Electrocardiography score based on the Minnesota code classification system predicts cardiovascular mortality in an asymptomatic low-risk population.

BACKGROUND: The use of a single abnormal finding on electrocardiography (ECG) is not recommended for stratifying the risk of cardiovascular (CV) events in low-risk general populations because of its low discriminative power. However, the value of a scoring system containing multiple abnormal ECG findings for predicting CV death has not been sufficiently evaluated. METHODS: In a prospective community-based cohort study, 8417 participants without atherosclerotic CV diseases (ASCVDs) and any related symptoms were followed for 18 years. The standard 12-lead ECGs were recorded at baseline and the ECG findings were categorized using the Minnesota code classification. CV deaths were defined as death from myocardial infarction (MI), chronic ischemic heart disease, heart failure, fatal arrhythmia, cerebrovascular event, pulmonary thromboembolism, peripheral vascular disease and sudden cardiac arrest and identified using the Korean National Statistical Office (KOSTAT) database. RESULTS: In a multivariate Cox proportional hazard (CPH) model, major and minor ST-T wave abnormalities, atrial fibrillation (AF), Q waves in the anterior leads, the lack of Q waves in the posterior leads, high amplitudes of the left and right precordial leads, left axis deviation and sinus tachycardia were associated with higher risks of CV deaths. The ECG score consisted of these findings showed modest predictive values represented by C-statistics that ranged from 0.632 to 760 during the follow-up and performed better in the early follow-up period. The ECG score independently predicted CV death after adjustment for relevant covariates in a multivariate model, and improved the predictive performance of the 10-year ASCVD risk estimator and a model of conventional risk factors including age, diabetes and current smoking. The combined ECG score (Harrell's C-index: 0.852, 95% confidence interval [CI], 0.828-0.876) composed of the ECG score and the conventional risk factors outperformed the 10-year ASCVD risk estimator (Harrell's C-index: 0.806; 95% CI, 0.780-0.833) and the model of the conventional risk factors (Harrell's C-index: 0.841, 95% CI, 0.817-0.865) and exhibited an excellent goodness of fit between the predicted and observed probabilities of CV death. CONCLUSIONS: The ECG score could be useful to predict CV death independently and may add value to the conventional CV risk estimators regarding the risk stratification of CV death in asymptomatic low-risk general populations.

The ECG score based on the Minnesota code classification can independently predict CV death and significantly improve the predictive power of the conventional CV risk estimators in asymptomatic low-risk general population.The combined ECG score comprised the ECG score, age and the presence of diabetes and current smoking predicted CV mortality more accurately than the conventional SV risk estimators.ECG may still be a viable CV risk stratification tool for population-based health screening projects.

Association of anaemia with long-term mortality among patients with hypertensive crisis in the emergency department.

BACKGROUND: Anaemia is frequent in patients with cardiovascular disease and is significantly associated with poor prognosis. However, the prognostic significance of anaemia in hypertensive crisis remains unknown. We conducted this study to determine whether anaemia is a risk factor for all-cause mortality in patients with hypertensive crisis visiting the emergency department (ED). METHODS: This retrospective study included patients who visited the ED between 2016 and 2019 for hypertensive crisis, which was defined as systolic blood pressure ≥180 mmHg or diastolic blood pressure ≥110 mmHg. A total of 5,512 patients whose serum haemoglobin levels were checked were included in this study and were classified into three groups according to their serum haemoglobin levels at admission to the ED: moderate/severe anaemia (haemoglobin <11 g/dL), mild anaemia (haemoglobin 11 to <13 g/dL in men and 11 to <12 g/dL in women), and non-anaemia (haemoglobin ≥13 g/dL in men and ≥12 g/dL in women). RESULTS: Among 5,512 patients, 665 (12.1%) and 668 (12.1%) were classified into the moderate/severe anaemia and mild anaemia groups, respectively. The three-year all-cause mortality rates in the moderate/severe anaemia, mild anaemia, and non-anaemia groups were 46.0, 29.2, and 12.0%, respectively. After accounting for relevant covariates, patients with moderate/severe anaemia group (hazard ratio [HR], 2.15; 95% confidence interval [CI], 1.75-2.64) and mild anaemia group (HR, 1.32; 95% CI, 1.07-1.63) had a higher risk of 3-year all-cause mortality than the non-anaemia group. CONCLUSION: Anaemia is independently associated with 3-year all-cause mortality in patients with hypertensive crisis. A comprehensive therapeutic approach through more in-depth examination and close follow up are required for patients with hypertensive crisis with anaemia.KEY MESSAGESAnaemia is independently associated with 3-year all-cause mortality in patients with hypertensive crisis.A comprehensive therapeutic approach through more in-depth examination and close follow up are required for patients with hypertensive crisis with anaemia.

