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1.
J Laryngol Otol ; 137(7): 749-756, 2023 Jul.
Article in English | MEDLINE | ID: mdl-35916274

ABSTRACT

OBJECTIVE: For low-grade intraepithelial neoplasia cases, pharyngolaryngeal lesions equal to or less than 5 mm in size do not generally progress to invasive carcinoma. However, micro-superficial lesions equal to or less than 5 mm that showed rapid growth have been recently encountered. This study aimed to identify the characteristics of preferential progression of lesions equal to or less than 5 mm in size. METHOD: Gross findings, endoscopic findings and pathological results of 55 lesions measuring equal to or less than 5 mm in diameter were retrospectively reviewed to identify factors that distinguish squamous cell carcinoma or high-grade intraepithelial neoplasia from low-grade intraepithelial neoplasia or non-atypia lesions. RESULTS: The overall sensitivity, specificity, accuracy, and positive and negative predictive value of background colouration and intrapapillary capillary loop pattern in differentiation of squamous cell carcinoma or high-grade intraepithelial neoplasia from low-grade intraepithelial neoplasia or non-atypia lesions were all 100 per cent. CONCLUSION: Diagnosis based on background colouration and the intrapapillary capillary loop pattern on narrow-band imaging facilitates the pathological examination of lesions measuring equal to or less than 5 mm.


Subject(s)
Carcinoma in Situ , Carcinoma, Squamous Cell , Humans , Retrospective Studies , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/pathology , Narrow Band Imaging/methods , Predictive Value of Tests , Carcinoma in Situ/diagnostic imaging , Carcinoma in Situ/pathology
2.
J Laryngol Otol ; 135(9): 773-778, 2021 Sep.
Article in English | MEDLINE | ID: mdl-33928889

ABSTRACT

OBJECTIVE: Severe acute respiratory syndrome coronavirus-2 uses angiotensin-converting enzyme-2 as a primary receptor for invasion. This study investigated angiotensin-converting enzyme-2 expression in the sinonasal mucosa of patients with chronic rhinosinusitis, as this could be linked to a susceptibility to severe acute respiratory syndrome coronavirus-2 infection. METHODS: Ethmoid sinus specimens were obtained from 27 patients with eosinophilic chronic rhinosinusitis, 18 with non-eosinophilic chronic rhinosinusitis and 18 controls. The angiotensin-converting enzyme-2 and other inflammatory cytokine and chemokine messenger RNA levels were assessed by quantitative reverse transcription polymerase chain reaction. Angiotensin-converting enzyme-2 positive cells were examined immunohistologically. RESULTS: The eosinophilic chronic rhinosinusitis patients showed a significant decrease in angiotensin-converting enzyme-2 messenger RNA expression. In the chronic rhinosinusitis patients, angiotensin-converting enzyme-2 messenger RNA levels were positively correlated with tumour necrosis factor-α and interleukin-1ß (r = 0.4971 and r = 0.3082, respectively), and negatively correlated with eotaxin-3 (r = -0.2938). Angiotensin-converting enzyme-2 immunoreactivity was mainly localised in the ciliated epithelial cells. CONCLUSION: Eosinophilic chronic rhinosinusitis patients with type 2 inflammation showed decreased angiotensin-converting enzyme-2 expression in their sinus mucosa. Angiotensin-converting enzyme-2 regulation was positively related to pro-inflammatory cytokines, especially tumour necrosis factor-α production, in chronic rhinosinusitis patients.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Nasal Mucosa/enzymology , Rhinitis/enzymology , Sinusitis/enzymology , Adult , COVID-19/etiology , Chronic Disease , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Reverse Transcriptase Polymerase Chain Reaction , Rhinitis/complications , Rhinitis/metabolism , SARS-CoV-2/metabolism , Sinusitis/complications , Sinusitis/metabolism
3.
J Laryngol Otol ; 129(12): 1194-200, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26487482

