Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing.

The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.

Involvement of protein phosphatase 2A nuclear accumulation and subsequent inactivation of activator protein-1 in leptomycin B-inhibited cyclin D1 expression.

Leptomycin B (LMB) is a Streptomyces metabolite that causes the specific inhibition of the nuclear export of proteins containing a nuclear export signal (NES). LMB was reported to inhibit cell cycle progression in fission yeast and mammalian cells, however, the mechanism underlying LMB-induced cell cycle arrest is still obscure. In this study, we found that in serum-starved NIH3T3 cells, LMB inhibited serum-induced cyclin D1 expression at the level of transcription. However, this inhibition was reversed by inhibitors of protein phosphatase 2A (PP2A). Furthermore, we found that PP2A accumulated in the nucleus upon treatment with LMB. The finding prompted us to identify the functional NES in PP2A catalytic subunit alpha. These results indicated that LMB inhibited the chromosomal region maintenance 1 (CRM1)-dependent nuclear export of PP2A, resulting in sustained dephosphorylation in the nucleus. Although phosphorylation of c-Jun at Ser-63 is required for activator protein 1 (AP-1)-dependent expression of cyclin D1, it decreased in LMB-treated cells compared to untreated cells. Moreover, the inhibitors of PP2A restored the levels of c-Jun phosphorylated at Ser-63. We propose that inhibition of cyclin D1 expression by LMB is mediated by the LMB-induced nuclear accumulation of PP2A, leading to sustained dephosphorylation of c-Jun at Ser-63, which leads to inactivation of the transcription of the AP-1-responsive cyclin D1 gene.

Flow cytometric analysis of hemetopoietic progenitor cells in peripheral blood stem cell harvest from patients with CD34 positive acute leukemia.

We analyzed CD34 positive cells in peripheral blood stem cell harvest (PBSCH) using flow cytometry. PBSCH from CD34 positive acute myelogeous leukemia (AML-M2) patient contained 1.87% CD34 positive cells, of which 1.21% was represented by MRD.PBSCH from CD34 positive acute lymphoblast leukemia (ALL) patient contained 3.14% CD34 positive cells, of which 0.11% was accounted for by minimal residual disease (MRD). If PBSCH from CD34 positive acute leukemia patient is analyzed for CD34 monoclonal antibody alone, the presence of CD34 positive MRD may escape attention so that CD34 positive hematopoietic progenitor cells may be overestimated. To avoid this risk, it is necessary to analyze PBSCH using both CD34 monoclonal antibody and characteristic markers of leukemia cells that were found pre-treatment.

The regression of left ventricular hypertrophy by imidapril and the reduction of serum procollagen type III amino-terminal peptide in hypertensive patients.

Angiotensin-converting enzyme (ACE) inhibitors are known to be the most effective antihypertensive drugs for reducing left ventricular mass in hypertensives when compared to other classes of drugs. In the present study, we evaluated the effects of imidapril, an ACE inhibitor, on serum procollagen type III amino-terminal peptide (PIIIP) levels as well as the left ventricular mass index (LVMI). The subjects consisted of 15 patients (12 men and 3 women) in the outpatient clinic of our hospital who were diagnosed as essential hypertensives and who had not been treated with any antihypertensive medication prior to the study. Left ventricular hypertrophy was observed in all of the patients, ie., LVMI >110 g/m2 in men and >106 g/m2 in women. Blood pressure, LVMI, and serum PIIIP levels were measured before and after treatment with imidapril for 6 months. The starting dose of imidapril was 5 mg, and this was increased to 10 mg. Finally, 1 mg of trichlormethiazide was added to obtain adequate control of blood pressure. Blood pressure significantly decreased in 12 patients, and the mean LVMI decreased significantly from 153.1 +/- 9.0 to 135.4 +/- 6.3 (p< 0.01) after treatment. The changes in LVMI and PIIIP levels with treatment had significant correlation (r=0.639, p< 0.05). The present study showed that imidapril reduces the left ventricular mass in hypertensives after 6 months of treatment, and that this may at least in part be due to a decrease in the collagen content of the hypertrophied heart, suggesting that serum PIIIP levels are a useful marker of the regression of left ventricular hypertrophy.

Herbimycin A induces G1 arrest through accumulation of p27(Kip1) in cyclin D1-overexpressing fibroblasts.

