Expression of MMP-2, MMP-9 and TIMP-1 in the Wall of Abdominal Aortic Aneurysms

An impaired mechanism of regulatory feedback by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) has been implicated in the development of abdominal aortic aneurysms (AAAs). This study examined the pathogenesis of AAAs with respect to pathological characteristics and expressions of MMP-2, MMP-9 and TIMP-1. Their expressions were evaluated by immunohistochemistry, competitive polymerase chain reaction (PCR) and Western blotting in a total of 23 consecutive AAAs. The AAA specimens were obtained by surgery, while control specimens were obtained at autopsy. Specimens consisted of 6 patients with small-diameter AAAs (30?45 mm), 17 with medium-large-diameter AAAs (> 45 mm) and 11 controls (17?25 mm). Immunohistochemistry showed MMP-2- and TIMP-1-positive cells mainly in the intima, and MMP-9-positive cells in the intima and adventitia. Competitive PCR showed a significantly higher expression of MMP-2 messenger RNA (mRNA) in the small-diameter AAAs, and higher expressions of MMP-9 mRNA in the small-diameter and medium-large-diameter AAAs than in the controls. The mRNA levels significantly correlated between TIMP-1 and MMP-9, and between MMP-2 and MMP-9 in the AAAs, especially in the medium-large-diameter AAAs. Western blotting revealed the expression of MMPs and TIMP-1 variably in all the specimens examined. These results indicated that MMP-2 and MMP-9 might act cooperatively and play a crucial role in the development of AAAs, and that TIMP-1 inhibits MMP-9 in the AAAs, especially in those medium-large-diameter AAAs

A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3–96.8%. Additionally, we observed homogenous gene expression in 77.3–87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression

Frequent Mutations in the β-Catenin Gene in Cholangiocarcinoma

The molecular pathogenesis of cholangiocarcinoma (CC) remains unclear. β-Catenin functions in both intercellular adhesion and signal transduction. As a signaling molecule, mutations in exon 3 of the β-catenin gene encoding the regions phosphorylated by glycogen synthase kinase (GSK)-3β stabilize this protein in cytoplasm. Subsequently, accumulated β-catenin protein translocates to nuclei and up-regulates the transcriptional activity of genes involved in oncogenesis. Recently, mutations in exon 3 of the β-catenin gene were detected in various carcinomas. Using polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) analysis, direct sequencing and subcloning-sequencing, we investigated mutations of exon 3 of the β-catenin gene in CC. Mutations were found in 26 out of 33 (78.8%) CC tumor samples. All of the mutations were heterozygous 1-base deletions at codon 15, resulting in a stop codon at codon 46. This is the first study demonstrating the presence of β-catenin gene mutations in CC. However, it was suggested that this mutation might not be involved in deregulation of β-catenin signaling, because no correlation was observed between the β-catenin mutation and immunolocalization of β-catenin protein

Cord Blood from SGA Preterm Infants Exhibits Increased GLUT4 mRNA Expression

[Background] Insulin and insulin-like growth factor (IGF) signaling plays an important role in prenatal and postnatal growth and glucose metabolism. Both small-for-gestational age (SGA) and preterm infants have abnormal growth and glucose metabolism. However, the underlying mechanism remains unknown. Recently, we showed that term SGA infants have abnormal insulin/IGF signaling in cord blood. In this study, we examined whether preterm infants show similar aberrations in cord blood insulin/IGF signaling. [Methods] A total of 41 preterm cord blood samples were collected. Blood glucose, insulin, IGF-1, and C-peptide concentrations were measured, and mRNA expression of IGF1R, INSR, IRS1, IRS2, and SLC2A4 (i.e., GLUT4) was analyzed by quantitative reverse-transcription PCR. [Results] This study included 34 appropriate-for-gestational age (AGA) and 7 SGA preterm neonates. No hyperinsulinemia or any differences in IGF1R or INSR mRNA expression were detected between the two groups. However, GLUT4 mRNA levels were increased in preterm SGA. Moreover, the expression level in hypoglycemic preterm SGA was significantly higher than that in hypoglycemic preterm AGA. IRS2 mRNA expression did not show a statistically significant difference between preterm SGA and AGA neonates. [Conclusion] SGA preterm birth does not induce hyperinsulinemia; however, it modifies insulin/IGF signaling components such as GLUT4 in umbilical cord blood. Our study suggests that prematurity or adaptation to malnutrition alters the insulin/IGF signaling pathway

A new heterozygous compound mutation in the CTSA gene in galactosialidosis

Galactosialidosis is an autosomal recessive lysosomal storage disease caused by the combined deficiency of lysosomal β-galactosidase and neuraminidase due to a defect in the protective protein/cathepsin A. Patients present with various clinical manifestations and are classified into three types according to the age of onset: the early infantile type, the late infantile type, and the juvenile/adult type. We report a Japanese female case of juvenile/adult type galactosialidosis. Clinically, she presented with short stature, coarse facies, angiokeratoma, remarkable action myoclonus, and cerebellar ataxia. The patient was diagnosed with galactosialidosis with confirmation of impaired β-galactosidase and neuraminidase function in cultured skin fibroblasts. Sanger sequencing for CTSA identified a compound heterozygous mutation consisting of NM_00308.3(CTSA):c.746 + 3A>G and c.655-1G>A. Additional analysis of her mother’s DNA sequence indicated that the former mutation originated from her mother, and therefore the latter was estimated to be from the father or was a de novo mutation. Both mutations are considered pathogenic owing to possible splicing abnormalities. One of them (c.655-1G>A) is novel because it has never been reported previously

Association between Vitamin D Receptor Gene Polymorphisms and Renal Osteodystrophy in Patients on Maintenance Hemodialysis

