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OBJECTIVE -- To determine whether plasma concentrations of thrombin-activatable fibrinolysis inhibitor (TAFI) in patients with type 2 diabetes were associated with components of metabolic syndrome (MS), including high-sensitivity C-reactive protein (hs-CRP), plasminogen activator inhibitor (PAI)-1, and LDL cholesterol. RESEARCH DESIGN AND METHODS -- We studied 136 consecutive patients with type 2 diabetes. Diagnosis of MS was diagnosed by current criteria. Hypercholesterolemia (HC) was defined as serum LDL cholesterol >140 mg/dl (3.6 mmol/l) or treatment with a statin. For comparisons, diabetic patients were divided into four groups: those with no MS and no HC (n = 38), with MS but not HC (n = 39), with no MS but with HC (n = 26), and with both MS and HC (n = 33). RESULTS -- Considering all patients with type 2 diabetes, plasma PAI-1 was strongly associated with MS components such as BMI, triglyceride, alanine aminotransferase, a homeostasis model assessment of insulin resistance, and hs-CRP. Plasma TAFI only correlated positively and independently with LDL cholesterol. Plasma concentrations of plasmin-α2-antiplasmin complex (PAP), a measure of fibrinolytic activity in blood, showed a significant negative correlation with plasma PAI-1 but not TAFI. Diabetic patients with both MS and HC had the highest serum hs-CRP concentrations and the lowest plasma PAP concentrations. CONCLUSIONS -- LDL cholesterol is a main determinant of plasma TAFI in patients with type 2 diabetes. Coexistence of MS and HC synergistically accelerates inflammation and impairment of fibrinolysis via elevated concentrations of both TAFI and PAI-1, which inhibit fibrinolysis. [ABSTRACT FROM AUTHOR]

Objective: To investigate whether cardiac autonomic activity, particularly sympathovagal balance as estimated by power spectral analysis (PSA) of heart rate variation (HRV), is associated with serum adiponectin concentrations in patients with type 2 diabetes.Research Design and Methods: We studied 105 patients with type 2 diabetes (51 women and 54 men). Serum adiponectin concentrations were measured by sandwich enzyme-linked immunosorbent assay. HRV was determined automatically every 5 min over 24 h using Holter electrocardiographic recording. PSA of R-R intervals was performed using fast Fourier transformation. Low-frequency (both sympathetic and parasympathetic activities), high- frequency (pure parasympathetic activity), and the ratio of low-frequency-to-high-frequency power (LF-to-HF ratio), an index of sympathovagal balance, were used as indexes of cardiac autonomic activity.Results: We found no significant correlation between serum adiponectin and low-frequency or high-frequency power in patients with diabetes. Serum adiponectin concentration correlated negatively with the 24-h LF-to-HF ratio (r = -0.343, P = 0.0009) and creatinine clearance (r = -0.411, P < 0.0001). Serum adiponectin concentrations were significantly higher in patients with overt albuminuria than in those with normoalbuminuria or microalbuminuria. In multivariate analysis controlling for sex, BMI, glycemic control, lipid profile, and renal function, serum adiponectin concentration showed an independent negative association with 24-h LF-to-HF ratio (beta = -0.332, P = 0.020). Furthermore, sex, HDL cholesterol, and renal function retained significant influence on the serum adiponectin concentration in patients with diabetes.Conclusions: Sympathovagal balance favoring relative sympathetic activation was associated with low serum concentrations of adiponectin in patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

Examines the effect of warfarin on the secretion of recombinant protein C expressed in human kidney 293 or BHK cells. Details of materials and methods; Results and discussion.

Abstract: An increase of serum ferritin, an indicator of body iron store, is associated with insulin resistance and with an increased risk of type 2 diabetes in the general population. A low serum adiponectin is also associated with insulin resistance. Recently, hepcidin was identified as a regulator of iron metabolism. We investigated whether serum adiponectin was associated with serum ferritin or prohepcidin, a precursor of hepcidin, in healthy subjects and patients with type 2 diabetes. We studied 65 healthy subjects and 104 patients with type 2 diabetes. A serum ferritin concentration ≥300ng/ml for men or ≥150ng/ml for women was defined as hyperferritinemia. Serum ferritin was significantly higher and serum prohepcidin was significantly lower in diabetic patients than in control subjects. Serum total and high molecular weight (HMW) adiponectin correlated negatively with serum ferritin in control subjects or diabetic patients, while serum total and HMW adiponectin correlated positively with serum prohepcidin in diabetic patients, but not in control subjects. Serum total and HMW adiponectin were lower in patients with hyperferritinemia than in those without it. In conclusion, serum ferritin was increased in type 2 diabetic patients, while serum prohepcidin was decreased. A high serum ferritin was associated with insulin resistance, and with low serum total and HMW adiponectin in patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

