Effect of nitric oxide on VnfA, a transcriptional activator of VFe-nitrogenase in Azotobacter vinelandii.

The transcriptional activator, VnfA, is necessary for the expression of the structural genes encoding vanadium-dependent nitrogenase in Azotobacter vinelandii. We have previously reported that VnfA harbours a Fe-S cluster as a prosthetic group, presumably a 3Fe-4S type, which is vital for the transcriptionally active VnfA. A plausible effector molecule is a reactive oxygen species (ROS), which disassembles the Fe-S cluster switching the active VnfA to become fully inactive. This finding prompted us to investigate the effect of nitric oxide (NO), another physiologically important radical species on the VnfA activity. Unlike ROS, the VnfA activity was moderately inhibited and converged to 70% of the maximum by NO irrespective of its concentration. The Fe-S cluster of VnfA was found to react with NO to form a dinitrosyl-iron complex, either in the dimeric or monomeric form, dependent on the relative stoichiometry of NO to the Fe-S cluster. The VnfA species harbouring the dinitrosyl-iron complexes in each form exhibited 50% ATPase activity compared to the active VnfA. The findings of this study would open an argument about a biological effect of NO on nitrogenase in light of its transcriptional regulatory system.

High-normal diastolic blood pressure is a risk for development of microalbuminuria in the general population: the Watari study.

OBJECTIVE: Although hypertension is a well known risk factor for microalbuminuria, it is unclear whether blood pressure less than 140/90 mmHg could be a risk for microalbuminuria. We assessed the relationship between baseline blood pressure and the future onset of microalbuminuria in the general population. METHODS: We studied 2603 town inhabitants of Watari, located in the southeastern part of Miyagi prefecture, Japan. Demographic data, medical history, sitting blood pressure, fasting blood chemistry, and urinary albumin-creatinine ratio were measured at baseline and were followed annually during the next 3 years. RESULTS: Among 2338 individuals who were normoalbuminuric at baseline (albumin-creatinine ratio <30 mg/g Cr), 161 developed microalbuminuria (albumin-creatinine ratio 30-299 mg/g Cr) during a mean follow-up period of 2.4 years. Incident microalbuminuria patients were older (63.7 ± 8.6 vs. 61.4 ± 10.5 years; P < 0.01), included fewer men (31.1 vs. 40.1%; P < 0.05), had a greater BMI (23.8 ± 3.6 vs. 23.1 ± 3.1 kg/m²; P < 0.01), higher blood pressures (133.5 ± 18.1/75.8 ± 11.9 vs. 127.6 ± 18.1/73.4 ± 11.0 mmHg; P < 0.01 for both systolic and diastolic), higher triglycerides (median 1.18 vs. 1.02 mmol/l; P < 0.01), higher fasting blood glucose (median 5.05 vs. 4.94 mmol/l; P < 0.01), higher urinary albumin excretion (median 13.0 vs. 5.9 mg/g Cr; P < 0.001), and lower serum creatinine concentrations (59.2 ± 12.8 vs. 61.4 ± 13.2 µmol/l; P < 0.05) compared to persistent normoalbuminuric individuals. Multivariate Cox proportional hazards analysis including all covariates revealed that only baseline urinary albumin excretion was an independent predictor for future microalbuminuria, whereas high-normal DBP, triglyceride, and fasting blood glucose concentrations were all significant predictors in the model excluding urinary albumin excretion. CONCLUSIONS: High-normal DBP associated with metabolic disorders could initiate glomerular damage, leading to future microalbuminuria.

Suppression of advanced glycation and lipoxidation end products by angiotensin II type-1 receptor blocker candesartan in type 2 diabetic patients with essential hypertension.

OBJECTIVE: This study investigated whether the angiotensin II type-1 receptor blocker (ARB) candesartan affects markers of oxidative stress in type 2 diabetes mellitus (DM) patients with essential hypertension (EH). METHODS: Urinary excretion of pyrraline (PR), pentosidine (PT), acrolein (AC), and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and microalbuminuria were assessed in patients with DM complicated by EH who were treated with candesartan 4 mg/day for 3 months. RESULTS: In a total of 25 patients urinary excretion of PR (nmol/g · cr), PT (pmol/g · cr), and 8-OH-dG (ng/mg · cr) was significantly (all P < 0.05) decreased from (mean ± SEM) 11.9 ± 1.9, 30.6 ± 2.4, and 7.9 ± 0.6, respectively, at baseline to 8.4 ± 1.4, 27.1 ± 2.0, and 6.9 ± 0.6, respectively, at 3 months. Meanwhile, excretion of AC was unaltered from 209.6 ± 40.0 to 189 ± 24.8 nmol/mgcr (P = NS). Urinary albumin excretion was significantly (P < 0.05) reduced from 27.7 ± 4.6 to 14.1 ± 1.1 mg/g · cr. There were weak but statistically significant positive correlations between the change of urinary 8-OH-dG excretion and that of albumin (r = 0.414; P < 0.05) and change of hemoglobin (Hb) A1c (r = 0.45; P < 0.05). CONCLUSION: Candesartan exerts protective effect(s) on the cardiovascular system by suppression of oxidative stress--mainly through inhibiting production of advanced glycation end products (AGEs) rather than of advanced lipoxidation end products (ALEs)--in type 2 DM patients with EH.

