Fabrication of a Gelatin-Based Microdevice for Vascular Cell Culture.

This study presents a novel technique for fabricating microfluidic devices with microbial transglutaminase-gelatin gels instead of polydimethylsiloxane (PDMS), in which flow culture simulates blood flow and a capillary network is incorporated for assays of vascular permeability or angiogenesis. We developed a gelatin-based device with a coverslip as the bottom, which allows the use of high-magnification lenses with short working distances, and we observed the differences in cell dynamics on gelatin, glass, and PDMS surfaces. The tubes of the gelatin microfluidic channel are designed to be difficult to pull out of the inlet hole, making sample introduction easy, and the gelatin channel can be manipulated from the cell introduction to the flow culture steps in a manner comparable to that of a typical PDMS channel. Human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) were successfully co-cultured, resulting in structures that mimicked blood vessels with inner diameters ranging from 10 µm to 500 µm. Immunostaining and scanning electron microscopy results showed that the affinity of fibronectin for gelatin was stronger than that for glass or PDMS, making gelatin a suitable substrate for cell adhesion. The ability for microscopic observation at high magnification and the ease of sample introduction make this device easier to use than conventional gelatin microfluidics, and the above-mentioned small modifications in the device structure are important points that improve its convenience as a cell assay device.

Pre-stroke habitual prolonged sleep as a predictor for post-stroke sleep quality, stroke-related quality of life, and lifestyle values.

BACKGROUND: Prolonged sleep is a higher stroke risk, but post-stroke prolonged sleep facilitates stroke recovery. No study has explored the relationship between pre- and post-stroke prolonged sleep and their involvement in stroke-related quality of life (QOL).This study aimed to clarify the role of pre- and post-stroke prolonged sleep in QOL and sleep quality during hospitalization. METHODS: Fifty-one subacute stroke inpatients were enrolled. QOL was assessed by the Stroke and Aphasia QOL Scale-39-J. Sleep quality and lifestyle values were assessed by original questionnaires. RESULTS: Patients in pre-stroke prolonged sleep > 8 h had a higher incidence of post-stroke poor sleep quality than those belonging to the normal or shorter hours (OR 5.33, 95% CI 1.30-21.84, p = 0.047). In addition, pre-stroke prolonged sleep was associated with lower scores of psychosocial QOL and lifestyle values of "accepting disability; caring about what other people think of what you do". In contrast, post-stroke prolonged sleep was associated with the lower risk of post-stroke poor sleep quality (OR 0.27, 95% CI 0.08-0.86, p = 0.045). Post-stroke high sleep quality had higher (better) scores of physical and energy QOL, and lifestyle values of "caring about what other people think of what you do; having some places to go out after discharge" compared with post-stroke poor sleep quality. Post-stroke prolonged sleep was derived from pre-stroke not prolonged sleep rather than pre-stroke prolonged sleep (p = 0.039, Chi-square test). CONCLUSIONS: Pre-stroke prolonged sleep is associated with a higher incidence of post-stroke poor sleep quality and lower scores of QOL and lifestyle values after stroke.

Persistent or permanent atrial fibrillation is associated with severe cardioembolic stroke in patients with non-valvular atrial fibrillation.

