Screening of α-amino acid ester acyl transferase variant with improved activity by combining rational and random mutagenesis.

Random and rational mutagenesis of an α-amino acid ester acyl transferase from Sphingobacterium siyangensis AJ2458 (SAET) was conducted to examine the production of aspartame, an α-l-aspartyl-l-phenylalanine methyl ester. We previously reported aspartame production via combination of enzymatic and chemical methods. However, the productivity of the aspartame intermediate by SAET was approximately one-fifth that of l-alanyl-l-glutamine (Ala-Gln), whose production method has already been established. Here, to improve the enzymatic activity of SAET, we performed random mutagenesis in the gene encoding SAET and obtained 10 mutations that elevated the enzymatic activity (1.2- to 1.7-fold increase) relative to that of wild-type SAET. To further improve the activity, we performed mutagenesis to optimize the combination of the obtained mutations and finally selected one SAET variant with 10 amino acid substitutions (M35-4 SAET). An Escherichia coli strain overexpressing M35-4 SAET displayed a 5.7-fold higher activity than that of the wild-type SAET, which was almost equal to that of Ala-Gln by an E. coli strain overexpressing wild-type SAET. The Vmax value of M35-4 SAET was 2.0-fold greater, and its thermostability was higher than those of wild-type SAET. These results suggest that the obtained SAET variants contribute to improvement in aspartame production.

A novel route for aspartame production by combining enzymatic and chemical reactions for industrial use.

Here, we report a novel industrial aspartame production route, involving the enzymatic production of α-l-aspartyl-l-phenylalanine ß-methylester from l-aspartic acid dimethylester and l-phenylalanine by α-amino acid ester acyl transferase. The route also involves the chemical transformation of α-l-aspartyl-l-phenylalanine ß-methylester to α-l-aspartyl-l-phenylalanine methylester hydrochloride (aspartame hydrochloride) in an aqueous solution with methanol and HCl, followed by HCl removal to form aspartame.

Sensibility Assessment For User Interface And Training Program Of An Upper-Limb Rehabilitation Robot, D-SEMUL.

Upper-limb rehabilitation training for hemiplegic patients has been primarily conducted by human therapists, and, hence, their use of training methods and conditions strongly depends on their expertise. The force control and motion sensing functions of rehabilitation robots are expected to be used for the qualitative training/assessment in the next-generation computerized rehabilitation. In this study, we developed a desktop rehabilitation robot for upper limbs (D-SEMUL). In addition, we also assessed the usability of its user interface and the affinity (acceptance) of the training program with a questionnaire for elderly hemiplegic/non-hemiplegic participants (nine hemiplegic, five males and four females and seven non-hemiplegic, two males and five females). The results indicated that the touchscreen is acceptable for the user interface, and the background music used significantly affects the affinity of the program.

The influence of bone cement and American Society of Anesthesiologists (ASA) class on cardiovascular status during bipolar hemiarthroplasty for displaced femoral-neck fracture: A multicenter, prospective, case-control study.

