Mutsushi Matsuyama

Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations.

Transforming activity of the c-ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A mutations was investigated by transfection of NIH 3T3 cells. Mutant c-ret genes driven by the simian virus 40 or cytomegalovirus promoter induced transformation with high efficiencies. The 170-kDa Ret protein present on the cell surface of transformed cells was highly phosphorylated on tyrosine and formed disulfide-linked homodimers. This result indicated that MEN 2A mutations induced ligand-independent dimerization of the c-Ret protein on the cell surface, leading to activation of its intrinsic tyrosine kinase. In addition to the MEN 2A mutations, we further introduced a mutation (lysine for asparaginic acid at codon 300 [D300K]) in a putative Ca(2+)-binding site of the cadherin-like domain. When c-ret cDNA with both MEN 2A and D300K mutations was transfected into NIH 3T3 cells, transforming activity drastically decreased. Western blot (immunoblot) analysis revealed that very little of the 170-kDa Ret protein with the D300K mutation was expressed in transfectants while expression of the 150-kDa Ret protein retained in the endoplasmic reticulum was not affected. This result also demonstrated that transport of the Ret protein to the plasma membrane is required for its transforming activity.

A GENETIC LOCUS SUSCEPTIBLE TO THE OVERT PROTEINURIA IN BUF/MNA RAT

Abstract. The BUF/Mna (BUF) strain is a high-proteinuria line of rats, and virtually all rats develop overt proteinuria by the age of 20 weeks. Genetic analysis revealed that proteinuria susceptibility was determined principally by two autosomal recessive genes. These findings prompted us to perform genetic mapping of the genes. (BUF/Mna × WKY/NCrj) F1× BUF/Mna backcross rats were raised and maintained for 40–60 weeks to detect proteinuria. DNAs were extracted from ears of these rats and were examined by linkage study with polymerase chain reaction (PCR) with 132 microsatellite markers. Fifty-three out of 167 rats developed proteinuria. DNAs of 51 out of these 53 rats showed homozygous BUF/BUF genotype in the D13Mgh4 and D13N1 markers located on Chromosome (Chr) 13. The D13Rat1, D13Mgh2, D13Rat13, D13Mgh3, Syt2, Ren, D13Rat25, D13Mit2, D13Mgh5, and D13N2 markers located on the chromosome also showed statistically significant linkage to the development of proteinuria, whereas the other 110 markers showed no linkage. Here we report that a proteinuria-susceptible gene, Pur1, resides on a region flanked by the loci D13Mgh3 and D13Mgh4 on Chr 13.

Phosphate overload induces podocyte injury via type III Na-dependent phosphate transporter

Uptake of P(i) at the cellular membrane is essential for the maintenance of cell viability. However, phosphate overload is also stressful for cells and can result in cellular damage. In the present study, we investigated the effects of the transgenic overexpression of type III P(i) transporter Pit-1 to explore the role of extracellular P(i) in glomerular sclerosis during chronic renal disease. Pit-1 transgenic (TG) rats showed progressive proteinuria associated with hypoalbuminemia and dyslipidemia. Ultrastructural analysis of TG rat kidney by transmission electron microscopy showed a diffuse effacement of the foot processes of podocytes and a thickening of the glomerular basement membrane, which were progressively exhibited since 8 wk after birth. TG rats died at 32 wk of age due to cachexia. At this time, more thickening of the glomerular basement membrane and segmental sclerosis were observed in glomeruli of the TG rats. Immunohistochemical examination using anti-connexin 43 and anti-desmin antibodies suggested the progressive injury of podocytes in TG rats. TG rats showed higher P(i) uptake in podocytes than wild-type rats, especially under low P(i) concentration. When 8-wk-old wild-type and TG rats were fed a 0.6% normal phosphate (NP) or 1.2% phosphate (HP) diet for 12 wk, HP diet-treated TG rats showed more progressive proteinuria and higher serum creatinine levels than NP diet-treated TG rats. In conclusion, our findings suggest that overexpression of Pit-1 in rats induces phosphate-dependent podocyte injury and damage to the glomerular barrier, which result in the progression of glomerular sclerosis in the kidney.

