Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3beta.

Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.

Prenatal and molecular diagnosis of beta-thalassemia major in Taiwan by naturally and amplified created restriction sites.

To characterize mutations rapidly in 43 patients with beta-thalassemia major in Taiwan, we utilized a method of natural and amplified created restriction site (ACRS) analysis for detection of beta-globin gene mutation. After analysis, eight different point mutations were found among 86 known chromosomes. IVS-2 nt 654 (C-->T), accounting for 40 of the 86 mutations with mutant beta-globin genes, is the most common mutation, followed by frameshift codons 41/42 (-TCTT) in 28 mutations, -28 mutation (A-->G) in 7 mutations, nonsense codon 17 (A-->T) in 5 mutations, frameshift codons 27/28 (insertion of C) in 2 mutations, IVS-1 nt 1 (G-->T) in 2 mutations, frameshift codons 71/72 (insertion of A) in 1 mutation, and IVS-1 3' end TAG-->GAG in 1 mutation. The first four mutations account for 80 of all 86 mutations of beta-thalassemia major in Taiwan. Furthermore, the beta-globin gene mutation was identified successfully in one chorionic villi biopsy for prenatal diagnosis and in specimen of blood from one patient who had received bone marrow transplantation (BMT). Complete diagnosis is possible in all of the Chinese families with beta-thalassemia in Taiwan, and the first trimester prenatal diagnosis can be achieved simply by using only 13 oligonucleotide primers and 10 restriction endonucleases. This non-radioactive assay was shown to be a rapid, sensitive, precise and safe method in detecting the mutations of beta-thalassemia in Taiwan.

Rac1 gene mutations in human brain tumours.

AIMS: Rac1 is a member of the Ras superfamily of small GTPase and plays a fundamental role in cytoskeleton reorganization, regulation of gene expression and cell proliferation, and cellular transformation. Though recent studies point to an involvement of rac1 in tumorigenesis, little is known about the alteration of rac1 gene in human brain tumours. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), TA cloning, and DNA sequencing were performed to detect rac1 gene mutations in the surgical specimens of 45 human brain tumours. RESULTS: Twelve of 45 cases had base changes in the rac1 gene. The frequency of rac1 alterations was seven of 18 meningiomas, three of 14 astrocytomas, one of seven pituitary adenomas, and one of four metastatic brain tumours. No mutation was detected in acoustic neurilemomas. The subtypes of seven meningiomas include three meningotheliomatous, two atypical, one transitional and one angioblastic meningioma. Three astrocytomas had rac1 gene mutation, including one grade II, one grade III, and one grade IV astrocytoma. All of single base changes were transitions, five of them being T to C transitions. Sites of rac1 mutation were found in codons 34, 41 (two cases), 42 (two cases), 43, 44, 46 and 58. These mutations are mainly localized in the putative effector-domain of rac1 gene and may enhance the activity of rac1, which increases the survival of brain tumours. CONCLUSION: Our results suggest that rac1 gene may play a role in some brain tumours of divergent histogenesis and that the alterations of rac1 gene may contribute to tumorigenesis and/or metastasis.

Expression and mutation analysis of the p53 gene in astrocytoma.

The role of p53 gene mutations in the formation or progression of human astrocytic tumors is controversial. We studied the distribution pattern of p53 immunoreactivity and analyzed p53 gene mutations to define the significance of p53 gene mutations in astrocytoma tumorigenesis or malignant progression. Twenty-three astrocytic tumors were evaluated with immunohistochemistry, single-strand conformation polymorphism (SSCP) analysis, and sequence analysis. We also searched MEDLINE to collect data on p53 gene mutation frequencies in astrocytic tumors in order to evaluate the association of p53 mutations and tumor grade. Strong immunoreactivity with a diffuse clustering pattern was found in three of five glioblastomas and seven of 12 anaplastic astrocytomas. Three of four low-grade astrocytomas were immunonegative. The p53 immunopositive cells in the only positively staining low-grade astrocytoma in our study appeared sparsely scattered. The results of immunostaining suggested that clonal expansion was associated with astrocytoma progression. Mutations of the p53 gene were detected in four of the 23 astrocytomas (one glioblastoma and three anaplastic astrocytomas). In the genetic data analysis, 76 of 367 astrocytomas had p53 gene mutations. A significantly greater p53 gene mutation frequency was found in anaplastic astrocytomas or glioblastomas than in the low-grade astrocytomas. The results of these immunohistochemical and genetic studies support the view that p53 gene mutation is associated with the malignant progression from low-grade to high-grade astrocytomas rather than with tumor initiation or promotion.

Clinical significance of distribution patterns of P53 immunoreactivity in astrocytic tumors.

The difference of prognosis in patients with the same WHO grade of astrocytic tumors suggests that such tumors comprise a heterogeneous group in biological behavior. The correlation between p53 immunoreactivity and prognosis has often been reported but remains controversial. From the perspective of clonal expansion of p53 immunopositive cells, the distribution patterns of p53 immunoreactivity can be divided into four types: negative, scattered, focally clustered, and diffusely clustered. The survival rate was evaluated by classifying the p53 immunoreactivity into two groups: the significantly immunopositive patterns (focally and diffusely clustered types) and the significantly immunonegative patterns (negative and scattered types). The survival analysis showed a highly significant difference between these two patterns within the same WHO grade of astrocytic tumors (p = 0.0185). Our studies demonstrate that the distribution patterns of p53 immunoreactivity, which reflect the trends of clonal expansion of p53 immunopositive cells, are significantly valuable in predicting the prognosis of patients with the same WHO grade of astrocytic tumors.

The expression of rac1 pseudogene in human tissues and in human brain tumors.

BACKGROUND: Recent studies have demonstrated that Rac is a regulator of cell morphology and growth. Rac1 gene appears to have involvement in tumorigenesis and metastatic potential. In our previous study of rac1 gene in 45 human brain tumors, rac1 pseudogene was found. The rac1 pseudogene is an intronless pseudogene and has a similarity of 86% with rac1 nucleotide sequence. The rac1 pseudogene contains 579 nucleotides and only 46 amino acids can be translated. Little is known about the expression of rac1 pseudogene in human tissues or tumors. MATERIALS AND METHODS: The expression of rac1 gene and rac1 pseudogene in different human tissues and brain tumors was investigated by the use of reverse transcriptase-polymerase chain reaction and Northern blotting. RESULTS: The rac1 gene is apparently expressed in these 8 human tissues. The rac1 pseudogene is also apparently expressed in human tissues except for brain tissue. The overexpression of rac1 gene in brain tumors was 8% (2/25) and the overexpression of rac1 pseudogene was 76.9% (20/26). Only two astrocytomas had overexpression of rac1 gene, compared with normal brain tissues. The overexpression of rac1 pseudogene was 6 of 9 in meningiomas, 7 of 9 in astrocytomas, and 7 of 8 in pituitary adenomas. CONCLUSIONS: High frequency of overexpression of rac1 pseudogene was detected in the human brain tumors when compared with that expressed in the normal brain tissues. Our study suggested that the rac1 pseudogene may play an important role of the tumorigenesis of brain.