BACKGROUND: Little is known about how bone cement and American Society of Anesthesiologists (ASA) classification influence the cardiovascular system in elderly patients with femoral-neck fractures treated with cemented hemiarthroplasty. Therefore, we performed a case-control study to investigate these questions and compared the following:≥ASA III with≤ASA II patients who underwent cemented hemiarthroplasty; and cemented with cementless hemiarthroplasty in≥ASA III patients. HYPOTHESIS: ASA classification influences the cardiovascular system during cemented hemiarthroplasty and bone cement influences intraoperative blood pressure [IBP] in patients rated≥ASA III. MATERIALS AND METHODS: This multicenter, prospective study included patients with acute displaced femoral-neck fractures. Baseline data, medical history, anesthesia, FiO2, vasopressor use, femoral component, IBP, SpO2, and complications were evaluated. Of 200 patients, 100 were cemented (mean age, 77±10 years), and 100 were cementless (mean age, 78±9 years). Cemented hemiarthroplasty employed a third-generation technique (plugging, irrigating, drying and filling the canal with cement under pressurization). RESULTS: Systolic blood pressure (SBP) decreased significantly during cementing, versus pre-rasping in≤ASA II patients (from 117.9±24.5 [range, 65-199] to 106.9±20.3 [range, 59-172]; p=0.007), in≥ASA III patients (from 129.5±21.0 [range, 90-169] to 110.4±17.9 [range, 79-157]; p=0.006), and post-stem-insertion, versus pre-rasping in≤ASA II patients (from 117.9±24.5 [range, 65-199] to 103.9±20.7 [range, 53-178]; p=0.0004), and in≥ASA III patients (from 129.5±21.0 [range, 90-169] to 111.2±24.6 [range, 70-156]; p=0.009). In≥ASA III patients, SBP decreased significantly during cementing or rasping, versus pre-rasping in cemented patients (from 129.5±21.0 [range, 90-169] to 110.4±17.9 [range, 79-157]; p=0.006), in cementless patients (from 115.0±17.7 [range, 85-150] to 100.7±15.7 [range, 75-142]; p=0.004), and post-stem-insertion, versus pre-rasping in cemented patients (from 129.5±21.0 [range, 90-169] to 111.2±SD [range]; p=0.009), and in cementless patients (from 115.0±17.7 [range, 85-150] to 89.4±17.5 [range, 58-140]; p<0.0001). There were no lethal complications. CONCLUSIONS: This study indicate a similar hemodynamic change intraoperatively between≤ASA II patients and≥ASA III patients in the cemented group, and between patients with cemented and cementless hemiarthroplasty in the≥ASA III patients. With modern hemiarthroplasty techniques, bone cement might be as safe as cementless techniques in elderly,≥ASA III patients. LEVEL OF EVIDENCE: III, multicenter case-control cohort study.

Ankle-foot orthosis using elastomer-embedded flexible joint.

We proposed a new ankle-foot orthosis using elastomer-embedded flexible joints (EEFJ), composed of C-shaped springs and 3D-printed circular elastomer. This orthosis was designed to reduce burden on the tibialis anterior muscle (TA) and to achieve clearance between the tip of the toe and the ground. Strength testing, and gait analysis were conducted for the orthosis. According to the results of strength testing, the combination of the C-spring with 0.3 mm and 0.5 mm thickness and the elastomer with 30% and 60% filling density performs a supporting torque of 0.7-2.3 Nm to plantarflexion. In contrast, torques in the other directions were relatively small. According to the results of gait experiments in seven healthy young subjects, the proposed orthosis successfully reduced the TA activation on initial contact and in the swing phase, and range of motion on initial contact.

Upper limb training/assessment program using passive force controllable rehabilitation system.

Upper limb rehabilitation training for Hemiplegie patients has been conducted mainly by human therapists. Training methods and conditions depend strongly on their experience because of the wide range of individual differences between patients. The force control and sensing functions of rehabilitation robots are expected to be used for the qualitative assessment of next-generation computational rehabilitation. In this study, we developed a simple exercise machine for upper limbs (SEMUL) using an electro-rheological fluid brake, as a safe brake-type robotic rehabilitation system. We also developed a new upper limb training/assessment program, called "WIPE," for the SEMUL. Furthermore, we conducted clinical tests on twelve subjects (six each for the SEMUL training and for general training). We adopted the ABA design methodology for the clinical research tests. The motor functions of upper limbs were assessed using five clinical scores: Fugl-Meyer Assessment (FMA), Brunnstrome recovery stage, Simple Test for Evaluating Hand Function (STEF), Stroke Impairment Assessment Set (SIAS), and Motor Activity Log. Improvements of the FMA, STEF, and SIAS in the SEMUL training groups were found to be higher than those in the general training groups.

Novel ankle orthosis with elastomer-embedded flexible joint.