Establishment and characterization of high- and low-lung-metastatic cell lines derived from murine colon adenocarcinoma 26 tumor line.

We established and characterized high‐ (LuM1) and low‐lung‐metastatic (NM11) cell lines derived from murine colon adenocarcinoma 26 tumor line. LuM1 cell line was established as a clonal cell line from a cultured cell mixture derived from a lung‐metastatic nodule after 7 sequential subcutaneous transplantations of lung‐metastatic tumors in the abdominal wall of BALB/c mice. NM11 cell line was established from a cultured cell mixture derived from a subcutaneous transplant of murine colon adenocarcinoma 26 tumor cells. LuM1 cells showed marked spontaneous lung metastases, but NM11 cells rarely did. High invasive potential of LuM1 cells was revealed by in vitro invasion assay using Matrigel reconstituted membranes. Rapid retraction was observed in monolayers of human umbilical vein endothelial cells and bovine aortic endothelial cells when LuM1 cells were added on the monolayers. Gelatin zymography and immunochemical examinations with monoclonal antibodies against gelatinase B (Mr 95,000 type IV collagenase) showed secretion of large amounts of the gelatinase by LuM1 cells.

Actin -related protein 3 (Arp3) is mutated in proteinuric BUF/Mna rats

The BUF/Mna strain of rat is a model of focal and segmental glomerulosclerosis (FSGS) in which a quantitative trait locus (QTL) for proteinuria, Pur1, has been identified. The aim of the present study was to identify candidates for the Pur1 gene. To narrow the Pur1 QTL, we performed fine QTL mapping and single nucleotide polymorphism (SNP) genotyping. To identify candidate genes, sequencing and gene-expression analyses of all genes contained in the narrowed locus were conducted. The narrowed Pur1 region contained 25 genes. Among these genes, only the Arp3 gene was mutated in the BUF/Mna strain; it contained a missense mutation that caused an L111F substitution. This leucine is conserved across species. Gene-expression analysis failed to identify any other candidate genes for Pur1. Arp3-mediated actin assembly abnormalities were visible in immunohistochemical and electron microscopic examinations of podocytes in old BUF/Mna rats. Taken together, these data suggest that Arp3 is a candidate for the Pur1 gene. This observation is consistent with our growing recognition that abnormal signaling-induced assembly of actin in podocytes leads to the development of FSGS.

Composite tumor of mucinous cystadenoma and somatostatinoma of the kidney.

Abstract  Approximately 30 cases of carcinoid tumor of the kidney have been reported in the English literature, including three cases found as components of teratomas. Renal composite tumors associated with somatostatinoma have not been described. A 53‐year‐old female presented with an incidentally found right renal cystic lesion. Computed tomography demonstrated a cystic lesion associated with a solid nodule in the right kidney and postcontrast dynamic MRI revealed enhancement of the solid nodule. The patient underwent radical nephrectomy for the kidney lesion and is now well without recurrence 21 months after the operation. From the histopathological findings we diagnosed the cystic lesion as a composite tumor composed of mucinous cystadenoma and carcinoid tumor. Immunohistochemistry demonstrated the majority of cells of in carcinoid portion to be positive for antisomatostatin staining. The present case is the first documented composite tumor of mucinous cystadenoma and somatostatinoma of the kidney.

Mapping of two genetic loci, Ten-1 and Ten-2, associated with thymus enlargement in BUF/Mna rats

The thymoma-prone BUF/Mna rat is a useful model for human thymoma. Thymoma develops spontaneously in these rats at an incidence of nearly 100%. At pre-thymoma age, BUF/Mna rats have extremely large thymuses when compared with those of rats of the other strains, suggesting the presence of genes that regulate the thymus enlargement. We performed linkage study to identify the genetic loci associated with thymus enlargement in (WKY/NCrj × BUF/Mna) F1 × BUF/Mna backcross rats. Linkage study showed the significant associations between thymus size and markers on Chromosomes (Chrs) 1 and 13, suggesting the presence of two genes, Ten-1 and Ten-2, which regulate the thymus enlargement. Ten-1 was located between myosin light chain, muscle 2 (MYL2) and DIMgh11 loci on Chr 1, and Ten-2 was located between synaptotagmin II (SYT2) and D13N2 loci on Chr 13.