ABSTRACT

OBJECTIVE: Although human paranasal sinuses are critical organs for nitric oxide production, little information is available regarding the role of arginase in alterations of arginine metabolism and nasal nitric oxide levels that may be informative for classifying chronic rhinosinusitis subtypes. METHODS: The expression and localisation of arginase and nitric oxide synthase isoforms in paranasal sinus mucosa were examined, and the fractional exhaled nitric oxide was measured in chronic rhinosinusitis without nasal polyps (n=18) and chronic rhinosinusitis with nasal polyps (n = 27) patients. RESULTS: Increased arginase-2 activities in chronic rhinosinusitis without nasal polyps patients were associated with significantly lower levels of nasal fractional exhaled nitric oxide. Chronic rhinosinusitis with nasal polyps patients showed significant NOS2 messenger RNA upregulation with concomitant higher levels of oral and nasal fractional exhaled nitric oxide. CONCLUSION: These results indicate that fractional exhaled nitric oxide is a valid marker for differentiating chronic rhinosinusitis phenotypes based on a delicate balance between arginase and nitric oxide synthase activities in nitric oxide production.


Subject(s)
Arginase/metabolism , Nasal Polyps/diagnosis , Nitric Oxide/metabolism , Rhinitis/metabolism , Sinusitis/metabolism , Adult , Arginase/genetics , Biomarkers/analysis , Breath Tests , Chronic Disease , Cross-Sectional Studies , Female , Humans , Immunohistochemistry , Male , Middle Aged , Multivariate Analysis , Nasal Mucosa/metabolism , Nasal Mucosa/pathology , Nitric Oxide Synthase/metabolism , Protein Isoforms/metabolism , Real-Time Polymerase Chain Reaction , Rhinitis/genetics , Rhinitis/pathology , Sensitivity and Specificity , Sinusitis/genetics , Sinusitis/pathology , Statistics, Nonparametric
4.
Placenta ; 29(8): 753-9, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18602690

ABSTRACT

Hyperplastic placentas have been reported in several experimental mouse models, including animals produced by somatic cell nuclear transfer, by inter(sub)species hybridization, and by somatic cytoplasm introduction to oocytes followed by intracytoplasmic sperm injection. Of great interest are the gross and histological features common to these placental phenotypes--despite their quite different etiologies--such as the enlargement of the spongiotrophoblast layers. To find morphological clues to the pathways leading to these similar placental phenotypes, we analyzed the ultrastructure of the three different types of hyperplastic placenta. Most cells affected were of trophoblast origin and their subcellular ultrastructural lesions were common to the three groups, e.g., a heavy accumulation of cytoplasmic vacuoles in the trophoblastic cells composing the labyrinthine wall and an increased volume of spongiotrophoblastic cells with extraordinarily dilatated rough endoplasmic reticulum. Although the numbers of trophoblastic glycogen cells were greatly increased, they maintained their normal ultrastructural morphology, including a heavy glycogen deposition throughout the cytoplasm. The fetal endothelium and small vessels were nearly intact. Our ultrastructural study suggests that these three types of placental hyperplasias, with different etiologies, may have common pathological pathways, which probably exclusively affect the development of certain cell types of the trophoblastic lineage during mouse placentation.


Subject(s)
Placenta Diseases/etiology , Placenta/pathology , Placenta/ultrastructure , Animals , Female , Hyperplasia , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron , Placenta Diseases/pathology , Pregnancy
5.
Cytogenet Genome Res ; 113(1-4): 24-30, 2006.
Article in English | MEDLINE | ID: mdl-16575159

ABSTRACT

Gene expression from both parental alleles (biallelic expression) is beneficial in minimizing the occurrence of recessive genetic disorders in diploid organisms. However, imprinted genes in mammals display parent of origin-specific monoallelic expression. As some imprinted genes play essential roles in mammalian development, the reason why mammals adopted the genomic imprinting mechanism has been a mystery since its discovery. In this review, based on the recent studies on imprinted gene regulation we discuss several advantageous features of a monoallelic expression mechanism and the necessity of genomic imprinting in the current mammalian developmental system. We further speculate how the present genomic imprinting system has been established during mammalian evolution by the mechanism of complementation between paternal and maternal genomes under evolutionary pressure predicted by the genetic conflict hypothesis.


