A strain of Alcaligenes faecalis T I , which was isolated from activated sludge, excreted an extracellular poly(3-hydroxybutyrate) depolymerase as it grew in a medium containing poly(3-hydroxybutyrate) as the sole carbon source. The molecular weight of the enzyme, purified from the culture medium to electrophoretic homogeneity, was 48 000 as determined by Sephadex G-I00 filtration, and 50000 by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The pH optimum for the enzyme reaction was 7.5. The purified enzyme depolymerized poly(3-hydroxybutyrate) purified from Zoogloea ramigeva 1-1 6-M, but did not attack the bacterial native poly(3-hydroxybutyrate)-containing granules. K, values were 13.3 pg/ml (= 0.78 pM, based on an estimated average molecular weight of 17000) for poly(3-hydroxybutyrate) and 5.4mM for the trimeric ester of I I ( --)-3-hydroxybutyric acid. Analysis of hydrolytic products of poly(3-hydroxybutyrate), several oligomeric esters of I )( --)-3-hydroxybutyric acid, and the methyl ester of the trimeric ester indicated that the enzyme hydrolyzed these substrates from the free hydroxyl terminus, releasing 1>(-)-3-hydroxybutyrate dimer units one at a time.Poly(3-hydroxybutyrate) is a unique intracellular reserve of organic carbon and/or chemical energy found in a wide variety of microorganisms [I, 21. In contrast to the fairly extensive studies on the biosynthesis of poly(3-hydroxybutyrate) [3-91, relatively little is known about the mechanism of intracellular degradation of poly(3-hydroxybutyrate) owing to its complexity, e.g. the dependence of the depolymerase activity on the structural integrity of poly(3-hydroxybutyrate)-containing granules and on the presence of a thermostable activator and a protein inhibitor [3][4][5] 101. On the other hand, some bacteria secrete extracellular poly(3-hydroxybutyrate) depolymerase which degrades purified poly(3-hydroxybutyrate) [I 1 -141 devoid of a granular membrane component [15].Recently we isolated a poly(3-hydroxybutyrate)-depolymerizing bacterium, Alcaligenes faecalis T which grew on a poly(3-hydroxybutyrate)-supplemented medium. It seems important to investigate the mechanisms of enzymatic depolymerization of the extremely hydrophobic poly(3-hydroxybutyrate) molecule. In this communication we describe the purification and some of the properties of poly(3-hydroxybutyrate) depolymerase from A . faecalis T,. EXPERIMENTAL PROCEDURES Poly(3-hydroxybutyrate)-Degrading BacteriumActivated sludge, obtained from the Toba sewagetreatment plant (Kyoto, Japan), was incubated in a mediumAbh~eviutions. Dimer, n(-)-3-[i~(-)-.l-hydroxybutyryloxy]butyric acid; trimer, trimeric ester of 1>(-)-3-hydroxybutyric acid; tetramer, tetrameric ester of 11(-)-3-hydrowybutyric acid; pentamer, pentameric ester of n(-)-3-hydroxybutyric acid; TEAE-cellulose, triethylaminoethyl cellulose.Enzymes.

Purpose: Colorectal carcinogenesis is thought to be related to abdominal obesity and insulin resistance. To investigate whether visceral fat accumulation contributes to colorectal carcinogenesis, we examined its accumulation and the levels of the adipose tissue^derived hormone adiponectin in Japanese patients with colorectal adenoma. Experimental Design: Fifty-one consecutive Japanese patients ages z40 years and with colorectal adenoma were subjected to measurement of visceral fat area by computed tomography scanning and plasma adiponectin concentration. The patients also underwent the 75-g oral glucose tolerance test. Insulin resistance was calculated by the homeostasis metabolic assessment (HOMA-IR) method. The controls were 52 Japanese subjects ages z40 years and without colorectal polyp. Cigarette smokers and subjects who consumed alcohol (z30 g ethanol/d) were excluded. Results: The patients with colorectal adenoma showed significantly more visceral fat area and significantly less plasma adiponectin concentration in comparison with the controls [odds ratio (OR), 2.19; 95% confidence interval (95% CI), 1.47-3.28; P < 0.001 and OR, 0.24; 95% CI, 0.14-0.41; P < 0.001, respectively] by logistic regression analysis. HOMA-IR index was also associated with colorectal adenoma (OR 2.60; 95% CI, 1.20-5.64; P = 0.040). Visceral fat area and adiponectin were associated with adenoma number (1, 2, z 3), the size of the largest adenoma (<10 and z10 mm), and adenoma histology (tubular and tubulovillous/villous). Conclusions: These results suggest an association of visceral fat accumulation and decreased plasma adiponectin concentration with colorectal adenoma in Japanese patients. This study may offer a new insight to understanding the relationship of colorectal carcinogenesis with abdominal obesity and insulin resistance.

