PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth

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Assessment of the methods used to detect HER2-positive advanced extramammary Paget’s disease

The human epidermal growth factor receptor 2 (HER2) is recognized as an oncogene as well as a therapeutic target in various cancers. Certain patients with advanced extramammary Paget’s disease (EMPD) have also been reported to express HER2, which is therefore considered a therapeutic target for EMPD. However, an accurate methodology to determine HER2-positive EMPD has not been established. To assess the optimal methods for detection of HER2-positive EMPD, 73 EMPD samples were analyzed by immunohistochemical (IHC) staining, fluorescence in situ hybridization (FISH), and the HER2 testing algorithm for breast cancer of the American Society of Clinical Oncology/College of American Pathologists, which combined the results of IHC staining and FISH. The results showed discordance in the rate of positive IHC staining and FISH results. While 68.6% (24/35) of the metastatic samples showed equivocal or positive IHC staining, only 37.1% (13/35) were positive by FISH. To assess the accuracy of these methods, the degree of HER2 expression detected by each method was correlated with the staining profiles of activated downstream signaling pathways involving phosphorylated p44/42 MAPK (Thr202/Tyr204) (p-ERK1/2) and phosphorylated AKT (Ser473) (p-AKT). Among 16 lymph node metastasis samples, all HER2-positive samples as determined by the testing algorithm stained positively for both p-ERK1/2 and p-AKT. On the other hand, 10-14.3% of the samples determined by FISH or IHC showed negative staining for p-ERK1/2 and p-AKT. The results showed that combining the results of IHC and FISH according to the HER2 testing algorithm is a useful method for accurately evaluating HER2-positive EMPD.     

Direct activation of endothelial NO pathway by Ba2+ in canine coronary artery

1. We have reported that Ba²⁺ causes endothelium-dependent relaxation of canine coronary arteries through NO synthesis in Ca²⁺-free and depolarizing solution. To determine the cellular mechanisms by which the endothelium-dependent relaxation occurs, we used fura-2 fluorometry (F₃₅₀ and F₃₉₀; excitation wavelengths, 350 and 390 nm, respectively) and estimated the intracellular Ba²⁺ concentration in endothelial and vascular smooth muscle cells.
2. Ba²⁺ (10⁻³ M) increased the fura-2 ratio (F₃₅₀/F₃₉₀) recorded from a combined preparation of smooth muscle and endothelium (0.445±0.073, n=4) and contracted the arteries in the presence of 80 mM K⁺ (0.22±0.06 g, n=4).
3. Diltiazem (3×10⁻⁶ M) blocks Ba²⁺ entry into vascular smooth muscle cells via L-type Ca²⁺ channels. In this condition, Ba²⁺ increased the fura-2 ratio in endothelial cells (0.141±0.014, n=5) and relaxed the underlying smooth muscle (0.08±0.01 g, n=5) by a mechanism which was sensitive to 10⁻⁴ M N^G-methyl-L-arginine (L-NMMA).
4. Ba²⁺-induced relaxation was not attenuated with repeated application and was elicited even after endothelium-dependent relaxations in response to 10⁻⁶ M bradykinin were abolished due to tachyphylaxis. Neither 10⁻² M caffeine nor 10⁻⁶ M thapsigargin had effect upon Ba²⁺-induced relaxation.
5. To further rule out changes in intracellular Ca²⁺ as a mechanism of Ba²⁺-induced relaxation, fura-2 fluorescence was measured at the isosbestic wavelengths for Ca²⁺ (360 nm) and Ba²⁺ (370 nm) in endothelium-intact arteries. Ba²⁺ altered F₃₆₀, but not F₃₇₀, suggesting little or no contribution of intracellular Ca²⁺ to the phenomenon of Ba²⁺-induced relaxation.
6. These results suggest that the Ba²⁺-induced relaxation is due to its direct activation of endothelial NO synthesis without mobilization of intracellular Ca²⁺.

