A 10-year longitudinal study of deep white matter lesions on magnetic resonance imaging.

PURPOSE: Deep white matter lesions (DWMLs), T2 high-intensity areas in the subcortical white matter on magnetic resonance imaging (MRI), are a clinical phenotype of cerebral small vessel disease. Factors such as age and hypertension have been reported to significantly contribute to the presence and severity of DWMLs in cross-sectional studies. We herein report a 10-year longitudinal study on DWMLs in elderly Japanese subjects to reveal the clinical variables contributing to the progression of DWMLs. METHODS: A total of 469 Japanese subjects were invited to participate in the study. Of the participants at baseline, 259 subjects completed the revisit MRI study 10 years later. In those 259 subjects, we evaluated the correlation between the progression of DWMLs and clinical variables, such as the gender, age, and overt vascular risk factors. To clarify the role of hypertension, 200 subjects with grade 1 DWMLs at baseline were categorized into three groups according to their status of hypertension and its treatment. RESULTS: Of the 200 subjects with grade 1 DWMLs, 47 subjects (23.5%) showed progression of DWMLs (progression group). In the progression group, the percentage of subjects with hypertension and the systolic blood pressure values were higher than in the non-progression group. In addition, subjects ≥ 60 years old at baseline tended to show deterioration of DWMLs in the group with hypertension without antihypertensive treatment. CONCLUSION: The results of this 10-year longitudinal study imply a positive correlation between long-standing hypertension and the progression of DWMLs.

Serum albumin to globulin ratio is related to cognitive decline via reflection of homeostasis: a nested case-control study.

BACKGROUND: Recent research suggests that several pathogenetic factors, including aging, genetics, inflammation, dyslipidemia, diabetes, and infectious diseases, influence cognitive decline (CD) risk. However, no definitive candidate causes have been identified. The present study evaluated whether certain serum parameters predict CD. METHODS: A total of 151 participants were assessed for CD using the Mini-Mental State Examination (MMSE), and 34 participants were identified as showing CD. RESULTS: Among CD predictive risk factors, Helicobacter pylori seropositivity was significantly predictive of CD risk, more so than classical risk factors, including white matter lesions and arterial stiffness [adjusted odds ratio (OR) = 4.786, 95% confidence interval (CI) = 1.710-13.39]. A multivariate analysis indicated that the albumin to globulin (A/G) ratio was the only factor that significantly lowered CD risk (OR = 0.092, 95% CI = 0.010-0.887). A/G ratio also was positively correlated with MMSE scores and negatively correlated with disruption of homeostatic factors (i.e., non-high-density lipoprotein, hemoglobin A1c, and high-sensitive C-reactive protein). CONCLUSIONS: The current study results suggest that the A/G ratio is related to cognitive decline and may reflect homeostatic alterations.

A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood.

BACKGROUND: Personalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis; however, the technical difficulties in isolating and analyzing rare CTCs have limited progress to date. In this preclinical study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology. METHODS: We developed a cell capture platform using On-chip Sort (On-Chip Biotechnologies), a novel bench-top cell sorter equipped with a disposable microfluidic chip. Spike-in experiments comprising a series of lung cancer cell lines with varying epithelial cell adhesion molecule (EpCAM) expression levels were conducted to assess the capture and purification efficiency of the platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by sample fixation and labeling. The enriched and labeled samples were then sorted by On-chip Sort based on cytokeratin, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification followed by mutation analysis using deep targeted sequencing, and copy number analysis using quantitative polymerase chain reaction (qPCR). RESULTS: Spike-in experiments revealed an excellent overall mean capture rate of 70.9%. A 100% success rate in the detection of EGFR, KRAS and BRAF mutations from captured cells was achieved using pyrosequencing and deep sequencing. The mutant variant detection rates were markedly higher than those obtained with the CellSearch profile kit. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number changes of the EGFR in captured tumor cells. CONCLUSIONS: Using a novel cell sorter, we established an efficient and convenient platform for the capture of CTCs. Results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Multicolor detection of rare tumor cells in blood using a novel flow cytometry-based system.

