Long-term weight-change slope, weight fluctuation and risk of type 2 diabetes mellitus in middle-aged Japanese men and women: findings of Aichi Workers' Cohort Study.

OBJECTIVE: This study aims to investigate the association of long-term weight-change slopes, weight fluctuation and the risk of type 2 diabetes mellitus (T2DM) in middle-aged Japanese men and women. METHODS: A total of 4234 participants of Aichi Workers' Cohort Study who were aged 35-66 years and free of diabetes in 2002 were followed through 2014. Past body weights at the ages of 20, 25, 30, 40 years, and 5 years before baseline as well as measured body weight at baseline were regressed on the ages. Slope and root-mean-square-error of the regression line were obtained and used to represent the weight changes and the weight fluctuation, respectively. The associations of the weight-change slopes and the weight fluctuation with incident T2DM were estimated by Cox proportional hazards models. RESULTS: During the median follow-up of 12.2 years, 400 incident cases of T2DM were documented. After adjustment for baseline overweight and other lifestyle covariates, the weight-change slopes were significantly associated with higher incidence of T2DM (hazard ratio (HR): 1.80, 95% confident interval (CI): 1.17-2.77 for men; and HR: 2.78, 95% CI: 1.07-7.23 for women), while the weight fluctuation was not (HR: 1.08, 95% CI: 1.00-1.18 for men and HR: 1.02, 95% CI: 0.84-1.25 for women). CONCLUSIONS: Regardless of the presence of overweight, the long-term weight-change slopes were significantly associated with the increased risk of T2DM; however, the weight fluctuation was not associated with the risk of T2DM in middle-aged Japanese men and women.

IGF-2 coated porous collagen microwells for the culture of pancreatic islets.

Islet transplantation, the only curative therapy for type I diabetes, requires isolation of the graft in highly specialized facilities for its later dispatch to remote transplantation centres. During transport and culture, many valuable cells are lost due to several factors such as mechanical stress, islet aggregation and dissociation. Here, we evaluate a porous microwell array sheet made of natural collagen type I extracellular matrix (ECM) protein as a novel islet culture substrate. This culture platform can be coated with IGF-2, a growth factor favorable for islet survival, and allows segregation of the islets within the porous microwell sheet, preventing aggregation. This design shows promising results for improving human pancreatic islets viability and function during culture and could form a novel paradigm for the transport of islets between isolation and transplantation centres.

Bcl-2 protein inhibits bufalin-induced apoptosis through inhibition of mitogen-activated protein kinase activation in human leukemia U937 cells.

In a previous study, we demonstrated that bufalin, which is an active principle of Chinese medicine, chan'su, caused apoptosis in human leukemia U937 cells by anomalous activation of mitogen-activated protein kinase (MAPK) via the signaling pathway of Ras, Raf-1, and MAPK kinase-1. Here, we report the effect of overexpression of bcl-2 in U937 cells on the signaling pathway of apoptosis that is induced by bufalin. The results indicated that the apoptosis induced by bufalin in U937 cells was significantly inhibited by overexpression of the Bcl-2 protein. No significant difference was detected in the activation of MAPK kinase-1 that is induced by bufalin in wild-type or Bcl-2-overexpressed U937 cells; however, the activation of MAPK by bufalin was significantly attenuated in the cells overexpressing Bcl-2. Bufalin treatment activated activator protein-1 transcriptional activity; however, this activation was decreased to 40% in bcl-2-overexpressed U937 cells. These results indicate that Bcl-2 acts downstream of MAPK kinase-1 but upstream of MAPK and suggest that, in the signaling pathway of the apoptotic process induced by bufalin, the transcriptional activity of activator protein-1 may be down-regulated through the inhibition of MAPK activity by Bcl-2.

Tiam1 is involved in the regulation of bufalin-induced apoptosis in human leukemia cells.

