A/Leu-3a monoclonal antibody (mAb) and, to a lesser extent, OKT4 mAb to the CD4 cell receptor for human immunodeficiency virus type 1 (HIV-1). The effect was seen within 1 min at an ATA concentration of 10 microM in various T4+ cells (MT-4, U-937, peripheral blood lymphocytes, and monocytes). It was dose-dependent and reversible. ATA prevented the attachment of radiolabeled HIV-1 particles to MT-4 cells, which could be expected as the result of its specific binding to the HIV/CD4 receptor. Other HIV inhibitors such as suramin, fuchsin acid, azidothymidine, dextran sulfate, heparin, and pentosan polysulfate did not affect OKT4A/Leu-3a mAb binding to the CD4 receptor, although the sulfated polysaccharides suppressed HIV-1 adsorption to the cells at concentrations required for complete protection against HIV-1 cytopathogenicity. Thus, ATA is a selective marker molecule for the CD4 receptor. ATA also interfered with the staining of membrane-associated HIV-1 glycoprotein gp120 by a mAb against it. These unusual properties of a small molecule of nonimmunological origin may have important implications for the study of CD4/HIV/AIDS pathogenesis and possibly treatment.
Transformation to continuous growth of primary human T lymphocytes by human T-cell leukemia virus type I X-region genes transduced by a Herpesvirus saimiri vector.
Authors: Grassmann R , Dengler C , Müller-Fleckenstein I , Fleckenstein B , McGuire K , Dokhelar M C , Sodroski J G , Haseltine W A
Published: May 2006

The role of the X region of the genome of the human T-cell leukemia virus type I (HTLV-I) in the immortalization of lymphocytes has been difficult to distinguish from its role in viral replication as this region encodes at least two genes, tax and rex, required for replication and the expression of viral proteins. To determine whether the X region does encode immortalizing functions, a fragment of the HTLV-I provirus capable of expressing known X-region proteins was inserted into the genome of a transformation-defective, replication-competent Herpesvirus saimiri. Infection of fresh mitogen-activated human cord blood and thymocytes yielded immortal T-cell lines that had the same phenotype (CD4+, CD5+, HLA class II+, interleukin 2 receptor alpha-chain +) as lymphocytes transformed by cocultivation with HTLV-I. These experiments demonstrate that the X region encodes the functions of HTLV-I that immortalize a distinct subpopulation of human T cells. The experiments also demonstrate the utility of the H. saimiri vector for the transduction of heterologous genes into human T cells.
Role of endothelium-derived nitric oxide in the regulation of blood pressure.
Authors: Rees D D , Palmer R M , Moncada S
Published: May 2006

The role of endothelium-derived nitric oxide in the regulation of blood pressure in the anesthetized rabbit was studied with N omega-monomethyl-L-arginine (L-NMMA), a specific inhibitor of its formation from L-arginine. L-NMMA (3-100 mg.kg-1), but not its D-enantiomer, induced a dose-dependent long-lasting (15-90 min) increase in mean systemic arterial blood pressure. L-NMMA (100 mg.kg-1) also inhibited significantly the hypotensive action of acetylcholine, without affecting that of glyceryl trinitrate. Both these actions of L-NMMA were reversed by L-arginine (300 mg.kg-1), but not by D-arginine (300 mg.kg-1), indomethacin (1 mg.kg-1), prazosin (0.3 mg.kg-1), or by vagotomy. The effects of L-NMMA in vivo were associated with a significant inhibition of the release of nitric oxide from perfused aortic segments ex vivo. This inhibition was reversed by infusing L-arginine through the aortic segments. These results indicate that nitric oxide formation from L-arginine by the vascular endothelium plays a role in the regulation of blood pressure and in the hypotensive actions of acetylcholine.
An oligomer complementary to c-myb-encoded mRNA inhibits proliferation of human myeloid leukemia cell lines.
Authors: Anfossi G , Gewirtz A M , Calabretta B
Published: May 2006

