The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids.

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity.

Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3(Pro) of dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B.

Polyprotein processing of dengue virus type 2, a positive strand RNA virus, is carried out by the host signal peptidase and a novel two-component viral proteinase of the serine proteinase family, NS2B/NS3(Pro), in the endoplasmic reticulum. Using an in vitro processing system, we examined the cis and trans cleavages of the 2B/3 and 4B/5 sites by NS2B/NS3(Pro), respectively. Lysates of BHK-21 cells coexpressing NS2B and NS3(Pro) mediated trans cleavage of the 4B/5 site in vitro, and the protease activity was associated with the membrane fraction. To study the role of membranes in the protease activity of NS2B/NS3(Pro), labeled precursors, NS2B-NS3(Pro), and the mutant ndNS2B-NS3(Pro) in which the functional hydrophilic domain of NS2B was deleted, were analyzed using a coupled in vitro transcription/translation system (TnT). The results showed that cotranslational addition of microsomal membranes to the TnT reaction markedly enhanced the cis cleavage of the 2B/3 site in a dose-dependent manner. NS2B synthesized in the presence of membranes also facilitated trans cleavage of the 2B/3 site in the mutant precursor. The cleavage products, NS2B and NS3(Pro), were membrane-associated. Furthermore, this membrane requirement was dictated by the hydrophobic regions of NS2B. Deletion of hydrophobic regions of NS2B, leaving only the conserved hydrophilic domain of 40 amino acids, resulted in highly efficient processing of the 2B-3 site in vitro in the absence of microsomal membranes.

Association between NS3 and NS5 proteins of dengue virus type 2 in the putative RNA replicase is linked to differential phosphorylation of NS5.

Dengue virus type 2, a member of the family Flaviviridae, encodes a single polyprotein precursor consisting of 3391 amino acids residues that is processed to at least 10 mature proteins by host and viral proteases. The NS3 protein contains a domain commonly found in cellular serine proteinases that in cooperation with NS2B is involved in polyprotein processing. In addition, NS3 and NS5 proteins contain conserved motifs found in several RNA helicases and RNA-dependent RNA polymerases, respectively. Both enzymatic activities have been suggested to be involved in viral RNA replication. In this report, we demonstrate that the NS3 and NS5 proteins interact in vivo in dengue virus type 2-infected monkey kidney (CV-1) cells and in HeLa cells coinfected with recombinant vaccinia viruses encoding these proteins as shown by coimmunoprecipitations and immunoblotting methods. We also show by immunofluorescence, metabolic labeling, and two-dimensional peptide mapping that NS5 is a nuclear phosphoprotein and that phosphorylation occurs on serine residues at multiple sites. Furthermore, NS5 exists in differentially phosphorylated states in the nuclear and the cytoplasmic fractions, and only the cytoplasmic form of NS5 is found to coimmunoprecipitate with NS3, suggesting that differential phosphorylation may control the interaction between these proteins and its function in the viral RNA replicase.

Expression of the extracellular domain of the rat liver prolactin receptor and its interaction with ovine prolactin.

A clone of the extracellular domain of the rat liver prolactin receptor was generated by the RNA-based polymerase chain reaction, and the NH2-terminal 210 amino acids were expressed in HeLa cells using a vaccinia virus/T7 hybrid expression system. The protein was isolated from serum-free culture medium directly by chromatography on an ovine prolactin affinity column and yielded approximately 1.5 mg of protein/liter of suspension culture. The extracellular domain of the rat prolactin receptor inhibited the ovine prolactin-dependent mitogenesis of rat lymphoma Nb2 cells with an IC50 of 7.1 pM and bound 125I-labeled ovine prolactin with a Kd of 1.21 +/- 0.19 nM. In contrast, the binding of the 125I-labeled extracellular domain to ovine prolactin exhibited positive cooperativity with a Hill coefficient of 1.73. High pressure gel filtration chromatography was used to demonstrate the formation of a complex consisting of one molecule of ovine prolactin and two molecules of the extracellular domain of the rat prolactin receptor. Complex formation occurred with human growth hormone, but not with ovine growth hormone, a non-lactogen.

Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen.

The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically.

Interactions of antisense peptides with ovine prolactin.

Two antisense peptides were synthesized to a sense peptide corresponding to amino acid residues 23-35 of ovine prolactin. Both of the antisense peptides formed a saturable complex with the sense peptide and ovine prolactin. The sense peptide inhibited the interaction of ovine prolactin with the antisense peptides. Both of the antisense peptides have a common core sequence VMNV which can bind to ovine prolactin. The lactogenic hormones, rat prolactin and human growth hormone, compete with the binding of ovine prolactin to an antisense peptide whereas a nonlactogen, ovine growth hormone, does not compete indicating a degree of specificity in the interaction.