Bufalin, a component of the Chinese medicine chan'su, induces apoptosis in various lines of human tumor cells, such as leukemia HL60 and U937 cells, by altering the expression of apoptosis-related genes, for example, bcl-2 and c-myc. In this study, we characterized a gene that is involved in bufalin-induced apoptosis by the differential display (DD) technique. The partial nucleotide sequence of one of the differentially expressed clones obtained after treatment with bufalin was identical to that of the human gene for Tiam1. When U937 cells were treated with 10(-7) M bufalin, expression of both Tiam1 mRNA and the protein was induced 1 h after the start of the treatment. The increase of Tiam1 mRNA was transient but the level of Tiam1 protein continued to increase at least for 6 h. In addition, the activities of Rac1 and p21-activated kinase (PAK) were also stimulated by bufalin treatment. To evaluate the role of Tiam1 in the apoptotic process, we examined the effects of the expression of sense and antisense RNA for Tiam1 in U937 cells. Apoptosis was strongly induced by bufalin in cells that expressed sense RNA for Tiam1 as compared to apoptosis in control cells treated with bufalin only. Cells expressing antisense RNA for Tiaml were significantly more resistant than the control bufalin-treated cells to induction of DNA fragmentation in response to bufalin. Moreover, sense transformants had elevated activities of PAK and c-Jun NH2-terminal kinase (JNK). These results suggest that Tiaml might play a critical role in bufalin-induced apoptosis through the activation of Rac1, PAK, and JNK pathway.

Blood pressure and its regulation in spontaneously hypertensive rats bred on the lowest sodium diet for normal growth.

To investigate the effects of dietary sodium restriction from conception to adulthood on blood pressure and its regulatory mechanisms, male offspring were derived from inbreeding in spontaneously hypertensive rats fed a diet containing sodium of 175 mumol/g food (control) or 22 mumol/g (low sodium), which is the least sodium content for normal growth. While urinary sodium excretion was markedly less, the low sodium diet did not inhibit body growth and failed to blunt the development of hypertension. Neither plasma catecholamine concentration nor depressor response to hexamethonium was different between the two groups at any age examined (8, 12, and 20 weeks). Plasma renin concentration was not elevated, whereas urinary excretion of aldosterone was increased at any age in the low sodium group compared with that in the control group. Other sets of rats were fed a diet containing sodium of 175 mumol/g plus mefruside (a diuretic) of 0.001% in the same manner as in the other two groups. Urinary sodium excretion per creatinine was higher than in the other groups. The diuretic treatment inhibited body growth and suppressed adult blood pressure. While the sympathetic function was not affected, both plasma renin concentration and urinary excretion of aldosterone were elevated. These results indicate that dietary sodium restriction with the least sodium for normal growth from conception cannot blunt either the sympathetic nervous function or the development of hypertension in spontaneously hypertensive rats. Aldosterone appears to play an important role in maintaining sodium homeostasis under the dietary sodium restriction.

Neural effects on renal blood flow during acute hypotension vary with antihypertensive drugs.

To examine the neural effects of antihypertensive drugs on renal blood flow, we measured blood flow and renal sympathetic nerve activity simultaneously in conscious spontaneously hypertensive rats aged 13 to 15 weeks. One to two days after surgery, intravenous administration of manidipine (calcium antagonist, n = 10), doxazosin (alpha 1-adrenergic receptor antagonist, n = 9), and clonidine (n = 7) lowered mean arterial pressure by at least 20% from baseline levels. Manidipine initiated a reduction of renal blood flow when mean pressure decreased by 20 +/- 2 mm Hg. At the maximal decrease in renal blood flow (mean pressure, -33 +/- 2 mm Hg), percent decrease in flow (-27 +/- 2%) significantly correlated with percent increase in renal nerve activity (+205 +/- 40%, r = -.878). Doxazosin began to decrease renal blood flow at a level of arterial pressure similar to that in manidipine treatment, whereas the maximal decrease in flow (-19 +/- 2%; mean pressure, -33 +/- 2 mm Hg; nerve activity, +225 +/- 44%) was significantly less than that in manidipine treatment. Although clonidine decreased arterial pressure and renal nerve activity, renal blood flow did not decrease even at the maximal decrease in mean pressure of 29 +/- 1 mm Hg. The addition of clonidine to manidipine treatment suppressed reflexly enhanced renal nerve activity and restored blood flow to the pretreatment level despite pronounced hypotension. These results clearly demonstrate that antihypertensive drugs with blocking action on renal nerve activity are capable of maintaining renal blood flow and that those associated with reflex-induced enhancement of nerve activity exert deteriorating effects on renal blood flow. Furthermore, a decrease in renal blood flow induced by calcium antagonists is mainly attributed to reflexly enhanced renal nerve activity.

