Calcium-dependent Ret activation by GDNF and neurturin.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) define a new family of neurotrophic factors that play crucial roles in survival and differentiation of various neurons. Recent studies demonstrated that GDNF and NTN use a multicomponent receptor system in which glycosyl-phosphatidylinositol (GPI)-linked cell surface proteins and Ret receptor tyrosine kinase function as the ligand-binding and signalling components, respectively. In the present study, we investigated the role of Ca2+ ions for biochemical and biological activities of Ret because Ret has a unique structure of the extracellular domain with the cadherin-like motif. The results demonstrated that Ca2+ ions might be required for the complex formation of Ret and GDNF or NTN that induces Ret oligomerization and autophosphorylation. Full morphological differentiation of neuroblastoma cells by these neurotrophic factors was also Ca2+-dependent. These findings thus suggested that, in addition to GPI-linked cell surface proteins, Ca2+ ions are components of the signal transducing complex formed by Ret and GDNF protein family.

Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens.

A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.

Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells.

Recombinant baculoviruses that produce a putative non-structural protein 1 (NS1) of hepatitis C virus (HCV), predicted to be the second envelope glycoprotein, were constructed. The recombinant NS1 protein (re-NS1) produced in infected insect cells was localized on the cell surface and was apparently glycosylated, because it was susceptible to treatment with both tunicamycin and N-glycanase. Furthermore, re-NS1 was effectively secreted into the culture supernatant when the putative NS1 signal peptide (SP) was replaced by the SP of rabies virus G protein, and the C-terminal hydrophobic region was eliminated. The secreted re-NS1 was tagged with six His residues at the C terminus and purified simply by native Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) affinity column chromatography. An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of HC using purified re-NS1. Anti-NS1 antibody (Ab) was detected in 55 of 60 patients (92%) with chronic HC liver diseases. Thus, this ELISA for Ab directed against HCV re-NS1 produced in insect cells is useful for the detection of chronic HC patients.

Expression of herpes simplex virus glycoprotein B gene in yeast.

The DNA sequence coding for herpes simplex virus type 1 glycoprotein B was placed under control of the acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of replicating in both yeast and Escherichia coli. Yeast transformed by the plasmid synthesized immunologically active glycoprotein B polypeptide.

Conserved immunogenic region of a major core protein (p24) of human and simian immunodeficiency viruses.

A murine monoclonal antibody (MoAb), VAK 4, has been known to specifically react with a major core protein (p24) as well as with its precursor (p55-57) and intermediate precursor (p40) of human immunodeficiency virus strain IIIB (HTLV-IIIB). Radioimmunoprecipitation assays revealed that VAK 4 MoAb precipitated a major core protein and its precursors from a variety of strains of HIV and also from simian immunodeficiency virus (SIV), although the molecular weights of the precursor proteins in each viral strain were slightly different. A protein synthesized by transfected Escherichia coli containing amino acid sequences corresponding to residues 121-436 of the HTLV-IIIB gag gene was reactive with VAK 4 MoAb, but the protein carrying only residues 121-309 was not reactive, suggesting that the epitope recognized by VAK 4 MoAb resides at the carboxyl terminus of p24 protein. A competitive enzyme-linked immunosorbent assay showed that patient sera containing anticore protein antibody inhibited the binding of VAK 4 to HTLV-IIIB. These findings suggested that VAK 4 MoAb recognized an immunogenic and conserved epitope belonging to a major core protein of HIV-related viruses.

Prognostic impact of telomerase activity in patients with neuroblastoma.

Neuroblastoma is one of the most common malignant neoplasms occurring among children. The prognosis for this disease is strongly associated with age, disease stage, histology, and some biologic features. It has been reported that telomerase, a ribonucleoprotein enzyme, which maintains the telomere length in immortal cells, is related to disease stage and other biologic features. The purpose of this study was to evaluate the prognostic value of telomerase activity compared to TrkA expression in 65 patients with neuroblastoma. Telomerase activity and TrkA expression were examined in tissue samples collected between 1980 and 1994 from 65 patients by polymerase chain reaction-based telomerase activity. TrkA expression was examined by immunoblotting using a rabbit anti-gp140 proto-trk polyclonal antibody. Low telomerase activity was found in 22 of 30 (73.3%) patients with Stages 1, 2, or 4S neuroblastomas; 7 of 13 (53.8%) with Stage 3; and 8 of 22 (36.3%) with stage 4; no telomerase activity was detected in 7 of 22 (31.8%) patients with Stage 4 neuroblastoma. The 5-year event-free survival (EFS) rate was 86.5% for patients with low telomerase activity, while it was 53.8% for patients with high telomerase activity. By the combination of telomerase activity and TrkA expression, the 5-year EFS rate was highest among patients with a high TrkA expression and a low or non-existent telomerase activity (91.7%), and it was lowest among patients with a low TrkA expression and a high telomerase activity (29.6%). Thus, it appears that telomerase activity would be a useful prognostic factor for neuroblastoma, especially when used in combination with the TrkA expression.