The presence and number of circulating tumor cells (CTCs) in the blood of patients with solid tumors are predictive of their clinical outcomes. To date, the CellSearch system is the only US Food and Drug Administration-approved CTC enumeration system for advanced breast, prostate, and colon cancers. However, sensitivity issues due to epithelial cellular adhesion molecule (EpCAM)-based enrichment and limited capability for subsequent molecular analysis must be addressed before CTCs can be used as predictive markers in the clinical setting. We have developed a multicolor CTC detection system using cross-contamination-free flow cytometry, which permits the enumeration and characterization of CTCs for multiple molecular analyses. Tumor cell lines with different expression levels of EpCAM were spiked into peripheral blood obtained from healthy donors. Spike-in samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, and this was followed by fixation and labeling with CD45-Alexa Fluor 700, EpCAM-phycoerythrin, cytokeratin (CK)-fluorescein isothiocyanate antibodies, and/or 7-aminoactinomycin D for nuclei staining. Excellent detection (slope = 0.760-0.888) and a linear performance (R(2) = 0.994-0.998) were noted between the observed and expected numbers of tumor cells, independent of EpCAM expression. The detection rate was markedly higher than that obtained using the CellSearch system, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Additionally, the incorporation of an epithelial-mesenchymal transition (EMT) marker allowed us to detect EpCAM-/CK- cells and EMT-induced tumor cells. Taken together, our multicolor CTC detection system may be highly efficient in detecting previously unrecognized populations of CTCs.

Risk of cancer in patients with autoimmune pancreatitis.

OBJECTIVES: Although simultaneous occurrences of autoimmune pancreatitis (AIP) and cancer are occasionally observed, it remains largely unknown whether cancer and AIP occur independently or these disorders are interrelated. The aim of this study was to examine the relationship between AIP and cancer. METHODS: We conducted a multicenter, retrospective cohort study. One hundred and eight patients who met the Asian diagnostic criteria for AIP were included in the study. We calculated the proportion, standardized incidence ratio (SIR), relative risk, and time course of cancer development in patients with AIP. We also analyzed the clinicopathological characteristics of AIP patients with cancer in comparison with those without cancer. RESULTS: Of the 108 AIP patients, 18 cancers were found in 15 patients (13.9%) during the median follow-up period of 3.3 years. The SIR of cancer was 2.7 (95% confidence interval (CI) 1.4-3.9), which was stratified into the first year (6.1 (95% CI 2.3-9.9)) and subsequent years (1.5 (95% CI 0.3-2.8)) after AIP diagnosis. Relative risk of cancer among AIP patients at the time of AIP diagnosis was 4.9 (95% CI 1.7-14.9). In six of eight patients whose cancer lesions could be assessed before corticosteroid therapy for AIP, abundant IgG4-positive plasma cell infiltration was observed in the cancer stroma. These six patients experienced no AIP relapse after successful cancer treatment. CONCLUSIONS: Patients with AIP are at high risk of having various cancers. The highest risk for cancer in the first year after AIP diagnosis and absence of AIP relapse after successful treatment of the coexisting cancers suggest that AIP may develop as a paraneoplastic syndrome in some patients.

Real-time cell viability assays using a new anthracycline derivative DRAQ7®.

The exclusion of charged fluorescent dyes by intact cells has become a well-established assay for determining viability of cells. In search for a noninvasive fluorescent probe capable of long-term monitoring of cell death in real-time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be nontoxic to a panel of cancer cell lines grown continuously for up to 72 h and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. The DRAQ7 provided a sensitive, real-time readout of cell death induced by a variety of stressors such as hypoxia, starvation, and drug-induced cytotoxicity. The overall responses to anticancer agents and resulting pharmacological dose-response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real-time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨ(m) ) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multiparameter and kinetic fingerprint of anticancer drug action.

High-purity Isolation for Genotyping Rare Cancer Cells from Blood Using a Microfluidic Chip Cell Sorter.

BACKGROUND/AIM: A multistep sorting method for enrichment of rare cells, such as circulating tumor cells, in the blood without cumbersome pretreatments required by most flow cytometry-based methods, which lead to high cost and decreased detection efficiency, was developed. MATERIALS AND METHODS: After only hemolysis and cell staining, cancer cells are enriched by repetitive sorting (3×) based on nuclear-positive, cytokeratin-positive, and CD45-negative expression. RESULTS: Experiments using spikes of PC-9 cells showed a mean recovery of 65% and mean purity of 83%, which was retained up to 72 hours after blood draw using preservative tubes. Significant differences in expression level of programmed death-ligand 1 or vimentin were observed between high- and low-expressing cell lines, concurrently with enrichment. Next-generation sequencing analysis of recovered PC-9, A549, and MDA-MB231 cells successfully detected all known mutations. CONCLUSION: This novel isolation method applicable for preserved samples with sufficient recovery and purity may be substantially beneficial for recovering cells for subsequent molecular analysis.