The ansamycin antibiotic herbimycin A is a potent tyrosine kinase inhibitor and reduces the growth rate of various types of mammalian cells. When quiescent Rat6 fibroblast cells were treated with herbimycin A, serum-induced expression of cyclin D1 was inhibited, and this was associated with inhibition of G1 phase progression. However, herbimycin A also inhibited serum-induced G1 progression in derivatives of the Rat6 fibroblast cell line that stably overexpress a human cyclin D1 cDNA (R6ccnD1#4 cells), without affecting the expression levels of G1 cyclins. We found that herbimycin A prevented serum-induced downregulation of the cyclin-dependent kinase inhibitor p27(Kip1), thereby leading to inactivation of the protein kinase activity of CDK2. These results suggest that herbimycin A inhibits a tyrosine kinase(s) that plays a role in degradation of the p27(Kop1) protein.

Reduction in interleukin-2-producing cells but not Th1 to Th2 shift in moderate and advanced stages of human immunodeficiency virus type-1-infection: direct analysis of intracellular cytokine concentrations in CD4+ CD8- T cells.

Previous studies have suggested that CD4+ T lymphocytes shift from the Th1 type to the Th2 type during disease progression in patients with the human immunodeficiency virus type-1 (HIV-1). In the present study, we used a modified method that allowed a direct measurement of intracellular cytokines in CD4+ CD8- T cells. A total of 48 HIV-1-infected (HIV+) and 16 HIV-1-uninfected (HIV-) individuals were studied. The percentages of CD4+ CD8- T cells producing interleukin-2 (IL-2), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), or interleukin-5 (IL-5) in HIV+ and HIV- subjects were 23.6% versus 34.9% (P < 0.01), 13.7% versus 13.2%, 1.3% versus 1.0%, and 1. 2% versus 0.9%, respectively. The population of IL-2-producing cells decreased proportionately with reductions in CD4 counts (< 200/mm3, 200-500/mm3, and > 500/mm3 to 18.0%, 23.5%, and 30.5%, P < 0.05, respectively). There was an inverse correlation between the percentage of IL-2-producing cells and plasma viral load (r = - 0. 446, P < 0.05). However, the percentages of CD4+ CD8- T cells producing other cytokines were not different between HIV+ and HIV-. Our cross-sectional study demonstrated a decrease in IL-2-producing cells but not the Th1 to the Th2 shift in the CD4+ CD8- T cell population in the moderate and advanced stages of HIV-1-infection.

Caspase-3 activation is not responsible for vinblastine-induced Bcl-2 phosphorylation and G2/M arrest in human small cell lung carcinoma Ms-1 cells.

Vinblastine arrests cells in the G2/M phase of the cell cycle and subsequently induces cell death by apoptosis. We found that treatment of cells with vinblastine induced phosphorylation of Bcl-2, resulting in the dissociation of Bcl-2 and Bax. Moreover, vinblastine-induced apoptosis was suppressed by an inhibitor of caspase-3, Ac-DEVD-CHO; and a 17-kDa active fragment of caspase-3 was detected following vinblastine treatment, suggesting that caspase-3 is involved in vinblastine-induced apoptosis. However, Ac-DEVD-CHO affected neither vinblastine-induced Bcl-2 phosphorylation nor vinblastine-induced G2/M arrest. Vinblastine caused G2/M arrest prior to apoptosis, whereas vinblastine-induced apoptosis was not dependent on the duration of the G2/M phase. Thus, vinblastine-induced apoptosis might be mediated by the phosphorylation of Bcl-2, resulting in Bcl-2 inactivation, and by subsequent activation of caspase-3.

Potentiation of paclitaxel cytotoxicity by inostamycin in human small cell lung carcinoma, Ms-1 cells.

In the present study, we found that inostamycin increased the ability of paclitaxel to induce apoptosis in Ms-1 cells. A considerably higher concentration of paclitaxel was required for the induction of apoptosis in Ms-1 cells than in other cell lines tested. Treatment of Ms-1 cells with inostamycin, an inhibitor of phoshatidylinositol (PI) synthesis, reduced the dosage of paclitaxel required to induce cell death by apoptosis. This effect of inostamycin is specific to Ms-1 cells, and inostamycin did not increase the cytotoxicity of other antitumor drugs such as adriamycin, vinblastine, methotrexate, cisplatin, etoposide, or camptothecin in Ms-1 cells. Addition of inostamycin to paclitaxel-treated cells caused a significant increase in the sub G1 peak, representing apoptosis, which was accompanied by a decrease in the G2/M peak seen in paclitaxel-treated Ms-1 cells, without affecting paclitaxel-inhibited tubulin depolymerization. Moreover, paclitaxel did not enhance inostamycin-inhibited PI synthesis. The expression levels of Bcl-2, Bax, and Bcl-XL were not changed following the co-treatment with inostamycin plus paclitaxel, whereas the activated form of caspase-3 was markedly increased. Thus, inostamycin is a chemosensitizer of paclitaxel in small cell lung carcinoma Ms-1 cells.

