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1.
An. pediatr. (2003, Ed. impr.) ; 64(supl.2): 15-22, mayo 2006. ilus
Article in Spanish | IBECS | ID: ibc-145278

ABSTRACT

La regeneración del corazón dañado ha sido objeto de intensa investigación durante la década pasada. Las diferentes estrategias que han sido desarrrolladas (como por ejemplo la terapia celular basada en células madre (ES)), esclarecen la información acerca de las moléculas y los factores de transcripción involucrados en las cardiomiogénesis. Sin embargo, todavía no es posible programar eficientemente las ES para que se desarrollen a cardiomiocitos. Los mecanismos celulares y moleculares inherentes en el desarrollo embrionario del corazón, así como las interconexiones entre ellos, pueden aportar datos acerca de las rutas bioquímicas necesarias para la diferenciación de las ES embrionarias a células cardíacas. Nosotros proponemos que un modelo cuantitativo que puede servir para descifrar las elaboradas rutas involucradas en la cardiomiogénesis. Esta aproximación podría revelar la etiología de los defectos cardíacos y permitiría producir cardiomiocitos con propósitos clínicos en la regeneracíon y la toxicología entre otros (AU)


No disponible


Subject(s)
Humans , Heart/embryology , Cell- and Tissue-Based Therapy/methods , Myocytes, Cardiac , Calcium Signaling/physiology , Regeneration/physiology , Embryonic Stem Cells , Gene Expression , Cell Differentiation/physiology
2.
Cell Death Differ ; 13(7): 1170-80, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16311512

ABSTRACT

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.


Subject(s)
Phosphoprotein Phosphatases/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acids/metabolism , Apoptosis/physiology , Cell Cycle Proteins/metabolism , Cell Line , Cell Nucleus/metabolism , Checkpoint Kinase 2 , DNA Damage , HCT116 Cells , Humans , Immunoblotting , Mutation , Phosphoprotein Phosphatases/genetics , Phosphorylation , Protein Phosphatase 1 , Protein Phosphatase 2C , RNA, Small Interfering/genetics , Serine/metabolism , Threonine/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
3.
East Afr Med J ; 82(12): 609-13, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16619704

ABSTRACT

OBJECTIVE: To determine the pattern of opportunistic infections such as TB and Candida species in HIV infected patients in Northern Kenya. DESIGN: Cross-sectional study. SETTING: Five health facilities in Moyale (n=224), Mandera (n=121) and Turkana Kakuma; (n=83), Lopiding; (n=94) districts during different periods in 2003. SUBJECTS: Five hundred and fifty two patients. RESULTS: In total 94 (18%) patients were found to be HIV positive (Moyale=42, Mandera=13, Turkana; Kakuma=8, Lopiding=31). Only 65 of 94 HIV positive patients provided saliva samples. Of these, 11 (17%) were TB smear positive and 19 (29.2%) were colonized by oral Candida species. The Candida isolates were as follows; Co-infection of Candida species and TB (n=4), C. albicans only (n=12), C. tropicalis only (n=1), C. albicans and C. glabarata (n=1) and C. albicans, C. glabarata and C. tropicalis. co-infection (n=1). CONCLUSION: The findings provides an important insight into the differences in mucosal susceptibility to bacteria (TB) infection and fungal (Candida species) colonization during HIV immunosuppression, based on collected blood, sputum and saliva specimens. Further studies are needed to elucidate the comparative transmission dynamics and pathogenetic mechanisms of these opportunistic infections-in different regions of Kenya. Such studies would improve the efficiency of directly observed preventive therapy programme (DOPT-P) whose implementation involves screening by tuberculin skin testing.


