Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe

We demonstrate a novel rapid direct detection method for immunohistochemistry, using a bioluminescent probe. An anti‐CEA antibody‐fused far‐red bioluminescent protein can monitor the accumulation of this type of probe in tumour tissues. The bimodal spectrum (λ_max = 460 and 675 nm) of this bioluminescent probe is extremely stable under different conditions of pH and ion concentration. The sensitivity of our bioluminescent labelling was at the same level of enzymatic labelling, e.g. peroxidase, as an indirect system. Our novel technique is simple and can shorten the pretreatment time of paraffin sections to around 30 min. The utility of our bioluminescent labelling covers all imaging in vitro, in vivo and ex vivo, suggesting that our antibody‐fused bioluminescent probe has the potential to detect tumour antigens with a high sensitivity in routine immune histological examinations. Copyright © 2012 John Wiley & Sons, Ltd.     

Circadian disruption accelerates tumor growth and angio/stromagenesis through a Wnt signaling pathway

Epidemiologic studies show a high incidence of cancer in shift workers, suggesting a possible relationship between circadian rhythms and tumorigenesis. However, the precise molecular mechanism played by circadian rhythms in tumor progression is not known. To identify the possible mechanisms underlying tumor progression related to circadian rhythms, we set up nude mouse xenograft models. HeLa cells were injected in nude mice and nude mice were moved to two different cases, one case is exposed to a 24-hour light cycle (L/L), the other is a more "normal" 12-hour light/dark cycle (L/D). We found a significant increase in tumor volume in the L/L group compared with the L/D group. In addition, tumor microvessels and stroma were strongly increased in L/L mice. Although there was a hypervascularization in L/L tumors, there was no associated increase in the production of vascular endothelial cell growth factor (VEGF). DNA microarray analysis showed enhanced expression of WNT10A, and our subsequent study revealed that WNT10A stimulates the growth of both microvascular endothelial cells and fibroblasts in tumors from light-stressed mice, along with marked increases in angio/stromagenesis. Only the tumor stroma stained positive for WNT10A and WNT10A is also highly expressed in keloid dermal fibroblasts but not in normal dermal fibroblasts indicated that WNT10A may be a novel angio/stromagenic growth factor. These findings suggest that circadian disruption induces the progression of malignant tumors via a Wnt signaling pathway.     

Switch of histamine receptor expression from H2 to H1 during differentiation of monocytes into macrophages

It is known that histamine suppresses gene expression and synthesis of tumor necrosis factor alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in human peripheral blood mononuclear monocytes (HPM) or alveolar macrophages via histamine H2 receptors. We investigated the effect of histamine and differentiation in macrophages on the expression and secretion of TNF-alpha, TNF-alpha-converting enzyme (TACE), and histamine H1 and H2 receptors by use of a leukemia cell line, U937, and HPM. Differentiation of U937 and HPM cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the H1 receptor expression and rather suppressed the H2 receptor, resulting in up-regulation of the histamine-induced expression and secretion of TNF-alpha, modulated via TACE. Therefore, histamine failed to inhibit up-regulated expression of TNF-alpha induced by LPS in macrophages. The switch from H2 to H1 receptors during differentiation in the monocyte/macrophage lineage could participate in the pathogenic processes of atherosclerosis and inflammatory reactions in the arterial wall.     

Genetic manipulation of the γ‐aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ‐aminobutyric acid transaminase (GABA‐T) lead to sustained and high levels of GABA accumulation in rice kernels

Gamma‐aminobutyric acid (GABA) is a non‐protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA‐transaminase, GABA‐T), we attempted seed‐specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB‐1) or rice embryo globulin promoters (REG) and GABA‐T‐based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2–100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA‐T expression was relatively weak. In these two lines both with two T‐DNA copies, their starch, amylose, and protein levels were slightly lower than non‐transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75–350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.     