Subject(s)
Gene Expression Regulation , Genomic Imprinting , Mammals/genetics , Models, Genetic , Animals , Chromosome Mapping , Female , Genetic Complementation Test , Germ Cells/physiology , Life Cycle Stages/genetics , Male , Mammals/growth & development , Mice , Placenta/physiology , Pregnancy
6.
Cytogenet Genome Res ; 113(1-4): 223-9, 2006.
Article in English | MEDLINE | ID: mdl-16575184

ABSTRACT

The imprinted region on mouse distal chromosome 12 covers about 1 Mb and contains at least three paternally expressed genes (Pegs: Peg9/Dlk1, Peg11/Rtl1, and Dio3) and four maternally expressed genes (Megs: Meg3/Gtl2, antiPeg11/antiRlt1, Meg8/Rian, and Meg9/Mirg). Gtl2(lacZ) (Gene trap locus 2) mice have a transgene (TG) insertion 2.3 kb upstream from the Meg3/Gtl2 promoter and show about 40% growth retardation when the TG-inserted allele is paternally derived. Quantitative RT-PCR experiments showed that the expression levels of Pegs in this region were reduced below 50%. These results are consistent with the observed phenotype in Gtl2lacZ mice, because at least two Pegs(Peg9/Dlk1 and Dio3) have growth-promoting effects. The aberrant induction of Megs from silent paternal alleles was also observed in association with changes in the DNA methylation level of a differentially methylated region (DMR) located around Meg3/Gtl2 exon 1. Interestingly, a 60 approximately 80% reduction in all Megs was observed when the TG was maternally derived, although the pups showed no apparent growth or morphological abnormalities. Therefore, the paternal or maternal inheritance of the TG results in the down-regulation in cis of either Pegs or Megs, respectively, suggesting that the TG insertion influences the mechanism regulating the entire imprinted region.


Subject(s)
Genomic Imprinting , Proteins/genetics , Animals , Base Sequence , Chromosome Aberrations , Chromosome Mapping , DNA Primers , Female , Gene Expression Regulation , Growth Disorders/genetics , Male , Mice , Mice, Transgenic , Mutagenesis, Insertional , RNA, Long Noncoding , Reverse Transcriptase Polymerase Chain Reaction , beta-Galactosidase/genetics
7.
Am J Med Genet ; 104(3): 225-31, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11754049

ABSTRACT

Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.


Subject(s)
Abnormalities, Multiple/genetics , Growth Disorders/pathology , Proteins/genetics , 5' Flanking Region/genetics , Abnormalities, Multiple/pathology , Alternative Splicing , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Methylation , Exons , Genes/genetics , Humans , Introns , Molecular Sequence Data , Mutation , Sequence Analysis, DNA , Syndrome
8.
Proc Natl Acad Sci U S A ; 98(21): 12109-13, 2001 Oct 09.
Article in English | MEDLINE | ID: mdl-11593023

ABSTRACT

Vertebrate cells have evolved two major pathways for repairing DNA double-strand breaks (DSBs), homologous recombination (HR) and nonhomologous DNA end-joining (NHEJ). To investigate the role of DNA ligase IV (Lig4) in DSB repair, we knocked out the Lig4 gene (LIG4) in the DT40 chicken B-lymphocyte cell line. The LIG4(-/-) cells showed a marked sensitivity to X-rays, bleomycin, and VP-16 and were more x-ray-sensitive in G(1) than late S or G(2)/M, suggesting a critical role of Lig4 in DSB repair by NHEJ. In support of this notion, HR was not impaired in LIG4(-/-) cells. LIG4(-/-) cells were more x-ray-sensitive when compared with KU70(-/-) DT40 cells, particularly at high doses. Strikingly, however, the x-ray sensitivity of KU70(-/-)/LIG4(-/-) double-mutant cells was essentially the same as that of KU70(-/-) cells, showing that Lig4 deficiency has no effect in the absence of Ku. These results indicate that Lig4 is exclusively required for the Ku-dependent NHEJ pathway of DSB repair and that other DNA ligases (I and III) do not substitute for this function. Our data may explain the observed severe phenotype of Lig4-deficient mice as compared with Ku-deficient mice.