Subject(s)
Candidiasis, Oral/complications , HIV Infections/complications , Opportunistic Infections/complications , Tuberculosis/complications , Female , HIV Infections/epidemiology , Humans , Kenya/epidemiology , Male , Mass Screening , Opportunistic Infections/epidemiology , Opportunistic Infections/microbiology , Population Surveillance , Prevalence , Tuberculin Test , Tuberculosis/diagnosis , Tuberculosis/prevention & control
4.
Cell Death Differ ; 11 Suppl 2: S153-61, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15153943

ABSTRACT

The tumor suppressor Chk2 kinase plays crucial roles in regulating cell-cycle checkpoints and apoptosis following DNA damage. We investigated the expression levels of the genes encoding Chk2 and several cell-cycle regulators in nine cell lines from lymphoid malignancies, including three Hodgkin's lymphoma (HL) lines. We found that all HL cell lines exhibited a drastic reduction in Chk2 expression without any apparent mutation of the Chk2 gene. However, expression of Chk2 in HL cells was restored following treatment with the histone deacetylase inhibitors trichostatin A (TsA) and sodium butyrate (SB), or with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza-dC). Chromatin-immunoprecipitation (Chip) assays revealed that treatment of HL cells with TsA, SB or 5Aza-dC resulted in increased levels of acetylated histones H3 and H4, and decreased levels of dimethylated H3 lysine 9 at the Chk2 promoter. These results indicate that expression of the Chk2 gene is downregulated in HL cells via epigenetic mechanisms.


Subject(s)
Azacitidine/analogs & derivatives , Epigenesis, Genetic/physiology , Gene Expression Regulation, Neoplastic , Hodgkin Disease/genetics , Lymphoma/genetics , Protein Serine-Threonine Kinases/genetics , Acetylation , Apoptosis/drug effects , Apoptosis/physiology , Apoptosis/radiation effects , Azacitidine/pharmacology , Butyrates/pharmacology , Cell Line, Tumor , Checkpoint Kinase 2 , Decitabine , Histones/drug effects , Histones/metabolism , Humans , Hydroxamic Acids/pharmacology , Methylation , Protein Serine-Threonine Kinases/drug effects , RNA, Messenger/genetics
5.
Antivir Chem Chemother ; 13(5): 283-8, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12630676

ABSTRACT

Pf-gp6, a 6 kDa anti-degranulation glycoprotein purified from the extract of Perilla frutescens, was examined for its antiviral activity against HIV-1 and HIV-2 in vitro. HIV-1-induced cytopathic effect and proviral DNA synthesis were inhibited in the presence of Pf-gp6. The 50% inhibitory concentrations of Pf-gp6 for various HIV-1 strains, including clinical isolates and CCR5-using (R5) HIV-1, ranged between 1.3 and 71.0 microg/ml, depending on the combination of viral strain and host cell. Furthermore, Pf-gp6 did not directly inactivate infectious viral particles. A time-of-addition experiment revealed that Pf-gp6 lost its activity before zidovudine but after the CXCR-4 antagonist AMD3100 during the early stage of viral infection. Although the pinpoint target of Pf-gp6 remains to be elucidated, it may interfere with a step between viral entry and reverse transcription.


Subject(s)
Anti-HIV Agents/isolation & purification , Anti-HIV Agents/pharmacology , Glycoproteins/isolation & purification , HIV-1/drug effects , HIV-1/physiology , Perilla frutescens/chemistry , Virus Replication/drug effects , Adsorption/drug effects , Anti-HIV Agents/chemistry , Dose-Response Relationship, Drug , Giant Cells/drug effects , Giant Cells/virology , Glycoproteins/chemistry , Glycoproteins/pharmacology , HIV-1/classification , HIV-1/genetics , HIV-2/drug effects , HIV-2/physiology , Receptors, HIV/metabolism , Time Factors
6.
BMC Microbiol ; 1: 24, 2001.
Article in English | MEDLINE | ID: mdl-11710959

ABSTRACT

BACKGROUND: Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein. RESULTS: In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody. CONCLUSION: The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.