Comparative biochemistry of disintegrins isolated from snake venom: consideration of the taxonomy and geographical distribution of snakes in the genus Echis.

Species in the genus Echis have been classified mainly based on their morphological appearance and the analytical patterns of their serum. However, re-classification of the genus Echis has recently been suggested by taxonomists, toxicologists, and clinicians, since there have been problems with the current classification, such as the efficacy of antivenoms used for treating bites and the broad geographical distribution of Echis snakes. In this study, we purified five novel disintegrins, the platelet aggregation inhibitors pyramidin A and B from the venom of Echis pyramidum, ocellatin from the venom of Echis ocellatus, and leucogastin A and B from the venom of Echis leucogaster, to compare their sequences and allow us to re-evaluate the classification of various species in the genus Echis. Comparison of the amino acid sequences of five new and four known isolated disintegrins from snake venoms of six Echis species and their distribution strongly support the recent re-classification of the genus Echis.

A novel human metalloprotease synthesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease?

We identified a novel metalloprotease, which could be responsible for cleaving the Tyr842-Met843 peptide bond of von Willebrand factor (vWF). This metalloprotease was purified from Cohn Fraction-I precipitate of human pooled plasma by the combination of gel filtration, DEAE chromatography, and preparative polyacrylamide gel electrophoresis in the presence of SDS. The NH2-terminal amino acid sequence of the isolated protein was: AAGGILHLELLVAVGPDVFQAHQEDTRRY. Based on this sequence, we searched human genomic and EST databases, and identified compatible nucleotide sequences. These results suggested that this protein is a novel metalloprotease, a member of the family of a disintegrin and metalloprotease with thrombospondin type-1 motifs (ADAMTS), and its genomic DNA was mapped to human chromosome 9q34. Multiple human tissue northern blotting analysis indicated that the mRNA encoding this protease spanned approximately 5 kilobases and was uniquely expressed in the liver. Furthermore, we determined the cDNA sequence encoding this protease, and found that this protease was comprised of a signal peptide, a proregion followed by the putative furin cleavage site, a reprolysin-type zinc-metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (TSP1) motif, a cysteine-rich region, a spacer domain, and COOH-terminal TSP1 motif repeats.

An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin.

Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.

Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and in vitro DNA amplification-restricted transcription-translation system.

Three kinds of hepatitis C virus (HCV) core peptides were produced directly and efficiently in E. coli: 1-120 aa of the C region as NCC, 1-157 aa as NCCT and 1-190 aa as NCCL. These peptides were estimated to be 16, 22 and 24 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The processing to produce p22 core protein observed in insect cells and mammalian systems did not occur in E. coli. These peptides were similarly reactive with serum antibody from patients with hepatitis C. A mutant clone of NCC recombinant plasmid pKNCC4 was obtained, whose product, NCC4, was more stable in the E. coli lysate and was highly immunoreactive with sera of hepatitis C patients. This stable immunoreactive core peptide produced by pKNCC4 is useful for the detection of anti-HCV core antibody. Immunoreactive core peptides were also produced by DNA amplification-restricted transcription-translation. Five kinds of cDNA from C to E1 region were amplified and transcribed in vitro, and these five transcripts were then translated in vitro using rabbit reticulocyte lysate: 1-120 aa as 17 kDa of C1, 1-155 aa as 21 kDa of C2, 1-174 aa as 22 kDa of C3, 1-192 aa as 24 kDa of C4, and 1-213 aa as 26 kDa of C5. Cotranslational processing using microsomal membranes occurred in peptides C4 and C5 to produce p22 the same size as C3. These results indicate that the C-terminus of the mature core protein p22 may be generated at around aa 174 by cleavage with the signal peptidase.

Induction of acquired factor IX inhibitors in cynomolgus monkey (Macaca fascicularis): a new primate model of hemophilia B.