Subject(s)
Antigens, Nuclear , DNA Damage , DNA Helicases , DNA Ligases/physiology , DNA Repair , DNA-Binding Proteins/physiology , Nuclear Proteins/physiology , Animals , B-Lymphocytes , Base Sequence , Cell Line , Chickens , DNA Ligase ATP , DNA Ligases/genetics , DNA, Complementary , Gene Targeting/methods , Ku Autoantigen , Molecular Sequence Data , Radiation, Ionizing
9.
Clin Cancer Res ; 7(9): 2636-42, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11555573

ABSTRACT

Although the prognostic impact of PTEN mutation in endometrial carcinoma is beginning to be analyzed, the prognostic significance of mutated PTEN exons has not ever been described. Sixty-seven endometrial carcinomas were analyzed for PTEN mutations using single-strand conformation polymorphism analysis and DNA sequencing. First, survival rates were compared according to PTEN status and mutated PTEN exons. Subsequently, univariate and multivariate analyses of various favorable prognostic factors for survival were conducted. The associations between PTEN mutation and clinicopathological features were also statistically evaluated. PTEN mutations were detected in 37 of 67 (55%) specimens. Among 47 mutations, frameshifts (57%) and mutations in exon 8 (38%) were most frequent. In univariate analysis, a factor of PTEN mutation only outside exons 5-7 was associated with significantly better survival (P = 0.02), although mutation in any exon of PTEN was not (P = 0.33). Subsequent multivariate analysis revealed that factors of mutation only outside exons 5-7 of PTEN, stage I/II, and G1 were significant and independent prognostic indicators for favorable survival (P = 0.004, 0.004, and 0.0006, respectively). In the subset of advanced-stage disease, mutation only outside exons 5-7 was associated with a trend toward better survival (P = 0.13). No significant correlation was observed between PTEN mutation and estrogen-related clinicopathological features. In conclusion, we find that PTEN mutation located only outside exons 5-7 is a significant and independent positive prognostic indicator for survival. The current observation has prognostic and therapeutic implications for the management of patients with endometrial carcinoma.


Subject(s)
Endometrial Neoplasms/pathology , Exons/genetics , Phosphoric Monoester Hydrolases/genetics , Tumor Suppressor Proteins , Adult , Aged , DNA Mutational Analysis , DNA, Neoplasm/chemistry , DNA, Neoplasm/genetics , Endometrial Neoplasms/genetics , Female , Humans , Middle Aged , Mutation , Neoplasm Staging , PTEN Phosphohydrolase , Polymorphism, Single-Stranded Conformational , Predictive Value of Tests , Prognosis , Survival Analysis
10.
Biochemistry ; 40(8): 2387-96, 2001 Feb 27.
Article in English | MEDLINE | ID: mdl-11327859

ABSTRACT

The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.


Subject(s)
Bacterial Proteins/chemistry , Proteins/chemistry , Ribosomes/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Escherichia coli/chemistry , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Conformation , Protein Structure, Tertiary , Ribosomal Proteins , Sequence Homology, Amino Acid , Solutions , Thermodynamics , Thermotoga maritima/chemistry
11.
Genomics ; 73(2): 232-7, 2001 Apr 15.
Article in English | MEDLINE | ID: mdl-11318613

ABSTRACT

A novel paternally expressed imprinted gene, PEG10 (Paternally Expressed 10), was identified on human chromosome 7q21. PEG10 is located near the SGCE (Sarcoglycan epsilon) gene, whose mouse homologue was recently shown to be imprinted. Therefore, it is highly possible that a new imprinted gene cluster exists on human chromosome 7q21. Analysis of two predicted open reading frames (ORF1 and ORF2) revealed that ORF1 and ORF2 have homology to the gag and pol proteins of some vertebrate retrotransposons, respectively. These data suggest that PEG10 is derived from a retrotransposon that was previously integrated into the mammalian genome. PEG10 is likely to be essential for understanding how exogenous genes become imprinted.