     

Nature or nurture? Steady‐state lymphocyte formation in adults does not recapitulate ontogeny

Summary:  Substantial progress has been made in determining developmental relationships between lymphocyte precursors and those corresponding to other blood cell lineages. Indeed, exploitation of RAG1/GFP knock-in mice has recently made it possible to chart the entire sequence of lymphocyte differentiation events in adult bone marrow and thymus. However, the differentiation pathways proposed for fetal life are very different from this model. We review many examples where the results of gene targeting experiments are substantially dependent on developmental age. In mice, adult patterns of gene expression and corresponding properties of lymphocyte precursors are not fully established until several weeks after birth, and the same might be true for humans. Furthermore, examples are cited where fetal hematopoietic cells did not efficiently acquire those properties when transplanted to an adult environment. There are several important implications of these findings. Cognizance of developmental age-related changes might resolve apparent conflicts in the literature. Hematopoietic stem cells and their lymphoid lineage progeny appear in waves, and a direct connection is yet to be established between fetal stem cells and ones that sustain adult blood cell formation. There is the possibility that adult stem cells derive from founders with an unknown origin.     

Laryngeal neuroma in multiple endocrine neoplasia type 2B

Multiple endocrine neoplasia (MEN) type 2 syndrome is an autosomal dominant inherited disease caused by mutations of the RET proto-oncogene, and is clinically divided into three phenotypes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. Although multiple mucosal neuromas are commonly observed in patients with MEN2B, there are only a few reports of laryngeal neuroma. We present here a rare case of laryngeal mucosal neuromas with MEN2B.     

Lubricin Contributes to Homeostasis of Articular Cartilage by Modulating Differentiation of Superficial Zone Cells

Lubricin encoded by the proteoglycan 4 (Prg4) gene is produced from superficial zone (SFZ) cells of articular cartilage and synoviocytes, which is indispensable for lubrication of joint surfaces. Loss-of-function of human and mouse Prg4 results in early-onset arthropathy accompanied by lost SFZ cells and hyperplastic synovium. Here, we focused on increases in the thickness of articular cartilage in Prg4-knockout joints and analyzed the underlying mechanisms. In the late stage of articular cartilage development, the articular cartilage was thickened at 2 to 4 weeks and the SFZ disappeared at 8 weeks in Prg4-knockout mice. Similar changes were observed in cultured Prg4-knockout femoral heads. Cell tracking showed that Prg4-knockout SFZ cells at 1 week of age expanded to deep layers after 1 week. In in vitro experiments, overexpression of Prg4 lacking a mucin-like domain suppressed differentiation of ATDC5 cells markedly, whereas pellets of Prg4-knockout SFZ cells showed enhanced differentiation. RNA sequencing identified matrix metalloproteinase 9 (Mmp9) as the top upregulated gene by Prg4 knockout. Mmp9 expressed in the SFZ was further induced in Prg4-knockout mice. The increased expression of Mmp9 by Prg4 knockout was canceled by IκB kinase (IKK) inhibitor treatment. Phosphorylation of Smad2 was also enhanced in Prg4-knockout cell pellets, which was canceled by the IKK inhibitor. Expression of Mmp9 and phosphorylated Smad2 during articular cartilage development was enhanced in Prg4-knockout joints. Lubricin contributes to homeostasis of articular cartilage by suppressing differentiation of SFZ cells, and the nuclear factor-kappa B-Mmp9-TGF-β pathway is probably responsible for the downstream action of lubricin. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Phosphatidylcholine (18:0/20:4), a potential biomarker to predict ethionamide-induced hepatic steatosis in rats