Clinical characteristics, practice patterns, and outcomes of patients with acute severe hypertension visiting the emergency department.

OBJECTIVES: Data regarding acute severe hypertension, a life-threatening condition encountered in the emergency department, are limited. We aimed to identify the characteristics, practice patterns, and outcomes of patients with acute severe hypertension in the emergency department. METHODS: This cross-sectional study at a tertiary referral centre included patients aged at least 18 years who were admitted to the emergency department between January 2016 and December 2019 for acute severe hypertension, which was defined as SBP at least 180 mmHg and/or DBP at least 100 mmHg. RESULTS: Of 172 105 patients who visited the emergency department, 10 219 (5.9%) had acute severe hypertension. Of them, 2506 (24.5%) patients had acute hypertension-mediated organ damage (HMOD), and these patients had more cardiovascular risk factors than did patients without HMOD. Additionally, 4137 (40.5%) patients were admitted, and nine (0.1%) died in the emergency department. The overall 3-month, 1-year, and 3-year mortality rates were 4.8, 8.8, and 13.9%, respectively. In patients with HMOD, the 1-year mortality rate was 26.9%, and patients lost to follow-up had a significantly higher 1-year mortality rate than those who were followed up (21.3 vs. 10.5%, respectively, P < 0.001). CONCLUSION: The mortality rate in patients with acute severe hypertension in the emergency department is high, especially in patients with HMOD. Evaluation of HMOD, investigating the underlying causes, and adequate follow-up are mandatory to improve the outcomes in these patients. This study emphasizes the need for disease-specific guidelines that include detailed acute treatment strategies and follow-up management for acute severe hypertension.

Potential pancreatic lipase inhibitory activity of phenolic constituents from the root bark of Morus alba L.

Detailed phytochemical investigation from the root bark of Morus alba resulted in the isolation of eleven new compounds, including seven 2-arylbenzofuran derivatives (morusalfurans A-G), three flavonoids (morusalnols A-C), and one geranylated stilbene (morusibene A), as well as 22 known compounds. The structures of the identified compounds were elucidated based on a comprehensive analysis of spectroscopic data and Mosher's method. Compounds 2, 3, 6-8, 11, 23, 24, and 29 showed potent inhibition of PL in comparison with the positive control treatment (orlistat, IC50=0.012µM), with IC50 values ranging from 0.09 to 0.92µM.

Human monoclonal antibodies against glucagon receptor improve glucose homeostasis by suppression of hepatic glucose output in diet-induced obese mice.

AIM: Glucagon is an essential regulator of hepatic glucose production (HGP), which provides an alternative therapeutic target for managing type 2 diabetes with glucagon antagonists. We studied the effect of a novel human monoclonal antibody against glucagon receptor (GCGR), NPB112, on glucose homeostasis in diet-induced obese (DIO) mice. METHODS: The glucose-lowering efficacy and safety of NPB112 were investigated in DIO mice with human GCGR for 11 weeks, and a hyperinsulinemic-euglycemic clamp study was conducted to measure HGP. RESULTS: Single intraperitoneal injection of NPB112 with 5 mg/kg effectively decreased blood glucose levels in DIO mice for 5 days. A significant reduction in blood glucose was observed in DIO mice treated with NPB112 at a dose ≥5 mg/kg for 6 weeks, and its glucose-lowering effect was dose-dependent. Long-term administration of NPB112 also caused a mild 29% elevation in glucagon level, which was returned to the normal range after discontinuation of treatment. The clamp study showed that DIO mice injected with NPB112 at 5 mg/kg were more insulin sensitive than control mice, indicating amelioration of insulin resistance by treatment with NPB112. DIO mice treated with NPB112 showed a significant improvement in the ability of insulin to suppress HGP, showing a 33% suppression (from 8.3 mg/kg/min to 5.6 mg/kg/min) compared to the 2% suppression (from 9.8 mg/kg/min to 9.6 mg/kg/min) in control mice. In addition, no hypoglycemia or adverse effect was observed during the treatment. CONCLUSIONS: A novel human monoclonal GCGR antibody, NPB112, effectively lowered the glucose level in diabetic animal models with mild and reversible hyperglucagonemia. Suppression of excess HGP with NPB112 may be a promising therapeutic modality for the treatment of type 2 diabetes.