Prognostic significance of the brachial-ankle pulse wave velocity in patients with essential hypertension: final results of the J-TOPP study.

Brachial-ankle pulse wave velocity (baPWV) is a new tool for measuring arterial stiffness. The prognostic significance of this measure, however, is not fully established. We initiated a multicenter cohort study to examine the prognostic significance of baPWV in patients with essential hypertension in 2002. After baseline measurements were obtained, 662 previously untreated patients (mean age 60±12 years, mean blood pressure 156±19/94±12 mm Hg, 45% men) underwent long-term follow-up according to the current hypertension treatment guidelines. During the follow-up period (mean: 3 years, range: 3 months-8 years), 24 cardiovascular events were observed. The subjects were divided into high and low baPWV groups according to the median value (1750, cm s(-1)). Patients in the high baPWV group were older and had a lower body mass index, higher blood pressure, faster heart rate and higher fasting glucose and plasma creatinine concentrations compared with those in the low baPWV group. Cardiovascular morbidities per 1000 person-years for the high and low baPWV groups were 17.48 and 6.38, respectively (P<0.05), and the 8-year cardiovascular event-free survival rates were 78.2% and 93.5%, respectively (log-rank test, P=0.01). A multivariate Cox proportional hazard analysis showed that high baPWV compared with low baPWV was associated with a significantly poorer outcome (hazard ratio (HR) 2.97; 95% CI: 1.006-9.380). In conclusion, baPWV is an independent risk factor for future cardiovascular events in patients with essential hypertension.

The role of the GAF and central domains of the transcriptional activator VnfA in Azotobacter vinelandii.

VnfA is a transcriptional activator that is required for the expression of the structural genes encoding nitrogenase-2 in Azotobacter vinelandii. VnfA consists of three domains: an N-terminal regulatory domain termed GAF, including a Cys-rich motif; a central domain from the AAA+ family; and a C-terminal domain for DNA binding. Previously, we reported that transcriptionally active VnfA harboring an Fe-S cluster (presumably of the 3Fe-4S type) as a prosthetic group and the Cys-rich motif were possibly associated with coordination of the Fe-S cluster. In the present study, we have investigated the roles of the GAF and central domains in the regulatory function of VnfA using truncated variants: ΔN15(VnfA) and ΔGAF(VnfA) that lack the N-terminal 15 residues and whole GAF domain, respectively, and GAF(VnfA) consisting of only the GAF domain. ΔN15(VnfA) and ΔGAF(VnfA) lost the ability to bind the Fe-S cluster, whereas GAF(VnfA) was still able to bind to the cluster, consistent with the hypothesis that the Cys-rich motif is essential for Fe-S cluster binding. The GAF domain showed an inhibitory effect on the transcriptional activity of VnfA, which was reversed in the presence of the Fe-S cluster, and reactivated upon disassembly of the cluster. The inhibitory activity of the GAF domain acts on the NTPase activity of the central domain, whereas the binding ability of VnfA to DNA was not significantly affected, when VnfA retains its tetrameric conformation. The results imply that a major pathway, by which VnfA function is regulated, operates via the control of NTPase activity by the GAF domain.

High-normal blood pressure is associated with microalbuminuria in the general population: the Watari study.