Enumeration, characterization, and collection of intact circulating tumor cells by cross contamination-free flow cytometry.

Circulating tumor cells (CTC) are an important biomarker for several solid cancers. Most of the commercially available systems for enumeration of CTC are based on immunomagnetic enrichment of epithelial cell adhesion molecule (EpCAM/CD326)-expressing CTC before microscopic cell imaging or reverse-transcription PCR (RT-PCR). The aim of this study was to establish a practical method for enumeration of CTC using a novel flow cytometer that has a disposable microfluidic chip, which is designed to realize absolute cross contamination-free measurements and to collect the analyzed cell sample. Although the process of enumeration and labeling of CTC was optimized for this device, the simplified protocol described here could be applied to other flow cytometers. Cultured cancer cells spiked into normal blood were enriched using MACS® EpCAM-MicroBeads following cell labeling with an allophycocyanin (APC)-conjugated EpCAM mAb, instead of by intracellular staining of cytokeratins (CK). The EpCAM double-positive selection/labeling method allows enumeration of intact CTC, maintenance of cellular integrity, and the concomitant performance of a CTC viability test. The combination of the fine-tuned CTC enrichment process and the cytometric multicolor analysis resulted in a linear relationship between the output cell count and the input cell number from zero to hundreds of cells. In particular, a satisfactory signal/noise ratio was obtained by gate-exclusion of leukocyte signals using an anti-CD45 mAb. The entire process had little influence on the viability of the spiked lung cancer cell PC-9. Measured PC-9 and breast cancer MCF-7 cells bearing EpCAM-MicroBeads, APC-conjugated EpCAM mAb, and the DNA staining dye SYTO9 grew normally, demonstrating the potential usefulness of the collected samples for further studies. This intact CTC enumeration and analysis procedure (iCeap) would be of great benefit to clinicians by providing them with rapid stratification of antitumor therapy, and to basic researchers by permitting further molecular and cellular characterization of CTC.

Activities of occupational physicians for occupational health services in small-scale enterprises in Japan and in the Netherlands.

OBJECTIVES: Occupational health service (OHS) for small-scale enterprises (SSEs) is still limited in many countries. Both Japan and the Netherlands have universal OHS systems for all employees. The objective of this survey was to examine the activities of occupational physicians (OPs) in the two countries for SSEs and to investigate their proposals for the improvement of service. METHODS: Questionnaires on types and sizes of the industries they serve, allocation of service hours (current and desired), sources of information for occupational health activities etc. were mailed in 2006 to 461 and 335 Japanese and Dutch OPs, respectively, who have served in small- and medium-scale enterprises. In practice, 107 Japanese (23%) and 106 Dutch physicians (32%) replied, respectively. RESULTS AND CONCLUSIONS: Total service time per month was longer for OPs in the Netherlands than OPs in Japan. Japanese OPs spent more hours for health and safety meetings, worksite rounds, and prevention of overwork-induced ill health (14-16% each). Dutch OPs used much more hours for the guidance of absent workers (48%). Thus, service conditions were not the same for OPs in the two countries. Nevertheless, both groups of OPs unanimously considered that employers are the key persons for the improvement of OHS especially in SSEs and their education is important for better OHS. The conclusions should be taken as preliminary, however, due to study limitations including low response rates in both groups of physicians.

Genetic factors associated with elevation of uric acid after treatment with thiazide-like diuretic in patients with essential hypertension.