To study the role of the protooncogene c-myb in regulating myeloid leukemia cell proliferation and differentiation, we exposed cells of the human leukemia lines HL-60, ML-3, KG-1, and KG-1a to an oligodeoxynucleotide complementary to an 18-base-pair (bp) sequence of c-myb-encoded mRNA. This treatment resulted in a significant decrease in cell proliferation in all of the lines, which was most marked in HL-60 cells. After 5 days in culture, in several separate experiments with different oligomer preparations, 75% growth inhibition was observed in c-myb antisense treated cells in comparison to untreated HL-60 cells. Two c-myb antisense oligomers of identical length with either 2- or 4-bp mismatches had no effect on cell growth nor did an 18-bp c-myb sense or myeloperoxidase antisense oligomer. The effect of a c-myc antisense oligomer (18 bp) on the growth of HL-60, KG-1, and KG-1a cells was also studied. This oligomer had much less inhibitory effect on cell proliferation than did the c-myb antisense sequence. Interestingly, although c-myc antisense treatment induced maturation of HL-60 cells while it inhibited cell proliferation, such an effect was not noted in c-myb antisense treated cells. These studies indicate that the nuclear protein encoded by the c-myb protooncogene is required for maintenance of proliferation in certain leukemia cell lines. In compared to c-myc protein suggest that, at least in HL-60 cells, c-myc amplification or N-ras activation may not be sufficient to maintain the leukemic growth in the absence of c-myb protein. These findings support the hypothesis that development and maintenance of a malignant phenotype requires a multiplicity of interrelated genetic events.
Angiotensin II induces oscillations of intracellular calcium and blocks anomalous inward rectifying potassium current in mouse renal juxtaglomerular cells.
Authors: Kurtz A , Penner R
Published: May 2006

Simultaneous patch-clamp and fura-2 measurements were used to investigate the electrical properties and receptor-mediated changes of intracellular calcium in renal juxtaglomerular cells. Here we report the presence of voltage-activated inward and outward rectifying potassium currents and the inhibition of the anomalous inward rectifying potassium current by angiotensin II (ANG-II). This action of ANG-II was mimicked by guanosine 5'-[gamma-thio]triphosphate but not by cAMP, cGMP, inositol 1,4,5-trisphosphate, or phorbol ester, suggesting that ANG-II inhibits the potassium channel directly by means of a guanine nucleotide-binding regulatory protein or by means of an unusual type of second messenger. Blocking of the inward rectifier was paralleled by membrane depolarization, but we obtained no evidence for calcium entry due to voltage-gated calcium channels in juxtaglomerular cells. Instead, under voltage clamp, ANG-II and guanosine 5'-[gamma-thio]triphosphate induced release of calcium from intracellular stores followed by a sustained phase of transmembrane calcium influx and oscillations of intracellular Ca2+ concentrations. Changes in intracellular Ca2+ concentrations were found to depend on the extracellular Ca concentration--i.e., the sustained elevation was abolished in absence of extracellular Ca, and the frequency of repetitive calcium release was directly related to the extracellular concentration of calcium. Moreover, an elevation of extracellular Ca concentration by itself induced release of intracellular calcium in the absence of other stimuli. Changes in intracellular Ca2+ concentrations were accompanied by prominent calcium-activated chloride currents, and this mechanism is inferred to be responsible for the inhibitory role of calcium in renin secretion. Intracellular application of cAMP but no cGMP inhibited ANG-II and guanosine 5'-[gamma-thio]triphosphate induced calcium mobilization in juxtaglomerular cells, being consistent with the facilitatory effects of elevated cAMP levels of renin release. The frequency of ANG-II induced oscillations was also markedly attenuated at depolarized membrane potentials suggesting effective negative feedback control of ANG-II-induced depolarization on repetitive Ca2+ transients induced by the hormone.
Parathyroid hormone-related peptide gene is expressed in the mammalian central nervous system.
Authors: Weir E C , Brines M L , Ikeda K , Burtis W J , Broadus A E , Robbins R J
Published: May 2006

A parathyroid hormone-related peptide (PTHRP) has been identified in human tumors associated with the syndrome of humoral hypercalcemia of malignancy. While parathyroid hormone (PTH) gene expression appears to be limited to the parathyroid glands, PTHRP mRNA has been identified in a variety of normal tissues. To investigate the apparent expression of the PTHRP in the central nervous system, we examined extracts of whole rat brain for PTHRP bioactivity by measuring adenylate cyclase-stimulating activity (ACSA) in a PTH-sensitive assay. Extracts consistently contained ACSA and this activity was completely inhibited by a PTHRP antiserum but was unaffected by a PTH antiserum. ACSA was found in a number of anatomic subregions of rat brain, being greatest in the cortex and telencephalon. RNase protection analysis revealed PTHRP transcripts in total RNA prepared from whole rat brain and from the same anatomic subregions. By in situ hybridization histochemistry, we found that the highest levels of PTHRP gene expression occurred in neurons of the cerebral cortex, hippocampus, and cerebellar cortex. These studies demonstrate that both PTHRP mRNA and biological activity are present in a number of regions of rat brain. The widespread expression of this peptide by multiple types of neurons suggests that the PTHRP may play a general role in neuronal physiology.
Type II hereditary angioneurotic edema that may result from a single nucleotide change in the codon for alanine-436 in the C1 inhibitor gene.
Authors: Levy N J , Ramesh N , Cicardi M , Harrison R A , Davis A E
Published: May 2006