Subject(s)
Capsid/immunology , Escherichia coli/immunology , Norwalk virus/immunology , Antibodies, Monoclonal , Antigens, Viral/immunology , Epitopes/immunology , Protein Structure, Tertiary , Recombinant Proteins/immunology
7.
Mol Cell Biol ; 21(24): 8329-35, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11713269

ABSTRACT

The mammalian Ror family of receptor tyrosine kinases consists of two structurally related proteins, Ror1 and Ror2. We have shown that mRor2-deficient mice exhibit widespread skeletal abnormalities, ventricular septal defects in the heart, and respiratory dysfunction, leading to neonatal lethality (S. Takeuchi, K. Takeda, I. Oishi, M. Nomi, M. Ikeya, K. Itoh, S. Tamura, T. Ueda, T. Hatta, H. Otani, T. Terashima, S. Takada, H. Yamamura, S. Akira, and Y. Minami, Genes Cells 5:71-78, 2000). Here we show that mRor1-deficient mice have no apparent skeletal or cardiac abnormalities, yet they also die soon after birth due to respiratory dysfunction. Interestingly, mRor1/mRor2 double mutant mice show markedly enhanced skeletal abnormalities compared with mRor2 mutant mice. Furthermore, double mutant mice also exhibit defects not observed in mRor2 mutant mice, including a sternal defect, dysplasia of the symphysis of the pubic bone, and complete transposition of the great arteries. These results indicate that mRor1 and mRor2 interact genetically in skeletal and cardiac development.


Subject(s)
Bone and Bones/abnormalities , Heart Defects, Congenital/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/physiology , Animals , Animals, Newborn , Bone and Bones/metabolism , In Situ Hybridization , Lung/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Genetic , Mutation , Phenotype , Protein Structure, Tertiary , Receptor Protein-Tyrosine Kinases , Receptor Tyrosine Kinase-like Orphan Receptors , Time Factors
8.
Mech Dev ; 105(1-2): 153-6, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11429290

ABSTRACT

In mammals, the Ror-family receptor tyrosine kinases consist of two structurally related proteins, Ror1 and Ror2, characterized by the extracellular Frizzled-like cysteine-rich domain and membrane proximal kringle domains. As an attempt to gain insights into their roles in mouse development, expression patterns of Ror1 and Ror2 during early embryogenesis were examined and compared. Interestingly, at early stages, Ror1 and Ror2 exhibit similar expression patterns in the developing face, including the frontonasal process and pharyngeal arches, which are derived from cephalic neural crest cells. On the other hand, they exhibit different expression patterns in the developing limbs and brain, where the expression of Ror2 was detected broadly compared with that of Ror1. At a later stage, both genes are expressed in a similar fashion in the developing heart and lung, yet in a distinct manner in the brain and eye.


Subject(s)
Gene Expression Regulation, Developmental , Receptors, Cell Surface/biosynthesis , Animals , Brain/embryology , Cysteine/chemistry , Extremities/embryology , Eye/embryology , In Situ Hybridization , Mice , Protein Structure, Tertiary , RNA/metabolism , Receptor Protein-Tyrosine Kinases , Receptor Tyrosine Kinase-like Orphan Receptors , Time Factors
9.
Vaccine ; 19(20-22): 3019-32, 2001 Apr 06.
Article in English | MEDLINE | ID: mdl-11282214