We investigated changes in blood pressure (BP) and metabolic adverse effects, especially elevation of uric acid (UA), after treatment with a thiazide-like diuretic (TD) in patients with essential hypertension. Furthermore, the role of genetic factors in the elevation of UA by TD was assessed by a 500 K SNP DNA microarray. The subjects included 126 hypertensive patients (57 women and 69 men, mean age 59 ± 12 years) who registered for the GEANE (Gene Evaluation for ANtihypertensive Effects) study. After one month of the nontreatment period, TD, indapamide, angiotensin II receptor antagonist valsartan, and Ca channel blocker amlodipine were administered to all patients for 3 months each in a randomized crossover manner. BP, renal function, serum UA level, and electrolytes were measured at baseline and at the end of each treatment period. Single nucleotide polymorphisms (SNPs) associated with UA elevation after treatment with indapamide were investigated by a genome-wide association study (GWAS). Indapamide significantly decreased both office and home BP levels. Treatment with indapamide also significantly reduced the estimated glomerular filtration rate and serum potassium and increased serum UA. Patients whose UA level increased more than 1 mg/dl showed significantly higher baseline office SBP and plasma glucose and showed greater decline in renal function compared with those who showed less UA increase (<1 mg/dl). Some SNPs strongly associated with an increase in UA after treatment with indapamide were identified. This study is the first report on SNPs associated with UA elevation after TD treatment. This information may be useful for the prevention of adverse effects after treatment with TD.

COMPARISON OF COSMIC-RAY ENVIRONMENTS ON EARTH, MOON, MARS AND IN SPACECRAFT USING PHITS.

Estimation of cosmic-ray doses is of great importance not only in aircrew and astronaut dosimetry but also in evaluation of background radiation exposure to public. We therefore calculated the cosmic-ray doses on Earth, Moon and Mars as well as inside spacecraft, using Particle and Heavy Ion Transport code System PHITS. The same cosmic-ray models and dose conversion coefficients were employed in the calculation to properly compare between the simulation results for different environments. It is quantitatively confirmed that the thickness of physical shielding including the atmosphere and soil of the planets is the most important parameter to determine the cosmic-ray doses and their dominant contributors. The comparison also suggests that higher solar activity significantly reduces the astronaut doses particularly for the interplanetary missions. The information obtained from this study is useful in the designs of the future space missions as well as accelerator-based experiments dedicated to cosmic-ray research.

Association between α-Klotho and Deep White Matter Lesions in the Brain: A Pilot Case Control Study Using Brain MRI.

BACKGROUND: The anti-aging protein, α-Klotho, may be involved in cognitive decline and has potential as a surrogate marker that reflects dementia. However, the role of α-Klotho in the brain has not been sufficiently investigated. OBJECTIVE: Here, we investigated the association between α-Klotho and cognitive decline that is associated with cerebral deep white matter lesions (DWMLs). METHODS: Two hundred-eighty participants (187 males and 93 females, mean age: 70.8 years old) were evaluated for DWMLs, and the Fazekas scale (Grade) was assessed following brain magnetic resonance imaging. A questionnaire concerning lifestyle and neuropsychological tests was administered, and their associations with the blood α-Klotho level were retrospectively investigated. RESULTS: The α-Klotho level was 685.1 pg/mL in Grade 0 (68 subjects), 634.1 in G1 (134), 596.0 in G2 (62), and 571.6 in G3 (16), showing that the level significantly decreased with advanced grades. Significant correlations were noted between the α-Klotho level and higher brain function tests including the Mini-Mental State Examination and word fluency tests (p < 0.05). When a 90th percentile value of the level in the G0 group (400 pg/mL) or lower was defined as a low α-Klotho level, the odds ratio of the high-grade G3 group was 2.9 (95% confidence interval: 1.4-7.8) (after correction for age, sex, hypertension, and chronic kidney disease), which was significant. CONCLUSION: A reduced blood α-Klotho level was correlated with grading of cerebral DWMLs and was accompanied by cognitive decline as an independent risk factor. The α-Klotho level may serve as a useful clinical index of vascular cognitive impairment.

Possible beneficial effects of health counseling, given less frequently than ordinary, on blood pressure.

This study was initiated to investigate the effects of health counseling under Total Health Promotion Plan (THP). The study populations consisted of 1,655 working men who had an initial THP-based counseling and a follow-up 3 yr later (the THP group; the counseling was given on a once-three year basis), and 1,655 age- and body mass index (BMI)-matched controls (working men who had no health counseling; the control group). In the THP group, systolic blood pressure (SBP) and the prevalence of excessive alcohol drinking were decreased, and nutritional score was improved, although BMI was increased. In contrast, SBP, BMI and the ratio of excessive drinkers were all increased in the controls. Multiple regression analysis of the THP group showed that the reduction in SBP was positively associated with the increase in maximal oxygen consumption (VO(2max)) and physical activity scores, as well as the decrease in BMI. In sub-group analyses of the THP group, VO(2max) and physical activity scores were increased, and BMI did not change in the sub-group with marked BP decrease. In the sub-group with marked BP increase, however, BMI was increased, and VO(2max) was decreased. The ratio of excessive drinkers was reduced only in the sub-group with marked BP decrease. The obese-hypertensive subjects in the THP group showed decreases in BMI and BP, and increases in VO(2max). Thus, these results appeared to suggest that health counseling given even at a low frequency of once in three years prevented the age-associated increase in BP through improvement of physical endurance, decrease in alcohol intake and reduction in body weight.