Adiponectin (Acrp30), an adipocyte-derived protein, exists in serum as a trimer, a hexamer, and a high-molecular weight (HMW) form, including 12-18 subunits. Because HMW adiponectin may be biologically active, we measured it in serum using a novel enzyme-linked immunosorbent assay (ELISA) confirmed by gel filtration chromatography that the ELISA detected mainly adiponectin with 12-18 subunits, and we compared HMW with total adiponectin concentration in patients with type 2 diabetes. We next investigated the relationship between serum HMW and coronary artery disease (CAD) in 280 consecutive type 2 diabetic patients, including 59 patients with angiographically confirmed CAD. Total adiponectin was measured in serum by a commercially available ELISA. Like serum total adiponectin, HMW adiponectin correlated positively with HDL cholesterol and negatively with triglyceride, insulin sensitivity, creatinine clearance, and circulating inflammatory markers. Total and HMW adiponectin were significantly higher in women than in men, as was the HMW-to-total adiponectin ratio. Serum HMW and the HMW-to-total adiponectin ratio were significantly lower in men with than without CAD (P < 0.05, respectively). In women, the ratio, but neither total nor HMW adiponectin, tended to be lower when CAD was present. In conclusion, determination of HMW adiponectin, especially relative to total serum adiponectin, is useful for evaluating CAD in type 2 diabetic patients. Diabetes 55:1954-1960, 2006 [ABSTRACT FROM AUTHOR]

A method for the accurate evaluation of the organic carbon (OC) content of phytoplankton by soft X-ray microscopy (XM) was developed and applied to the picophytoplanktonSynechococcussp., whose cells are covered by extracellular polysaccharides (EPS). Based on the X-ray absorption coefficients and gray levels of the XM images, the OC content of EPS-covered cells ofSynechococcussp. (0.39–0.47 pg/cell) was found to be 2.0–2.4 times larger than that of EPS-removed cells (0.20 pg/cell). These findings suggest that soft XM could be a useful tool for evaluating the OC content of picophytoplankton and EPS without pretreatment steps. [ABSTRACT FROM PUBLISHER]

Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100°C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95°C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 µmol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 µmol/min/mg, respectively. [ABSTRACT FROM AUTHOR]

Retinal binding protein (RBP)-4 is a newly identified adipocytokine that can cause insulin resistance. The main purpose of the current study was to investigate the relationship between RBP4 and various markers related to insulin resistance, including triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), blood pressure (BP), endothelial function, oxidative stress, inflammation, and diabetic complications in patients with type 2 diabetes. The effect of pioglitazone (a thiazolidinedione) on RBP4 was also examined. Circulating RBP4 levels were measured in 101 hospitalized patients with type 2 diabetes and in 22 non-hospitalized control subjects. Endothelial function and oxidative stress were evaluated using flow-mediated vasodilatation (FMD) and urinary 8-iso-prostaglandin-F2α, respectively. Pioglitazone was administered for 12 weeks at a dose of 30 mg/day in a further 22 patient with type 2 diabetes in outpatient department. There was a significant elevation of RBP4 levels in diabetic patients compared with healthy subjects. RBP4 showed significant positive correlations with TG, systolic BP and log urinary albumin excretion (UAE), and significant negative correlations with HDL-C and FMD. By stepwise regression analysis log UAE, TG and gender showed a significant association with RBP4. There was a significant elevation of RBP4 in patients with proliferative-diabetic retinopathy compared with non-diabetic retinopathy and simple-diabetic retinopathy patients. Pioglitazone therapy for 12 weeks did not significantly affect the RBP4 concentration. In conclusion, this current study indicates the possibility that circulating RBP4 levels are associated with variables related to insulin resistance and diabetic complications. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. d-Amino acid dehydrogenase is a flavoenzyme that digests free neutral d-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 d-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial d-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to d-proline. The enzyme mediated electron transport from d-proline to coenzyme Q1, thus distinguishing it from d-amino acid oxidase. The apparent Km and Vmax values were 40.2 mM and 25.0 μmol min−1 mg−1, respectively, for dehydrogenation of d-proline, and were 8.2 μM and 12.3 μmol min−1 mg−1, respectively, for reduction of Q1. The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian d-amino acid oxidase than other bacterial d-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from d-proline to a c-type cytochrome was suggested spectrophotometrically. [ABSTRACT FROM AUTHOR]