Bufalin induces apoptosis and influences the expression of apoptosis-related genes in human leukemia cells.

A low concentration of bufalin, a component of bufadienoides in the traditional Chinese medicine chan'su, was shown previously to induce differentiation of a broad range of human leukemia cell lines. In the present study, we found that bufalin at concentrations of 10(-7) M and higher induced apoptosis in human leukemia cells, such as HL60, ML1, but not in mouse leukemia M1 cells. A mere 15 min pretreatment of HL60 cells with 10(-6) M bufalin, followed by incubation for 15 h without bufalin, caused fragmentation of DNA and a decrease in cell viability, indicating that the signal for induction of apoptosis is triggered rapidly upon treatment with bufalin. Bufalin-induced apoptosis in HL60 cells was inhibited by ZnCl2, an inhibitor of endonuclease, but not by cycloheximide, an inhibitor of protein synthesis. Northern blot analysis revealed that the levels of expression of the c-myc and bcl-2 genes in HL60 cells decreased with time after treatment with bufalin. These results suggest that bufalin induces apoptosis specifically in human leukemia cells by altering the expression of these genes involved in apoptosis.

Bufalin reduces the level of topoisomerase II in human leukemia cells and affects the cytotoxicity of anticancer drugs.

When human leukemia HL-60 cells were treated with 10(-7) M bufalin, the amounts of both topoisomerase (topo) II alpha and II beta and the activity of topo II decreased markedly and were almost undetectable 18 h after the start of treatment. The level of topo II mRNA started to decrease immediately after the start of treatment with bufalin, with a subsequent decrease in the amount of topo II alpha protein. These changes preceded the fragmentation of DNA, a typical feature of apoptosis. The results suggest that bufalin caused a marked decrease in the steady-state level of topo II alpha mRNA, which led to a decrease in the amount and activity of the enzyme and to the induction of apoptosis. A reduction in the level of topo II alpha by bufalin was also observed in other lines of human leukemia cells such as ML1 and U937. The results were exploited to potentiate the effects of cisplatin and retinoic acid (RA) on HL-60 cells: pretreatment of HL-60 cells with 10(-7) M bufalin for 6 h increased the inhibitory effects of cisplatin and RA on cell growth and enhanced the induction of cell death.

Induction of apoptosis by bufalin in human tumor cells is associated with a change of intracellular concentration of Na+ ions.

In an attempt to characterize the mechanisms that are operative at the early stages of the induction of apoptosis by bufalin, a component of the traditional Chinese medicine chan'su, we examined the effects of bufalin on plasma membrane potential, as determined by monitoring the uptake by cells of rhodamine 123. Bufalin induced apoptosis in human monocytic leukemia THP-1 cells, in human lymphoblastic leukemia MOLT-3 cells, and in human colon adenocarcinoma COLO320DM cells but not in normal human leukocytes, for example, polymorphonuclear cells and lymphocytes, and not in murine leukemia P388D1 and M1 cells. Treatment for 3 h with bufalin at 10(-6) M caused a decrease in the plasma membrane potential in several lines of human tumor cells but not in murine leukemia cells. No changes in mitochondrial membrane potential, as monitored with the fluorescent dye JC-1, and no release of cytochrome c were observed within at least 6 h after the start of treatment with bufalin. Moreover, overexpression of bcl-2 in human leukemia HL60 cells that had been transfected with cDNA for bcl-2 prevented bufalin-induced apoptosis but had no significant effect on the change in plasma membrane potential induced by bufalin. Since bufalin specifically inhibits the Na+,K(+)-ATPase of human but not murine tumor cells, and since this inhibition leads to a change in intracellular concentration of Na+ ions, our findings suggest that bufalin induces apoptosis in human tumor cells selectively via inhibition of the Na+,K(+)-ATPase, which acts upstream of the bcl-2 protein.