Ethionamide (ETH), a second-line drug for multidrug-resistant tuberculosis, is known to cause hepatic steatosis in rats and humans. To investigate predictive biomarkers for ETH-induced steatosis, we performed lipidomics analysis using plasma and liver samples collected from rats treated orally with ETH at 30 and 100 mg/kg for 14 days. The ETH-treated rats developed hepatic steatosis with Oil Red O staining-positive vacuolation in the centrilobular hepatocytes accompanied by increased hepatic contents of triglycerides (TG) and decreased plasma TG and total cholesterol levels. A multivariate analysis for lipid profiles revealed differences in each of the 35 lipid species in the plasma and liver between the control and the ETH-treated rats. Of those lipids, phosphatidylcholine (PC) (18:0/20:4) decreased dose-dependently in both the plasma and liver. Moreover, serum TG-rich very low-density lipoprotein (VLDL) levels, especially the large particle fraction of VLDL composed of PC containing arachidonic acid (20:4) involved in hepatic secretion of TG, were decreased dose-dependently. In conclusion, the decreased PC (18:0/20:4) in the liver, possibly leading to suppression of hepatic TG secretion, was considered to be involved in the pathogenesis of the ETH-induced hepatic steatosis. Therefore, plasma PC (18:0/20:4) levels are proposed as mechanism-related biomarkers for ETH-induced hepatic steatosis.     

Ventricular wall stress and silent myocardial damage are associated with pulse pressure in the general population

Pulse pressure (PP) is a risk factor for cardiovascular diseases and is associated with increased afterload and myocardial oxygen demand. Brain natriuretic peptide (BNP) and heart‐type fatty acid–binding protein (H‐FABP) are known as biomarkers indicating ventricular wall stress and silent myocardial damage. However, the association between PP and ventricular wall stress and silent myocardial damage in the general population is unclear. The authors enrolled 3504 patients who participated in a community‐based annual health check. Serum levels of BNP and H‐FABP were measured as markers of ventricular wall stress and silent myocardial damage. Patients were divided into four groups according to the quartiles of PP. Patients in the highest PP group showed higher serum BNP and H‐FABP levels than that of the other groups. Multivariate logistic analysis showed that high PP was independently associated with ventricular wall stress and silent myocardial damage on the basis of BNP and H‐FABP levels. Compared with systolic blood pressure, diastolic blood pressure, and mean blood pressure, PP was superior in predicting ventricular wall stress and silent myocardial damage evaluated according to BNP and H‐FABP levels, which was reflected by the receiver operating characteristic analysis. Screening of healthy patients revealed that high PP was related to high BNP and H‐FABP levels, suggesting that an asymptomatic general population with high PP may be exposed to ventricular wall stress and myocardial damage and might be susceptible to silent heart failure.     

Development of and Clinical Experience with a Simple Device for Performing Intraoperative Fluorescein Fluorescence Cerebral Angiography: Technical Notes

To perform intraoperative fluorescence angiography (FAG) under a microscope without an integrated FAG function with reasonable cost and sufficient quality for evaluation, we made a small and easy to use device for fluorescein FAG (FAG filter). We investigated the practical use of this FAG filter during aneurysm surgery, revascularization surgery, and brain tumor surgery. The FAG filter consists of two types of filters: an excitatory filter and a barrier filter. The excitatory filter excludes all wavelengths except for blue light and the barrier filter passes long waves except for blue light. By adding this FAG filter to a microscope without an integrated FAG function, light from the microscope illuminating the surgical field becomes blue, which is blocked by the barrier filter. We put the FAG filter on the objective lens of the operating microscope correctly and fluorescein sodium was injected intravenously or intra-arterially. Fluorescence (green light) from vessels in the surgical field and the dyed tumor were clearly observed through the microscope and recorded by a memory device. This method was easy and could be performed in a short time (about 10 seconds). Blood flow of small vessels deep in the surgical field could be observed. Blood flow stagnation could be evaluated. However, images from this method were inferior to those obtained by currently commercially available microscopes with an integrated FAG function. In brain tumor surgery, a stained tumor on the brain surface could be observed using this method. FAG could be performed with a microscope without an integrated FAG function easily with only this FAG filter.