Plasma renin activity could be a useful predictor of left ventricular hypertrophy in essential hypertensives.

To clarify the ability of clinical and laboratory parameters to reflect target organ damage, especially left ventricular hypertrophy (LVH), we investigated which of these parameters might correlate to LVH as determined by electrocardiographic voltage at the first clinic visit in 108 (53 males and 55 females, average age 52 +/- 10 years) untreated essential hypertensives. The sum of the amplitude of the S wave in lead V1 plus that of the R wave in lead V5 or V6 (SV1 + R(V5, V6)) was correlated with blood pressure in both males and females. In subjects with LVH (SV1 + R(V5, V6) > or = 3.5mV), a stepwise multiple regression analysis revealed that SV1 + R(V5, V6) was associated with plasma renin activity (PRA) in both males and females, and with creatinine concentration (Cr) in males. These results suggest that PRA at the first visit could be a useful predictor of LVH in patients with essential hypertension.

Mild exercise activates renal dopamine system in mild hypertensives.

OBJECTIVE: The role of renal dopamine in the early depressor effect of exercise was evaluated in hypertensives. METHODS: After a general clinical observation period of 4 weeks, 29 essential hypertensives were divided into two groups. The exercise group (n=16) underwent blood lactate threshold exercise using a cycle ergometer for 60 min three times a week for 4 weeks. RESULTS: In the non-exercise group (n=13), blood pressure (BP) and humoral variables did not change significantly (from 150+/-3/93+/-2 to 145+/-2/94+/-1 mm Hg). In the exercise group (n=16), resting BP was significantly reduced from 158+/-2/92+/-2 at week 0 to 145+/-3/85+/-3 mm Hg at week 4. The increase in urinary free dopamine excretion (from 248+/-14 to 276+/-24 ng/mg Cr) at week 4 was significantly higher than that in the non-exercise group (from 220+/-31 to 196+/-27 ng/mg Cr). In the exercise group, urinary kallikrein activity also increased significantly from 173.0+/-35.4 at week 0 to 320.3+/-63.3 ng bradykinin/min/mg Cr at week 4. These changes in urinary free dopamine excretion and urinary kallikrein activity were negatively correlated with the change in BP. The change in urinary sodium excretion was also negatively correlated with the change in plasma volume index. Moreover, the change in urinary free dopamine excretion was positively correlated with the changes in urinary kallikrein activity and urinary sodium excretion. The change in renal decarboxylation rate of DOPA (3,4-dihydroxyphenylalanine) positively correlated with the changes in urinary free dopamine excretion and urinary sodium excretion, and was negatively correlated with the change in systolic BP. CONCLUSION: These results suggest that exercise triggered renal dopamine generation and activation of renal kallikrein-kinin system, resulting in natriuresis and BP reduction in the early phase (4 weeks) of mild exercise.

Inhibition of cyclin D1 expression and induction of apoptosis by inostamycin in small cell lung carcinoma cells.

Previously, we demonstrated that inostamycin, an inhibitor of phosphatidylinositol turnover, caused cell cycle arrest at the G1 phase, inhibiting the expression of cyclins D1 and E in normal cells. In the present study, we examined the effects of inostamycin on cell cycle progression and apoptosis in human small cell lung carcinoma Ms-1 cells. Treatment of exponentially proliferating Ms-1 cells with low concentrations of inostamycin caused cells to accumulate in the G1 phase. We found that inostamycin decreased cyclin D1, and increased cyclin-dependent kinase inhibitors such as p21WAF1 and p27KIP1 in Ms-1 cells. On the other hand, higher concentrations of inostamycin induced morphological apoptosis and DNA fragmentation in Ms-1 cells without affecting the expression of p53, Bcl-2 and Bax. Inostamycin-induced apoptosis was suppressed by an inhibitor of caspase-3, and a 17 kDa fragment of activated caspase-3 was detected following inostamycin treatment. Therefore, caspase-3(-like) would appear to be involved in inostamycin-induced apoptosis. On the other hand, an inhibitor of caspase-3(-like) proteases did not affect the inhibitory effect of inostamycin on cyclin D1 expression, suggesting that caspase-3(-like) proteases were not responsible for inostamycin-induced G1 arrest.

Role of endogenous ouabain-like substance during graded exercise in hypertensive individuals.