Modulation of baroreflex function by angiotensin II endogenous to the caudal ventrolateral medulla.

Neurons in the ventrolateral medulla (VLM) mainly determine the tonic sympathetic activity. The caudal VLM (CVLM) relays baroreflex signals to the rostral VLM. We have reported that endogenous angiotensin II (ANG II) contributes to the ongoing activity of the VLM neurons. In the present study, we examined if ANG II endogenous to the CVLM modulates the baroreflex function in anesthetized normotensive Sprague-Dawley rats. Changes in renal sympathetic nerve activity (RSNA) in response to changes in mean arterial pressure (MAP) induced by i.v. infusion of phenylephrine and nitroglycerin were recorded before and after bilateral microinjection of [Sar1, Thr8]-ANG II, an ANG II antagonist, into the CVLM. The ANG II antagonist injection into the CVLM significantly increased MAP and RSNA by 17.6 +/- 8.0 mmHg (mean +/- S.D.) and 36.3 +/- 18.1%, respectively. It also significantly increased the baroreflex sensitivity (BS) from -0.49 +/- 0.38 to -0.74 +/- 0.37%/mmHg during nitroglycerin infusion. In contrast, the BS examined by phenylephrine infusion was not altered by the pretreatment with ANG II antagonist. Injection of artificial CSF affected neither the baseline values of MAP and RSNA nor the BS. These results suggest that ANG II endogenous to the CVLM exert a modulating role in baroreflex control of RSNA.

Fifteen-year experience in axillofemoral bypass with externally supported knitted Dacron prosthesis in a Japanese hospital.

BACKGROUND: Recently, several reports in the United States have demonstrated remarkable improvement in the patency of axillofemoral (AXF) bypass with an externally supported prosthesis. The purpose of this study was to review the prognoses regarding graft patency, limb salvage, and survival of patients who underwent AXF bypass grafting with an externally supported, knitted Dacron prosthesis (EXS) in a Japanese hospital and to analyze what factors affected the graft patency. METHODS: The clinical records of 81 patients with arteriosclerosis obliterans (ASO) who underwent 47 axillounifemoral bypasses and 34 axillobifemoral bypasses with EXS were retrospectively checked and, by uni- and multivariate analysis, perioperative factors were evaluated. RESULTS: The cumulative primary and secondary patency rates of AXF bypass grafts were 81% and 88%, 73% and 80%, and 70% and 77% at 3, 5, and 7 years, respectively, with no change thereafter. Limb salvage rate was 100%. The operative mortality was 3.7% and the survival rate was 63%, 41%, and 35% at 3, 5, and 7 years, respectively. The risk factors adversely affecting the patency were age (younger than 75 years), poor distal runoff, and preoperative leukocyte count (more than 8,000/microL) by univariate analysis, none of which were significant by stepwise multivariate analysis. CONCLUSIONS: AXF bypass using EXS was an acceptable procedure in ASO patients at high risk for conventional anatomic bypass or with limited life expectancy, and there was no significant risk factor that independently affected the patency.

Circadian rhythms of urinary excretions of water and electrolytes in patients receiving total parenteral nutrition (TPN).

In order to determine whether the usual feeding pattern actually modifies the circadian rhythms of urinary excretion of water and electrolytes, we compared the circadian rhythm characteristics in patients receiving total parenteral nutrition (TPN group) with those in patients on an ordinary hospital diet (control group). Statistically significant circadian rhythms were detected in all of the urinary variables investigated herein by using the population mean-cosinor method in both groups. In addition, there were no statistically significant differences of the mesor, the %-amplitude and the acrophase between the two groups. These results suggest that the usual feeding pattern is not a main determinant in forming the circadian rhythm characteristics of human urinary variables.

Bioassay using a labeled oligonucleotide obtained by in vitro selection.

