Highly sensitive method for genomewide detection of allelic composition in nonpaired, primary tumor specimens by use of affymetrix single-nucleotide-polymorphism genotyping microarrays

Loss of heterozygosity (LOH), either with or without accompanying copy-number loss, is a cardinal feature of cancer genomes that is tightly linked to cancer development. However, detection of LOH is frequently hampered by the presence of normal cell components within tumor specimens and the limitation in availability of constitutive DNA. Here, we describe a simple but highly sensitive method for genomewide detection of allelic composition, based on the Affymetrix single-nucleotide-polymorphism genotyping microarray platform, without dependence on the availability of constitutive DNA. By sensing subtle distortions in allele-specific signals caused by allelic imbalance with the use of anonymous controls, sensitive detection of LOH is enabled with accurate determination of allele-specific copy numbers, even in the presence of up to 70%-80% normal cell contamination. The performance of the new algorithm, called "AsCNAR" (allele-specific copy-number analysis using anonymous references), was demonstrated by detecting the copy-number neutral LOH, or uniparental disomy (UPD), in a large number of acute leukemia samples. We next applied this technique to detection of UPD involving the 9p arm in myeloproliferative disorders (MPDs), which is tightly associated with a homozygous JAK2 mutation. It revealed an unexpectedly high frequency of 9p UPD that otherwise would have been undetected and also disclosed the existence of multiple subpopulations having distinct 9p UPD within the same MPD specimen. In conclusion, AsCNAR should substantially improve our ability to dissect the complexity of cancer genomes and should contribute to our understanding of the genetic basis of human cancers.     

Factors responsible for glucose intolerance in Japanese subjects with impaired fasting glucose.

Impaired fasting glucose (IFG) represents risk of development of diabetes (DM) and its complications. We investigated insulin secretion and insulin sensitivity in 403 IFG subjects divided into three levels of 2-hour postchallenge glucose (2-h PG) to clarify the factors responsible in the development of glucose intolerance in Japanese IFG. Nearly 60% of the subjects at annual medical check-up with FPG of 6.1-7.0 mmol/l at the first screening were diagnosed by 75 g oral glucose tolerance test (OGTT) to have impaired glucose tolerance (IGT; FPG <7.0 mmol/l and 7.8 mmol/l <2-h PG <11.1 mmol/l) or DM (isolated postchallenge hyperglycemia (IPH); FPG <7.0 mmol/l and 11.1 mmol/l <2-h PG level). The primary factor in the decreased glucose tolerance was a decrease in early-phase insulin, with some contribution of increasing insulin resistance. In addition, IFG/IGT and IFG/IPH subjects showed a compensatory increase in basal insulin secretion sufficient to keep FPG levels within the non-diabetic range. IFG is composed of three different categories in basal, early-phase insulin secretion, and insulin sensitivity.     

Escherichia coli dnaJ deletion mutation results in loss of stability of a positive regulator, CRP.

The dnaJ deletion mutant K7052(lambda dnaK) has a temperature-sensitive defect in the synthesis of beta-galactosidase. We confirmed this operon-specific and temperature-sensitive defect in cell-free extracts prepared from the mutant cells and found that the missing factor was CRP. In the mutant, the cellular concentration of CRP was too low to allow the expression of the lac operon at a nonpermissive temperature. Introduction of a CRP over-producing plasmid into the dnaJ deletion mutant suppressed the defect of beta-galactosidase synthesis. The lower content of CRP in the mutant was found to result from extreme instability of the protein. These results strongly suggested that the heat shock protein dnaJ is involved in the stabilization (or degradation) of CRP.     

Induction of tumour differentiation and apoptosis and LeY antigen expression in treatment with differentiation-inducing agent, vesnarinone, of a patient with salivary adenoid cystic carcinoma

A patient with locally-advanced submandibular adenoid cystic carcinoma with poorly differentiated solid type, was treated with differentiation-inducing agent, vesnarinone, per os at a dose of 60 mg/day daily for 8 weeks. The vesnarinone administration caused marked regression of the tumour. In addition to conversion into the well-differentiated tubular type from the poorly differentiated solid type, the induction of apoptosis and LeY antigen was observed in the treated tumour. These findings indicate that vesnarinone might be a useful therapeutic agent for treatment of salivary cancer. Since we found the new expression of LeY antigen in the well-differentiated tubular lesion in the salivary adenoid cystic carcinoma treated with vesnarinone, we examined the LeY antigen expression in relation to tumour differentiation in five cases of salivary adenoid cystic carcinoma. Consequently, tissue sections from all of the adenoid cystic carcinoma examined showed no positive LeY staining, except for some areas in the tumour lesion with the tubular pattern including the histologically normal-appearing tissue adjacent to the tumour tissue. These findings suggest that there is the intimate relationship between the LeY antigen expression and tumour differentiation in human salivary adenoid cystic carcinoma.     