Abstract: Histidine-rich glycoprotein (HRG) promoted the adhesion and morphological changes of human T-cell line MOLT-4 in a Con A-dependent manner. This morphological change-promoting activity was specific for HRG and the Arg23–Lys66 glycopeptide from human HRG. The carbohydrate chain at Asn45 was essential for this activity. The morphological changes of MOLT-4 cells caused by HRG and Con A (HRG/Con A) were not inhibited by phosphatidylinositol 3-kinase inhibitor, wortmannin or LY294002, while the changes by Con A alone were completely inhibited by these reagents, suggesting that HRG/Con A cooperate to activate leukocytes via a signaling pathway distinct from that by Con A alone. The morphological changes by Con A were associated with pseudopodia like structure. On the other hand, the morphological changes caused by HRG/Con A were associated not only with pseudopodia like structure but also with an increase of the F-actin-rich surface protrusions. Wortmannin inhibited only the formation of pseudopodia like structure. [Copyright &y& Elsevier]

Abstract: Histidine-rich glycoprotein (HRG) is a plasma protein implicated in the innate immune system. In recent studies, we showed that either HRG, or the Arg23-Lys66 glycopeptide derived from HRG, in concert with concanavalin A (Con A), promotes a morphological change and adhesion of the human leukemic T-cell line MOLT-4 to culture dishes, and that cell surface glycosaminoglycan or Fcγ receptors do not participate in this cellular event. In the present study, we identified the α-subunit of ATP synthase as one of the HRG-binding proteins on the surface of T-cells by HRG-derived glycopeptide affinity chromatography and by a peptide mass finger printing method. HRG specifically interacted with mitochondrial ATP synthase with a dissociation constant of 66 nM. The presence of α- and β-subunits of ATP synthase on the plasma membrane of MOLT-4 cell was demonstrated by immunofluorescent staining and FACS analysis. The HRG/Con A-induced morphological changes of MOLT-4 cells were specifically inhibited by a monoclonal antibody against the β-subunit of ATP synthase. These results strongly suggest that the cell surface ATP synthase functions as a binding protein for HRG on MOLT-4 cells, which is required for the morphological changes observed in MOLT-4 cells following treatment with HRG/Con A. [Copyright &y& Elsevier]

Abstract: The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K m and V max values of 100 mM and 909 μmol min−1 mg−1, respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS–PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 °C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5′-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells. [Copyright &y& Elsevier]

Objective: We investigated the correlation between acute-phase reactants, ie, high-sensitivity C-reactive protein (hsCRP) or fibrinogen and diabetic complications. Methods: In 73 patients with type 2 diabetes, we investigated associations between both markers and carotid artery intimal medial complex thickness (IMT), heart rate variability, or urinary albumin excretion (UAE). Results: Log hsCRP and fibrinogen correlated significantly with each other (r = 0.3701, P = 0.0013). Fibrinogen correlated negatively with the coefficient of variation of RR intervals (CV(RR)) and positively with log UAE (r = -0.2433, P = 0.0381; r = 0.4815, P < 0.0001), while log hsCRP did not. Furthermore both log hsCRP and fibrinogen did not correlate with IMT. Conclusion: We concluded that despite their close correlation, fibrinogen compared with hsCRP might be closely associated with diabetic microangiopathy and that both markers might not correlate with IMT as a marker of macroangiopathy. [ABSTRACT FROM AUTHOR]

Objective: To determine whether plasma concentrations of thrombin-activatable fibrinolysis inhibitor (TAFI) in patients with type 2 diabetes were associated with components of metabolic syndrome (MS), including high-sensitivity C-reactive protein (hs-CRP), plasminogen activator inhibitor (PAI)-1, and LDL cholesterol.Research Design and Methods: We studied 136 consecutive patients with type 2 diabetes. Diagnosis of MS was diagnosed by current criteria. Hypercholesterolemia (HC) was defined as serum LDL cholesterol >140 mg/dl (3.6 mmol/l) or treatment with a statin. For comparisons, diabetic patients were divided into four groups: those with no MS and no HC (n = 38), with MS but not HC (n = 39), with no MS but with HC (n = 26), and with both MS and HC (n = 33).Results: Considering all patients with type 2 diabetes, plasma PAI-1 was strongly associated with MS components such as BMI, triglyceride, alanine aminotransferase, a homeostasis model assessment of insulin resistance, and hs-CRP. Plasma TAFI only correlated positively and independently with LDL cholesterol. Plasma concentrations of plasmin-alpha2-antiplasmin complex (PAP), a measure of fibrinolytic activity in blood, showed a significant negative correlation with plasma PAI-1 but not TAFI. Diabetic patients with both MS and HC had the highest serum hs-CRP concentrations and the lowest plasma PAP concentrations.Conclusions: LDL cholesterol is a main determinant of plasma TAFI in patients with type 2 diabetes. Coexistence of MS and HC synergistically accelerates inflammation and impairment of fibrinolysis via elevated concentrations of both TAFI and PAI-1, which inhibit fibrinolysis. [ABSTRACT FROM AUTHOR]