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells.

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.

Cell engineering and cultivation of chinese hamster ovary (CHO) cells.

Mammalian cell lines are important host cells for the industrial production of pharmaceutical proteins owing to their capacity for correct folding, assembly and post-translational modification. In particular, Chinese hamster ovary (CHO) cells are the most dependable host cells for the industrial production of therapeutic proteins. Growing demand for therapeutic proteins promotes the development of technologies for high quality and productivity in CHO expression systems. The following are fundamentally important for effective production. 1) Construction of cultivation process. The CHO-based cultivation process is well established and is a general platform of therapeutic antibody production. The cost of therapeutic protein production using CHO cells is equivalent to that using microbial culture. 2) Cell line development. Recent developments in omics technologies have been essential for the development of rational methods of constructing a cell line. 3) Cell engineering for post-translational steps. Improvement of secretion, folding and glycosylaiton is an important key issue for mammalian cell production systems. This review provides an overview of the industrial production of therapeutic proteins using a CHO cell expression system.

Glycosylation pattern of humanized IgG-like bispecific antibody produced by recombinant CHO cells.

The glycosylation pattern of a humanized anti-EGFRxanti-CD3 bispecific single-chain diabody with an Fc portion (hEx3-scDb-Fc) produced by recombinant Chinese hamster ovary cells was evaluated and compared with those of a recombinant humanized anti-IL-8 antibody (IgG1) and human serum IgG. N-Linked oligosaccharide structures were estimated by two-dimensional high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. No sialylation was observed with purified hEx3-scDb-Fc and the anti-IL-8 antibody. From the analysis of neutral oligosaccharides, approximately more than 90% of the N-linked oligosaccharides of hEx3-scDb-Fc and the anti-IL-8 antibody were alpha-1,6-fucosylated. The galactosylated biantennary oligosaccharides comprise over 40% of the total N-linked oligosaccharides in both hEx3-scDb-Fc and the anti-IL-8 antibody. The fully galactosylated biantennary oligosaccharides from hEx3-scDb-Fc and the anti-IL-8 antibody accounted for only 10% of the N-linked; however, more than 20% of the N-linked oligosaccharides were fully galactosylated biantennary oligosaccharides in human serum IgG. The glycosylation pattern of hEx3-scDb-Fc was quite similar to that of the anti-IL-8 antibody.

The tetramer structure of the glycoside hydrolase family 27 alpha-galactosidase I from Umbelopsis vinacea.

The crystal structure of Umbelopsis vinacea alpha-galactosidase I, which belongs to glycoside hydrolase family 27, was determined at 2.0 A resolution. The monomer structure was well conserved with those of glycoside hydrolase family 27 enzymes. The biological tetramer structure of this enzyme was constructed by the crystallographic 4-fold symmetry, and tetramerization appeared to be caused by three inserted peptides that were involved in the tetramer interface. The quaternary structure indicated that the substrate specificity of this enzyme might be related to the tetramer formation. Three N-glycosylated sugar chains were observed, and their structures were found to be of the high-mannose type.

Expression of rice (Oryza sativa L. var. Nipponbare) alpha-galactosidase genes in Escherichia coli and characterization.

Two putative alpha-galactosidase genes from rice (Oryza sativa L. var. Nipponbare) belonging to glycoside hydrolase family 27 were cloned and expressed in Escherichia coli. These enzymes showed alpha-galactosidase activity and were purified by Ni Sepharose column chromatography. Two purified recombinant alpha-galactosidases (alpha-galactosidase II and III; alpha-Gal II and III) showed a single protein band on SDS-PAGE with molecular mass of 42 kDa. These two enzymes cleaved not only alpha-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose, and stachyose, but also liberated the galactosyl residues attached to the O-6 position of the mannosyl residue at the reducing-ends of mannobiose and mannotriose. In addition, these enzymes clipped the galactosyl residues attached to the inner-mannosyl residues of mannopentaose. Thus, alpha-Gal II catalyzes efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.