Inhibitory effect of ferulic acid and isoferulic acid on the production of macrophage inflammatory protein-2 in response to respiratory syncytial virus infection in RAW264.7 cells

We investigated the effect of ferulic acid (FA) and isoferulic acid (IFA), which are the main active components of the rhizoma of Cimicifuga heracleifolia (CH), an anti-inflammatory drug used frequently in Japanese traditional medicine, on the production of macrophage inflammatory protein-2 (MIR-2) in a murine macrophage cell line, RAW264.7, in response to respiratory syncytial virus (RSV) infection. Following the exposure of cells to RSV for 20h, the MIP-2 level in condition medium was increased to about 20 ng/ml, although this level in mock-infected cells was negligible. In the presence of either FA or IFA, RSV-infected cells reduced MIP-2 production in a dose-dependent manner. These data suggest that FA and IFA might be responsible, at least in part, for the anti-inflammatory drug effect of CH extract through the inhibition of MIP-2 production.     

Immunohistochemical localization of transforming growth factor α in the major salivary glands of male and female rats

Summary The three major salivary glands of normal male and female Fischer 344 rats of different ages were examined for the localization of epidermal growth factor (EGF) and transforming growth factor (TGF) by immunohistochemical staining. EGF was demonstrated only in the granulated convoluted tubule (GCT) cells of the submandibular gland, the results confirming the previous reports, and most abundantly in adult males and pregnant females. TGF stain was localized in all three glands and was found throughout the entire duct system, excluding acinar cells. The myoepithelial cells of the sublingual gland were also reactive with the TGF antibody. The specificity of the staining was confirmed by negative staining reaction with the absorbed antibody and by radio-immunoassay and Western blot methods. This is the first report describing the presence of TGF in the rat salivary glands.     

Amyloid β protein activates PKC-δ and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia

The increased accumulation of activated microglia containing amyloid β protein (Aβ) around senile plaques is a common pathological feature in subjects with Alzheimer's disease (AD). Much less is known, however, of intracellular signal transduction pathways for microglial activation in response to Aβ. We investigated intracellular signaling in response to Aβ stimulation in primary cultured rat microglia. We found that the kinase activity of PKC-δ but not that of PKC- or - is increased by stimulation of microglia with Aβ, with a striking tyrosine phosphorylation of PKC-δ. In microglia stimulated with Aβ, tyrosine phosphorylation of PKC-δ was evident at the membrane fraction without an overt translocation of PKC-δ. PKC-δ co-immunoprecipitated with MARCKS from microglia stimulated with Aβ. Aβ induced translocation of MARCKS from the membrane fraction to the cytosolic fraction. Immunocytochemical analysis revealed that phosphorylated MARCKS accumulated in the cytoplasm, particularly at the perinuclear region in microglia treated with Aβ. Taken together with our previous observations that Aβ-induced phosphorylation of MARCKS and chemotaxis of microglia are inhibited by either tyrosine kinase or PKC inhibitors, our results provide evidence that Aβ induces phosphorylation and translocation of MARCKS through the tyrosine kinase-PKC-δ signaling pathway in microglia.     

Lysyl-tRNA synthetase is a target for mutant SOD1 toxicity in mitochondria

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting the motor neurons. The majority of familial forms of ALS are caused by mutations in the Cu,Zn-superoxide dismutase (SOD1). In mutant SOD1 spinal cord motor neurons, mitochondria develop abnormal morphology, bioenergetic defects, and degeneration. However, the mechanisms of mitochondrial toxicity are still unclear. One possibility is that mutant SOD1 establishes aberrant interactions with nuclear-encoded mitochondrial proteins, which can interfere with their normal trafficking from the cytosol to mitochondria. Lysyl-tRNA synthetase (KARS), an enzyme required for protein translation that was shown to interact with mutant SOD1 in yeast, is a good candidate as a target for interaction with mutant SOD1 at the mitochondrion in mammals because of its dual cytosolic and mitochondrial localization. Here, we show that in mammalian cells mutant SOD1 interacts preferentially with the mitochondrial form of KARS (mitoKARS). KARS-SOD1 interactions occur also in the mitochondria of the nervous system in transgenic mice. In the presence of mutant SOD1, mitoKARS displays a high propensity to misfold and aggregate prior to its import into mitochondria, becoming a target for proteasome degradation. Impaired mitoKARS import correlates with decreased mitochondrial protein synthesis. Ultimately, the abnormal interactions between mutant SOD1 and mitoKARS result in mitochondrial morphological abnormalities and cell toxicity. mitoKARS is the first described member of a group of mitochondrial proteins whose interaction with mutant SOD1 contributes to mitochondrial dysfunction in ALS.