Inherited hemophilia dog and other transient hemophilic animal models have been used for evaluation of hemostatic agents for use in treatment of hemophilia. We established the first nonhuman primate hemophilic model by immunizing cynomolgus monkeys with human FIX (hFIX) in adjuvants. FIX activities of all three hFIX-immunized monkeys decreased transiently to less than 10% in accordance with prolongation of activated partial thromboplastin time (APTT). Forty micrograms of human factor VIIa (hFVIIa) per kilogram body weight (that was reported to be clinically effective) was administered to the monkey with the highest inhibitor titer to evaluate its usefulness as a hemophilia inhibitor model. Results of thromboelastography (TEG) after the injection demonstrated that the hemostatic effect of FVIIa in this model would be similar to that in hemophiliacs with inhibitors. The antibodies purified from the monkey's plasma by hFIX-immobilized gel were composed of two types: Ca(2+)-dependent and -independent antibodies, with features of IgG(1) and IgG(4). Both types of antibodies reacted to cynomolgus FIX, and only Ca(2+)-dependent antibodies also expressed inhibitory activity against cynomolgus FIX. Immunoblotting analyses of Ca(2+)-dependent antibodies using hFIX and its derivatives suggested that they recognized the Ca(2+)-dependent conformation related to the gamma-carboxyglutamic acid (Gla) domain. Comparison of FIX cDNA from human, cynomolgus monkey, and other species, and the results of immunization of various animals (goats, beagle dogs, rabbits, and rats) with hFIX in adjuvants strongly suggested that the development of acquired FIX inhibitors in the monkeys might be due to high cross-reactivity of the antibodies to molecular mimic antigens, hFIX, and cynomolgus FIX.

Increased interleukin-10 associated with low IL-6 concentration correlated with greater survival rates in mice infected by rabies virus vaccinated against it and immunomodulated with P. acnes.

Macrophage activity, cytokines serum concentration, serum neutralizing antibodies and lethality by rabies were evaluated in swiss mice experimentally infected with street rabies virus and submitted or not to antirabies vaccination and immunomodulation with P. acnes. Animals were killed at different times and serum was collected in order to evaluate cytokines concentration; peritonial and splenic macrophages were collected for macrophage activity evaluation. Greater survival rates higher IL-10 and low IL-6 serum concentration were observed in vaccinated animals treated using P. acnes.

Construction and characterization of phage libraries displaying artificial proteins with random sequences.

Three phage libraries, PL1, PL2, and PL3, displaying artificial proteins with random sequences were constructed. The artificial proteins, which are model of ancestral proteins, are derivatives of the 25 kinds of random proteins with about 140 amino acid residues produced via random mutagenesis and combinatorial recombination. The random proteins were displayed on the surface of filamentous bacteriophage as fusion protein with the pIII coat protein at an estimated average number on the phage particles in PL1, PL2, and PL3 of 0.32, 0.32, and 0.08, respectively. Each library was shown to express 10(5) to 10(6) kinds of random proteins. With the phage libraries displaying long random peptides, we now have an effective selection system to observe in vitro evolution of new functional proteins from artificial proteins with random sequences.

In vivo properties of KNT-127, a novel δ opioid receptor agonist: receptor internalization, antihyperalgesia and antidepressant effects in mice.

BACKGROUND AND PURPOSE: Activation of δ opioid (DOP) receptors regulates pain and emotional responses, and also displays ligand-biased agonism. KNT-127 (1,2,3,4,4a,5,12,12a-octahydro-2-methyl-4aß,1ß-([1,2]benzenomethano)-2,6-diazanaphthacene-12aß,17-diol) is a novel DOP receptor agonist inducing analgesia and antidepressant effects in mice. Here, we have assessed KNT-127 for (i) analgesia against chronic inflammatory pain; (ii) effects on depression, locomotion and DOP receptor internalization; and (iii) for cross-tolerance to analgesic and antidepressant effects of acute treatment by other DOP receptor agonists. EXPERIMENTAL APPROACH: Inflammatory pain was induced by complete Freund's adjuvant injection into tail or hindpaw, and thermal and mechanical sensitivities were determined in mice. Locomotor and antidepressant-like effects were measured using actimetry and forced swim test respectively. In vivo KNT-127 selectivity and internalization were assessed using DOP receptor knockout mice and knock-in mice expressing fluorescent-tagged DOP receptors. KNT-127 was injected acutely at 0.1-10.0 mg·kg(-1) or administered chronically at 5 mg·kg(-1) daily over 5 days. KEY RESULTS: Acute treatment with KNT-127 reversed inflammatory hyperalgesia, produced an antidepressant-like effect but induced neither hyperlocomotion nor receptor sequestration. Chronic treatment with KNT-127 induced tolerance and cross-tolerance to SNC80-induced analgesia, but no tolerance to SNC80-evoked hyperlocomotor or antidepressant-like effects. CONCLUSIONS AND IMPLICATIONS: The DOP receptor agonist KNT-127 induced agonist-specific acute and chronic responses, at both behavioural and cellular levels. It displays activities similar to the other recently reported DOP agonists, AR-M1000390, ADL5747 and ADL5859, and differs from SNC80. SNC80 differs from the other DOP receptor agonists including KNT-127, by exhibiting ligand-biased tolerance at this receptor.