Microalbuminuria, for which hypertension and diabetes are well-known risk factors, has recently been used to detect individuals at risk for cardiovascular and chronic kidney diseases in the general population. We aimed to determine the thresholds of blood pressure or blood glucose concentration at which the odds ratio of having microalbuminuria begins to increase. An annual public health checkup was conducted on 3166 participants aged 29-84 years (mean, 61±11 years; 40% men) living in Watari town, Japan. We studied their demographic data, medical history of hypertension, diabetes, dyslipidemia, sitting blood pressure and fasting blood samples. Urinary albumin excretion was examined in terms of the albumin/creatinine ratio in spot urine samples. Microalbuminuria was defined as 30-299 mg albumin per gram creatinine. Final analyses included 2133 participants under no anti-hypertensive or anti-diabetic medication. Microalbuminuria was detected in 118 subjects (5.5%). We used the adjusted odds ratios for microalbuminuria in different blood pressure and blood glucose groupings as the reference. The adjusted odds ratio increased linearly with increase in systolic blood pressure. A similar trend, although not as linear, was observed with diastolic blood pressure. The odds ratio increased significantly with high-normal levels of both systolic and diastolic blood pressure. For blood glucose, the adjusted odds ratio increased significantly from the impaired fasting glucose level. Therefore, the odds ratio for microalbuminuria begins to increase from high-normal blood pressure and impaired fasting glucose levels in comparison with the reference. Early intervention in such cases may be useful for reducing cardiovascular and renal risks.

Higher brachial-ankle pulse wave velocity as an independent risk factor for future microalbuminuria in patients with essential hypertension: the J-TOPP study.

OBJECTIVE: Brachial-ankle pulse wave velocity (baPWV) is a new measure of arterial stiffness. We have shown that a higher baPWV is associated with more severe albuminuria in patients with essential hypertension. However, it is still unclear as to whether baPWV predicts the future albuminuria status in patients under standard care. METHODS: A total of 321 nondiabetic patients with essential hypertension who had normoalbuminuria or microalbuminuria and were receiving treatment under the current hypertension guidelines were followed up for 2 years. Resting BP, fasting blood glucose, urinary albumin excretion and baPWV were measured before treatment, and periodically thereafter for 2 years. RESULTS: A 2-year treatment regimen lowered BP from 156/93 to 135/81 mmHg (P < 0.0001) and reduced the incidence of microalbuminuria from 25.8 to 14.0%. To determine the predictors for future microalbuminuria status, we compared the baseline data between patients with normoalbuminuria (n = 276) and those with microalbuminuria (n = 45) 2 years after the treatment. The microalbuminuric patients had a significantly higher baPWV and urinary albumin excretion and a significantly lower high-density lipoprotein concentration than the normoalbuminuric patients. Furthermore, the frequency of the use of renin-angiotensin system inhibitors was significantly lower in the microalbuminuric patients than in the normoalbuminuric patients. A multiple logistic regression analysis showed that higher baPWV was an independent risk factor for microalbuminuria 2 years after treatment of hypertension. When we restricted our analysis to a cohort of patients without microalbuminuria at the baseline, we found that higher baPWV was an independent predictor of the development of microalbuminuria after 2 years. CONCLUSION: Higher baPWV could be an independent risk factor for future microalbuminuria in patients with essential hypertension.

A retroperitoneal sympathetic paraganglioma invading the duodenum and mimicking a submucosal tumor.

We report a case of an autopsy of unusual retroperitoneal sympathetic paraganglioma (SPG) that directly invaded the duodenum and showed expansive growth mimicking a submucosal tumor. The tumor was clinically suspected to be a gastrointestinal stromal tumor (GIST) of the duodenum because of its location and extension to the retroperitoneum without catecholamine-associated symptoms. However, a small biopsy specimen of the tumor showed diffuse proliferation of large basophilic cells that were negative for C-kit and CD34, ruling out GIST and indicating an epithelial malignancy. An autopsy revealed that the tumor was mainly in the retroperitoneum, measuring 7.5 x 9.5 cm, weighing 600 g and extending into the duodenum, adjacent to the pancreas but free of the adrenal glands. On cut section, the tumor involved the entire wall of the duodenum. There were no metastases in any organs. For differential diagnosis, endocrine tumors of the duodenum or pancreas and extra-adrenal SPG were considered. The tumor cells were immunohistochemically strongly positive for chromogranin A and were surrounded by cells positive for S100 protein. The Ki67-labeling index was under 1%. The four catecholamine-synthesizing enzymes were detected in the tumor cells. We report this case of SPG with emphasis on differential diagnosis and the significance of its local invasion.

Progress report on The Hypertension Objective Treatment Based on Measurement by Electrical Devices of Blood Pressure (HOMED-BP) study: status at February 2004.