BACKGROUND: Little is known about the difference in the severity of cardioembolic (CE) stroke between patients with paroxysmal atrial fibrillation (PAF) and persistent/permanent AF (PerAF). We assessed stroke severity in patients with CE stroke divided by the type of AF. METHODS: Three hundred and fifty-eight consecutive patients with CE stroke within 48 h of onset and with a modified Rankin Scale (mRS) score ≤ 1 before onset were studied. We compared basic characteristics, stroke severity, and functional outcome between patients with PAF (n = 127) and PerAF (n = 231). RESULTS: Patients with PerAF were more likely to take oral anticoagulants (OACs) than those with PAF (37% vs. 13%, P <  0.0001), even though still underuse of OAC in both patients. Regarding stroke severity on admission, patients with PerAF exhibited a tendency toward a higher score on the National Institutes of Health Stroke Scale (NIHSS) compared with patients with PAF (12 [5-20] vs. 9 [4-18]; P = 0.12). Mortality and mRS score at discharge were higher in the PerAF than in the PAF group (13% vs. 4%; P = 0.005, and 3 [1-5] vs. 2 [1-4]; P = 0.01, respectively). Multivariate analyses confirmed that PerAF was a significant determinant of severe stroke (NIHSS score > 8) on admission (odds ratio [OR] to PAF = 1.80; 95% confidence interval [CI] 1.08-2.98; P = 0.02) and of an mRS score ≥ 3 at discharge (OR = 2.07; 95% CI 1.24-3.46; P = 0.006). Patients with PerAF had three times more internal carotid artery occlusion evaluated by magnetic resonance angiography, which indicated a more severe cerebral embolism compared with patients with PAF. CONCLUSIONS: We found underuse of OAC in high risk AF patients with CE stroke. PerAF is significantly associated with severe stroke on admission and an unfavorable functional outcome at discharge in Japanese patients with CE stroke.

Low Body Mass Index is a Poor Prognosis Factor in Cardioembolic Stroke Patients with NonValvular Atrial Fibrillation.

BACKGROUND: The relationship between body mass index (BMI) and the severity of cardioembolic stroke (CES) remains poorly understood. METHOD: A total of 419 consecutive CES patients with nonvalvular atrial fibrillation (NVAF), and with a modified Rankin Scale (mRS) score of 0 or 1 before onset admitted within 48hours after onset to the Hirosaki Stroke and Rehabilitation Center were studied. The patients were divided into three groups, low BMI (L-BMI; n = 36, BMI < 18.5 kg/m2), normal BMI (N-BMI; n = 284, 18.5 ≤ BMI < 25.0), and high BMI (H-BMI; n = 99, BMI ≥ 25.0). We compared stroke severity and functional outcome among the three groups. RESULTS: Stroke severity on admission, assessed by the National Institutes of Health Stroke Scale (NIHSS) showed that patients with L-BMI had the highest NIHSS score (median, 16 [11-25]), followed by N-BMI and H-BMI (11 [5-19] and 9 [3-19], P = .002). Functional outcome at discharge, assessed by mRS, was most severe in L-BMI patients (5 [3-5]), followed by N-BMI and H-BMI (3 [1-4] and 2 [1-4], P = .001). Multivariate analyses revealed that L-BMI was a significant determinant of severe stroke (NIHSS scores ≥8) at admission (odds ratio [OR] to N-BMI = 2.79, 95% confidence interval [CI], 1.17-7.78, P = .02) and poor functional outcome (mRS scores ≥3) at discharge (OR = 2.53, 95% CI, 1.12-6.31, P = .02). However, H-BMI did not affect stroke severity at admission or functional outcome at discharge. CONCLUSION: Low BMI is a risk factor for severe stroke on admission and unfavorable functional outcome at discharge in Japanese CES patients with NVAF.

Impact of Atrial Natriuretic Peptide Value for Predicting Paroxysmal Atrial Fibrillation in Ischemic Stroke Patients.