Subject(s)
Chromosomes, Human, Pair 7 , Genomic Imprinting , Proteins/genetics , Retroelements , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins , Choriocarcinoma/genetics , DNA-Binding Proteins , Female , Genes, gag/genetics , Genes, pol/genetics , Humans , Male , Mice , Molecular Sequence Data , Nuclear Proteins/genetics , Open Reading Frames , Physical Chromosome Mapping/methods , Polymorphism, Genetic , RNA-Binding Proteins , Radiation Hybrid Mapping/methods , Sequence Homology, Amino Acid , Syndrome , Transcription Factors/genetics
12.
Genes Cells ; 6(3): 237-47, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11260267

ABSTRACT

BACKGROUND: Mouse imprinted gene Peg3 encodes a large C2H2 type zinc finger protein with unique characteristics. Peg3 knockout mice were found to show an impairment in maternal behaviour of the adult female. Mouse Peg3 is located on the proximal region of chromosome 7 which is syntenic to the long arm of human chromosome 19. It has been reported that a loss of heterozygosity (LOH) of chromosome 19q occurs frequently in several glioma types. RESULTS: We isolated human PEG3 cDNA. Both human and mouse PEG3 were strongly expressed in the adult brain and the Peg3 protein was localized in the nuclei of both neurones and glial cells. A significant decrease in PEG3 expression was more commonly observed in glioma cell lines as compared with that in primary cultures of astrocytes. Transfection of PEG3 cDNA in a glioma cell line resulted in a loss of tumorigenicity in nude mice. CONCLUSIONS: The human PEG3 gene is a paternally expressed imprinted gene. Introduction of PEG3 cDNA into the glioma cells suggests that human PEG3 protein functions as a tumour suppressor. Human PEG3 is located on 19q13.4 and is one of the candidates for tumour suppressor genes that are predicted to be sited in gliomas.


Subject(s)
Genes, Tumor Suppressor , Genomic Imprinting/genetics , Glioma/genetics , Protein Kinases , Proteins/genetics , Transcription Factors , 5' Untranslated Regions/genetics , Alternative Splicing/genetics , Animals , Brain/growth & development , Brain/metabolism , Brain Neoplasms/chemistry , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Female , Glioma/chemistry , Glioma/pathology , Humans , Kruppel-Like Transcription Factors , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Nude , Middle Aged , Molecular Sequence Data , Protein Biosynthesis , Proteins/metabolism , Proteins/physiology , Tumor Cells, Cultured
13.
Methods Mol Biol ; 181: 101-12, 2001.
Article in English | MEDLINE | ID: mdl-12843444

ABSTRACT

Imprinted genes show monoallelic expression from either the paternal or maternal genome (1,2), and their regulated expression is usually associated with the existence of parentally differentially methylated regions on genomic DNAs (3,4). Because of this, essentially two different approaches, using either cDNA or genomic DNA as starting material (5) have been developed for systematic isolation of imprinted genes. In this chapter, we describe a subtraction-hybridization method (6-8) as an example of the former approach. Both parthenogenetic embryos and androgenetic embryos (9,10) are the most suitable biological materials for the subtraction or detection of imprinted genes. However, it is difficult to obtain a large amount of such special materials because only a small number of these embryos develop to the d 10 stage (9,10). Thus, polymerase chain reaction (PCR)-based techniques, such as the differential display (11-13) and subtraction-hybridization methods, are necessary to accomplish this experiment. The subtraction-hybridization method has been successfully applied for isolation of both paternally expressed genes (Pegs) (6,14,15) and maternally expressed genes (Megs) (7), and it allows cDNA libraries to be made from a very small amount of biological material. We are convinced that this method can be applied in many fields of biological science.