ABSTRACT

The viral accessory gene product Nef has been shown to play an important role in human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis. Only little information is available regarding the differences in the host immune responses against Nef protein and its function in vivo among different subtypes of HIV-1. In the present study, we showed marked differences in the immune responses to Nef protein between subtypes B and E. The amino acid sequence in subtype E Nef showed 72% homology with that in subtype B. Most murine monoclonal antibodies obtained by immunization with subtype B or E Nef protein showed cross-reactivity with both Nef proteins (80 and 67%, respectively). Next, we focused on the immune responses among infected Japanese and Thai individuals. Subtyping of the individuals into B and E was carried out by enzyme-linked immunosorbent assay (ELISA) using synthetic peptides corresponding to the V3 loop representing the principal neutralizing domain. Most of the sera from these individuals reacted strongly with Gag p24 proteins derived from subtypes B and E at similar levels. However, the immune responses among these individuals to Nef protein were markedly different. Some subtype B-infected Japanese and Thai individuals (40 and 35%, respectively) showed higher levels of anti-Nef antibodies, although these antibodies preferentially recognized epitopes specific to subtype B. On the other hand, most of the subtype E-infected Japanese and Thai individuals showed low or no antibody responses to Nef proteins. Thus, immune responses to Nef were markedly different between subtypes B- and E-infected carriers, suggesting different function(s) for Nef in AIDS pathogenesis. Further, vaccine design must take into account the different subtypes of HIV-1.


Subject(s)
Acquired Immunodeficiency Syndrome/immunology , Gene Products, nef/immunology , HIV Antibodies/blood , HIV-1/classification , HIV-1/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Carrier State , Cross Reactions , Epitopes , Gene Products, env/immunology , Gene Products, gag/immunology , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Recombinant Proteins/immunology , T-Lymphocytes, Cytotoxic/immunology , nef Gene Products, Human Immunodeficiency Virus
10.
Mol Cell Biol ; 21(4): 1329-35, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11158318

ABSTRACT

Although chromosomal segregation at meiosis I is the critical process for genetic reassortment and inheritance, little is known about molecules involved in this process in metazoa. Here we show by utilizing double-stranded RNA (dsRNA)-mediated genetic interference that novel protein kinases (Ce-CDS-1 and Ce-CDS-2) related to Cds1 (Chk2) play an essential role in meiotic recombination in Caenorhabditis elegans. Injection of dsRNA into adult animals resulted in the inhibition of meiotic crossing over and induced the loss of chiasmata at diakinesis in oocytes of F(1) animals. However, electron microscopic analysis revealed that synaptonemal complex formation in pachytene nuclei of the same progeny of injected animals appeared to be normal. Thus, Ce-CDS-1 and Ce-CDS-2 are the first example of Cds1-related kinases that are required for meiotic recombination in multicellular organisms.


Subject(s)
Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Meiosis/genetics , Meiosis/physiology , Protein Kinases/genetics , Protein Kinases/physiology , Protein Serine-Threonine Kinases , Recombination, Genetic , Amino Acid Sequence , Aneuploidy , Animals , Base Sequence , Checkpoint Kinase 2 , DNA Primers/genetics , DNA, Helminth/genetics , Female , Genes, Helminth , Male , Molecular Sequence Data , Phenotype , RNA, Double-Stranded/genetics , RNA, Helminth/genetics , Sequence Homology, Amino Acid
11.
Kansenshogaku Zasshi ; 74(8): 638-45, 2000 Aug.
Article in Japanese | MEDLINE | ID: mdl-11019511

ABSTRACT

It is well-known that the biological characteristics of HIV-1 persistently infected in the host have often changed into a rapid growth in vitro, T-cell line tropism and marked syncytium inducing (SI) ability accompanied by the progress of clinical stages from AC to ARC or AIDS. We have developed a follow-up diagnostic test using the clinical markers based on the virus phenotypes mentioned above, and reported that the test was significantly useful for determining the clinical status of HIV infected individuals. Recently, highly active antiretroviral therapy (HAART) was introduced into HIV-1 chemotherapy, and has been reported to be a markedly effective treatment for HIV infected individuals. In this study, we carried out an investigation to see whether the follow-up diagnosis was useful even after introducing the HAART in Japan in 1997. The results by the laboratory diagnosis on 139 HIV infected individuals who were clinically observed over a long period showed that the positive rate of virus isolation and MT-4 cell tropism in the isolates during the two years between 1997 and 1998 were significantly lower than that of the nine years from 1988 to 1996 before the implementation of HAART. In addition, we obtained data that the effect of HAART reflects the biological profiles of virus isolation more than the CD4+ T cell counts. These results suggest that data of clinical examination using virus isolation as a parameter are useful not only for predicting the development of AIDS but also for evaluating the effects of HAART, particularly in patients showing no association between the CD4+ T cell counts and the plasma viral load.


Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Acquired Immunodeficiency Syndrome/virology , HIV-1/isolation & purification , Anti-HIV Agents/therapeutic use , CD4 Lymphocyte Count , Drug Evaluation , Humans
12.
Kansenshogaku Zasshi ; 74(5): 450-7, 2000 May.
Article in Japanese | MEDLINE | ID: mdl-10860357

ABSTRACT

We have analyzed the sequences of HIV-1 reverse transcriptase and protease genes in peripheral blood mononuclear cells obtained from patients receiving antiretroviral therapy to evaluate the drug resistance-associated mutations. Of 84 HIV-1-infected individuals treated with reverse transcriptase inhibitors, 43 (51.2%) have been found to carry amino acid substitutions predicted to acquire drug-resistances. One to 3 mutations at amino acid residues reported to be associated with protease inhibitor-resistance were detected in more than 80% of protease inhibitor-naive patients. However, these pre-existing mutations did not seem to raise a real resistance after the initiation of therapy with protease inhibitors. Phenotypic resistance assay was performed with 6 clinical isolates to compare with genotypic resistance. In most of the cases, phenotype was correlated with genotypic changes, however, two strains which were isolated from patients having no experience of chemotherapy showed a decrease in susceptibility to several drugs without any resistance-related mutations detected in their genes. Taken together, determination of phenotypic resistance is necessary, especially when a newly-infected patients starts antiviral therapy.


Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , HIV-1/isolation & purification , Acquired Immunodeficiency Syndrome/virology , Drug Resistance, Microbial/genetics , Genotype , HIV-1/drug effects , HIV-1/genetics , Humans , Phenotype , Protease Inhibitors/therapeutic use
13.
J Med Virol ; 61(1): 125-31, 2000 May.
Article in English | MEDLINE | ID: mdl-10745244

ABSTRACT

We previously reported that human astrovirus type 6 (HAstV T6) was the etiologic agent of a large-scale outbreak of acute gastroenteritis that occurred in 1991 in Katano City, Osaka, Japan [Oishi et al., 1994]. The two representative strains, Katano virus K23 and K24, have been analyzed by sequencing the open reading frame 2 (ORF2) region after amplification by reverse transcription-polymerase chain reaction (RT-PCR). The ORF2 region of HAstV T6 strains, including K23, was found to be about 20 bp smaller than those of other types. There was 94% nucleotide sequence identity and 95% amino acid sequence identity between K23 and K24, with the Oxford strains belonging to HAstV T6. The high homology of the ORF2 region between the Katano and Oxford strains shows intratype genomic stability, irrespective of time and place of virus isolation. Comparing sequences of ORF2 of different HAstV serotypes, we established a rapid and highly sensitive detection system for HAstV types using RT-PCR with the AC230/AC1' primer set designed from the 5'-terminal end region of ORF2. This RT-PCR system seems very useful in detecting at least two different viruses in a single PCR test tube using AC230/AC1' in addition to the NV81/82, SM82 primer sets. Thus, our rapid and effective detection system may contribute to the epidemiologic characterization of astrovirus infections as well as Norwalk-like viruses.