INTRODUCTION: The impact of atrial natriuretic peptide (ANP) value for predicting paroxysmal atrial fibrillation (pAF) in ischemic stroke patients remains uncertain. METHODS: The consecutive 222 ischemic stroke patients (median 77 [IQR 68-83] years old, 93 females) within 48 hours after onset were retrospectively studied. Plasma ANP and brain natriuretic peptide (BNP) levels were simultaneously measured at admission. Of all, 158 patients had no evidence of atrial fibrillation (AF) (sinus rhythm [SR] group), 25 patients had pAF (pAF group), and the other 39 patients had chronic AF (cAF group). We investigated predicting factors for pAF, with focus on ANP, BNP, and ANP/BNP ratio. RESULTS: ANP value was significantly higher in the pAF than in the SR group (97 [50-157] mg/dL versus 42 [26-72] mg/dL, P < .05) and further increased in the cAF group (228 [120-392], P < .05 versus pAF and SR groups). Similarly, the BNP value was higher in the pAF than in the SR group (116 [70-238] mg/dL versus 34 [14-72] mg/dL, P < .05) and further increased in the cAF group (269 [199-423], P < .05 versus pAF and SR groups). ANP/BNP ratio was lower in the pAF and cAF groups than in the SR group (.6 [.5-1.2] and .7 [.5-1.0] versus 1.3 [.8-2.4], both P < .05]. Multivariate analysis in the SR and pAF groups (n = 183) demonstrated that age, congestive heart failure, ANP, and BNP, but not ANP/BNP ratio, were independent predictors for detecting pAF. Receiver operating characteristic curve analysis further showed that area under the curve was similar between ANP and BNP (.76 and .80). CONCLUSIONS: ANPmay be clinically useful for detecting pAF in ischemic stroke patients as well as BNP.

Effect of rivaroxaban on prothrombin fragment 1+2 compared with warfarin in patients with acute cardioembolic stroke: Insight from its serial measurement.

INTRODUCTION: Patients with intracerebral hemorrhage during rivaroxaban treatment have small hematoma and favorable outcomes compared with those with warfarin. We investigated its possible mechanism, focusing on prothrombin fragment 1+2 (F1+2), a marker of thrombin generation. MATERIALS AND METHODS: In 65 patients with acute cardioembolic stroke (median 77years), rivaroxaban was initiated at 5days after the onset. Plasma F1+2 level (normal range, 69-229pmol/L), prothrombin time (PT), and rivaroxaban concentration evaluated by anti-Xa activity were serially measured. RESULTS: Median plasma F1+2 was 276 (IQR, 195-454) pmol/L before starting rivaroxaban, and significantly decreased to 196 (141-267) and 192 (151-248) on 7 and 28days after rivaroxaban, respectively (both p<0.05). Serial measurements of PT and rivaroxaban concentration at trough, 2, 4, and 6h after taking rivaroxaban showed a positive correlation (R2=0.69, p<0.01). PT at 4h after rivaroxaban was significantly prolonged compared with trough (16.6 versus 11.5s, p<0.0001). F1+2 at 4h was also decreased compared with trough (160 (123-245.5) versus 196 (141-266.5), p=0.04), but no patients showed F1+2 below the normal range at 4h. In other 34 patients with warfarin treatment (77years), median PT-INR and F1+2 were 2.06 (1.75-2.50) and 75 (48-111) (p<0.0001 versus 4h after rivaroxaban). Notably, of those with PT-INR≥2.0 (18/34), 12 (12/18, 67%) showed F1+2 below the normal range. CONCLUSIONS: Rivaroxaban retains a normal thrombin generation even at its peak level with prolonged PT, whereas warfarin at therapeutic levels inhibits thrombin generation. This may partly explain different outcomes in patients complicated with bleeding events.

Multi-reassortant G3P[3] group A rotavirus in a horseshoe bat in Zambia.

Group A rotavirus is a major cause of diarrhoea in humans, especially in young children. Bats also harbour group A rotaviruses, but the genetic backgrounds of bat rotavirus strains are usually distinct from those of human rotavirus strains. We identified a new strain of group A rotavirus in the intestinal contents of a horseshoe bat in Zambia. Whole genome sequencing revealed that the identified virus, named RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3], possessed the genotype constellation G3-P[3]-I3-R2-C2-M3-A9-N2-T3-E2-H3. Several genome segments of LUS12-14 were highly similar to those of group A rotaviruses identified from humans, cows and antelopes, indicating interspecies transmission of rotaviruses between bats and other mammals with possible multiple genomic reassortment events.

Impact of Sex Difference on Severity and Functional Outcome in Patients with Cardioembolic Stroke.