In this study, we propose a new ankle orthosis with elastomer-embedded flexible joints (EEFJ), composed of C-shaped springs and 3D-printed circular elastomer. This orthosis was designed to reduce burden on the tibialis anterior muscle (TA) and to achieve clearance between the tip of the toe and the ground. Fabrication method, strength testing, and gait analysis were conducted. According to the results of strength testing, the combination of the C-spring with 0.3 mm and 0.5 mm thickness and the elastomer with 30% and 60% filling density performs a supporting torque of 0.7-2.3 Nm to plantarflexion. In contrast, torques in the other directions were relatively small. According to the results of gait experiments in seven healthy young subjects, the proposed orthosis successfully reduced activation of TA on initial contact and in the swing phase, and range of motion on initial contact.

Spinal anesthesia increases the risk of venous thromboembolism in total arthroplasty: Secondary analysis of a J-PSVT cohort study on anesthesia.

Clinical guidance on the choice of anesthetic modality vis-à-vis the risk of perioperative venous thromboembolism (VTE) is largely lacking because of a paucity of recent evidence. A comparative effect of general anesthesia and neuraxial blockade on the perioperative incidence of VTE has not been well-investigated.We compared the effects of different types of anesthetic modalities on the risk of VTE after total hip arthroplasty (THA) and total knee arthroplasty (TKA).This is a secondary analysis of the Japanese Study of Prevention and Actual Situation of Venous Thromboembolism after Total Arthroplasty (J-PSVT). Data pertaining to a total of 2162 patients who underwent THA and TKA at 34 hospitals were included in this analysis. We compared the different anesthetic modalities with respect to the incidence of VTE. The composite end-point was asymptomatic/symptomatic deep vein thrombosis detected using scheduled bilateral ultrasonography up to postoperative day (POD) 10 and fatal/non-fatal pulmonary embolism up to POD 10.The study groups were as follows: general anesthesia (n = 646), combined epidural/general anesthesia (n = 1004), epidural anesthesia (n = 87), and spinal anesthesia (n = 425). On multivariate analysis, only spinal anesthesia was associated with a significant increase in the risk of VTE as compared with that associated with general anesthesia. Propensity score-matched analysis for "combined epidural/general anesthesia group" versus "spinal anesthesia group" demonstrated a 48% higher incidence of VTE (relative risk = 1.48, 95% confidence interval [CI] 1.18-1.85) in the latter.Spinal anesthesia was associated with a higher risk of postoperative VTE, as compared with that associated with combined epidural/general anesthesia, in patients undergoing total arthroplasty.

Intraoperative blood pressure changes during cemented versus uncemented bipolar hemiarthroplasty for displaced femoral neck fracture: a multi-center cohort study : The effect of bone cement for bipolar hemiarthroplasty in elderly patients.

INTRODUCTION: The purpose of this study was to compare the cemented bipolar hemiarthroplasty with uncemented about the change of intraoperative blood pressure and the incidence of major complications in elderly patients with femoral neck fracture. MATERIALS AND METHODS: This multiple center prospective cohort study included only patients with acute displaced femoral neck fracture (Garden stage III or IV). All patients were treated with cemented or uncemented bipolar hemiarthroplasty using modified Hardinge or Watson-Jones approach in the lateral decubitus position. Baseline data, medical history, type of anesthesia, FiO2 value, the number of vasopressor using during operation, femoral component, intraoperative blood pressure, SaO2, and major complications were evaluated. RESULTS: Of 164 patients (45 males and 119 females), 86 underwent cemented and 78 underwent uncemented bipolar hemiarthroplasty. Baseline medical histories were similar in both groups. In both the cemented and uncemented groups, intraoperative systolic blood pressure significantly decreased during cementing or rasping (106.3 and 103.6 mmHg) and after femoral component insertion (103.3 and 99.1 mmHg) compared to before rasping (120.7 and 116.4 mmHg) (p < 0.0001, respectively). Donaldson's grade seemed more favorable in uncemented group than in cemented group during cementing or rasping, during stem insertion; however, no patients experienced the lethal complication in both groups. CONCLUSIONS: Intraoperative blood pressure did not change during cemented and uncemented bipolar hemiarthrplasty for displaced femoral neck fracture. If the standard modern cement technique was performed during operation, bone cement is a safe and acceptable for elderly patients who have a lot of medical histories.