Identical single-base changes in the C1 inhibitor gene that may result in dysfunctional inhibitor proteins are described in two different families with type II hereditary angioneurotic edema. Initially, a restriction fragment length polymorphism was defined that resulted from loss of a Pst I site within exon VIII, which encodes the region containing the reactive center. Exon VIII from the normal and abnormal allelles was amplified by the polymerase chain reaction. Amplified DNA product was cloned into plasmid pUC18; clones representing normal and mutant allelles were distinguished by the presence and absence, respectively, of the Pst I restriction site. DNA sequence analysis revealed a G----A mutation in the codon for alanine-436, which would result in replacement with a threonine residue. This position is nine amino acid residues amino-terminal to the reactive-center arginylthreonine peptide bond. In contrast, previously defined mutations in type II hereditary angioneurotic edema result in replacement of the reactive-center arginine.
Production of abiotic receptors by molecular imprinting of proteins.
Authors: Braco L , Dabulis K , Klibanov A M
Published: May 2006

When a protein is dissolved in a concentrated aqueous solution of a multifunctional organic compound, freeze-dried, and washed with an anhydrous organic solvent to remove the ligand, the resultant "imprinted" protein preparation binds up to 30-fold more of the template compound in anhydrous solvents than the nonimprinted protein in the same solvent (and both proteins in water). These artificial receptors exhibit marked ligand selectivity as well as stability in anhydrous media. This phenomenon of molecular imprinting, demonstrated for several unrelated proteins and ligands, may be helpful in the development of unique bioadsorbents and, potentially, new biocatalysts.
Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation.
Authors: Springman E B , Angleton E L , Birkedal-Hansen H , Van Wart H E
Published: May 2006

Latent human fibroblast collagenase (HFC) can be activated by a variety of seemingly disparate means. In addition to the well-characterized activation by trypsin and organomercurial compounds, the enzyme can be activated to various extents by surfactants such as sodium dodecyl sulfate, by chaotropic ions such as SCN-, by disulfide compounds such as oxidized glutathione, by sulfhydryl alkylating agents such as N-ethylmaleimide, and by oxidants such as NaOCl. The underlying basis for these activations is the modification, exposure, or proteolytic release of the Cys73 residue from its habitat in the latent enzyme where it is thought to be complexed to the active-site zinc atom. This residue is not accessible for reaction with small molar excesses of dithionitrobenzoate in native, latent HFC. However, on addition of EDTA, this residue becomes fully exposed and is quantitatively labeled. All modes of activation of latent HFC are believed to involve the dissociation of Cys73 from the active-site zinc atom and its replacement by water, with the concomitant exposure of the active site. This is thought to be the primary event that precedes the well-known autolytic cleavages that are observed following the appearance of collagenase activity. The dissociation of Cys73 from the zinc atom in the latent enzyme "switches" the role of the zinc from a noncatalytic to a catalytic one. This "cysteine switch" mechanism of regulation may be applicable to the entire collagenase gene family.
Point substitutions in albumin genetic variants from Asia.
Authors: Arai K , Madison J , Shimizu A , Putnam F W
Published: May 2006

Despite their rarity and physiologically neutral character, more inherited structural variants of serum albumin (alloalbumins) are known than for any other human protein except hemoglobin. Including three previously unreported examples described here, we have identified 13 different point substitutions in alloalbumins of Japanese origin. Of these only albumin B and two proalbumins have been reported in other ethnic groups, and these are the most common variants of European origin. Some alloalbumins of Asiatic origin, but not yet identified in Japanese, are present in diverse ethnic groups. An alloalbumin found in indigenes of New Guinea (lysine----asparagine at position 313) is also present in Caucasians of various European descents. Albumin Lambadi, occurring in a tribal group in south India, has a mutation (glutamic acid----lysine at position 501) also found as a rare variant in individuals of diverse ethnic origin resident on four continents. These results suggest that some alloalbumins with the same substitution may have originated by independent mutations in various populations. This, together with the apparent clustering of point substitutions in the protein structure, may reflect hypermutability of the albumin gene.
Mechanism of L-leucyl-L-leucine methyl ester-mediated killing of cytotoxic lymphocytes: dependence on a lysosomal thiol protease, dipeptidyl peptidase I, that is enriched in these cells.
Authors: Thiele D L , Lipsky P E
Published: May 2006