A new bioassay system using an oligodeoxyribonucleotide (DNA) obtained by the in vitro selection method is described. The DNA aptamer which selectively binds to a target molecule, Reactive Green 19 (RG19), was labeled with fluorescein. The circular dichroism spectra revealed that the fluorescein labeling did not significantly affect the conformation of the DNA. The binding affinity toward RG19 of the labeled DNA was the same as that of the nonlabeled DNA. Using the labeled DNA and the RG19-immobilized gel, a semiquantitative assay of RG19 concentration was performed.

Long-term prognosis of malignant hypertension; difference between underlying diseases such as essential hypertension and chronic glomerulonephritis.

A long-term prognosis was studied in 69 patients with malignant hypertension, followed for an average of 56 months. Overall survival rate was 90% for a 5-year period, although the prognosis was different between two major underlying diseases, namely the 5-year survival was 79% for 33 essential hypertension (EHT) and 100% for 26 chronic glomerulonephritis (CGN); the difference is significant (p less than 0.01). In contrast, the 5-year rate for renal survival, defined as the probability of surviving without hemodialysis, was 37% in all cases. However, there was significant difference in the renal survival between EHT (60% for a 5-year period) and CGN (4% for a 18-month period). Multivariate analyses of the Cox's proportional hazards model revealed that the long-term change in renal function was different between the two groups, namely more rapid deterioration in the CGN group. These results indicate that a long-term prognosis of malignant hypertension is influenced by the underlying diseases and hemodialysis besides antihypertensive treatment may increase survival in malignant hypertension associated with severely damaged renal function.

[Relationship between the Ki-67 nuclear antigen content and cell-cycle perturbation on WiDr cells treated with 5-FU].

The profile of Ki-67 nuclear antigen content according to the change in the cell-cycle of WiDr cells affected by 5-fluorouracil (5-FU) was studied by flow cytometry. The cases who received 10 micrograms/ml for 1 hr and showed reproliferation in the growth curve presented significant accumulation at G2M phases. However, significant increase of Ki-67 antigen content was not observed in G2M phases. Meanwhile, the cases who continuously received 10 micrograms/ml for 72 hr and showed the suppression of cellular proliferation without reproliferation presented gradual accumulation into S phase and Ki-67 antigen content was found to be significantly increased. This increase occurred in G1 phase, and the cells with Ki-67 antigen content of the largest increase in G1 phase shifted to S phase. It indicates a possibility that Ki-67 nuclear antigen content is destined to be controlled specifically in G1 phase of the cell-cycle. 5-FU seems to damage this regulation system and, as a result, increase Ki-67 nuclear antigen.

[Study of fluorescence in situ hybridization for detection of chromosome aberration].

The authors applied fluorescence in situ hybridization (FISH) technique for the detection of chromosome aberration in interphase nuclei using the probe specific to alphoid repeats on chromosome 11 and X. Chromosome 11 specific probe showed two major spots in lymphocyte nuclei, while X specific probe showed single spot in male and double spots in female respectively. On the other hand three spots were detected in most of the nuclei from HeLa cells with 11 and X specific probes. We concluded that FISH with the use of chromosome specific probe may become a useful and reliable tool for the detection of chromosome aberration in interphase nuclei.

Pathophysiology in malignant hypertension: with special reference to the renin-angiotensin system.

Pathophysiology of malignant hypertension, of which underlying disease was essential hypertension (EHT) in 33 cases and chronic glomerulonephritis (CGN) in 26 cases, was studied with reference to the renin-angiotensin system. Plasma renin activity (PRA) was significantly higher in the EHT than in the CGN group, and angiotensin II antagonist [Sar1, Ile8]angiotensin II (AIIA) induced a significant lowering of blood pressure only in the former group. PRA was linearly correlated with both pretreatment mean blood pressure (MBP, r = 0.474, n = 29, p less than 0.01) and serum creatinine (r = 0.540, n = 29, p less than 0.01) in the EHT group but not in CGN patients, although there was an inverse correlation between PRA and serum sodium in both groups. Multiple regression analysis revealed that PRA was independently related to MBP, serum creatinine, and serum sodium in the EHT group, but not in the CGN group. These results suggest that the renin-angiotensin system plays a significant role in elevating blood pressure and deteriorating renal function in malignant hypertension derived from EHT, but it is less important in CGN related hypertension.