Association between Mid-Regional Proadrenomedullin Levels and Progression of Deep White Matter Lesions in the Brain Accompanying Cognitive Decline.

BACKGROUND: Adrenomedullin (ADM) is a vasoreactive physiological peptide with anti-inflammatory effects and vasodilative and immunomodulatory actions that is widely distributed throughout the vascular system of the brain. OBJECTIVE: To investigate mid-regional proADM (MR-proADM), a stable fragment of the ADM precursor, and cerebral deep white matter lesions (DWMLs) in association with cognitive decline. METHODS: The study participants were 288 patients (194 men, 94 women) who gave consent to participate in a 5-year longitudinal survey on arteriosclerosis from 2008 to 2013. The Fazekas classification system (Grade [G] 0 [normal] to G3 [severe]) was used for the evaluation of DWMLs on brain magnetic resonance imaging (MRI). In addition, all participants were asked to undergo cognitive function tests regarding word/letter fluency, the results of which were assessed for correlations with MR-proADM levels. RESULTS: MR-proADM levels significantly increased with DWML grade progression. The odds ratio for high MR-proADM levels was 3.08 (95% confidence interval: 1.49-5.17) in the groups graded G3 on brain MRI, suggesting that a high level of MR-proADM is an independent risk factor for DWMLs. A significant inverse correlation was observed between MR-proADM levels and cognitive test scores. MR-proADM levels were significantly increased in the G3 group in 2013 compared with 2008. CONCLUSION: MR-proADM levels were significantly different between the DWML groups and inversely correlated with cognitive function test scores, suggesting that high MR-proADM levels and DWMLs are associated with cognitive decline. Therefore, the MR-proADM level may be an effective candidate as a potential diagnostic surrogate marker of cognitive decline.

Glucose area under the curve during oral glucose tolerance test as an index of glucose intolerance.

HbA1c and fasting plasma glucose (FPG) levels are commonly recognized as diagnostic indices for diabetes and glucose intolerance. However, they are not sufficient for clear detection of glucose intolerance in the early stage unless an oral glucose tolerance test (OGTT) is performed. Moreover, even in case of an OGTT, 2-h postprandial plasma glucose (PG) levels, a criterion for glucose intolerance in OGTTs, may not provide complete information regarding glucose tolerance. Whole glucose excursion after OGTT is considered to represent glucose tolerance well, and the glucose area under the curve (AUC) can be an index of glucose excursion. However, few studies have investigated measurement of the glucose AUC in glucose intolerance screening. In the present study, data from 520 OGTTs were analyzed to define the cutoff value for the glucose AUC for glucose intolerance screening. Our results showed that a cutoff value of 290 mg h/dl for the glucose AUC was highly sensitive and specific (90 and 93 %, respectively) for detecting diabetes, impaired glucose tolerance (IGT), and group at increased risk of diabetes (normal glucose tolerance with 1-h PG levels of ≥180 mg/dl after glucose load) and showed a better concordance rate than the use of HbA1c, FPG, or 2-h PG levels. Moreover, the cutoff value for the glucose AUC calculated using the diagnostic criteria in the OGTT (305 mg h/dl) was consistent with the value determined from OGTT analysis. These data suggest a possibility that glucose intolerance screening using a glucose AUC cutoff value of 290 mg h/dl could be useful.

Oral Cnm-positive Streptococcus Mutans Expressing Collagen Binding Activity is a Risk Factor for Cerebral Microbleeds and Cognitive Impairment.