Exposure of murine or human lymphocytes to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) results in selective killing of cytotoxic lymphocytes, whereas helper T cells and B cells remain functionally intact. Cytolytic lymphocytes incubated in the presence of toxic concentrations of Leu-Leu-OMe were found to contain membranolytic metabolites of the structure (Leu-Leu)n-OMe, where n greater than or equal to 3. The sensitivity of cytotoxic lymphocytes to Leu-Leu-OMe was found to be dependent upon production of these metabolites by a lysosomal thiol protease, dipeptidyl peptidase I, which is present at far higher levels in cytotoxic lymphocytes than in cells without cytolytic potential or not of bone marrow origin. Thus, this granule enzyme is required for the unique effects of Leu-Leu-OMe and may provide a target for the development of other immunotherapeutic agents designed to delete cytotoxic lymphocyte responses.
Purification and properties of Myxococcus xanthus C-factor, an intercellular signaling protein.
Authors: Kim S K , Kaiser D
Published: May 2006

C-factor, a Myxococcus xanthus protein that restores the developmental defects of a class of nonautonomous mutants resulting from mutation of the csgA gene, has been purified approximately 1000-fold from starved wild-type cells. The monomeric form of C-factor is a single polypeptide with a molecular mass of 17 kDa that can be solubilized by detergent from membrane components. Characterization by gel filtration and denaturing gel electrophoresis suggests that biologically active C-factor is a dimer composed of two 17-kDa monomers. Antibodies against a form of the M. xanthus csgA gene product overexpressed in Escherichia coli react with purified C-factor.
Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells.
Authors: Zenke M , Steinlein P , Wagner E , Cotten M , Beug H , Birnstiel M L
Published: May 2006

Most current gene transfer methods function satisfactorily in specialized systems involving established cell lines but are often not applicable with nonadherent, primary hematopoietic cells, which are notoriously difficult to transfect. To approach this problem, we have investigated an alternative method of gene transfer, "transferrinfection," in which DNA complexed to transferrin-polycation conjugates is introduced into cells by receptor-mediated endocytosis [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We show here that transferrin-polylysine and transferrin-protamine, when complexed to plasmid DNA containing a luciferase reporter gene, is efficiently bound and moved into avian erythroblasts by endocytosis. Successful transfer and expression of the luciferase reporter gene depends on specific interaction of the transferrin-polylysine-DNA complex with the transferrin receptor and occurs in a significant fraction (greater than 95%) of the cells. Gene transfer efficiency by transferrinfection is lower than with an optimized DEAE-dextran transfection method but reaches similar efficiencies when the cells are treated with chloroquine. Because the procedure in the absence of chloroquine is completely nontoxic to cells, a constant expression level of transferred genes may be maintained by repeated additions of transferrin-polylysine-DNA complex. In addition, the usefulness of transferrinfection for gene transfer into primary hematopoietic cells is demonstrated.
Transfection of beta-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells.
Authors: Yoshimura M , Oka T
Published: May 2006

To study the regulatory sequence elements responsible for casein gene expression, we constructed a chimeric gene containing 5.3 kilobases (kb) of the 5'-flanking sequence and 1.6 kb of the 3'-flanking sequence of the mouse beta-casein gene fused to the bacterial chloramphenicol acetyl-transferase (CAT) gene. The chimeric gene was transfected by the calcium phosphate-precipitation procedure into primary mouse mammary epithelial cells prepared from pregnant mice. The transfection procedure had negligible effect on expression of the endogenous beta-casein gene. Expression of the beta-casein-CAT chimeric gene required the synergistic actions of insulin, hydrocortisone, and prolactin. Expression of the chimeric gene also depended on the appropriate substratum because the degree of hormonal induction of the chimeric gene was much higher in cells cultured on a reconstituted basement membrane (Matrigel) than in cells cultured on either type I collagen gel or plastic. On the other hand, the expression of a simian virus 40-CAT chimeric gene in which the CAT gene was driven by the early promoter of the virus was not influenced by the hormonal milieu and occurred at the highest level in cells cultured on plastic. Additional transfection experiments with a series of beta-casein-CAT constructs suggested the existence of regulatory elements responsible for hormonal induction and negative regulatory elements.
The gamma subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting.
Authors: Flower A M , McHenry C S
Published: May 2006

