Production of prostacyclin-like substance in stroke-prone and stroke-resistant spontaneously hypertensive rats

Activities of aortae to produce prostaglandin (PG) I₂-like substance in stroke-prone spontaneously hypertensive rats (SHRSP), stroke-resistant SHR (SHRSR) and normotensive control rats from the Wistar-Kyoto (WK) colony were compared. PGI₂-like substance was produced by the incubation of the aortic ring in pH 9.0 borate-buffered saline and the amount produced was estimated by comparison of its anti-aggregatory activity with that produced by known amounts of the sodium salt of synthetic PGI₂. Before the development of stroke, amounts of this substance generated in SHRSP and SHRSR were significantly higher than those in WK rats (p<0.01 and p<0.02, respectively). Remarkably reduced capacity to generate PGI₂-like substance was observed in some SHRSP after the development of stroke.     

Impact of Histone H4 Lysine 20 Methylation on 53BP1 Responses to Chromosomal Double Strand Breaks

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.     

Analysis of Factors That Determine Weight Gain during Smoking Cessation Therapy

Cigarette smokers are generally known to gain weight after quitting smoking, and such weight gain is thought to contribute to the worsening of glucose tolerance. While smoking cessation therapy such as nicotine replacement is useful to minimize post-cessation weight gain, substantial gain occurs even during the therapy. The purpose of the present study was to identify factors associated with weight gain during smoking cessation therapy. We evaluated 186 patients(132 males and 54 females)who visited our outpatient clinic for smoking cessation, and successfully achieved smoking abstinence. We performed gender-adjusted regression analysis for the rate of BMI increase from the beginning of cessation to 3 months after initiation. Furthermore, we performed multivariate analysis to investigate factors that determine the BMI increase after smoking cessation. The mean BMI significantly (p<0.0001) increased from 23.5±3.6 kg/m² at the initial consultation to 23.9±3.8 kg/m² at 3 months after the start of therapy. There was no significant difference in the extent of BMI increase between nicotine patch and varenicline therapy groups. Factors significantly correlated with the %BMI increase at 3 months after the start of therapy were triglyceride (p = 0.0006, βa = 0.260), high-density lipoprotein cholesterol (p = 0.0386, βa = −0.168), daily cigarette consumption (p = 0.0385, βa = 0.154), and the Fagerström Test for Nicotine Dependence (FTND) score (p = 0.0060, βa = 0.203). Stepwise multivariate analysis demonstrated that triglyceride and the FTND score were the factors determining the post-cessation BMI increase and that the FTND score was the strongest one. The present study demonstrated that smokers with a high FTND score are more likely to gain weight during smoking cessation therapy. Thus, smokers with a high nicotine dependency may require intervention against weight gain in the cessation clinic.     

Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes

Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the bphA1 gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of bphA1 of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400, a number of evolved Bph Dox enzymes were created. Among them, an Escherichia coli clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.     

Distinct characteristics of circulating vascular endothelial growth factor-a and C levels in human subjects

The mechanisms that lead from obesity to atherosclerotic disease are not fully understood. Obesity involves angiogenesis in which vascular endothelial growth factor-A (VEGF-A) plays a key role. On the other hand, vascular endothelial growth factor-C (VEGF-C) plays a pivotal role in lymphangiogenesis. Circulating levels of VEGF-A and VEGF-C are elevated in sera from obese subjects. However, relationships of VEGF-C with atherosclerotic risk factors and atherosclerosis are unknown. We determined circulating levels of VEGF-A and VEGF-C in 423 consecutive subjects not receiving any drugs at the Health Evaluation Center. After adjusting for age and gender, VEGF-A levels were significantly and more strongly correlated with the body mass index (BMI) and waist circumference than VEGF-C. Conversely, VEGF-C levels were significantly and more closely correlated with metabolic (e.g., fasting plasma glucose, hemoglobin A1c, immunoreactive insulin, and the homeostasis model assessment of insulin resistance) and lipid parameters (e.g., triglycerides, total cholesterol (TC), low-density-lipoprotein cholesterol (LDL-C), and non-high-density-lipoprotein cholesterol (non-HDL-C)) than VEGF-A. Stepwise regression analyses revealed that independent determinants of VEGF-A were the BMI and age, whereas strong independent determinants of VEGF-C were age, triglycerides, and non-HDL-C. In apolipoprotein E-deficient mice fed a high-fat-diet (HFD) or normal chow (NC) for 16 weeks, levels of VEGF-A were not significantly different between the two groups. However, levels of VEGF-C were significantly higher in HFD mice with advanced atherosclerosis and marked hypercholesterolemia than NC mice. Furthermore, immunohistochemistry revealed that the expression of VEGF-C in atheromatous plaque of the aortic sinus was significantly intensified by feeding HFD compared to NC, while that of VEGF-A was not. In conclusion, these findings demonstrate that VEGF-C, rather than VEGF-A, is closely related to dyslipidemia and atherosclerosis.     