Subject(s)
Astroviridae Infections/virology , Mamastrovirus/genetics , Amino Acid Sequence , Astroviridae Infections/epidemiology , Child , Child, Preschool , Feces/virology , Female , Gastroenteritis/virology , Humans , Japan/epidemiology , Male , Mamastrovirus/classification , Mamastrovirus/isolation & purification , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Homology, Amino Acid , Serotyping
14.
J Med Virol ; 60(4): 475-81, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10686033

ABSTRACT

The capsid protein of Norwalk-like virus (NLV) isolates NLV-36 (Mexico virus type, genogroup II [GII]), NLV-21 (Lordsdale virus type, GII), NLV-114 (untyped GII virus), and NLV-96-908 (KY89 virus type, GI) have been expressed in an Escherichia coli system. The expressed recombinant NLV capsid proteins, fused with maltose binding protein (MBP-rV) and thioredoxin (TRX-rV) in E. coli lysate, were analyzed using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Rabbit IgG (R-IgG) in hyperimmune serum has been raised against MBP-rV-36 capsid protein and was purified before further study. Detection of TRX-rVs using an enzyme-linked immunosorbent assay (ELISA) showed that R-IgG had immunologic reactivity to GII as well as to the GI rV capsid proteins TRX-rV-36, TRX-rV-21, TRX-rV-114, and TRX-rV-96-908. Results of Western immunoblot (WB) analysis showed the same broad recognition of R-IgG when using the same samples. The results of the ELISA tests on serum samples obtained from patients involved in confirmed outbreaks of NLV proved that expressed NLV capsid proteins in E. coli can be detected by NLV-infected human serum. In addition, purified NLVs (LD virus types) derived from patients' stool could be detected using anti-NLV R-IgG, whereas normal R-IgG did not react when using WB. Our results strongly suggest that the immunologic detection of NLV antigens using anti-rV R-IgG is possible and seems a significant step toward simplification of an NLV detection test.


Subject(s)
ATP-Binding Cassette Transporters , Caliciviridae Infections/immunology , Capsid Proteins , Capsid/immunology , Disease Outbreaks , Escherichia coli Proteins , Gastroenteritis/immunology , Monosaccharide Transport Proteins , Norwalk virus/immunology , Animals , Antibodies, Viral/immunology , Base Sequence , Blotting, Western/methods , Caliciviridae Infections/blood , Caliciviridae Infections/epidemiology , Caliciviridae Infections/virology , Capsid/genetics , Capsid/isolation & purification , Carrier Proteins/genetics , Carrier Proteins/immunology , Cloning, Molecular , DNA, Viral , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli , Feces/virology , Gastroenteritis/blood , Gastroenteritis/epidemiology , Gastroenteritis/virology , Humans , Immunoglobulin G/immunology , Immunoglobulins/blood , Immunoglobulins/immunology , Japan/epidemiology , Maltose-Binding Proteins , Molecular Sequence Data , Norwalk virus/genetics , Norwalk virus/isolation & purification , Peptide Fragments/genetics , Peptide Fragments/immunology , Peptide Fragments/isolation & purification , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Thioredoxins/genetics , Thioredoxins/immunology
15.
J Vet Med Sci ; 62(1): 65-8, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10676892

ABSTRACT

Ejaculated sperm collected from 12 beagle dogs were incubated in canine capacitation medium (CCM), supplemented with 5 microg/ml chondroitin sulfate A (CS), 5 microg/ml hyaluronic acid (HA), or 5 microg/ml heparin (HP) for 7 hr at 38 degrees C in a 5% CO2 in air atmosphere to investigate the effects of glycosaminoglycans (GAGs) on dog sperm capacitation. The percentages of motile sperm, hyperactivated sperm (%HY), and acrosome-reacted sperm (%AR) in all media were examined after 4 hr and 7 hr of incubation. The oviducts and uteri of 9 anestrous and 18 estrous beagle bitches were removed under halothane inhalation anesthesia to measure the total GAG amounts in oviductal and uterine fluids. The lumens of the ampulla of the oviducts, isthmus of the oviducts, and the uterine horns were each flushed with 1 ml HEPES-EDTA fluid. Total GAG amounts in the flush fluids obtained were measured with a spectrophotometer. Sperm motility (51-59%), %HY (79-86%), and %AR (31-36%) in CCM supplemented with CS, HA, or HP were significantly higher after 7 hr of incubation than when incubated in CCM without GAGs (P<0.01 or 0.05). The mean total GAG amounts in the fluids from the ampulla and isthmus of the oviducts and the uterine horns in the estrous bitches were higher than in the anestrous bitches. These results indicate that GAGs in the oviductal and uterine fluids in estrous bitches are associated with in vivo sperm capacitation.