The tau and gamma subunits of DNA polymerase III holoenzyme are both products of the dnaX gene. Since tau and gamma are required as stoichiometric components of the replicative complex, a mechanism must exist for the cell to coordinate their synthesis and ensure that both subunits are present in an adequate quantity and ratio for assembly. We have proposed that gamma is produced by a translational frameshift event. In this report, we describe the use of dnaX-lacZ fusions in all three reading frames to demonstrate that gamma, the shorter product of dnaX, is generated by ribosomal frameshifting to the -1 reading frame of the mRNA within an oligo(A) sequence that is followed by a sequence predicted to form a stable secondary structure. Immediately after frameshifting a stop codon is encountered, leading to translational termination. Mutagenesis of the oligo(A) sequence abolishes frameshifting, and partial disruption of the predicted distal secondary structure severely impairs the efficiency. Comparison of the expression of lacZ fused to dnaX distal to the site of frameshifting in the -1 and 0 reading frames indicates that the efficiency of frameshifting is approximately 40%.
Identification of a serpin-enzyme complex receptor on human hepatoma cells and human monocytes.
Authors: Perlmutter D H , Glover G I , Rivetna M , Schasteen C S , Fallon R J
Published: May 2006

Formation of the covalently stabilized complex of alpha 1-antitrypsin (alpha 1-AT) with neutrophil elastase, the archetype of serine proteinase inhibitor serpin-enzyme complexes, is associated with structural rearrangement of the alpha 1-AT molecule and hydrolysis of a reactive-site peptide bond. An approximately 4-kDa carboxyl-terminal cleavage fragment is generated. alpha 1-AT-elastase complexes are biologically active, possessing chemotactic activity and mediating increases in expression of the alpha 1-AT gene in human monocytes and macrophages. This suggested that structural rearrangement of the alpha 1-AT molecule, during formation of a complex with elastase, exposes a domain that is recognized by a specific cell surface receptor or receptors. To test this hypothesis, the known three-dimensional structure of alpha 1-AT and comparisons of the primary structures of the serpins were used to select a potentially exteriorly exposed and highly conserved region in the complexed form of alpha 1-AT as a candidate ligand (carboxyl-terminal fragment, amino acids 359-374). We show here that synthetic peptides based on the sequence of this region bind specifically and saturably to human hepatoma cells and human monocytes (Kd = 4.0 X 10(-8) M, 4.5 X 10(5) plasma membrane receptors per cell) and mediate increases in synthesis of alpha 1-AT. Binding of peptide 105Y (Ser-Ile-Pro-Pro-Glu-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Tyr-Leu-Ile) is blocked by alpha 1-AT-elastase complexes, antithrombin III (AT III)-thrombin complexes, alpha 1-antichymotrypsin (alpha 1-ACT)-cathepsin G complexes, and, to a lesser extent, complement component C1 inhibitor-C1s complexes, but not by the corresponding native proteins. Binding of peptide 105Y is also blocked by peptides with sequence corresponding to carboxy-terminal fragments of the serpins AT III and alpha 1-ACT, but not by peptides having the sequence of the extreme amino terminus of alpha 1-AT. The results also show that peptide 105Y inhibits binding of 125I-labeled alpha 1-AT-elastase complexes. Thus, these studies demonstrate an abundant, relatively high-affinity cell surface receptor which recognizes serpin-enzyme complexes (SEC receptor). This receptor is capable of modulating the production of at least one of the serpins, alpha 1-AT. Since the ligand specificity is similar to that previously described for in vivo clearance of serpin-enzyme complexes, the SEC receptor may also be involved in the clearance of certain serpin-enzyme complexes.
Transforming growth factor beta 1 suppression of c-myc gene transcription: role in inhibition of keratinocyte proliferation.
Authors: Pietenpol J A , Holt J T , Stein R W , Moses H L
Published: May 2006