INTRODUCTION: Female sex is a risk factor for thromboembolic events in Caucasian, but not in Japanese, patients with nonvalvular atrial fibrillation. However, it remains unclear whether the female sex is also a risk factor for severe stroke and unfavorable functional outcome in patients with cardioembolic (CE) stroke. METHODS: Three hundred fifty-five consecutive patients with CE stroke within 48 hours after onset and with a modified Rankin Scale (mRS) score of 1 or lower before onset were studied. We compared basic characteristics, stroke severity, and functional outcome between female (n = 157) and male (n = 198) patients. RESULTS: The mean age was higher in female than in male patients (80 ± 8 versus 75 ± 9 years, P < .00001). The congestive heart failure, hypertension, age [≥ 75 years], diabetes, stroke/transient ischemic attack [TIA] (CHADS2) score before onset was similar between the two groups (median, 3 [2-4] in both groups). Stroke severity on admission, assessed by the National Institutes of Health Stroke Scale (NIHSS), was higher in female than in male patients (13 [5-20] versus 8 [3-16], P = .0009). Functional outcome at discharge, assessed by mRS, was unfavorable in female than in male patients (3 [1-5] versus 2 [1-4], P = .005). An mRS score of 3 or higher at discharge was found more in female than in male patients (59% versus 39%, P = .0001). Multivariate analyses confirmed that female sex was a significant determinant of severe stroke (NIHSS ≥ 8) on admission (odds ratio [OR] to male = 1.97; 95% confidence interval [CI]; 1.24-3.15, P = .004) and for the mRS score of 3 or higher at discharge (OR = 1.83; 95% CI, 1.16-2.89; P = .01). Similar results were obtained by propensity-score matching analysis. CONCLUSIONS: Female sex is a risk factor for severe stroke on admission and unfavorable functional outcome at discharge in Japanese patients with CE stroke.

Severity and Functional Outcome of Patients with Cardioembolic Stroke Occurring during Non-vitamin K Antagonist Oral Anticoagulant Treatment.

BACKGROUND: Severity and functional outcome of patients with cardioembolic stroke (CE) occurring during non-vitamin K antagonist oral anticoagulant (NOAC) treatment remain uncertain. METHODS: The consecutive 355 CE patients within 48 hours after onset and with modified Rankin Scale (mRS) score of 1 or less before onset were studied. Of all, 262 patients were treated with no anticoagulants (non-AC), 63 with warfarin below therapeutic range of prothrombin time-international normalized ratio (PT-INR) on admission (PT-INR <1.6 [WF-Lo]), 16 with warfarin within therapeutic range (PT-INR ≥1.6 [WF-Tp]), and 14 with NOACs (9 dabigatran and 5 rivaroxaban [NOAC-DR]). We compared severity and functional outcome of CE patients among these 4 groups, especially focusing on patients during NOAC treatment. RESULTS: Stroke severity on admission, assessed by the National Institutes of Health Stroke Scale, was lower in WF-Tp (median, 5 [1-15]) and NOAC-DR (5 [3-6]) than in non-AC (11 [5-19]) and WF-Lo (12 [5-19]; P = .006). Functional outcome at discharge, assessed by mRS, was favorable in WF-Tp (median, 1 [0-4]) and NOAC-DR (1 [1-2]) compared with that in non-AC (2 [1-4]) and WF-Lo (3 [1-5]; P = .02), and ratios of the patients with mRS score of 1 or less were 63% and 64% versus 31% and 33%, respectively (P = .005). Multivariate analysis also showed a favorable functional outcome at discharge in WF-Tp and NOAC-DR groups. Drug management was likely associated with NOAC-associated CE. CONCLUSIONS: Stroke severity and functional outcome of CE patients treated with warfarin within therapeutic range and with NOACs are similar to each other, and are more favorable than those with no anticoagulants and with warfarin below therapeutic range.

Characteristics of intracerebral hemorrhage during rivaroxaban treatment: comparison with those during warfarin.

