Genetic susceptibility testing for hereditary breast and ovarian cancer--approach from a commercial clinical laboratory

Genetic testing for hereditary cancers and other common diseases are still considered as the research testing, not for the clinical testing in Japan. One of the major reason of this situation is related to the guidelines regarding the human genetic testing issued successively in the spring of 2001, one by joint work of the eight learned societies and the other by the Japan clinical laboratories association. Both of these guidelines warn the condition of the clinical application of genetic testing after research stage must have the evidence data for clinical utility. We describe the situation of the genetic testing in the U.S. focusing the social background of increasing breast cancer cases and the contribution of Myriad Genetic Laboratories, Inc. for the genetic testing industry. We also describe the Japanese situation of the genetic testing and problems to be solved before spreading widely.     

Active Cyclin A-CDK2 Complex, a Possible Critical Factor for Cell Proliferation in Human Primary Lung Carcinomas

Expression of cyclins A and E and cyclin-dependent kinase 2 (CDK2) was examined immunohistochemically in 190 cases of human lung carcinoma. Cyclin A and CDK2 were expressed in the majority of squamous cell carcinomas, small cell carcinomas, and large cell carcinomas, but in significantly fewer cases of adenocarcinomas. Cyclin E was expressed in a minority of all subtypes. In particular, well differentiated cells in squamous cell carcinoma stained positively for cyclin E; in contrast, cyclin A was expressed in the nonkeratinized proliferating areas of the tumor nests. Immunoblotting revealed that all these proteins were expressed at higher levels in tumor tissues than in adjacent normal tissues. Immunoprecipitation also revealed higher levels of cyclin A and cyclin E associated with CDK2 in tumor tissues. Furthermore, tumor tissues which exhibited higher cyclin A and CDK2 expression also had higher CDK2 kinase activity. However, cyclin E-associated kinase activity was barely detectable even in tumor samples exhibiting higher cyclin E expression. Consistent with these data, elevated expression of cyclin A correlated to shorter survival periods in contrast to expression of cyclin E, which correlated to longer survival periods. These results suggest that in human lung carcinomas, elevated expression of active cyclin A-CDK2 complexes with associated higher CDK2 kinase activity is critical for promoting cell cycle progression and unrestrained proliferation of tumor cells and can be a predictive marker for patients’ prognosis. On the other hand, immunohistochemical detection of cyclin E-CDK2 reflects accumulation of inactive forms of protein complexes, implying differentiation or senescence of the tumor and the better prognosis.     

ATP-sensitive K+ channels in the hypothalamus are essential for the maintenance of glucose homeostasis

Glucose-responsive (GR) neurons in the hypothalamus are thought to be critical in glucose homeostasis, but it is not known how they function in this context. Kir6.2 is the pore-forming subunit of K(ATP) channels in many cell types, including pancreatic beta-cells and heart. Here we show the complete absence of both functional ATP-sensitive K+ (K(ATP)) channels and glucose responsiveness in the neurons of the ventromedial hypothalamus (VMH) in Kir6.2-/- mice. Although pancreatic alpha-cells were functional in Kir6.2-/-, the mice exhibited a severe defect in glucagon secretion in response to systemic hypoglycemia. In addition, they showed a complete loss of glucagon secretion, together with reduced food intake in response to neuroglycopenia. Thus, our results demonstrate that KATP channels are important in glucose sensing in VMH GR neurons, and are essential for the maintenance of glucose homeostasis.     

Continuous measurement of fluid mobilization from ICF space following acute dehydration by dialyzation in dogs

The changes of the distribution of body fluid following acute isotonic dialyzation were studied from the results of continuous monitoring of extracellular fluid volume and physiological parameters. An indicator of extracellular space, 51Cr-EDTA, was injected into splenonephrectomized dogs. After the equilibrium of tracer dilution was attained, 10 ml/kg of plasma water was isotonically withdrawn by means of a dialyzer of hollow fiber type. The volumes of extracellular fluid (ECF) and plasma (PV) were continuously monitored for 80 min and plasma osmolality was measured at 10 min intervals. At the 50-60th min after the fluid modification, the reduction of PV was only 3.8 ml/kg and that of ECF was 4.2 ml/kg. The continuous profile of ECF change showed a significant mobilization of water from intracellular fluid (ICF) soon after the dialyzation. It is concluded that, in the state of hypovolemia, plasma fluid is replenished with the transvascular fluid absorption from interstitial space and that, concomitantly, the reduction of ISF is restituted with the fluid mobilization from ICF into extracellular space within the early stage of 1 hr. Good linear correlation was found between the amount of mobilized water from ICF and the increment of plasma osmolality. An increase in osmotic force was considered the mechanism which caused the fluid shift. These findings suggest that the change in plasma osmolality is a good predictor of mobilized volume from ICF in hypovolemia. The effects of inverse fluid modification, i.e., isotonic infusion, were also compared.     