Subject(s)
Genomic Imprinting/genetics , Nucleic Acid Hybridization/methods , Animals , DNA, Complementary , Humans , Models, Genetic , Polymerase Chain Reaction
15.
Genes Cells ; 5(3): 211-20, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10759892

ABSTRACT

BACKGROUND: The paternal duplication of mouse distal chromosome 12 leads to late embryonal/neonatal lethality and growth promotion, whereas maternal duplication leads to late embryonal lethality and growth retardation. Human paternal or maternal uniparental disomies of chromosome 14q that are syntenic to mouse distal chromosome 12 have also been reported to show some imprinting effects on growth, mental activity and musculoskeletal morphology. For the isolation of imprinted genes in this region, a systematic screen of maternally expressed genes (Megs) was carried out by our subtraction-hybridization method using androgenetic and normally fertilized embryos. RESULTS: We have isolated seven candidate clones of the mouse Meg gene. Among them, we identified a novel maternally expressed imprinted gene, Meg3, on mouse distal chromosome 12 and showed that it was identical to the Gtl2 gene. We also found that the human homologue MEG3 on chromosome 14q was also monoallelically expressed. CONCLUSIONS: This is the first identification of the imprinting gene, both on mouse distal chromosome 12 and on human chromosome 14q, respectively. Because there are no obvious open reading frames in either the mouse Meg3/Gtl2 or human MEG3, the function of these genes remains unclear. However, this result will provide a good basis for the further investigation of several important imprinted genes in this chromosomal region.


Subject(s)
Chromosomes, Human, Pair 14 , Genome, Human , Genomic Imprinting , Animals , Chromosome Mapping , Genome , Humans , Mice
16.
J Biochem ; 127(3): 475-83, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10731720

ABSTRACT

A large imprinted gene cluster in human chromosome 11p15.5 has been implicated in Beckwith-Wiedemann syndrome and Wilms' tumor. We have identified a paternally expressed imprinted gene, PEG8/IGF2AS, in this locus. It is transcribed in the opposite direction to the IGF2 transcripts and some genomic regions are shared with the IGF2 gene, as in the case of the mouse imprinted Igf2as gene reported previously by T. Moore et al. As to the relationship between these genomic regions, the human and mouse genes are very similar but there is no homology in their middle parts. Interestingly, PEG8/IGF2AS and IGF2 were found to be overexpressed in Wilms' tumor samples, at levels over ten and a hundred times higher than that in normal kidney tissues neighboring the tumors, respectively. These findings indicate that PEG8/IGF2AS is a good marker of Wilms' tumor and also suggest the possibility of PEG8/IGF2AS being one of the candidate Wilms' tumor genes.


Subject(s)
Biomarkers , DNA, Antisense/metabolism , Genomic Imprinting , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Proteins/genetics , Wilms Tumor/genetics , Wilms Tumor/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Chorionic Villi/metabolism , Chromosomes, Human, Pair 11 , Embryo, Mammalian/metabolism , Exons , Fathers , Genes, Wilms Tumor/genetics , Humans , Kidney/embryology , Mice , Models, Genetic , Molecular Sequence Data , Polymorphism, Genetic , Promoter Regions, Genetic , Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Transcription, Genetic
17.
Genes Cells ; 5(12): 953-63, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11168582

ABSTRACT

BACKGROUND: Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does. RESULTS: The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF-50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G. CONCLUSIONS: These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor.