Isolation and characterization of a transferrin binding protein from rat plasma.

A transferrin binding protein was isolated from normal rat placenta and from iron-deficient rat plasma using a human transferrin affinity column. The yield of the isolated pure protein from iron-deficient rat plasma was about 0.5 micrograms/ml plasma. The major protein had a molecular mass of 85 kDa and contained carbohydrate. Reduction with mercaptoethanol did not change the molecular mass of the plasma transferrin binding protein whereas the native placental transferrin receptor of 180 kDa was reduced to 90 kDa. The transferrin binding protein reacted with both monoclonal and polyclonal antibodies raised against rat transferrin receptor. Immunoblotting of both normal and iron deficient rat plasma showed that the transferrin binding protein had a molecular mass of 85 kDa. In vitro digestion of purified rat placental transferrin receptor and red blood cells with trypsin provided an identical peptide profile, suggesting that the transferrin binding protein in rat plasma is derived from proteolysis of the extracellular portion of the transferrin receptor of the erythroid tissues.

Induction of c-fos mRNA in rat lymphoma Nb-2 cells.

Cyclosporin A, immunosuppressive agent, reversibly blocks the mitogenic effect of prolactin in rat lymphoma Nb-2 cells and removal from the medium leads to a rapid and transient induction of c-fos mRNA. Activators of protein kinase C, such as TPA, mellitin and phospholipase C and the calcium ionophore, A23187, induced c-fos mRNA in the presence or absence of cyclosporin A. Activators of the cAMP pathway such as forskolin, dBcAMP and cholera toxin failed to induce c-fos mRNA in the presence or absence of cyclosporin A. These results suggest that cyclosporin A may act at the level of protein kinase C.

Cloning and nucleotide sequence of ovine prolactin cDNA.

A cDNA expression library was constructed in the lambda gt 11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5' region of this cDNA. Translation initiation could occur at the 5' proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283-292)], with its substitution of the A at -3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3'-untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.

An immunochemical study of avian pancreatic polypeptide: the nature of the principal epitope.

Structural features contributing to the antigenic recognition of the small globular hormone avian pancreatic polypeptide (APP) by a polyclonal antiserum have been defined using a solution phase radioimmunoassay technique. Cross-reactivity studies with PP homologues suggest that the surface residues within the alpha-helix of the peptide may be antigenic, whereas hydrophilicity and atomic mobility predictive methods implicate the molecules beta-turn region. Immunochemical data and circular dichroism measurements on a timed trypsin digest of APP indicate that the secondary structure of the alpha-helix is vital for the molecule's immunological competence. Immunoreactivities of iodinated derivatives of APP, as well as that of peptide fragments of APP and its homologues, support the importance of teritary structure involving the interaction of the polyproline and alpha helices. The highly mobile C-terminal residues 34-36 (His-Arg-Tyr-NH2) have been found by immunological analysis to be unimportant. Arginine residue 33, which has been conserved through vertebrate evolution, is a major antigenic contributor, since a large decrease in immunoreactivity, not accompanied by a significant change in conformation, was observed upon specific removal of this residue by carboxypeptidase B. These results are consistent with a "discontinuous" epitopic model for APP in which Arg-33 and exposed residues in the alpha-helix are principal components of an epitope or epitopes mediated by the secondary and tertiary structures of the molecule.

The effect of cyclosporin A on the growth and prolactin binding to Nb-2 rat lymphoma cells.

Cyclosporin A, an immunosuppressive agent, inhibited the prolactin stimulated growth of rat lymphoma Nb-2 cells. In the presence of 1 ng/ml of prolactin, 50% inhibition of growth was at 5 x 10(-6) M and the inhibition was reversible. The Kd of cyclosporin A binding to the Nb-2 cells was 10(-7) M and was independent of prolactin. The Kd of prolactin binding to the Nb-2 cells was 2 x 10(-10) M. Cyclosporin A did not influence the binding of prolactin to the cells and vice versa. The inhibitory effect of cyclosporin A on the growth of Nb-2 cells is due to some step other than the binding of prolactin to the cells.

Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin, and two glucagon-like peptides.

Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals.

Isolation of alligator gar (Lepisosteus spatula) glucagon, oxyntomodulin, and glucagon-like peptide: amino acid sequences of oxyntomodulin and glucagon-like peptide.

Oxyntomodulin, glucagon, and a glucagon-like peptide (GLP) have been isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish. The three peptides were isolated by gel filtration and HPLC and were identified by size, composition, and glucagon-like immunoreactivity. The amino acid sequences of the oxyntomodulin and GLP were determined. The oxyntomodulin contains 36 amino acid residues and its sequence is H S Q G T F T N D Y S K Y L D T R R A Q D F V Q W L M S T K R S G G I T. The composition of the glucagon is identical to the N-terminal 29 residues of the gar oxyntomodulin. The single form of GLP found contains 34 amino acid residues in the following sequence: H A D G T Y T S D V S S Y L Q D Q A A K K F V T W L K Q G Q D R R E. These findings suggest that all three peptides are derived from a common precursor.