Background: Diabetic nephropathy, especially when advanced, is associated with high prevalence of atherosclerotic cardiovascular disease in which inflammation and coagulation may play pathogenic roles. We investigated the relationships between diabetic nephropathy and coagulation, fibrinolysis, or inflammation in patients with Type 2 diabetes. Methods: We evaluated markers of inflammation and coagulation in 105 Type 2 diabetic patients with various grades of nephropathy and 49 healthy control subjects, in association with plasma total homocysteine (tHcy) measurements. Results: Plasma tHcy concentrations were significantly higher in diabetic patients than in controls (8.96±3.04 vs. 6.92±1.36 μmol/l, P<0.0001). Plasma concentrations of interleukin (IL)-6 were significantly higher in diabetic patients than in control subjects (P<0.0001). In diabetic patients, plasma tHcy correlated positively with urinary albumin, fibrinogen, IL-6 and plasmin-α2-antiplasmin complex (PAP), while plasma tHcy correlated negatively with creatinine clearance (Ccr) and protein C activity. After adjustment for Ccr, IL-6 and protein C activity were significantly associated with plasma tHcy. Plasma tHcy concentrations were significantly higher in patients with overt albuminuria than in those with normoalbuminuria or microalbuminuria, as were plasma concentrations of fibrinogen, prothrombin F1+2, and interleukin-6. Conclusions: Diabetic nephropathy is associated with elevated markers for both coagulation and inflammation. High plasma homocysteine may be a link between diabetic nephropathy and both chronic inflammation and hypercoagulability, increasing cardiovascular risk. [Copyright &y& Elsevier]

The authors describe a 52-year-old woman diagnosed with microscopic polyangiitis. She presented with abnormal liver function tests accompanied by fever, headache, and fatigue. Two months later, rapidly progressive necrotizing glomerulonephritis developed together with seropositivity for perinuclear antineutrophil cytoplasmic antibody. Although liver dysfunction from microscopic polyangiitis is very rare, especially at presentation, this diagnostic possibility should be kept in mind to permit prompt consideration of steroid therapy. [ABSTRACT FROM AUTHOR]

Objective: Three blood markers of inflammation (high-sensitivity C-reactive protein [hsCRP], interleukin [IL]-6, and fibrinogen) were compared with markers of atherosclerotic cardiovascular disease (CVD) (history of stroke or cardiac ischemia and measured toe-brachial index [TBI]) to determine whether inflammatory markers are associated with atherosclerosis in patients with type 2 diabetes.Research Design and Methods: Of 103 patients with type 2 diabetes, 26 had CVD. TBI was plethysmographically determined in both great toes. Serum hsCRP was immunonephelometrically determined. Plasma IL-6 was measured by an enzyme immunoassay.Results: Both ABI and TBI were lower in diabetic patients with CVD than in those without CVD (1.05 +/- 0.19 vs. 1.14 +/- 0.09, P < 0.05, and 0.75 +/- 0.20 vs. 0.95 +/- 0.21, P < 0.001, respectively). By linear regression, right TBI but not right ABI showed a significant negative correlation with serum hsCRP (r = -0.372, P < 0.01) and plasma fibrinogen (r = -0.224, P < 0.05). Serum hsCRP was also negatively correlated with lower TBI, but not lower ABI. We found no significant correlation between plasma IL-6 and ABI or TBI.Conclusions: TBI was strongly associated with CVD, serum hsCRP, and plasma fibrinogen. Of these inflammatory markers, serum hsCRP may be the most promising marker for vascular inflammation. [ABSTRACT FROM AUTHOR]