BACKGROUND AND PURPOSE: Neuroradiological characteristics and functional outcomes of patients with intracerebral hemorrhage (ICH) during novel oral anticoagulant treatment were not well defined. We examined these in comparison with those during warfarin treatment. METHODS: The consecutive 585 patients with ICH admitted from April 2011 through October 2013 were retrospectively studied. Of all, 5 patients (1%) had ICH during rivaroxaban treatment, 56 (10%) during warfarin, and the other 524 (89%) during no anticoagulants. We focused on ICH during rivaroxaban and warfarin treatments and compared the clinical characteristics, neuroradiological findings, and functional outcomes. RESULTS: Patients in the rivaroxaban group were all at high risk for major bleeding with hypertension, abnormal renal/liver function, stroke, bleeding history or predisposition, labile international normalized ratio, elderly, drugs/alcohol concomitantly (HAS-BLED) score of 3 and higher rate of past history of ICH. Moreover, multiple cerebral microbleeds (≥4) were detected more frequently in rivaroxaban group than in warfarin (80% versus 29%; P=0.04). Hematoma volume in rivaroxaban group was markedly smaller than that in warfarin (median: 4 versus 11 mL; P=0.03). No patient in the rivaroxaban group had expansion of hematoma and surgical treatment. Rivaroxaban group showed lower modified Rankin Scale at discharge relative to warfarin, and the difference between modified Rankin Scale before admission and at discharge was smaller in rivaroxaban than in warfarin (median: 1 versus 3; P=0.047). No patient in the rivaroxaban group died during hospitalization, whereas 10 (18%) warfarin patients died. CONCLUSIONS: Rivaroxaban-associated ICH occurs in patients at high risk for major bleeding. However, they had a relatively small hematoma, no expansion of hematoma, and favorable functional and vital outcomes compared with warfarin-associated ICH.

Streptavidin-aequorin fusion protein for bioluminescent immunoassay.

The fusion protein of streptavidin to aequorin (STA-AQ) was highly purified from inclusion bodies in Escherichia coli cells and applied to a bioluminescent sandwich immunoassay. α-Fetoprotein (AFP), which is a serological marker of liver cancer, was used as a model analyte to test STA-AQ in an immunoassay. The measurable range of AFP by the sandwich immunoassay, using the complex of STA-AQ and the biotinylated anti-AFP antibody, was 0.02-200 ng/mL with an average coefficient of variation of 4.9%. The detection sensitivity with the complex of STA-AQ and the biotinylated anti-AFP antibody was similar to that with the complex of biotinylated aequorin, streptavidin and the biotinylated anti-AFP antibody. STA-AQ would be a useful reporter protein for immunoassays.

p122 protein enhances intracellular calcium increase to acetylcholine: its possible role in the pathogenesis of coronary spastic angina.

OBJECTIVE: Phospholipase C-δ1 activity is enhanced in patients with coronary artery spasm, and a p122 protein was recently cloned to potentiate phospholipase C-δ1 activity. To investigate the role of p122 in enhanced vasomotility, we examined p122 expression in the cultured skin fibroblasts obtained from patients with and without coronary spasm, intracellular Ca(2+) concentration ([Ca(2+)]i) [corrected] at baseline and after stimulation with acetylcholine in the cells transfected with p122, and promoter in genomic DNA. METHODS AND RESULTS: [corrected] p122 protein and gene expression levels in patients with coronary spasm (n=11) were enhanced compared with levels in control subjects (n=9) (P<0.01 for both). [Ca(2+)](i) at baseline and the peak increase in [Ca(2+)](i) in response to acetylcholine were both 2 times higher in cells transfected with p122 than in those without p122. Conversely, knockdown of p122 resulted in diminished [Ca(2+)](i) response. In the p122 promoter analysis, the -228G/A and -1466C/T variants revealed the increase in luciferase activity. Although the -1466C/T variant was similar between 144 patients with coronary spasm and 148 controls, the -228G/A variant was more frequent in male patients than in male controls (P<0.05). CONCLUSIONS: The p122 protein is upregulated in patients with coronary spasm, causing increased [Ca(2+)](i) to acetylcholine, and thereby seems to be related to enhanced coronary vasomotility.