Changes in plasma endogenous ouabain-like substance (EOLS) and plasma noradrenaline, along with changes in blood pressure (BP), heart rate, and blood lactate concentration, were investigated in hypertensive individuals during strenuous exercise testing. Thirteen (4 men and 9 women) middle-aged (34-63 years, mean 50 +/- 2 years) patients with mild essential hypertension underwent graded multistage submaximal exercise testing on a cycle ergometer. The workload in each exercise test was increased depending on the individual's physical fitness until they reached 75-80% of the predicted age-adjusted maximal heart rate prescribed by the American College of Sports Medicine. Systolic (S) BP rose by 67 +/- 9 mm Hg (P < 0.001), mean (M) BP by 28 +/- 3 mm Hg (P < 0.001), diastolic (D) BP by 9 +/- 2 mm Hg (P < 0.005) and heart rate by 79 +/- 6 beats/min (P < 0.001) after submaximal graded exercise. The blood lactate concentration and plasma noradrenaline increased significantly (+3.40 +/- 0.34 mmol/l and +895 +/- 94 pg/ml respectively, P < 0.001). Although the change in EOLS was not significant, it showed a strong positive correlation with the change in plasma noradrenaline (R = 0.760, P < 0.001). These results suggest that EOLS may participate in modifying sympathetic vasoconstriction during submaximal graded exercise.

Urinary kallikrein activity is increased during the first few weeks of exercise training in essential hypertension.

OBJECTIVE: To determine whether the renal kallikrein-kinin and dopamine systems participate in lowering blood pressure during mild exercise in hypertensives. DESIGN: After a general clinical observation period of 4 weeks, 27 essential hypertensives were divided into two groups. The exercise group underwent blood lactate threshold exercise, using a cycle ergometer for 60 min three times a week for 10 weeks. The non-exercise group was observed at the outpatient clinic. Blood pressure and humoral parameters were measured at weeks 0, 1, 2, 4 and 10 in both groups. METHODS: Blood pressure was measured indirectly with an automatic blood pressure recorder. Twenty-four-hour urinary kallikrein activity (by kininogenase assay), total or free dopamine and total noradrenaline (by high-performance liquid chromatography) were also measured. RESULTS: In the non-exercise group blood pressure and humoral parameters did not change. In the exercise group the change in resting blood pressure between weeks 0 and 10 was statistically significant. The change in 24-h urinary kallikrein activity of the exercise group was significantly greater than that of the non-exercise group between weeks 0 and 1 and weeks 0 and 2. Moreover, the change in systolic blood pressure (SBP) between weeks 0 and 2 was negatively correlated with the change in urinary kallikrein activity between weeks 0 and 2, the change in total dopamine between weeks 0 and 2 was negatively correlated with the change in diastolic blood pressure in the same period, and the change in SBP between weeks 0 and 10 was positively correlated with the change in total noradrenaline in the same period in the exercise group. Subjects with a relatively high baseline urinary kallikrein activity had a significantly greater change in SBP between weeks 0 and 10 than subjects with a relatively low baseline activity. CONCLUSIONS: The renal kallikrein-kinin and dopamine systems may participate in lowering blood pressure during the first few weeks of exercise training. The subsequent reduction of sympathetic activity may be involved in maintaining the lowered blood pressure. Mild exercise is more effective in reducing blood pressure in hypertensives who have a relatively high basal renal kallikrein-kinin system activity.

[Cognitive ability regarding dental plaque and oral bacteria in schoolchildren: results from an interview research of primary and junior high school students].

The cognitive abilities regarding dental health terms, especially of dental plaque, were examined among schoolchildren. An interview of 112 primary and junior high school students was performed to test knowledge of the cause of dental caries and meaning of some dental health terms. Results indicate that most schoolchildren had difficulty understanding the correct meaning of dental plaque and oral bacteria. Some of them did not recognize the existence of oral bacteria, much less dental plaque. From these results, factors which may interfere with correct understanding of dental plaque were examined. Among them, inaccurate knowledge of existence of oral bacteria appears to be an important factor.

Crossover comparison between the depressor effects of low and high work-rate exercise in mild hypertension.