Blood pressure control status and relationship between salt intake and lifestyle including diet in hypertensive outpatients treated at a general hospital.

The purpose of the present study was to investigate blood pressure (BP) control and salt intake in hypertensive outpatients treated at a general hospital and to examine the relationship between their lifestyles and amount of salt intake. Subjects comprised 429 hypertensive patients (206 males, 223 females, and average age of 71 ± 11 years). We estimated 24-hour salt excretion using spot urine samples and assessed lifestyle using a self-description questionnaire. Average clinic BP and the number of antihypertensive drugs were 132 ± 11/73 ± 8 mmHg and 1.8 ± 0.9, respectively. In all subjects, average estimated salt intake was 9.2 ± 2.8 g/day and the rate of achievement of the estimated salt intake of <6 g/day was 11.2%. In patients with chronic kidney disease or cardiovascular disease, these values were 8.6 ± 2.6 g/day and 15.5%, and 9.1 ± 3.3 g/day and 18.2%, respectively. Estimated salt intake was lower in patients living alone than in those with a family. In a multivariate analysis, estimated salt intake correlated positively with body mass index and negatively with age. Among patients with an excessive salt intake (≥10 g/day), 75.2% answered that they made an effort to reduce their salt intake. The amount of food and processed food consumption correlated with estimated salt intake. In conclusion, the rate of achievement of salt restriction was low in hypertensive patients treated at a general hospital. It may be important to provide data on actual salt intake and guide salt restriction in the individual management of hypertension.

Venous thromboembolism after total joint arthroplasty: results from a Japanese multicenter cohort study.

INTRODUCTION: Real-world evidence of the effectiveness of pharmacological thromboprophylaxis for venous thromboembolism (VTE) is limited. Our objective was to assess the effectiveness and safety of thromboprophylactic regimens in Japanese patients undergoing joint replacement in a real-world setting. METHOD: Overall, 1,294 patients (1,073 females and 221 males) who underwent total knee arthroplasty (TKA) and 868 patients (740 females and 128 males) who underwent total hip arthroplasty (THA) in 34 Japanese national hospital organization (NHO) hospitals were enrolled. The primary efficacy outcome was the incidence of deep vein thrombosis (DVT) detected by mandatory bilateral ultrasonography up to post-operative day (POD) 10 and pulmonary embolism (PE) up to POD28. The main safety outcomes were bleeding (major or minor) and death from any cause up to POD28. RESULTS: Patients undergoing TKA (n = 1,294) received fondaparinux (n = 360), enoxaparin (n = 223), unfractionated heparin (n = 72), anti-platelet agents (n = 45), or no medication (n = 594). Patients undergoing THA (n = 868) received fondaparinux (n = 261), enoxaparin (n = 148), unfractionated heparin (n = 32), anti-platelet agents (n = 44), or no medication (n = 383). The incidence rates of sonographically diagnosed DVTs up to POD10 were 24.3% in patients undergoing TKA and 12.6% in patients undergoing THA, and the incidence rates of major bleeding up to POD28 were 1.2% and 2.3%, respectively. Neither fatal bleeding nor fatal pulmonary embolism occurred. Significant risk factors for postoperative VTE identified by multivariate analysis included gender (female) in both TKA and THA groups and use of a foot pump in the TKA group. Only prophylaxis with fondaparinux reduced the occurrence of VTE significantly in both groups. Propensity score matching analysis (fondaparinux versus enoxaparin) showed that the incidence of DVT was lower (relative risk 0.70, 95% confidence interval (CI) 0.58 to 0.85, P = 0.002 in TKA and relative risk 0.73, 95% CI 0.53 to 0.99, P = 0.134 in THA) but that the incidence of major bleeding was higher in the fondaparinux than in the enoxaparin group (3.4% versus 0.5%, P = 0.062 in TKA and 4.9% versus 0%, P = 0.022 in THA). CONCLUSIONS: These findings indicate that prophylaxis with fondaparinux, not enoxaparin, reduces the risk of DVT but increases bleeding tendency in patients undergoing TKA and THA. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trials Registry: UMIN000001366. Registered 11 September 2008.