Development of a 5' fluorogenic nuclease-based real-time PCR assay for quantitative detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

A 5' nuclease TaqMan PCR was developed for the quantitative detection of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The relative numbers of bacteria were measured by the comparative threshold cycle method. This simplified method is a way of obtaining the relative quantities of these organisms from specimens and of monitoring the effect of therapy.     

Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice

Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3beta and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD).     

Reduction of a vascular endothelial growth factor receptor, fetal liver kinase-1, by antisense oligonucleotides induces motor neuron death in rat spinal cord exposed to hypoxia

Vascular endothelial growth factor (VEGF) is reported to play a neuroprotective role through a VEGF receptor, fetal liver kinase-1 (Flk-1) in vitro. We investigated whether reduction of Flk-1 could induce motor neuron loss in rat spinal cord by inhibiting the expression of Flk-1 in rat spinal cord using antisense oligodeoxynucleotides (ODNs) against the Flk-1 receptor. Rat spinal cord was repetitively exposed to 12% hypoxia, and the change of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the mitogen-activated protein kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathway was examined. Intrathecal infusion of Flk-1 antisense ODNs for 7 days suppressed almost completely Flk-1 expression in the lumbar segment of the spinal cord and was followed by a hypoxic challenge with 12% oxygen for 1 h that was repeated for 7 more days. In the lumbar segment, we observed that reduced Flk-1 expression and hypoxic challenge for 7 days resulted in approximately 50% loss of motor neurons, in which the activation of Akt and ERK, that is, increased levels of phosphorylated-Akt and of phosphorylated-ERK by hypoxia, was markedly inhibited. In contrast, the reduction of Flk-1 expression alone did not induce motor neuron loss. These results suggest that VEGF exerts its protective effect on motor neurons against hypoxia-induced toxicity by the Flk-1 receptor through the PI3-K/Akt and the MEK/ERK signaling pathways.     

Studies on fine structure and location of lipids in quick-freeze replicas of atherosclerotic aorta of WHHL rabbits

Summary The fine structure of intracellular and extracellular lipids in the atherosclerotic aorta of Watanabe-heritable hyperlipidemic (WHHL) rabbits was demonstrated by a quick-freeze etching technique. Many lipid droplets, with and without a membrane, were observed in the foam cells. Membrane-free droplets were observed as onionlike structure with a concentric lamellar structure surrounded by 10 nm filaments. Droplets surrounded by a limited membrane probably correspond to lipid-laden lysosomes.In the extracellular connective tissue space, marked accumulation of lipids with a vesicular structure was seen among collagen fibers. The appearance of these lipids was similar to that of lipids in lysosomes of foam cells.     

Cataractogenesis in neonatal Sprague-Dawley rats by N-methyl-N-nitrosourea

Cataract was induced by a single intraperitoneal injection of 100 mg/kg N-methyl-N-nitrosourea (MNU) to 0-, 5-, 10-, 15-, or 20-day-old male and female Sprague-Dawley rats. In day 0, 5, 10, and 15 MNU-treated rats, mature cataracts were constantly seen 7, 14, 14, and 30 days after dosing, respectively. In the day 20 MNU-treated rats, only subcapsular cataract was seen 30 days after dosing. Therefore, the rats exposed to MNU at an earlier age caused cataract more rapidly and severely. In the day 0 MNU-treated rats, 7-methyldeoxyguanosine DNA adduct was detected in the lens epithelial nuclei 12 hours after MNU dosing, followed by apoptosis, which was confirmed by morphology, by TUNEL signals, and by DNA ladder and peaked 3 days after MNU dosing. In the apoptosis cascade, upregulation of Bax, downregulation of Bcl-2, and increased CPP32 protease (caspase-3) activity were seen 12 hours after MNU dosing. Therefore, the pathogenesis of MNU-induced cataract was associated with DNA adduct formation in the lens epithelial cell nuclei leading to apoptosis by upregulation of Bax protein, downmodulation of Bcl-2 protein, and activation of caspase-3.