Genetic regulation of slowly progressing mild muscle atrophy in fast-twitch muscles of BUF/Mna rats

BUF/Mna strain rats spontaneously develop slowly progressing mild‐moderate muscle atrophy of extensor digitorum longus, tibialis, and extraocular muscles, which consist mainly of fast‐twitch type fibers, at nearly 100% incidence. They have lighter extensor digitorum longus muscles than soleus muscles, when alive for more than 6 weeks. Genetic segregation of the development of the muscle atrophy was studied by crossing the BUF/Mna strain with three other strains, ACI/NMs, WKY/NCrj, and BDIX, which were free of muscle atrophy. Two autosomal dominant susceptible genes, Mas‐1 and Mas‐2, determine the development of the muscle atrophy in these combinations of crosses.

Genetic mapping of genes regulating the thymus size in back‐cross rats between the laboratory BUF/Mna strain and the MITE strain derived from the wild rat, Rattus norvegicus

The thymoma prone BUF/Mna (B) rat is a useful model for Studying the genes responsible for thymus enlargement during the stage of young growth. Among the strains of rats, B rats have the largest thymuses at al stages of life. A locus, Ten‐1, which contributes to thymus enlargement in back‐cross (BC) rats between the B and WKY/NCrj (W) strains, was mapped on chromosome 1. To determine the precise location of the bus, (B×(B×MITE)F1) BC rats were generated by crossing the B strain with the Inbred MITE (M) strain, which was established from captured, Japanese wild rats, and were examined by linkage study using polymerase chain reaction with 67 microsatellite markers. Linkages with thymus enlargements were found In genotypes of seven markers, BSIS, LSN, MYL2, IGF2, PBPC2, D1Mgh11, and D1Mit6, by X2‐test and Students t‐test, which confirmed the presence of the genetic locus associated with thymus enlargement, Ten‐1, in this region. Paradoxically, a suppressive locus, Tsu‐1, to thymus enlargement was also found on chromosome 3, showing linkages of phenotype of the small thymus with genotypes of SCN2A, CAT D3Mit16, and D3Mit13. By analyses of mapmaker/exp and mapmaker/qtl, Ten‐1 was mapped at 4.6 cM proximal from IGF2 locus on chromosome 1 and Tsu‐1 at 4.0 cM proximal from CAT locus on chromosome 3, respectively.

Generation of polymorphic markers tightly linked to the thymus enlargement loci by phenotype-directed representational difference analysis

Abstract. To obtain genetic markers linked to a specific genetic locus, genomic subtraction with a DNA pool of backcross or F2 intercross animals with a specific genotype at the locus is known to be effective. To determine whether the pooling strategy is also effective for isolation of genetic markers linked to a quantitative phenotype that can potentially be controlled by multiple genetic loci, we tested the ability of representational difference analysis (RDA) to isolate genetic markers linked to the thymus enlargement observed in the BUF/Mna (BUF) rat. This is known to be controlled by single major and minor genes, Ten1 and Ten2, on Chromosomes (Chrs) 1 and 13, respectively, both of which have dose effects on the normal WKY/Ncj (WKY) allele. DNA from an inbred WKY rat was used as the tester, and the driver was prepared from a DNA pool of 12 (WKY × BUF)F1× BUF backcross rats with high thymus ratios (thymus weight/body weight), expected to have dominance of the BUF allele in the responsible loci. By two RDA series with the restriction enzymes BglII and BamHI, respectively, 28 polymorphic markers were isolated, and 8 of them were shown to be linked to Ten1, and one to Ten2. One of the 8 markers linked to Ten1 demonstrated no recombination in 18 rats with high thymus ratios. RDA with a DNA pool based on a quantitative phenotype (phenotype-directed RDA) can thus be considered an efficient approach for direct isolation of polymorphic markers linked to a quantitative trait.