Predictive factors for acetabular labral lesions in osteoarthritis of the hip with radial magnetic resonance imaging--a cross-sectional study.

OBJECTIVES: To document the reliability of Abe's classification and to clarify the predictive factors for acetabular labral lesions in osteoarthritis of the hip with radial magnetic resonance (MR) imaging. METHODS: Reliability trial for the classification of acetabular labral lesion was performed by six orthopedic surgeons, grading 20 radial MR images in a blinded fashion at an interval of 4-5 weeks. Radial MR images of 275 hips in 263 patients were prospectively analyzed to determine the relationship between acetabular labral lesions, their distribution, age, and the acetabular coverage. RESULTS: Cohen's quadratic weighted kappa of inter-observer reliability was 0.784 for the grade and 0.812 for the shape category. The weighted kappa of intra-observer reliability was 0.852 for the grade and 0.90 for the shape category. Multiple regression analysis revealed that both the grade and the shape were associated with age, acetabular coverage, and location of the labrum. CONCLUSIONS: Abe's classification of labral lesions was reliable for both the grade and shape categories. Aging, acetabular dysplasia, and the anterosuperior portion would be predictive factors for degeneration of the acetabular labrum using radial MR imaging.

Enzymatic production of L-alanyl-L-glutamine by recombinant E. coli expressing α-amino acid ester acyltransferase from Sphingobacterium siyangensis.

An enzymatic production method for synthesizing L-alanyl-L-glutamine (Ala-Gln) from L-alanine methyl ester hydrochloride (AlaOMe) and L-glutamine (Gln) was developed in this study. The cultivation conditions for an Escherichia coli strain overexpressing α-amino acid ester acyltransferase from Sphingobacterium siyangensis AJ 2458 (SAET) and reaction conditions for Ala-Gln production were optimized. A high cell density culture broth prepared by fed-batch cultivation showed 440 units/mL of Ala-Gln-producing activity. In addition, an Ala-Gln-producing reaction using intact E. coli cells overexpressing SAET under optimum conditions was conducted. A total Ala-Gln yield of 69.7 g/L was produced in 40 min. The molar yield was 67% against both AlaOMe and Gln.

Gene cloning and characterization of α-amino acid ester acyl transferase in Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458.

The gene encoding α-amino acid ester acyl transferase (AET), the enzyme that catalyzes the peptide-forming reaction from amino acid methyl esters and amino acids, was cloned from Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458 and expressed in Escherichia coli. This is the first report on the aet gene. It encodes a polypeptide composed of 616 (ATCC14234) and 619 (AJ2458) amino acids residues. The V(max) values of these recombinant enzymes during the catalysis of L-alanyl-L-glutamine formation from L-alanine methylester and L-glutamine were 1,010 U/mg (ATCC14234) and 1,154 U/mg (AJ2458). An amino acid sequence similarity search revealed 35% (ATCC14234) and 36% (AJ2458) identity with an α-amino acid ester hydrolase from Acetobacter pasteurianus, which contains an active-site serine in the consensus serine enzyme motif, GxSYxG. In the deduced amino acid sequences of AET from both bacteria, the GxSYxG motif was conserved, suggesting that AET is a serine enzyme.

Improvement of blood pressure control in a hypertension clinic in Japan: a 15-year follow-up study.