PAX4 mutation (R121W) as a prodiabetic variant in Okinawans

We previously reported that a missense mutation at codon 121 (CGG(Arg) to TGG(Trp), R121W) of PAX4 may be associated with the onset of type 2 diabetes in Japanese. In this study, we determined the frequency of the R121W mutation of PAX4 and characterized the prodiabetic phenotype in a population-based study. Healthy 372 residents participated in annual health check-ups in Nishihara (Okinawa, Japan) and unrelated 193 type 2 diabetic patients from the outpatient clinic of Ryukyu University Hospital were enrolled. Diagnosis of diabetes was based on the 1997 American Diabetes Association criteria. The R121W mutation in PAX4 was genotyped by PCR-RFLP analysis. In healthy residents, R121W mutation was detected in 12 of 372 residents (3.1%). The prevalence of newly diagnosed type 3 diabetes (25% vs. 5%, p=0.004) and HbA(1c) (5.6+/-1.9 vs. 5.1+/-0.7, p=0.026) was higher in the variants than in the wild-types. The odds ratio of diabetes in the R121W variants was 5.98 with 95% confidence interval from 1.50 to 23.9. The R121W mutation was observed in 12 of the 193 type 2 diabetic patients (6.2%). Onset-ages of diabetes were earlier (37+/-10 vs. 47+/-13 years, p=0.010) and the rate of insulin user was two times higher (83% vs. 41%, p=0.005) in the variants. The R121W mutation in PAX4 is a predisposing factor for the development of type 2 diabetes in Okinawans.     

Evidence for a deficient pancreatic beta-cell response in a rat model of hyperthyroidism

To clarify mechanism behind the abnormal glucose tolerance, observed in hyperthyroidism, we studied genomic and nongenomic effects of thyroid hormone on insulin secretion using a rat model of hyperthyroidism. Male Sprague-Dawley rats were intraperitoneally injected with vehicle, low (100 microg/kg) or high dose (600 microg/kg) of thyroxin (T(4)) for 2 weeks. Rats treated with high dose, but not low dose, of T(4), showed an increase in serum T(3) levels, and a decrease in body weight as compared to control rats. In rats treated with either dose of T(4), fasting blood glucose levels were increased, but serum insulin levels were similar to those of controls. After an oral glucose load, blood glucose levels were increased in rats treated with high dose, but not low dose, of T(4). Serum insulin levels after the oral glucose load were decreased in rats treated with either dose of T(4). After an intravenous glucose load, blood glucose levels were comparable among groups, but serum insulin levels tended to be low in T(4)-treated rats. Steady-state blood glucose levels were comparable among groups. The insulin secretory responses to high glucose (20mM) or arginine (10mM) of the isolated pancreas was decreased in rats treated with high dose, but not low dose, of T(4). Mean insulin secretory response to glucose and arginine were decreased by 40.1% and by 60.4% in high-dose-T(4)-treated rats. Addition of T(3) in the perfusion medium decreased glucose-induced insulin release. Ratios of proinsulin mRNA levels to beta-actin mRNA were decreased in the islets of T(4)-treated rats (0.45 +/- 0.07 vs control 0.61 +/- 0.03, p < 0.05). Levels of TR (thyroid hormone nuclear receptor) alpha1 + cErb Aalpha2 mRNA, but not TRbeta1, were decreased in the pancreatic islets of T(4)-treated rats. Calculated islet area was increased, but the number of beta-cells determined immunohistochemically was not increased in T(4)-treated rats, nor the volume density of insulin positive islets. We concluded that a deficient pancreatic beta-cell response to glucose, rather than insulin resistance, was responsible for abnormal glucose tolerance in this model of hyperthyroidism. Thyroid hormone causes a decrease in glucose-induced insulin secretion. We observed nongenomic and genomic effects of thyroid hormone on glucose-induced insulin secretion.     

R3: Invasive salivary duct carcinoma ex pleomorphic adenoma of the parotid gland: a teaching case giving rise to the genuine diagnostic difficulty on an inadequate cytology specimen

ABSTRACT: A history of a recent rapid increase in long-standing swelling mass was presented in the right parotid gland of an 85-year-old male. The inadequate cytologic specimens contained few small clusters of three-dimensional malignant epithelial cells having hyperchromatic pleomorphic nuclei and prominent nucleoli, adjacent to a cluster of benign monomorphic myoepithelial cells. We first interpreted it merely as an adenocarcinoma, not otherwise specified. A radical parotidectomy was performed, and gross examination revealed an encapsulated and firm tumor lesion, looking grayish-blue to yellowish-white, focally associated with extracapsular invasion. On microscopic examination, the tumor was predominantly composed of a proliferation of highly atypical epithelial cells having abundant eosinophilic cytoplasm, often arranged in a Roman-bridge appearance with foci of comedo necrosis, alternating with extensive infiltration to adjacent stroma in a trabecular or alveolar fashion with severe vessel permeation. Within the background of pleomorphic adenoma, the carcinoma cells sometimes replaced ductal luminal cells while retaining an intact-like myoepithelial layer. Therefore, we finally made a diagnosis of invasive salivary duct carcinoma ex pleomorphic adenoma. We should be aware that owing to its characteristic features, cytopathologists might be able to determine correct diagnosis, based on multiple and adequate samplings. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2126158270695815.