Reoperation for graft failure of femoropopliteal bypass with externally supported knitted Dacron prosthesis.

BACKGROUND: Aggressive reoperation for failing or failed femoropopliteal (FP) bypass has been affirmative despite graft material. There has been no report regarding results of reoperation for FP bypass with externally supported knitted Dacron prosthesis (EXS). The aim of this study is to justify aggressive reoperation of FP bypass with EXS and to examine risk factors affecting the result of reoperation. METHODS: A retrospective review was performed on 204 patients undergoing 212 FP bypasses (with EXS between January 1982 and December 1997 and 34 FP EXSes of 32 patients underwent reoperation for graft failure until March 1998. The cumulative graft patency (GP) and limb salvage (LS) rate after first reoperation for 34 FP bypass EXS were calculated and the importance of each perioperative factor on GP or LS was estimated by uni- and multivariate analysis. RESULTS: Reoperated 34 FP EXSes included 12 failing and 22 failed grafts and 14 limbs with failed grafts underwent reoperation for limb salvage. The GP of 34 grafts and LS of 14 limbs rate were 58 and 67% at 2 years, respectively. Univariate analysis identified graft thrombosis (2 years GP; failing graft: failed graft=78: 48%) and continuance of smoking (2 years LS; smoker: non-smoker=43: 100%) as a significant risk factor of GP and IS, respectively, neither of which was significant by multivariate analysis. CONCLUSIONS: Early diagnosis and treatment, before graft thrombosis, can lead to superior durability of GP and discontinuance of smoking is important for LS.

Para-anastomotic aneurysms: incidence, risk factors, treatment and prognosis.

BACKGROUND: The purpose of this retrospective study was to analyze the incidence, risk factors, treatment, and prognosis of para-anastomotic aneurysms. METHODS: During the period between January, 1980 and August, 1996, 511 patients underwent surgical operations for arterial diseases with grafts and were followed for more than 30 days (average: 3.5 years). The number of anastomoses was 1445 in all. Until October, 1996, 18 para-anastomotic aneurysms had been detected in 13 patients. By Kaplan-Meier's method, the incidence of para-anastomotic aneurysms at 5, 10, and 15 years was 0.8, 6.2, and 35.8%, respectively. Univariate analysis indicated that arteriosclerosis obliterans, hypertension, thromboendarterectomy and an anastomosis in the groin were significant risk factors, while stepwised multivariate analysis revealed only hypertension as significant. The mean interval from the primary operation to the diagnosis was 79 months. Ten aneurysms were operated and seven were produced by dehiscence of the anastomotic line, namety anastomotic aneurysms, and three were juxta-anastomotic aneurysms with intact anastomotic lines. Eight patients underwent resection or exclusion of the aneurysm and reconstruction with a new graft and two patients underwent a replacement of the aneurysmal autovein patch to a Dacron one and aneurysmorrhaphy of the parent aneurysmal artery. RESULTS: No recurrence has been detected. In eight patients who were followed conservatively, two died of rupture and renal failure following acute arterial occlusion. CONCLUSIONS: Since para-anastomotic aneurysms can lead to fatal complications, an enlarging or symptomatic aneurysm should be treated promptly.

A case of syndrome of inappropriate secretion of antidiuretic hormone (SIADH) with low plasma concentrations of antidiuretic hormone.

A 66-year-old Japanese man presented with persistent hyponatremia without polydipsia and polyuria. Laboratory examination showed serum sodium of 117 mEq/l, plasma osmolality 239 mosm/kg, urine sodium 108 mEq/l, urine osmolality 577 mosm/kg, and normal levels (less than 2.0 pg/ml) of serum antidiuretic hormone (ADH). ADH release was regulated normally with changes in plasma osmolality. No obvious cause for the syndrome of inappropriate secretion of ADH (SIADH) could be detected. However, 20 months later, the patient had bouts of hematuria and was found to have cancer of the urinary bladder. Increased renal sensitivity to ADH was suspected as the underlying mechanism of SIADH.