Characterization of raffinose synthase from rice (Oryza sativa L. var. Nipponbare).

The putative raffinose synthase gene from rice was cloned and expressed in Escherichia coli. The enzyme displayed an optimum activity at 45 degrees C and pH 7.0, and a sulfhydryl group was required for its activity. The enzyme was specific for galactinol and p-nitrophenyl-alpha-D-galactoside as galactosyl donors, and sucrose, lactose, 4-beta-galactobiose, N-acetyl-D-lactosamine, trehalose and lacto-N-biose were recognized as galactosyl acceptors.

Cloning and expression of the gene encoding Streptomyces coelicolor A3(2) alpha-galactosidase belonging to family 36.

The alpha-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from Thermus thermophilus was over 40%. The alpha-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 degrees C and pH 7. The enzyme hydrolyzed p-nitrophenyl-alpha-D -galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moiety but also other substrates.

Crystallization and preliminary X-ray crystallographic studies of alpha-galactosidase I from Mortierella vinacea.

alpha-Galactosidases catalyze the hydrolysis of a galactosyl residue from galactooligosaccharides and galactopolysaccharides. alpha-Galactosidase I from Mortierella vinacea was crystallized in two crystal forms using the hanging-drop vapour-diffusion method. Type 1 crystals belonged to space group I422, with unit-cell parameters a = b = 142.4, c = 131.5 A, and diffracted to beyond 2.1 A resolution, while type 2 crystals belonged to space group P4, with unit-cell parameters a = b = 100.9, c = 102.7 A, and diffracted to beyond 1.6 A resolution. This enzyme crystallized as a glycoprotein tetramer and the tetrameric structure was located around the crystallographic fourfold axis.

Purification and characterization of alpha-galactosidase from sunflower seeds.

From 100 g sunflower seeds, 1.2 mg purified alpha-galactosidase was obtained with an overall yield of 51%. The alpha-galactosidase acted on both terminal alpha-galactosyl residues and side-chain alpha-galactosyl residues of the galactomanno-oligosaccharides and galactomannans. The cDNA coding for sunflower alpha-galactosidase was cloned and the deduced amino acid sequence revealed that the mature enzyme consisted of 363 amino acid residues with a molecular weight of 40,263. Seven cysteine residues were found but no putative N-glycosylation sites were present in the sequence. The deduced amino acid sequences of mature enzyme and alpha-galactosidases from coffee, guar and Mortierella vinacea alpha-galactosidase II showed over 81%, 77%, and 47% homology, respectively. These enzymes are classified into the third group in which the enzyme has no insertion sequence and a broad specificity on galactomanno-oligosaccharides compared to the other groups.

alpha-Galactosidase from cultured rice (Oryza sativa L. var. Nipponbare) cells.

The alpha-galactosidase from rice cell suspension cultures was purified to homogeneity by different techniques including affinity chromatography using N-epsilon-aminocaproyl-alpha-D-galactopyranosylamine as the ligand. From 11 l of culture filtrate, 28.7 mg of purified enzyme was obtained with an overall yield of 51.9%. The cDNA coding for the alpha-galactosidase was cloned and sequenced. The enzyme was found to contain 417 amino acid residues composed of a 55 amino acid signal sequence and 362 amino acid mature alpha-galactosidase; the molecular weight of the mature enzyme was thus calculated to be 39,950. Seven cysteine residues were also found but no putative N-glycosylation sites were present. The observed homology between the deduced amino acid sequences of the mature enzyme and alpha-galactosidases from coffee (Coffea arabica), guar (Cyamopsis tetragonolooba), and Mortierella vinacea alpha-galactosidase II were over 73, 72, and 45%, respectively. The enzyme displayed maximum activity at 45 degrees C when p-nitrophenyl-alpha-D-galactopyranoside was used as substrate. The rice alpha-galactosidase and Mortierella vinacea alpha-galactosidase II acted on both the terminal alpha-galactosyl residue and the side-chain alpha-galactosyl residue of the galactomanno-oligosaccharides.