Release of RASSF1C from the nucleus by Daxx degradation links DNA damage and SAPK/JNK activation

Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) responds to a variety of stress stimuli and controls cell fates such as cell cycle entrance, apoptosis and senescence. Stimuli such as ultraviolet irradiation and chemical reagents that damage genomic DNA induce the activation of the SAPK/JNK signaling pathway. However, it is unclear how the signal arising in the nucleus owing to DNA damage is transmitted to SAPK/JNK in the cytoplasm. Here, we report that the nuclear components Daxx and Ras-association domain family 1C (RASSF1C) link DNA damage to SAPK/JNK activation in HeLa cells. In response to DNA damage, Daxx localized in promyelocytic leukaemia-nuclear bodies (PML-NBs) undergoes ubiquitination and degradation. RASSF1C, a tumor suppressor and newly identified binding partner of Daxx, is constitutively anchored by Daxx in PML-NBs but is released from the nucleus when Daxx is degraded. This released RASSF1C translocates to cytoplasmic microtubules and participates in the activation of SAPK/JNK. Our data define a novel mechanism by which the Daxx-RASSF1C complex in PML-NBs couples nuclear DNA damage to the cytoplasmic SAPK/JNK signaling pathway.     

Distinct DNA methylation activity of Dnmt3a and Dnmt3b towards naked and nucleosomal DNA

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. De novo type DNA methyltransferases Dnmt3a and Dnmt3b are responsible for creating DNA methylation patterns during embryogenesis and in germ cells. Although their in vitro DNA methylation properties are similar, Dnmt3a and Dnmt3b methylate different genomic DNA regions in vivo. In the present study, we have examined the DNA methylation activity of Dnmt3a and Dnmt3b towards nucleosomes reconstituted from recombinant histones and DNAs, and compared it to that of the corresponding naked DNAs. Dnmt3a showed higher DNA methylation activity than Dnmt3b towards naked DNA and the naked part of nucleosomal DNA. On the other hand, Dnmt3a scarcely methylated the DNA within the nucleosome core region, while Dnmt3b significantly did, although the activity was low. We propose that the preferential DNA methylation activity of Dnmt3a towards the naked part of nucleosomal DNA and the significant methylation activity of Dnmt3b towards the nucleosome core region contribute to their distinct methylation of genomic DNA in vivo.     

The design and synthesis of a new tumor-selective fluoropyrimidine carbamate, capecitabine

To identify an orally available fluoropyrimidine having efficacy and safety profiles greatly improved over those of parenteral 5-fluorouracil (5-FU: 1), we designed a 5-FU prodrug that would pass intact through the intestinal mucisa and be sequentially converted to 5-FU by enzymes that are highly expressed in the human liver and then in tumors. Among various N4-substituted 5'-deoxy-5-fluorocytidine derivatives, a series of N4-alkoxycarbonyl derivatives were hydrolyzed to 5'-deoxy-5-fluorocytidine (5'-DFCR: 8) specifically by carboxylesterase, which exists preferentially in the liver in humans and monkeys. Particularly, derivatives having an N4-alkoxylcarbonyl moiety with a C4-C6 alkyl chain were the most susceptible to the human carboxylesterase. Those were then converted to 5'-deoxy-5-fluorouridine (5'-DFUR: 4) by cytidine deaminase highly expressed in the liver and solid tumors and finally to 5-FU by thymidine phosphorylase (dThdPase) preferentially located in tumors. When administered orally to monkeys, a derivative having the N4-alkoxylcarbonyl moiety with a C5 alkyl chain (capecitabine: 6) The highest AUC and Cmax for plasma 5'-DFUR. In tests with various human cancer xenograft models, capecitabine was more efficacious at wider dose ranges than either 5-FU or 5'-DFUR and was significantly less toxic to the intestinal tract than the others in monkeys.