Cerebral microbleeds (CMBs) are an important risk factor for stroke and dementia. We have shown that the collagen binding surface Cnm protein expressed on cnm-positive Streptococcus mutans is involved in the development of CMBs. However, whether the collagen binding activity of cnm-positive S. mutans is related to the nature of the CMBs or to cognitive impairment is unclear. Two-hundred seventy nine community residents (70.0 years) were examined for the presence or absence of cnm-positive S. mutans in the saliva by PCR and collagen binding activity, CMBs, and cognitive function were evaluated. Cnm-positive S. mutans was detected more often among subjects with CMBs (p < 0.01) than those without. The risk of CMBs was significantly higher (odds ratio = 14.3) in the group with S. mutans expressing collagen binding activity, as compared to the group without that finding. Deep CMBs were more frequent (67%) and cognitive function was lower among subjects with cnm-positive S. mutans expressing collagen binding activity. This work supports the role of oral health in stroke and dementia and proposes a molecular mechanism for the interaction.

CD62-mediated activation of platelets in cerebral white matter lesions in patients with cognitive decline.

BACKGROUND: Vascular dementia is related to intracranial arteriosclerosis associated with deep white matter lesions (DWMLs). DWMLs have been linked to thrombogenesis due to sustained platelet activation; therefore, an accurate hematological marker is needed. This study was done to evaluate the usefulness of a new method to examine the function of activated platelets in order to assess DWMLs associated with cognitive decline. METHODS: A total of 143 individuals (70.4 ± 6.1 years old) who underwent hospital-based health screening using head MRI were evaluated. DWLs were evaluated on T2-weighted and FLAIR images by semi-quantitatively grading them from Grade 0 (none) to Grade 3 (severe) using the Fazekas classification. Cognitive function was evaluated using the MMSE and the word fluency test. Platelet activation was assessed using fluorescence-labeled anti-human platelet monoclonal antibodies and semi-quantitatively determining PAC-1- and CD62P-positive rates by flow cytometry. RESULTS: Significant increases in hypertension and CD62P levels were observed with increasing DWML grade (2.6% in Group 0, 3.1% in Group 1, 4.1% in Group 2, and 5.0% in Group 3). CD62P levels were defined as elevated when they were above the mean+2SD of the Grade 0 group, and the odds ratio of the Grade 2+3 group was 3.03. A significant negative correlation was observed between CD62P levels and word fluency tests or the MMSE score. CONCLUSION: Elevations in CD62P levels, which reflect platelet function activation, were associated with white matter lesions accompanied by a decline in cognitive function. CD62P levels may be useful as a sensitive clinical marker for the early detection of DWMLs with cognitive decline.

Functional role of the N-terminus of Na(+),K(+)-ATPase alpha-subunit as an inactivation gate of palytoxin-induced pump channel.

The N-terminus of the Na(+),K(+)-ATPase alpha-subunit shows some homology to that of Shaker-B K(+) channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na(+),K(+)-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the alpha-subunit appears to act like an inactivation gate and performs a critical step in the Na(+),K(+)-ATPase pumping function.

Low frequency regular exercise improves flow-mediated dilatation of subjects with mild hypertension.

Although exercise is recommended for the primary prevention of hypertension, and although it is generally known to have a beneficial effect on endothelial function, working individuals often find it difficult to maintain a consistent exercise regimen. In the present study, therefore, we examined the effects of infrequently performed exercise on flow-mediated dilatation (FMD), which is an index of endothelial function, in 15 subjects with hypertension (mild hypertensives) and 10 normotensive subjects (normotensives). All subjects performed mild bicycle exercise twice a week for 12 weeks. To assess the FMD, the diameter of the brachial artery was measured using ultrasound at baseline, during reactive hyperemia, and following sublingual administration of nitroglycerin. Measurement of these parameters was performed twice, at the beginning and the end of the exercise program. At the baseline, FMD was significantly lower in the mild hypertensives than in the normotensives. Nitroglycerin-mediated dilatation (NTG-D) was similar in the two groups. The exercise decreased blood pressure in the mild hypertensives, and increased high-density lipoprotein (HDL) cholesterol in both groups. The exercise improved FMD without altering NTG-D in the mild hypertensives, but did not result in any change in the normotensives. Multiple regression analysis revealed that the elevation in FMD was positively associated with changes in HDL cholesterol, and negatively associated with changes in plasma norepinephrine and systolic blood pressure. These findings suggest that regular exercise at a low frequency improves FMD, and thereby endothelial function, and lowers blood pressure in mild hypertensives.