BackgroundThe sodium-glucose co-transporter-2 (SGLT-2) inhibitors have been reported to increase both low-density lipoprotein (LDL) and high-density lipoprotein (HDL)-cholesterol (C). This study aimed to determine how SGLT-2 inhibitors affect LDL and HDL-C subspecies.MethodsThis single center, open-label, randomized, prospective study included 80 patients with type 2 diabetes taking prescribed oral hypoglycemic agents. Patients were allocated to receive dapagliflozin (n = 40) or sitagliptin (n = 40) as add-on treatment. Fasting blood samples were collected before and 12 weeks after this intervention. Small dense (sd) LDL-C, large buoyant (lb) LDL-C, HDL2-C, and HDL3-C levels were determined using our established homogeneous assays. Statistical comparison of blood parameters before and after treatment was performed using the paired t test.ResultsDapagliflozin and sitagliptin comparably decreased HbA1c (0.75 and 0.63%, respectively). Dapagliflozin significantly decreased body weight, systolic blood pressure, plasma triglycerides and liver transaminases, and increased adiponectin; sitagliptin did not alter these measurements. LDL-C and apolipoprotein (apo) B were not significantly changed by dapagliflozin, whereas HDL-C and apo AI were increased. Dapagliflozin did not alter concentrations of LDL-C, but sd LDL-C decreased by 20% and lb LDL-C increased by 18%. Marked elevation in lb LDL-C (53%) was observed in individuals (n = 20) whose LDL-C was elevated by dapagliflozin. However, sd LDL-C remained suppressed (20%). Dapagliflozin increased HDL2-C by 18% without affecting HDL3-C. Sitagliptin did not alter plasma lipids or lipoprotein subspecies.ConclusionsA SGLT-2 inhibitor, dapagliflozin suppresses potent atherogenic sd LDL-C and increased HDL2-C, a favorable cardiometabolic marker. Although LDL-C levels are elevated by treatment with dapagliflozin, this was due to increased concentrations of the less atherogenic lb LDL-C. However, these findings were not observed after treatment with dipeptidyl peptidase-4 inhibitor, sitagliptin. Trial registration UMIN Clinical Trials Registry (UMIN000020984)Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-016-0491-5) contains supplementary material, which is available to authorized users.

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The extracellular poly(3-hydroxybutyrate) depolymerase gene from Akaligenes faecalis Tl was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis Ti genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. (22), modified by the addition of achromopeptidase (TBL-1; 1.5 mg/ml) (12) to the lysozyme-EDTA solution to lyse the organism. Plasmid DNA was isolated from a chloramphenicol-amplified culture (18) of E. coli by the method of Bimboim and Doly (1) and then purified by gel filtration. A. faecalis Ti DNA was partially digested with Sau3AI, and fragments of 4 to 9 kilobase pairs (kbp) in size were isolated from the agarose gel by the glass powder method (28). The size-fractionated DNAs were ligated into BamHI-digested and alkaline phosphatase-treated pUC8, using T4 ligase. E. coli DH1 was transformed with recombinant plasmid DNA by the calcium chloride method of Mandel and Higa (17), and ampicillinresistant (Apr) transformants were selected and immunologically screened (9) with anti-PHB depolymerase immunoglobulin G raised in a rabbit.Restriction mapping and subcloning. Mapping of restriction sites was performed by the standard procedure (18). Deletion derivatives were constructed by digesting plasmids with a single restriction enzyme, followed by ligation of the products. Subcloning was performed by isolating DNA fragments from 0.8% agarose gels by electroelution onto DEAE-paper (6). These fragments were then ligated into pUC8 that had been cut with appropriate restriction enzymes. pUC8 with subcloned DNA fragments was introduced into the E. coli JM103 recipient by transformation, and white colonies containing recombinant plasmids were 184 JOURNAL

Aims We conducted a national survey to clarify the characteristics and clinical course of type 1 diabetes related to antiprogrammed cell death-1 therapy. Methods We analyzed the detailed data of 22 patients that were collected using a Japan Diabetes Society survey and a literature database search. Results Among the 22 patients, 11 (50.0%) met the criteria for fulminant type 1 diabetes and 11 (50.0%) met the criteria for acute-onset type 1 diabetes. The average patient age was 63 years. The mean duration between the date of the first anti-PD-1 antibody injection and development of type 1 diabetes was 155 days and ranged from 13 to 504 days. Flu-like symptoms, abdominal symptoms, and drowsiness were observed in 27.8, 31.6, and 16.7% patients, respectively. Mean ± standard deviation or median (first quartile-third quartile) glucose levels, HbA1c levels, urinary C-peptide immunoreactivity levels, and fasting serum C-peptide immunoreactivity levels were 617 ± 248 mg/dl, 8.1 ± 1.3%, 4.1 (1.4-9.4) μg/day, and 0.46 (0.20-0.70) ng/ml, respectively. Seventeen of 20 patients (85.0%) developed ketosis, and 7 of 18 patients (38.9%) developed diabetic ketoacidosis. Ten of 19 patients (52.6%) showed at least one elevated pancreatic enzyme level at the onset and two of seven patients showed this elevation before diabetes onset. Only one of 21 patients was anti-glutamic acid decarboxylase antibody positive. Conclusions Anti-programmed cell death-1 antibody-related type 1 diabetes varies from typical fulminant type 1 diabetes to acute-onset type 1 diabetes. However, diabetic ketoacidosis was frequently observed at the onset of diabetes. An appropriate diagnosis and treatment should be provided to avoid life-threatening metabolic alterations.

A decreased level of adiponectin is strongly associated with an increased risk of colorectal adenoma and early cancer. These data call for further investigation, including a controlled prospective study.

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