Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases

We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to l-proline and l-pipecolinic acid, we found that 3,4-dehydro-l-proline, l-azetidine-2-carboxylic acid, cis-3-hydroxy-l-proline, and l-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-l-proline, cis-3-hydroxy-l-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-l-proline.     

Investigations on the Influence of a Low Casein Diet on the Free Fatty Acids in Liver Homogenate by a Method with Some Devices in Purification Procedure Using KOH

Previously, some changes were noticed in energy metabolism of rats fed a low casein diet. In connection with these phenomena, influence of a low casein diet on the composition and amounts of free fatty acids in liver homogenate after autolyzing for a few hours was investigated. For the measurement of free fatty acids, they were purified by a method with some devices in purification procedure using KOH. It was found that amounts of free fatty acids in liver homogenate after autolyzing for a few hours were lower in rats fed a low casein diet.     

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.     

Age- and Sex-Dependency of Laser Speckle Flowgraphy Measurements of Optic Nerve Vessel Microcirculation

Purpose

To investigate the relationship between various characteristics of a normal population and laser speckle flowgraphy (LSFG) measurements of mean blur rate (MBR) in the optic nerve head (ONH).

Methods

A total of 189 eyes of 189 normal subjects (93 male, 96 female, mean age 45 ± 14 years old, age range: 20–72) without any history of hypertension, hyperlipidemia or diabetes were enrolled. ONH microcirculation was measured with LSFG and overall MBR (MA), vessel-area MBR (MV), and tissue-area MBR (MT) were derived from these measurements. The statistical association of these measurements with characteristics such as sex, age, intraocular pressure (IOP) and systolic blood pressure (SBP) was then determined.

Results

There was a trend towards decreased IOP and MV and increased SBP with age (P = 0.002, P = 0.035, and P = 0.006, respectively). Furthermore, IOP, MV and SBP were correlated with age (r = -0.23, P = 0.011; r = -0.24, P < 0.001; and r = 0.30, P < 0.001, respectively). Separate multiple regression analyses of independent contributing factors revealed that sex and IOP contributed to MA (P < 0.001 and P = 0.002, respectively), sex, IOP, and age contributed to MV (P < 0.001, P = 0.003, and P = 0.024, respectively), while only IOP contributed to MT (P = 0.003).

Conclusion

In a normal population, MBR was affected by IOP in both the large vessel and capillary areas of the ONH, but not by SBP. MV was also affected by age and sex, while MT was stable independent of age or sex.     

Age at Transition from Pediatric to Adult Care Has No Relationship with Mortality for Childhood-Onset Type 1 Diabetes in Japan: Diabetes Epidemiology Research International (DERI) Mortality Study

Objective

To follow up Japanese patients with type 1 diabetes for a maximum of 40 years to examine when they transitioned from pediatric care to adult care and to explore whether the attending physician, i.e., pediatrician or internist, was associated with prognosis.

Methods

Participants consisted of 1,299 patients who had been diagnosed as having type 1 diabetes at less than 15 years old between 1965 and 1979 identified through two nationwide surveys. Patients were classified as having received either pediatric care or adult care at the age of 15 and 30, and were compared for differences in mortality associated with the attending physician.

Results

The attending physicians were confirmed for a total of 1,093 patients at the age of 15. Of these patients, 43.8% and 40.3% received pediatric care and adult care, respectively. Of the 569 patients receiving pediatric care, 74.2%, 56.6%, 53.4%, and 51.3% continued with pediatric care at 20, 30, 40, and 50 years old, respectively. The attending physicians (pediatrician or internist) at the age of 15 and 30 had no significant impact on their survival (P = 0. 892, 0.411, respectively).

Conclusions

More than half of the patients who had received pediatric care at the age of 15 continued to receive pediatric care even after the age of 30, suggesting that their transition was far from smooth, while the attending physician at the age of both 15 and 30 was not a prognostic factor for mortality. Thus, the timing for transition to adult care in these patients has no relationship with mortality in Japan.     

Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1) Gene in Primary Breast Cancer

Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1) gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC) with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP) in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004). Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007). Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.     

Angiotensin II,as well as 5-hydroxytriptamine,is a potent vasospasm inducer of saphenous vein graft for coronary artery bypass grafting in patients with diabetes mellitus

Diabetes mellitus (DM) is an important risk factor for adverse outcomes of coronary artery bypass grafting. The bypass grafts harvested from patients with DM tend to go into spasm after their implantation into the coronary circulation. To clarify the contribution of 5-hydroxytriptamine (5-HT) and angiotensin II (AngII) in the bypass graft spasm, we examined the contractile reactivity to 5-HT or AngII of isolated human endothelium-denuded saphenous vein (SV) harvested from DM and non-DM patients. The 5-HT-induced constriction of the SV was significantly augmented in the DM group than in the non-DM group, which is similar to our previous report. AngII-induced constriction of the SV was also significantly augmented in the DM group than the non-DM group. Especially in the non-DM group, the AngII-induced maximal vasoconstriction was markedly lower than the 5-HT-induced one. Meanwhile, the increasing rates of AngII-induced vasoconstriction in the DM group to the non-DM group were significantly greater than those of 5-HT-induced vasoconstriction. These results indicate that 5-HT is a potent inducer of SV graft spasm in both DM and non-DM patients, while AngII is a potent inducer of SV graft spasm only in patients with DM. Furthermore, the protein level of AngII AT₁ receptor (AT₁R), but not the protein level of 5-HT_2A receptor, in the membrane fraction of the SV smooth muscle cells of DM patients was significantly increased as compared with that of the non-DM patients. These results suggest that the mechanism for hyperreactivity to AngII in the SV from DM patients is due to, at least in part, the increase in the amount of AT₁R on membrane of the SV smooth muscle cells.     

Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 μM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.