Subject(s)
Dogs/physiology , Fallopian Tubes/physiology , Glycosaminoglycans/pharmacology , Sperm Capacitation/physiology , Uterus/physiology , Acrosome Reaction/physiology , Alcian Blue/chemistry , Animals , Chondroitin Sulfates/analysis , Chondroitin Sulfates/pharmacology , Coloring Agents/chemistry , Estrus/physiology , Fallopian Tubes/chemistry , Female , Glycosaminoglycans/analysis , Heparin/analysis , Heparin/pharmacology , Hyaluronic Acid/analysis , Hyaluronic Acid/pharmacology , Male , Sperm Capacitation/drug effects , Spermatozoa/physiology , Uterus/chemistry
16.
Genes Cells ; 5(1): 71-8, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10651906

ABSTRACT

BACKGROUND: A mouse receptor tyrosine kinase (RTK), mRor2, which belongs to the Ror-family of RTKs consisting of at least two structurally related members, is primarily expressed in the heart and nervous system during mouse development. To elucidate the function of mRor2, we generated mice with a mutated mRor2 locus. RESULTS: Mice with a homozygous mutation in mRor2 died just after birth, exhibiting dwarfism, severe cyanosis, and short limbs and tails. Whole-mount in situ hybridization analysis showed that mRor2 was expressed in the branchial arches, heart and limb/tailbuds, in addition to the developing nervous system. The mutants had cardiac septal defects, mainly a ventricular septal defect. In addition, an examination of the skeletal systems revealed that the mutants had shorter limbs, vertebrae and facial structure, with a particular defect in their distal portions, and that almost no calcification was observed in their distal limbs. Histological examination showed abnormalities in the chondrocytes. CONCLUSIONS: Our findings suggest that mRor2 plays essential roles in the development of the heart and in limb/tail formation, in particular cardiac septal formation and ossification of distal portions of limbs and tails.


Subject(s)
Forelimb/embryology , Heart/embryology , Hindlimb/embryology , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Cell Surface/physiology , Animals , Animals, Newborn , Embryonic and Fetal Development , Forelimb/growth & development , Forelimb/pathology , Heart/growth & development , Hindlimb/growth & development , Hindlimb/pathology , Mice , Mice, Knockout , Receptor Protein-Tyrosine Kinases/genetics , Receptor Tyrosine Kinase-like Orphan Receptors , Receptors, Cell Surface/genetics , Skull/embryology , Skull/growth & development , Skull/pathology , Spine/embryology , Spine/growth & development , Spine/pathology
17.
Microbiol Immunol ; 44(11): 905-14, 2000.
Article in English | MEDLINE | ID: mdl-11145271

ABSTRACT

The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli. All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions. Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the N-terminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548). Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples. It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Capsid Proteins , Capsid/immunology , Norwalk virus/immunology , Amino Acid Sequence , Animals , Antigens, Viral/chemistry , Antigens, Viral/immunology , Caliciviridae Infections/diagnosis , Caliciviridae Infections/virology , Capsid/chemistry , Capsid/genetics , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Epitopes , Escherichia coli/genetics , Feces/virology , Female , Humans , Immunoblotting , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Norwalk virus/isolation & purification , Recombinant Proteins/immunology
19.
J Immunol ; 163(9): 4901-8, 1999 Nov 01.
Article in English | MEDLINE | ID: mdl-10528192

