Purification and Properties of Orotidine-5′-phosphate Pyrophosphorylase in Micrococcus glutamicus.

A coupled enzyme system of orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase has been purified approximately 30-fold from cell-free extract of Micrococcus glutamicus 534 Co–147 by means of acid treatment and fractionations with ammonium sulfate and ethanol addition, and properties of the enzyme system have been studied.

Optima of pH, temperature and substrate concentrations for the activity of the purified enzyme system have been investigated, and compared with those of the same enzyme system from dried brewer’s yeast. Furthermore, effects of various inhibitors on the enzyme activity have been examined and it has become evident that the enzyme system is completely inactivated by addition of chelating agent such as EDTA, and regenerated by further addition of magnesium ion.     

Polyethylene Glycol–Modified Papain Catalyzed Oligopeptide Synthesis from the Esters of l-Aspartic and l-Glutamic Acids in Benzene

Comparative studies were made of the polymerization of l-aspartic and l-glutamic acid dialkyl esters using polyethylene glycol–modified papain as a catalyst in phosphate buffer (pH 7.5) and in benzene. Changes in the substrate specificity of papain and in the composition of oligomerized products were observed. In the buffer, the diethyl and di-n-propyl esters of l-glutamic acid were sufficiently converted to high molecular weight oligomers with the accumulation of dimer and trimer, but l-aspartic acid esters were very poor substrates. In benzene, l-aspartic acid esters became more reactive than L-glutamic acid esters. In particular, from l-aspartic acid dimethyl ester the product, which was mainly composed of heptamer to decamer, was obtained in a 90% yield. The reaction in benzene required desalted substrates and a small amount of water to proceed extensively.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Impaired autophagy by soluble endoglin,under physiological hypoxia in early pregnant period,is involved in poor placentation in preeclampsia

In early pregnancy, trophoblasts and the fetus experience hypoxic and low-nutrient conditions; nevertheless, trophoblasts invade the uterine myometrium up to one third of its depth and migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. Here, we showed that autophagy, an intracellular bulk degradation system, occurred in extravillous trophoblast (EVT) cells under hypoxia in vitro and in vivo. An enhancement of autophagy was observed in EVTs in early placental tissues, which suffer from physiological hypoxia. The invasion and vascular remodeling under hypoxia were significantly reduced in autophagy-deficient EVT cells compared with wild-type EVT cells. Interestingly, soluble endoglin (sENG), which increased in sera in preeclamptic cases, suppressed EVT invasion by inhibiting autophagy. The sENG-inhibited EVT invasion was recovered by TGFB1 treatment in a dose-dependent manner. A high dose of sENG inhibited the vascular construction by EVT cells and human umbilical vein endothelial cells (HUVECs), meanwhile a low dose of sENG inhibited the replacement of HUVECs by EVT cells. A protein selectively degraded by autophagy, SQSTM1, accumulated in EVT cells in preeclamptic placental biopsy samples showing impaired autophagy. This is the first report showing that impaired autophagy in EVT contributes to the pathophysiology of preeclampsia.     

A second Warburg-like effect in cancer metabolism: The metabolic shift of glutamine-derived nitrogen

Carbon and nitrogen are essential elements for life. Glucose as a carbon source and glutamine as a nitrogen source are important nutrients for cell proliferation. About 100 years ago, it was discovered that cancer cells that have acquired unlimited proliferative capacity and undergone malignant evolution in their host manifest a cancer-specific remodeling of glucose metabolism (the Warburg effect). Only recently, however, was it shown that the metabolism of glutamine-derived nitrogen is substantially shifted from glutaminolysis to nucleotide biosynthesis during malignant progression of cancer—which might be referred to as a “second” Warburg effect. In this review, address the mechanism and relevance of this metabolic shift of glutamine-derived nitrogen in human cancer. We also examine the clinical potential of anticancer therapies that modulate the metabolic pathways of glutamine-derived nitrogen. This shift may be as important as the shift in carbon metabolism, which has long been known as the Warburg effect.     

Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33°C. A temperature shift to 37°C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.M. Matsuo and K. Koizumi contributed equally to this work. This study was supported in part by Grants-in-Aid for the 21st Century COE Program and for CLUSTER (Cooperative Link of Unique Science and Technology for Economy Revitalization) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.     

Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis.     

Proton-conductivity assay of plugged and unplugged MotA/B proton channel by cytoplasmic pHluorin expressed in Salmonella

MotA and MotB form the proton-channel complex of the proton-driven bacterial flagellar motor. A plug segment of Escherichia coli MotB suppresses proton leakage through the MotA/B complex when it is not assembled into the motor. Using a ratiometric pH indicator protein, pHluorin, we show that the proton-conductivity of a Salmonella MotA/B complex not incorporated into the motor is two orders of magnitude lower than that of a complex that is incorporated and activated. This leakage is, however, significant enough to change the cytoplasmic pH to a level at which the chemotaxis signal transduction system responds.     

Heritability of male mandible length in the stag beetle Cyclommatus metallifer

Numerous coleopteran species express male‐specific “weapon traits” that often show size variations among males, even within a single population. Many empirical studies have demonstrated that environmental conditions during development affect absolute weapon size. However, relatively few studies in horned beetles support the hypothesis that the relationship between weapon size and body size, also referred to as a “scaling relationship” or “static allometry”, is largely determined by genetic factors. In this study, the heritability of absolute mandible length and static allometry between mandible length and body size were estimated in the stag beetle Cyclommatus metallifer. While no significant heritable variation was observed in absolute mandible length, high heritability (h² = 0.57 ± 0.25) was detected in the static allometry between mandible length and body size. This is the first report on the genetic effect on male mandible size in Lucanidae, suggesting that absolute mandible size is largely determined by environmental conditions while the static allometry between weapon size and body size is primarily determined by genetic factors.