Many guidelines recommended strict blood pressure (BP) control to prevent cardiovascular events. However, BP control in a substantial majority of hypertensives remains to be insufficient. We have determined the trends of BP control of the same patients during 15 years in a hypertension clinic. One hundred three patients (age 32-91, mean 68 +/- 11 years in 2006), who were followed at our hypertension clinic between 1991-2006, were retrospectively investigated. We compared the clinical characteristics of the patients in 2006 to those in 1991, 1996, and 2001, using the averaged BP determined at two occasions of each year for our analysis. The average BP decreased from 144 +/- 17/87 +/- 10 mmHg to 132 +/- 12/75 +/- 10 mmHg (p < 0.01) during the 15 years between 1991 and 2006. When good BP control was defined as < 140/90 mmHg, the rate of patients with good BP control increased from 35% in 1991 to 45% in 1996, to 54% in 2001 (p < 0.01 vs. 1991), and to 72% in 2006 (p < 0.01 vs. 1991). The number of anti-hypertensive drugs used in 2006 significantly increased compared to those in 1991, 1996, and 2001. More specifically, the use of diuretics and alpha-blockers increased significantly during this period. Results suggest that BP control improved in the 15 years studied, and the increased use of the anti-hypertensive drugs, as well as the increased awareness of the importance of strict BP control, seems to have contributed to improve the BP control.

Increased expression of gp91phox homologues of NAD(P)H oxidase in the aortic media during chronic hypertension: involvement of the renin-angiotensin system.

Although vascular cells express multiple members of the Nox family of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase, including gp91phox, Nox1, and Nox4, the reasons for the different expressions and specific roles of these members in vascular injury in chronic hypertension have remained unclear. Thus, we quantified the mRNA expressions of these NAD(P)H oxidase components by real-time polymerase chain reaction and evaluated superoxide production and morphological changes in the aortas of 32-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wistar Kyoto rats (WKY). The aortic media of SHRSP had an approximately 2.5-fold greater level of Nox4 mRNA and an approximately 10-fold greater level of Nox1 mRNA than WKY. The mRNA expressions of gp91phox and p22phox in SHRSP and WKY were comparable. SHRSP were treated from 24 weeks of age for 8 weeks with either high or low doses of candesartan (4 mg/kg/day or 0.2 mg/kg/day), or a combination of hydralazine (30 mg/kg/day) and hydrochlorothiazide (4.5 mg/kg/day). The high-dose candesartan or the hydralazine plus hydrochlorothiazide decreased the blood pressure of SHRSP to that of WKY, whereas the low-dose candesartan exerted no significant antihypertensive action. Media thickening and fibrosis, as well as the increased production of superoxide in SHRSP, were nearly normalized with high-dose candesartan and partially corrected with low-dose candesartan or hydralazine plus hydrochlorothiazide. These changes by antihypertensive treatment paralleled the decrease in mRNA expression of Nox4 and Nox1. These results suggest that blood pressure and angiotensin II type 1 receptor activation are involved in the up-regulation of Nox1 and Nox4 expression, which could contribute to vascular injury during chronic hypertension.

Effects of fluvastatin on endothelium-derived hyperpolarizing factor- and nitric oxide-mediated relaxations in arteries of hypertensive rats.

Endothelial cells release endothelium-derived hyperpolarizing factor (EDHF), as well as nitric oxide (NO). It has recently been suggested that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) improve NO-mediated endothelial function, partially independently of their cholesterol-lowering effects. It is, however, unclear whether statins improve EDHF-mediated responses. Eight-month-old stroke-prone spontaneously hypertensive rats (SHRSP) were treated with fluvastatin (10 mg/kg per day) for 1 month. Age-matched, normotensive Wistar Kyoto (WKY) rats served as controls. Both EDHF- and NO-mediated relaxations were impaired in SHRSP compared with WKY rats. Fluvastatin treatment did not affect blood pressure and serum total cholesterol. The acetylcholine (ACh)-induced, EDHF-mediated hyperpolarization in mesenteric arteries did not significantly differ between fluvastatin-treated SHRSP and untreated SHRSP and the responses in both groups were significantly smaller compared with those of WKY rats. Endothelium-derived hyperpolarizing factor-mediated relaxations, as assessed by the relaxation to ACh in mesenteric arteries contracted with noradrenaline in the presence of N(G)-nitro-l-arginine and indomethacin, were virtually absent and similar in both SHRSP groups. In contrast, NO-mediated relaxation, as assessed by the relaxation in response to ACh in rings contracted with 77 mmol/L KCl, was improved in fluvastatin-treated SHRSP compared with untreated SHRSP (maximum relaxation in control and fluvastatin groups 42.0 +/- 5.2 and 61.2 +/- 3.8%, respectively; P < 0.05). Hyperpolarization and relaxation in response to levcromakalim, an ATP-sensitive K(+) channel opener, were similar between the two SHRSP groups. These findings suggest that fluvastatin improves NO-mediated relaxation, but not EDHF-mediated hyperpolarization and relaxation, in SHRSP. Thus, the beneficial effects of the statin on endothelial function may be mainly ascribed to an improvement in the NO pathway, but not EDHF.