ABSTRACT

Despite extensive studies on the crucial functions of Ras and c-Myc in cellular proliferation and transformation, their roles in regulating cell adhesion are not yet fully understood. Involvement of Ras in modulating integrin activity by inside-out signaling has been recently reported. However, in contrast to R-Ras, H-Ras was found to exhibit a suppressive effect. Here we show that ectopic expression of a constitutively active H-Rasv12, but not c-Myc alone, in a hemopoietic cell line induces activation of very late Ag-4 (VLA-4, alpha4beta1) integrin without changing its surface expression. Intriguingly, coexpression of H-Rasv12 and c-Myc in these cells results in not only the activation of VLA-4, but also the induction of expression of VCAM-1, the counterreceptor for VLA-4, thereby mediating a marked homotypic cell aggregation. In addition, H-Rasv12-induced VLA-4 activation appears to be partly down-regulated by coexpression with c-Myc. Our results represent an unprecedented example demonstrating a novel role for H-Rasv12 in the regulation of cell adhesion via c-Myc-independent and -dependent mechanisms.


Subject(s)
Integrins/metabolism , Proto-Oncogene Proteins c-myc/physiology , Proto-Oncogene Proteins p21(ras)/physiology , Receptors, Lymphocyte Homing/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Adjuvants, Immunologic/physiology , Animals , Cell Adhesion/immunology , Cell Line , Fibronectins/metabolism , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/physiology , Humans , Integrin alpha4beta1 , Integrins/physiology , Mice , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins p21(ras)/biosynthesis , Proto-Oncogene Proteins p21(ras)/metabolism , Receptors, Lymphocyte Homing/physiology , Receptors, Very Late Antigen/metabolism , Vascular Cell Adhesion Molecule-1/biosynthesis , Vascular Cell Adhesion Molecule-1/physiology
20.
J Neurochem ; 73(5): 2119-29, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10537072

ABSTRACT

Ca2+/calmodulin-dependent protein kinases (CaMKs) are believed to play important roles in the development and function of the nervous system. We report here the identification and expression of mouse CaMKIbeta (mCaMKIbeta), in particular mCaMKIbeta2, an isoform of mCaMKIbeta. During embryogenesis, the mCaMKIbeta2 gene is expressed mainly in the nervous system, including brain, spinal cord, trigeminal ganglion, and retina. Within the CNS, the expression of mCaMKIbeta2 is detected in the mantle zone, but not in the ventricular zone, suggesting its possible involvement in the differentiation of neurons. In the adult brain, mCaMKIbeta2 transcripts are detected at high levels in the anterior olfactory nuclei, piriform cortex, septal nuclei, bed nuclei of the stria terminalis, hippocampal pyramidal cells, dentate granule cells, amygdala, hypothalamic nuclei, parabrachial nucleus, and nucleus of the solitary tract. The distinct gene expression pattern suggests that mCaMKIbeta2 may also be involved in different mature neuronal functions from other CaMKs. In addition, mCaMKI/beta2 proteins are localized to the cytoplasm and nuclei, but not to nucleoli, suggesting that mCaMKIbeta2 proteins might be involved in the cytoplasmic and nuclear signal transduction of the nervous system.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/analysis , Isoenzymes/analysis , Nervous System/enzymology , Amino Acid Sequence , Animals , Base Sequence , Brain/enzymology , Calcium-Calmodulin-Dependent Protein Kinase Type 1 , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Cell Nucleus/enzymology , Cloning, Molecular , Cytoplasm/enzymology , DNA, Complementary/chemistry , DNA, Complementary/genetics , Isoenzymes/chemistry , Isoenzymes/genetics , Mice , Molecular Sequence Data , Nervous System/growth & development , PC12 Cells , RNA, Messenger/analysis , Rats , Retina/enzymology , Spinal Cord/enzymology , Tissue Distribution , Transfection , Trigeminal Ganglion/enzymology
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