Subject(s)
Escherichia coli/metabolism , Peptide Elongation Factor G/metabolism , Proteins/metabolism , Ribosomes/metabolism , Surface Plasmon Resonance/methods , Amino Acid Substitution/genetics , Aminoglycosides , Anti-Bacterial Agents/pharmacology , Arginine/genetics , Biological Transport, Active/drug effects , Biological Transport, Active/genetics , Fusidic Acid/pharmacology , Glycine/genetics , Histidine/genetics , Mutagenesis, Site-Directed , Polyribosomes/drug effects , Polyribosomes/genetics , Polyribosomes/metabolism , Protein Binding/drug effects , Protein Binding/genetics , Protein Synthesis Inhibitors/pharmacology , RNA Stability/drug effects , RNA Stability/genetics , RNA, Ribosomal/metabolism , Ribosomal Proteins , Ribosomes/drug effects , Ribosomes/genetics , Thiostrepton/pharmacology
18.
Genes Cells ; 5(12): 1029-37, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11168589

ABSTRACT

BACKGROUND: Genomic imprinting significantly influences development, growth and behaviour in mammals. Systematic screening of imprinted genes has been extensively carried out to identify the genes responsible for imprinted phenotypes and to elucidate the biological significance of this phenomenon. In this study, we applied DNA chip technology for isolating paternally expressed imprinted genes (Pegs). We compared the resulting expression profiles of parthenogenetic and fertilized control embryos to identify novel imprinted genes. RESULTS: A novel paternally expressed mouse imprinted gene, Peg9/Dlk1, was identified. Consistent with this finding, the paternal expression of its human homologue, PEG9/DLK1, was also confirmed. These two genes form imprinted gene clusters with the reciprocally imprinted mouse Meg3/Gtl2 and human MEG3 genes that we first identified on distal chromosome 12 and chromosome 14q32, respectively. CONCLUSIONS: As DNA chip technology allows us to quickly screen a large number of genes, using this technology to search for imprinted genes could accelerate the identification of genes responsible for human and mouse genetic diseases. Dlk1 and DLK1, which encode transmembrane proteins, have six EGF-like repeats and show homology to the Delta gene in Drosophila melanogaster. Because of its homology to mammalian Delta homologues, PEG9/DLK1 may contribute to the scoliosis phenotype observed in maternal uniparental disomy 14 (mUPD14) patients.


Subject(s)
Gene Expression Regulation, Developmental , Gene Order , Genomic Imprinting , Membrane Proteins/genetics , Proteins/genetics , Animals , Crosses, Genetic , Embryo, Mammalian , Female , Gene Expression Profiling , Humans , Intracellular Signaling Peptides and Proteins , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Parthenogenesis , RNA, Long Noncoding
20.
J Biol Chem ; 273(32): 20223-7, 1998 Aug 07.
Article in English | MEDLINE | ID: mdl-9685370

ABSTRACT

S-modulin controls rhodopsin phosphorylation in a calcium-dependent manner, and it has been suggested that it modulates the light sensitivity of the photoreceptor cell. S-modulin binds to the ROS membrane at high Ca2+ concentration, and N-terminal myristoylation is necessary for this property (the calcium-myristoyl switch). S-modulin has four EF-hand motifs, of which two (EF-2 and -3) are functional. Here, we report on the roles of EF-2 and -3 in S-modulin function (calcium binding, membrane association, and inhibition of rhodopsin phosphorylation) by site-directed mutants (E85M and E121M). Surprisingly, E121M, which has a mutation in EF-3, neither binds Ca2+ nor inhibits phosphorylation. In contrast, E85M binds one Ca2+ and has the same membrane affinity as wild-type S-modulin, but has lost the ability to inhibit rhodopsin phosphorylation. It is suggested that the binding of Ca2+ to EF-3 is probably required for EF-2 to be a functional Ca2+-binding site and to induce exposure of the myristoyl group; and that the binding of Ca2+ to EF-2 is important for the interaction with rhodopsin kinase.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium/physiology , Eye Proteins , Lipoproteins , Nerve Tissue Proteins , Binding Sites/physiology , G-Protein-Coupled Receptor Kinase 1 , Hippocalcin , Models, Molecular , Mutagenesis, Site-Directed/genetics , Myristic Acid/metabolism , Phosphorylation , Protein Binding/physiology , Protein Conformation , Protein Kinases/metabolism , Recombinant Proteins/genetics , Recoverin , Rhodopsin/metabolism , Rod Cell Outer Segment/physiology , Spectrometry, Fluorescence
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