Angiotensin II receptor blockade corrects altered expression of gap junctions in vascular endothelial cells from hypertensive rats.

Blockade of the renin-angiotensin system improves the impaired endothelium-dependent relaxations associated with hypertension and aging, partly through amelioration of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses. Although the nature of EDHF is still controversial, recent studies have suggested the involvement of gap junctions in EDHF-mediated responses. Gap junctions consist of connexins (Cx), and we therefore tested whether the expression of Cx in vascular endothelial cells would be altered by hypertension and antihypertensive treatment. Spontaneously hypertensive rats (SHR) were treated with either the angiotensin II type 1 receptor antagonist candesartan or the combination of hydralazine and hydrochlorothiazide for 3 mo from 5 to 8 mo of age. Confocal laser scanning microscopy after immunofluorescent labeling with antibodies against Cx37, Cx40, and Cx43 revealed that the expression of Cx37 and Cx40 in endothelial cells of the mesenteric artery was significantly lower in SHR than in WKY. Treatment with candesartan, but not the combination of hydralazine and hydrochlorothiazide, significantly increased the expression of Cx37 and Cx40, although blood pressure decreased similarly. On the other hand, the expression of Cx43, though scarce and heterogeneous, was increased in SHR compared with WKY, and candesartan treatment lowered the expression of Cx43. These findings suggest that renin-angiotensin system blockade corrects the decreased expression of Cx37 and Cx40 in arterial endothelial cells of hypertensive rats, partly independently of blood pressure, whereas the expression of Cx43 changed in the opposite direction. It remains to be clarified whether these changes in Cx37 and Cx40 are related to endothelial function, particularly that attributable to EDHF.

Risk factors for the progression of early carotid atherosclerosis in a male working population.

Increased intima-media thickness (IMT) of the carotid artery may represent early atherosclerosis. Although several studies have evaluated risk factors for carotid IMT, only limited information is available concerning risk factors for the progression of carotid IMT. The present study was designed to determine risk factors for the progression of carotid IMT in a male working population. Male employees of a regional transport company (n = 220, 50.9 +/- 4.4 years) underwent baseline physical and laboratory examinations, and ultrasonographic assessment of the maximum common carotid IMT between 1992 and 1994, and they were reexamined 5 years later. In a multivariate analysis at baseline, carotid IMT was positively associated with age, diastolic blood pressure and total cholesterol, and negatively with high density lipoprotein (HDL) cholesterol. During the follow-up period, carotid IMT increased from 0.669 +/- 0.135 mm to 0.784 +/- 0.229 mm, or at a rate of 0.023 +/- 0.039 mm/year. In a multivariate regression analysis using baseline values of carotid IMT, age, body mass index, diastolic blood pressure, total cholesterol, HDL cholesterol and HbA1c as independent variables, the progression of carotid IMT was associated only with baseline total cholesterol. These findings suggest that in middle-aged men, although age, blood pressure, and total cholesterol were associated with baseline carotid IMT, total cholesterol level appeared to be the strongest determinant of the progression of carotid IMT, a result which underscores the importance of maintaining lower cholesterol levels to prevent early atherosclerosis.