Objective: We compared plasma interleukin (IL)-18 concentrations in patients with type 2 diabetes with those in age-matched control subjects and investigated whether plasma IL-18 was associated with plasma total homocysteine (tHcy) concentration or carotid intimal-media wall thickness (IMT), an early marker of atherosclerosis, in these patients.Research Design and Methods: We measured plasma IL-18 in 103 type 2 diabetic patients and 45 age-matched control subjects. We also measured patients' plasma tHcy and serum high-sensitivity C-reactive protein (hs-CRP). IMT was evaluated for both common carotid arteries.Results: Plasma IL-18 was significantly higher in diabetic patients than in control subjects (203 +/- 153 vs. 118 +/- 37 pg/ml, P < 0.001). High IL-18 was defined as equaling or exceeding the mean + 2 SD of plasma IL-18 in control subjects (192 pg/ml). Patients with high IL-18 showed a greater carotid IMT than those with normal IL-18. Carotid plaques were more numerous in diabetic patients with high IL-18 than in those with normal IL-18. Plasma tHcy concentrations were significantly higher in patients with high IL-18 than in those with normal IL-18. Univariate and multivariate analyses showed a strong independent association between tHcy and IL-18. Plasma IL-18 also correlated positively with serum hs-CRP.Conclusions: In patients with type 2 diabetes, plasma IL-18 concentrations are greater than in nondiabetic subjects. Plasma IL-18 is an independent determinant of plasma tHcy, which is linked independently with atherosclerotic carotid wall thickening. [ABSTRACT FROM AUTHOR]

Complex III was purified from submitochondrial particles prepared from Euglena gracilis. The purified complex consisted of 10 subunits and lost antimycin sensitivity. The Euglena complex III showed an atypical difference absorption spectrum for cytochrome ct with its α-band maximum at 561 nm. The pyridine ferrohemochrome prepared from covalently bound heme in the Euglena complex III had an α-peak at 553 nm. This wavelength is the same as that of pyridine ferrohemochrome prepared from Euglena mitochondrial cytochrome c (c-558), the heme of which is linked to only a single cysteine residue through a thioether bond. Cytochrome c1 which was a heme-stained subunit with a molecular mass of 32.5 kDa was isolated from the purified complex iii and its N-terminal sequence of 46 amino acids was determined. On the basis of apparent homologies to cytochromes ct from other sources, this sequence included the heme-binding region. However, the amino acid at position 36, corresponding to the first cysteine involved in heme linkage in other cytochromes c1, was phenylalanine. Position 39, corresponding to the second cysteine, was not identified despite the treatment for removal of the heme and carboxymethylation of the expected cysteine. The unidentified amino acid is assumed to be a derivative of cysteine to which the heme is linked through a single thioether bond. The histidine-40 corresponding to the probable fifth ligand for heme iron was conserved in Euglena cytochrome c1. [ABSTRACT FROM AUTHOR]

Obejective. To investigate short-term effects of pioglitazone or voglibose on serum concentrations of both total and high-molecular-weight (HMW) adiponectin measured with a novel sandwich enzyme-linked immunosorbent assay (ELISA) and on plasma fibrinolysis indicators, in type 2 diabetic patients with a second failure of treatment with sulfonylureas. Methods. Thirty-four diabetic patients were randomized to receive pioglitazone or voglibose treatment for 12 weeks, after which serum HMW adiponectin was measured. Plasma plasminogen activator inhibitor (PAI)-1 and thrombin-activatable fibrinolysis inhibitor (TAFI), a recently identified inhibitor of fibrinolysis, were measured as fibrinolysis inhibitors. Results. At baseline, serum HMW adiponectin correlated negatively with plasma TAFI in all patients with type 2 diabetes (r=0.367, P=0.0423). Both groups showed similar improvements in glycemic control. Serum total and HMW adiponectin increased in patients treated with pioglitazone, while showing no significant changes in patients treated with voglibose. The HMW/total adiponectin ratio increased significantly after treatment with pioglitazone (P=0.0004). Changes in HbA1c showed significant negative correlations with changes in serum HMW adiponectin in patients treated with pioglitazone (r=-0.694, P=0.0034). Plasma PAI-1 tended to decrease after pioglitazone treatment (P=0.0908), while plasma TAFI did not change. Conclusion. Increased serum HMW adiponectin may contribute to the improvement in glycemic control after pioglitazone treatment. Plasma PAI-1 decreased somewhat after treatment with pioglitazone, while plasma TAFI was unchanged by either drug. [ABSTRACT FROM AUTHOR]