Transforming growth factor beta 1 (TGF-beta 1) is a potent growth inhibitor for many cell types, including most epithelial cells. However, the mechanism of growth inhibition is unknown. In skin keratinocytes, TGF-beta 1 has been shown to inhibit growth and to rapidly reduce c-myc expression. It has been demonstrated that protein synthesis is required for TGF-beta 1 regulation of c-myc in keratinocytes. Here we present evidence that treatment of mouse BALB/MK keratinocyte cells with either antisense c-myc oligonucleotides or TGF-beta 1 inhibited cell entry into S phase. These results suggest that TGF-beta inhibition of c-myc expression may be essential for growth inhibition by TGF-beta 1. The block in c-myc expression by TGF-beta 1 occurred at the level of transcriptional initiation. Studies with a series of 5' deletion c-myc/chloramphenicol acetyltransferase constructs indicated that a cis regulatory element(s), which resides between positions -100 and +71 relative to P1 transcription start site, is responsible for the TGF-beta 1 responsiveness. Based on these data, it is proposed that the mechanism of TGF-beta 1 growth inhibition involves synthesis or modification of a protein that may interact with a specific element(s) in the 5' regulatory region of the c-myc gene, resulting in inhibition of transcriptional initiation.
NICER elements: a family of nerve growth factor-inducible cAMP-extinguishable retrovirus-like elements.
Authors: Cho K O , Minsk B , Wagner J A
Published: May 2006

We have shown previously that the transcription of the gene designated d5 is induced by nerve growth factor (NGF) in rat adrenal pheochromocytoma PC-12 cells and that this NGF induction is repressed by cAMP. In this paper we demonstrate that d5 is a member of a gene family that contains several hundred members, which is closely related to retroviruses and retrotransposons, as demonstrated by the following observations: (i) the original d5 cDNA hybridized to numerous restriction fragments in genomic DNA; (ii) d5 cDNA hybridized to genomic clones with various intensities, and genomic clones can be isolated with a frequency suggesting that this family includes several hundred members; and (iii) there were minor sequence variations in four independently isolated cDNA clones that were homologous to d5 cDNA. Primer extension studies show that initiation of the 5.7-kilobase d5 mRNA(s) occurs at a unique site relative to a synthetic primer. The 5' end of the cDNA sequence was homologous to Rasheed rat sarcoma virus; and a genomic clone contained several elements that are typical of a long terminal repeat (LTR), including a CCAAT box, a TATA box, a primer binding site, a poly(A) addition signal, and a poly(A) addition site. Furthermore, there is a LTR at the 3' end of at least one of the genes in this family, and there appeared to be a four-base duplication at the probable site of integration into host DNA. Since several members of this family retain responses to NGF and cAMP, we conclude that the regulatory elements present in the LTR have been conserved in many members of this family. We have named this family of genes the NICER elements because they are a family of NGF-inducible cAMP-extinguishable retrovirus-like elements.
Human mast cell tryptase: multiple cDNAs and genes reveal a multigene serine protease family.
Authors: Vanderslice P , Ballinger S M , Tam E K , Goldstein S M , Craik C S , Caughey G H
Published: May 2006

Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the approximately 1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family.
DNA intercalation and cleavage of an antitumor antibiotic dynemicin that contains anthracycline and enediyne cores.
Authors: Sugiura Y , Shiraki T , Konishi M , Oki T
Published: May 2006

Dynemicin is a hybrid containing anthraquinone and enediyne cores, which contribute to binding and cleavage of DNA, respectively. DNA strand scission by the antitumor antibiotic is significantly enhanced by the addition of NADPH or thiol compounds. The preferential cutting site of dynemicin is on the 3' side of purine bases (i.e., 5'-GC, -GT, and -AG) and is clearly different from the cutting sites of esperamicin and calicheamicin. The double-stranded and the stem regions of single-stranded DNAs are preferentially cleaved by dynemicin. Therefore, dynemicin may be a useful reagent for probing secondary structures of DNA. Pretreatment of DNA with Adriamycin and actinomycin D alters the cutting mode of dynemicin. Dynemicin-mediated DNA breakage is strongly inhibited by pretreatment of the DNA with distamycin A and anthramycin, suggesting that dynemicin interacts with the minor groove of the DNA helix. Intercalation of the anthraquinone core into the DNA followed by the attack of the phenyl diradical formed from the enediyne core is considered as a possible mechanism of action of dynemicin.