Novel pro-atherogenic molecule coupling factor 6 is elevated in patients with stroke: a possible linkage to homocysteine.

BACKGROUND AND PURPOSE: Homocysteine (Hcy) is an independent predictor of stroke. Coupling factor 6 (CF6) is regulated by nuclear factor kappa B (NF-kappaB) signaling which is activated by Hcy. We tested the hypothesis that CF6 is elevated with Hcy in stroke. We also examined the effect of vitamin treatment on CF6 and Hcy levels. METHODS AND RESULTS: The 59 Japanese patients with a recent history of stroke were randomly assigned to a group without vitamin treatment (Group 1, n = 29) and to a group with treatment with both folic acid and vitamin B(12) for 2 months (Group 2, n = 30). The CF6 level was elevated in the patients with stroke compared with that in controls (n = 64) at admission. In a multiple regression model, the plasma CF6 level was weakly correlated to the total Hcy (tHcy) level. In Group 1, the plasma tHcy and CF6 levels were unchanged. In Group 2, however, they were both decreased, and there was a weak positive correlation between the decreases in plasma levels of CF6 and tHcy. CONCLUSION: CF6 is elevated in patients with stroke independently of risk factors. Since Hcy and vitamin treatment affect CF6 levels in stroke, CF6 appears to be a novel molecule for the pathogenesis and treatment of stroke.

Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris.

The luciferase secreted by the deep-sea shrimp Oplophorus consists of 19 and 35kDa proteins. The 19-kDa protein (19kOLase), the catalytic component of luminescence reaction, was expressed in Escherichia coli using the cold-shock inducted expression system. 19kOLase, expressed as inclusion bodies, was solubilized with 6M urea and purified by urea-nickel chelate affinity chromatography. The yield of 19kOLase was 16 mg from 400 ml of cultured cells. 19kOLase in 6M urea could be refolded rapidly by dilution with 50mM Tris-HCl (pH 7.8)-10mM EDTA, and the refolded protein showed luminescence activity. The luminescence properties of refolded 19kOLase were characterized, in comparison with native Oplophorus luciferase. Luminescence intensity with bisdeoxycoelenterazine as a substrate was stimulated in the presence of organic solvents. The 19kOLase is a thermolabile protein and is 98 % inhibited by 1muM Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.

[Clinical analysis of HLA-DR-negative non-M3 AML].

The type of leukemia was defined as HLA-DR(-) non-M3-AML, when HLA antigens were detected by flow cytometry at an incidence of < 20% of the blast population excluding M3-AML. Out of 109 patients with de novo acute myeloid leukemia, 8 patients had HLA-DR(-) non-AML-M3. According to the French-American-British criteria, 7 patients could be subdivided into 3 patients with M1, 4 patients with M2 and 1 patient with M4. The morphological features of bone marrow aspiration demonstrated no dysplasia and peroxidase stain positivity was noted in over 86% of the blast cells in all patients, the blast cells with fine granularity in 7 patients. The cytogenetic analysis revealed a normal karyotype. There was no expression marker of the blast antigens except CD13, CD14, CD33, CD34 and CD56. All of 7 patients who underwent induction therapy attained complete remission. Overall survival and disease-free survival showed no significant differences between the HLA-DR(-) non- M3-AML group and the HLA-DR(+) AML group.

Imidazole-assisted catalysis of luminescence reaction in blue fluorescent protein from the photoprotein aequorin.

Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a dissociable complex of Ca(2+)-bound apoaequorin and coelenteramide, and is identified as a luciferase that catalyzes the oxidation of coelenterazine by molecular oxygen to emit light. Based on the chemical luminescence of coelenterazine oxidation by an acid-base mechanism, we found that the luminescence activity of BFP-aq was stimulated by imidazole at concentrations of 30-300mM with coelenterazine and its analogues. The kinetic analyses indicate that imidazole has no effect on the binding affinity of coelenterazine to BFP-aq and may act as a catalytic base, accepting a proton from the -NH- group of coelenterazine and stimulating luminescence activity.