Species-specific nested PCR as a diagnostic tool for Brucella ovis infection in rams / Nested PCR espécie-específica para o diagnóstico da infecção por Brucella ovis em carneiros

The aim of the present study was to evaluate a species-specific nested PCR based on a previously described species-specific PCR for detection of B. ovis in semen and urine samples of experimentally infected rams. The performance of the species-specific nested PCR was compared with the results of a genus-specific PCR. Fourteen rams were experimentally infected with the Brucella ovis REO 198 strain and samples of semen and urine were collected every week up to 180 days post infection. Out of 83 semen samples collected, 42 (50.6%) were positive for the species-specific nested PCR, and 23 (27.7%) were positive for the genus-specific PCR. Out of 75 urine samples, 49 (65.3%) were positive for the species-specific nested PCR, whereas 11 (14.6%) were genus-specific PCR positive. Species-specific nested PCR was significantly more sensitive (P<0.001) than the genus-specific PCR in semen and urine from experimentally infected rams. In conclusion, the species-specific nested PCR developed in this study may be used as a diagnostic tool for the detection of B. ovis in semen and urine samples from suspected rams.

O presente estudo objetivou avaliar uma técnica de nested PCR espécie-específica delineada a partir de PCR espécie-específica descrita anteriormente para detecção de B. ovis em sêmen e urina de carneiros infectados experimentalmente. O desempenho da nested PCR espécie-específica foi comparado com os resultados de uma PCR gênero-específica. Quatorze carneiros foram infectados experimentalmente com Brucella ovis REO 198 e amostras de sêmen e de urina foram colhidas semanalmente até 180 dias após a infecção. De 83 amostras de sêmen, 42 (50,6%) foram positivas pela nested PCR espécie-específica, e 23 (27,7%) foram positivas pela PCR gênero-específica. De 75 amostras de urina, 49 (65,3%) foram positivas pela nested PCR espécie-específica, enquanto 11 (14,6%) foram positivas em PCR gênero-específica. A técnica de nested PCR espécie-específica foi significativamente mais sensível (P<0,001) do que a PCR gênero-específica no sêmen e na urina de carneiros infectados experimentalmente. Em conclusão, a nested PCR espécie-específica desenvolvida neste estudo pode ser utilizada como ferramenta de diagnóstico para detecção de B. ovis em sêmen e urina de carneiros suspeitos.

Adaptation and evaluation of polymerase chain reaction for Brucella ovis detection in semen, urine and organs of rams experimentally infected / Adaptação e avaliação da reação em cadeia da polimerase para detecção de Brucella ovis em sêmen, urina e órgãos de carneiros infectados experimentalmente

O objetivo do estudo foi adaptar e avaliar a PCR para detecção de Brucella ovis e comparar os resultados com aqueles obtidos por cultivo microbiológico do sêmen, urina e dos órgãos de carneiros infectados experimentalmente. Dos 31 animais infectados experimentalmente, amostras de PCR do sêmen apresentaram maior sensibilidade (21,6 por cento) do que o cultivo (8,0 por cento). Em amostras de urina, a sensibilidade das técnicas foi semelhante (10,1 por cento para a cultivo e 12,7 por cento para PCR). PCR detectou a presença do agente em 21,5 por cento das amostras testadas, enquanto os órgãos de cultivo detectaram em apenas 3,3 por cento das amostras. PCR detectou um maior número de amostras positivas do que o cultivo microbiológico.