Isolation and structures of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide.

Insulin and a 36-residue peptide with homology to pancreatic polypeptide (PP) were isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish, by gel filtration and HPLC. Heterologous radioimmunoassays were used to detect insulin-like and PP-like immunoreactivities during purification of the two peptides. The sequence of the 36-amino acid peptide containing a C-terminal tyrosinamide was identical at 31 of 36 positions to porcine neuropeptide Y (NPY). The amino acid sequence of this peptide is YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY-NH2. The second peptide, gar insulin, contains 52 amino acid residues and is composed of a 21-residue A chain and a 31-residue B chain. The sequence of the A chain is GIVEQCCHKPCTIYELENYCN. The sequence of the B chain is AANQHLCGSHLVEALYLVCGEKGFFYNPNKV.

Regulation of expression of c-fos and c-myc in rat lymphoma Nb-2 cells.

This study examined the effects of the mitogen, prolactin and the cell cycle inhibitors, cyclosporin A and neomycin sulfate, on expression of the proto-oncogenes c-fos and c-myc in the rat lymphoma Nb-2 cell line. Stimulation of quiescent cultures with prolactin resulted in a 2-3-fold increase in the constitutive levels of c-myc mRNA which peaked at 4 h and declined thereafter. c-Fos mRNA was not detected in quiescent or prolactin-stimulated cultures. Cyclosporin A or neomycin sulfate reversibly blocked the mitogenic effect of prolactin on Nb-2 cells, but had little effect on constitutive levels of c-myc. However, the release of Nb-2 cells from a cyclosporin A or a neomycin sulfate block resulted in a rapid transient induction of c-fos which peaked at 0.5-1 h and declined rapidly thereafter. These results indicate that the rapid transient expression of c-fos following release from cell cycle blockage was not sufficient to elicit cell division, but these cells were competent to respond to prolactin. Prolactin allows progression through the cell cycle and enhances c-myc mRNA levels.

Structure of a peptide from coho salmon endocrine pancreas with homology to neuropeptide Y.

The amino acid sequence of a peptide isolated from the Pacific salmon (Oncorhynchus kisutch) endocrine pancreas has been determined. This simple 36 residue peptide is a member of the pancreatic polypeptide family. It contains a C-terminal tyrosinamide and is more homologous with porcine neuropeptide Y (NPY) (83%) and peptide YY (75%) than any of the previously characterized pancreatic polypeptides (PP). This peptide appears to be the major but not the only representative of this family of peptides present in the endocrine pancreas of this fish. This peptide is referred to as salmon pancreatic polypeptide (salmon PP).

Effect of oxidizing agents on rabbit mammary prolactin receptors.

Treatment of rabbit mammary membrane-bound and solubilized prolactin receptors with the oxidizing agents N-chlorobenzene sulfonamide and N-bromosuccinimide resulted in total inactivation of the ability of the receptor to bind prolactin. A similar inactivation was obtained with 2-hydroxy-5-nitrobenzylbromide. The results suggest the possible importance of tryptophan in maintaining the activity of the prolactin receptor.

Inactivation of rabbit mammary prolactin receptor by N-acetylimidazole.

Treatment of rabbit mammary prolactin receptor with N-acetylimidazole resulted in loss of prolactin binding activity. Loss of activity of the particulate receptor was time and concentration dependent with 100 mM reagent causing total inactivation in 10 min. Similar results were obtained with solubilized receptor preparations, but at lower reagent concentrations. The loss of binding activity was due to loss in the number of binding sites. Incubation of the reagent inactivated membranes or soluble receptor with hydroxylamine for 3 hr resulted in 80-90% reactivation of the prolactin binding activity. These results indicate the possible involvement of tyrosyl residue(s) on the receptor in the prolactin-receptor interaction.

Cyclosporin A and rabbit mammary prolactin receptors.

The binding of cyclosporin A and ovine prolactin to rabbit mammary gland membranes was determined. CsA bound with a Kd of 2.2 X 10(-6)M whereas prolactin bound with a Kd of 2 X 10(-10)M. The binding of each ligand was an independent event and neither ligand influenced the binding of the other ligand showing that CsA does not inhibit the binding of prolactin to its specific receptor in this system.

The Cuisinart food processor efficiently disaggregates tissues.

The Cuisinart Model DLC-7 PRO food processor efficiently disaggregates plant and animal tissues resulting in time savings and often increased yields in the isolated material.