Mutational analysis of Kir6.1 in Japanese patients with coronary spastic angina.

The etiology of coronary spastic angina (CSA) remains uncertain. Mice lacking the gene encoding the inwardly rectifying K(+) channel Kir6.1 were developed as an animal model of CSA. We investigated whether mutation in the coding region of the Kir6.1 gene is detected in Japanese patients with CSA. The study population included 19 Japanese patients with CSA (10 men and 9 women with a mean age of 61+/-14 years). Mutational analysis of the coding region of Kir6.1 was performed by direct sequencing. We found no missense or nonsense mutations in these samples, but we found in one female CSA patient, a single base substitution (C to T) at nucleotide position 111 in exon 2 of the coding region, which was heterozygous and did not cause amino acid substitution (Ile37Ile, silent mutation). In the remaining 18 patients, no base substitution was detected in the coding region of the Kir6.1 gene. No mutation that alters primary structure of Kir6.1 was detected in Japanese patients with CSA. The results indicate that abnormality in the primary structure of Kir6.1 may not be involved in the genetic pathogenesis of CSA in humans.

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: catalytic properties for the oxidation of coelenterazine as an oxygenase.

Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a complex of Ca2+ -bound apoaequorin and coelenteramide, and shows luminescence activity like a luciferase, catalyzing the oxidation of coelenterazine with molecular oxygen. To understand the catalytic properties of BFP-aq, various fluorescent proteins (FP-aq) have been prepared from semi-synthetic aequorin and characterized in comparison with BFP-aq. FP-aq has luciferase activity and could be regenerated into native aequorin by incubation with coelenterazine. The results from substrate specificity studies of FP-aq using various coelenterazine analogues have suggested that the oxidation of coelenterazine by BFP-aq in the luciferase reaction and the regeneration process to aequorin might involve the same catalytic site of BFP-aq.

Effect of vasoconstrictor coupling factor 6 on gene expression profile in human vascular endothelial cells: enhanced release of asymmetric dimethylarginine.

BACKGROUND: Coupling factor 6 (CF6), a component of ATP synthase, inhibits phospholipase A2 and induces vasoconstriction. However, because arachidonic acid acts in the widespread fields of vascular biology, CF6 might exert profound effects in addition to vasoconstriction. We investigated the effect of CF6 on the gene expression profile in human umbilical vein endothelial cells. METHODS AND RESULTS: The increased gene expression after 24-h exposure to CF6 at 10 mol/l, assessed by cDNA microarray (n = 3), included neuregulin-1 (1.84 +/- 0.07 fold compared with control, P < 0.05) and relaxin-1 (1.74 +/- 0.20, P < 0.05), both relating to congestive heart failure, urokinase type plasminogen activator receptor (1.77 +/- 0.24, P = 0.06) and estrogen receptor beta (1.74 +/- 0.36, P = 0.08), both relating to vascular inflammation and cell infiltration, and protein arginine methyltransferase (PRMT-1; 1.73 +/- 0.20, P < 0.05). Out of these genes, the enzyme relating to the synthesis (PRMT-1) of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), was further examined concomitantly with the degradation enzyme, dimethylarginine dimethylaminohydrolase 2 (DDAH-2). The ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, measured by real-time quantitative reverse transcription-polymerase chain reaction, was increased by 9 +/- 2% (n = 10, P < 0.01) at 48 h after CF6 at 10 mol/l, whereas the ratio of DDAH-2 to GAPDH was decreased by 12 +/- 2% (n = 8, P < 0.01). DDAH-2 protein and activity were decreased by 28 +/- 5% (n = 5, P < 0.01) and 19 +/- 2% (n = 6, P < 0.01) by CF6, respectively. ADMA release was enhanced by 20 +/- 8% and NOS activity was decreased by 13 +/- 1% (both n = 8, P < 0.05) by CF6. CONCLUSIONS: CF6 changes the gene expression profile to be proatherogenic and functions as a novel stimulator for ADMA release by enhancing its synthesis and suppressing its degradation.