We examined the possible involvement of vitamin D receptor (VDR) gene polymorphisms in patients on maintenance hemodialysis and further investigated the relation between VDR genotypes and bone histology. Two hundred and nine patients undergoing regular hemodialysis (male/female ratio, 124/85) were included in this study. DNA was extracted from peripheral blood leukocytes. VDR genotypes were analyzed as restriction fragment length polymorphisms, by using BsmI, ApaI, TaqI and FokI. Lumbar bone mineral density was measured by dual-energy X-ray absorptiometry, and expressed as a Z-score. Serum 1,25(OH)2D3, osteocalcin and ntact-parathyroid hormone (i-PTH) were determined by an immunoradiometric assay. In 97 patients, bone biopsy as performed and the histology was divided into osteitis fibrosa, mild lesion, dynamic bone disease and osteomalacia. Serum levels of osteocalcin, i-PTH and bone mineral density were significantly lower in the presence of B, A, t and f alleles. However, in this study, we did not find any association between bone histology and the four VDR genotypes. We concluded that renal osteodystrophy in dialysis patients was modified by environmental factors such as medication with active vitamin D, age, gender and duration of chronic renal failure, and that the impact of the VDR allelic effect may play a small role in determining on bone histology

Variations in Clinical Findings of Patients with Identical Tuberous Sclerosis Gene Mutations

Colorectal carcinogenesis involves environmental factors and genetic predispositions. Recent studies have suggested the associations between colorectal neoplasm and functional polymorphism of matrix metalloproteinases (MMPs) and cytokine genes. In this study, we analyzed polymorphisms of MMPs and tumor necrosis factor (TNF)-alpha genes, focusing on the susceptibility to colorectal neoplasm and the tumor progression. The subjects were 186 patients (95 men and 91 women) who underwent total colonoscopy, and were classified into cancer, adenoma and non-neoplasm (control) groups of 47, 72 and 67 patients, respectively. The polymorphisms at the MMP-2 ?1306C/T, MMP-3 ?1171 5A/6A, MMP-7 ?181A/G, MMP-9 ?1562C/T and TNF-alpha ?308G/A loci were analyzed. Regarding background factors, significant differences were found in the age, sex ratio and alcohol-drinking and cigarette-smoking histories in the adenoma and cancer groups, compared to those in the control group. On these factors-adjusted logistic regression analysis of polymorphisms and disease susceptibility, no significant difference was noted in the frequency of any polymorphism in the adenoma and cancer groups, compared to those in the control group. The analysis of the involvement of polymorphisms in tumor progression in the adenoma and cancer groups revealed that the odds ratio for the MMP-3 5A allele was significantly higher in the cancer group (2.74; 95% confidence interval = 1.11?6.74, P = 0.02). The polymorphisms of MMP genes and TNF-alpha genes were not associated with the susceptibility to colorectal neoplasm, but the involvement of the MMP-3 5A allele in the progression of adenoma to cancer was suggested

Association of the Trp64Arg Mutation of the β3-Adrenergic Receptor with Diabetes Mellitus, Impaired Glucose Tolerance and Lifestyle in Japanese Workers

In order to investigate whether the Trp64Arg (a missense mutation of tryptophan for arginine at position 64 codon) polymorphism of the β3-adrenergic receptor (β3-AR) gene is related to the incidence of non-insulin-dependent diabetes mellitus (NIDDM) and impaired glucose tolerance (IGT), a retrospective cohort study among Japanese workers was conducted. The subjects were Japanese workers at an occupational site in Shimane Prefecture. Informed consent was obtained from 492 workers. The baseline data were obtained at the regular health examination in 1992 and a retrospective cohort study was performed for analyzing the incidence of NIDDM and IGT in 1998. The Trp64Arg polymorphism β3-AR gene for each worker was detected by the single strand conformation polymerase analysis. Relative risks were calculated by the logistic regression analysis. The rates of Trp64Trp (TT), Trp64Arg (TA) and Arg64Arg (AA) genotypes were 66.3%, 31.1% and 2.6%, respectively. The relative risk of (TA + AA) against TT for the incidence of NIDDM and IGT by univariate analysis was 1.37 (95% incidence of NIDDM and IGT adjusted for confounders in a multiple logistic regression model including age, gender, family history, body mass index, alcohol consumption, eating habits and exercise was 1.31 (95% confidence interval, 0.65-2.67). The present findings suggested that a weak association between Trp64Arg polymorphism of the β3-AR gene and the incidence of NIDDM and IGT

Tuberous Sclerosis 2 Gene Is Expressed at High Levels in Specific Types of Neurons in the Mouse Brain

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by mental retardation, epilepsy and hamartomatous growth in many tissues. The gene (TSC2) encoding a tumor suppressor protein whose mutations cause TSC, has been demonstrated to be expressed at high levels in the adult and developing brain, raising the question of whether or not the TSC2 gene product has unique roles in differentiation related to cytoskeletal interactions within the central nervous system, in addition to a tumor suppressor function. To determine the expression of TSC2 in functionally distinct neuron types of the mouse brain, we carried out in situ hybridization with digoxigenin-labeled riboprobes for the detection of TSC2 mRNA. High levels of the TSC2 gene were in neurons of the pyramidal and dentate granular layer in the hippocampus, cerebellar Purkinje cells, neurons of the piriform cortex, motor neurons in the medulla and interneurons in the striatum, while intermediate levels were in cortical neurons, striatal neurons, septal neurons, thalamic neurons and neurons in the substantia nigra compacta. Thus, the high expression of the TSC2 gene has restricted distribution in specific neuronal types which are characterized by well-developed dendrites and rich in use-dependent long-term changes in synaptic efficacy. These results suggest that the function of the TSC2 gene product may be involved on a cellular basis in neuronal plasticity and relevant to mental retardation observed in TSC patients