The Hypertension Objective Treatment Based on Measurement by Electrical Devices of Blood Pressure (HOMED-BP) Study is the first large-scale intervention trial in Japan using a PROBE design to determine an optimal target blood pressure (BP) level on the basis of self-measured BP at home and optimal initial antihypertensive medication. The registered patients are randomized to one of two different BP levels and to initial antihypertensive regimens (i.e., calcium antagonist [Ca-A], angiotensin-converting enzyme inhibitor [ACE-I], or angiotensin II receptor antagonist [ARB]). At the end of February 2004, 2729, 1687, and 971 patients were recruited, randomized, and followed-up for 12 months or more. Among the 971 patients, mean systolic/diastolic BP levels at randomization in the Ca-A, ACE-I, and ARB groups were 150/90, 152/89, and 151/89 mmHg, respectively. These values reduced after six months of Ca-A-, ACE-I-, and ARB-based treatment (133/81 mmHg, 135/80 mmHg, and 134/79 mmHg, respectively) and further reduced after 12 months (131/80 mmHg, 134/79 mmHg, and 132/79 mmHg, respectively) without any significant differences among groups. In more intensive and less intensive BP-lowering groups, mean systolic/diastolic BP levels at randomization were 151/90 and 150/89 mmHg, respectively. Although BP levels reduced after 6-month (135/80 mmHg and 133/80 mmHg, respectively) and 12-month treatment (133/79 mmHg and 132/80 mmHg, respectively) in each target group, no significant difference in BP values was observed between groups. These results indicate the necessity of additional interventional strategies to improve the achievement of target BP levels, especially in the more intensive BP-lowering group.

Histological grading of adrenal and extra-adrenal pheochromocytomas and relationship to prognosis: a clinicopathological analysis of 116 adrenal pheochromocytomas and 30 extra-adrenal sympathetic paragangliomas including 38 malignant tumors.

Pheochromocytomas and extra-adrenal sympathetic paragangliomas show varied histological patterns, and it is difficult to diagnose malignancy or predict the clinical course using current histological criteria. In the present study, we reviewed 146 sympathetic paragangliomas including 116 adrenal (102 unilateral, 14 bilateral) and 30 extra-adrenal tumors including 38 metastatic tumors. We developed a scoring scale according to the following six factors: histological pattern, cellularity, coagulation necrosis, vascular/capsular invasion, Ki-67 immunoreactivity, and types of catecholamine produced. The tumors were classified as well (WD), moderately (MD), and poorly differentiated (PD) types according to their scores. The frequency of these tumor types were 113 WD (77%), 27 MD (19%), and 6 PD (4%). Metastasis was observed in 15 of 113 WD (13%), 17 of 27 MD (63%), and all 6 PD (100%). Five-year survivals of patients with metastases were 92% with WD, 69% with MD, 0% with PD. Respective 10-yr survivals were 83%, 38%, and 0%. Differences between groups were statistically significant. The data show that using this grading scoring system for sympathetic paragangliomas correlates with both metastatic potential and patient survival.

The second progress report on the Hypertension Objective treatment based on Measurement by Electrical Devices of Blood Pressure (HOMED-BP) study.

BACKGROUND: The Hypertension Objective treatment based on Measurement by Electrical Devices of Blood Pressure (HOMED-BP) study is a large-scale intervention trial to determine both optimal target blood pressure (BP) on the basis of self-measured BP at home, and optimal initial antihypertensive medication. OBJECTIVE: To investigate the quality of randomization at the end of March 2003, and BP-controlled conditions during initial 6 months. METHODS: We evaluated the number of patients randomized by the end of March 2003, and BP changes during the initial 6 months based on intention-to-treat analysis. RESULTS: By the end of March 2003, a total of 1086 patients (12% of a planned randomization of 9000 patients) had been randomized. Among 653 patients who had been followed for > or =6 months after randomization, mean systolic/diastolic BPs at randomization in the calcium antagonist, angiotensin-converting enzyme and angiotensin II receptor blocker groups were 149/89, 150/89 and 149/88 mmHg, respectively. Blood pressures were reduced after 3 months (137/83, 139/82 and 136/82 mmHg, respectively), and further reduced after 6 months (134/81, 135/80 and 133/80 mmHg, respectively), with no significant differences identified between groups. In more- and less-intensive BP-lowering groups, mean systolic/diastolic BPs at randomization were 149/88 and 150/89 mmHg, respectively. Although BPs were reduced after treatment for 3 months (137/83 and 137/82 mmHg, respectively) and 6 months (134/80 and 135/80 mmHg, respectively) in each target group, significant differences were still not observed between groups. CONCLUSIONS: Good quality of randomization was observed at the end of March 2003, but rate of achievement for target BPs was insufficient.

Crucial roles of Brn1 in distal tubule formation and function in mouse kidney.

This study identifies a role for the gene for the POU transcription factor Brn1 in distal tubule formation and function in the mammalian kidney. Normal development of Henle's loop (HL), the distal convoluted tubule and the macula densa was severely retarded in Brn1-deficient mice. In particular, elongation and differentiation of the developing HL was affected. In the adult kidney, Brn1 was detected only in the thick ascending limb (TAL) of HL. In addition, the expression of a number of TAL-specific genes was reduced in the Brn1+/- kidney, including Umod, Nkcc2/Slc12a1, Bsnd, Kcnj1 and Ptger3. These results suggest that Brn1 is essential for both the development and function of the nephron in the kidney.

Dna ploidy of pheochromocytoma on cytology specimen by image analysis.

Pheochromocytoma usually shows prominent nuclear atypia, but the presence of such atypical cells is known to be an unreliable predictor of malignancy. DNA ploidy of pheochromocytomas has been analyzed by flow cytometry or photospectrometry on paraffinem-bedded tissue, but the results were controversial. We performed DNA analysis on cytology specimens of 11 pheochromocytomas using an image analysis system. All tumors had a mixed pattern of a large population of diploid cells and a small population of polyploid cells. DNA content correlated with nuclear size, and larger cells had more DNA content. Such larger tumor cells had polyploid nuclei, such as 4 C, 8 C, 16 C, and 32 C, in both malignant and benign pheochromocytomas. The larger polyploid nuclei may result from difficulty of duplication at the mitotic phase of the cell cycle.

Ki-67 is an indicator of progression of neuroendocrine tumors.

No current histological or cytological indices can distinguish reliably malignant from benign tumors in neuroendocrine tumors, including pheochromocytomas, pancreatic endocrine tumors, and carcinoid tumors. We investigated immunohistochemically the expression of Ki-67 in 52 neuroendocrine tumors, including 17 pheochromocytomas, 9 pancreatic endocrine tumors, 23 carcinoid tumors, 2 neuroendocrine carcinomas (NEC), and 1 neuroblastoma with liver metastasis. Of the 52 tumors, distant metastasis was observed in 4 pheochromocytomas, 2 pancreatic endocrine tumors, 4 carcinoids, 2 NEC, and 1 neuroblastoma. We classified these tumors into 3 groups; Groups A, B, and C, depending on the number of Ki-67-positive cells counted under a 200 x magnified field. Expression of Ki-67 was extremely high in group A (> 50 labeled nuclei/field), moderately high in group B (20-50 labeled nuclei), and very low in group C (< 10 labeled nuclei). There was a significant correlation between expression of Ki-67 and tumor progression. The tumors in group A progressed rapidly with the worst outcome; the tumors in group B progressed slowly but with a bad outcome; and the tumors in group C had no metastasis and a good prognosis. Ki-67 is an excellent indicator to assess progression of neuroendocrine tumors.

Adrenal and retroperitoneal mixed neuroendocrine-neural tumors.

Four cases of mixed neuroendocrine-neural tumors composed of pheochromocytoma and neuroblastoma elements (including ganglioneuroma and ganglioneuroblastoma) were studied for the presence of catecholamine-synthesizing enzymes, neuroendocrine markers, and peptide hormones with clinicopathological correlations. Paroxysmal hypertension with hypercatech olaminemia was observed in 3 patients. One patient had an extremely elevated level of dopamine. The location of the tumor was in the adrenal glands in 2 patients and in the retroperitoneum in the other 2. Numerous electron-dense granules in the cytoplasm and neural processes with abundant neurotubules were characteristic of mixed neuroendocrine-neural tumors. Immunohistochemical study revealed that catecholamine-synthesizing enzymes were present in both components of the pheochromocytoma and neuroblastoma group, but phenylethanolamine N-methyltransferase was detectable only in epinephrine-producing tumors. Chromogranin and neurofilament immunoreactivities were present in both components; however, the intensity of chromogranin immunoreactivity was stronger in pheochromocytoma than in the other components. In contrast, neurofilament positivity was stronger in the neuroblastoma group than it was in pheochromocytoma. Multiple peptide hormones were immunoreactive in both components. Neuropeptide Y and met-enkephalin-positive cells were numerous in both; cells containing vasoactive intestinal peptide and somatostatin were less common but were comparatively more frequently found in ganglion cells than in pheochromocytoma cells.