1. The relationship between work-rate and the antihypertensive effect of exercise in hypertensives, and the mechanism of that effect, were investigated by a crossover clinical trial. 2. Ten mild hypertensives were randomly divided into two groups. One group performed low work-rate exercise (LWE) on a cycle ergometer for 10 weeks (blood lactate threshold; approximately 50% of maximum oxygen consumption [Vo2max]). After a 10 week interval without exercise training, these subjects were then switched to a high work-rate exercise (HWE) regimen (4 mmol/L of blood lactate; approximately 75% of Vo2max) for another 10 weeks. In the other group, the order of exercise training was reversed. Since two patients withdrew from the protocol during HWE periods, statistical analysis was performed on the data from the remaining eight patients. There were no order effects observed in any of the data from the two groups. 3. During both LWE and HWE, resting blood pressure (BP) fell significantly after the initiation of exercise therapy (P < 0.05). Furthermore, the overall effects of 10 weeks of LWE and HWE on BP were not significantly different. 4. The work-rate at the lactate threshold, which reflects physical fitness, had increased significantly by 16 W (P < 0.01) after the LWE period and by 11 W (P < 0.01) after the HWE. 5. During the LWE period, changes in haemodynamic and humoral variables were not significant, except for a reduction in plasma norepinephrine at week 10 (P < 0.05). In the HWE period, changes in haemodynamic and humoral variables were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

[Acute basophilic leukemia: a case report].

A 56-year-old woman was admitted to our hospital in January, 1990 because of fever and petechiae. Leukocyte count of peripheral blood showed 41,000/microliters with 89% immature cells, and bone marrow was normocellular with 96.2% immature cells. They were medium to large in size, positive for peroxidase staining, CD-13 and CD-33. Half of them contained azurophilic granules. They showed metachromasia by toluidine blue, contained basophilic granules in electron microscopic examination and reacted to G-CSF, G-CSF and IL-3. She was diagnosed as acute basophilic leukemia and treated with BHAC-DMP and B triple-V regimen, but remission was not attained. She died of peritonitis due to gastrointestinal tract perforation and pneumonia in March, 1990. This is the fifteenth case of acute basophilic leukemia reported in Japan, and the hematological examinations performed in this patient were demonstrated.

[A case of idiopathic hyperaldosteronism diagnosed by adrenal imaging].

We report a case of idiopathic hyperaldosteronism (IHA) which was differentiated from an aldosterone producing adenoma by the adrenal imaging techniques with computed tomography (CT) and scintigraphy. In this patient, the high basal aldosterone level with the suppressed plasma renin activity typically indicated the diagnosis of primary aldosteronism. However, the differentiation from an aldosterone producing adenoma by responses of plasma aldosterone levels to upright posture, captopril or adrenocorticotropic hormone (ACTH) administration was not definitive. Abdominal CT revealed bilateral adrenal swelling. Adrenal scintillation scanning with 131I-iodocholesterol showed bilateral uptake even after the administration of dexamethasone. Blood sampling from the right adrenal vein was unsuccessful. Blood pressure and serum potassium levels remained unchanged during dexamethasone administration (2 mg/day) over ten days. After the administration of spironolactone and nisoldipine blood pressure and serum potassium levels were normalized. Adrenal imaging is considered to be very useful for the diagnosis of IHA.

Influence of workload on the antihypertensive effect of exercise.

1. The relation between workload and the antihypertensive effect of exercise therapy in hypertensive patients, and the mechanism of that effect, were investigated. 2. Twenty-six patients participated in the study and were randomly assigned to 10 weeks of either low or high workload exercise. In the low workload group, 16 mild hypertensive patients were treated with bicycle ergometer exercise at approximately 50% of their maximum oxygen consumption (VO2max) for 60 min three times a week for 10 weeks. In the high workload group, 10 mild hypertensive patients exercised on the same schedule, but at approximately 75% of VO2max. 3. After 10 weeks of exercise, the low workload group had significantly lower systolic (9 mmHg), mean (6 mmHg) and diastolic (6 mmHg) blood pressures. In the high workload group, decreases in systolic (3 mmHg), mean (4 mmHg) and diastolic (5 mmHg) blood pressure were not statistically significant. 4. In the low workload group, changes in haemodynamic and humoral variables were not significant, except for a reduction in plasma norepinephrine at week 7. Cardiac index and plasma norepinephrine tended to decrease. In the high workload group, plasma norepinephrine and the renin-angiotensin system were transiently stimulated after 4 weeks of exercise. Stroke volume significantly increased (+26.4%) after 10 weeks of high workload exercise. 5. Based on these results and better patient compliance with the exercise programme in the low workload group than in the high workload group, low workload exercise therapy was recommended to mild hypertensive patients.

Cardiac effects of local angiotensin-converting enzyme inhibition in hypertensive patients.

1. The direct cardiac effects of angiotensin-converting enzyme (ACE) inhibitor in 22 hypertensive patients were investigated. Radionuclide ventriculography and echocardiography were performed to measure left ventricular function before and 60 min after administration of a subdepressor dose of captopril. 2. Since the response to ACE inhibitor is not uniform, patients were classified into 12 patients without significant blood pressure change following captopril (group I) and 10 patients with reduction of blood pressure (group II). 3. Clinical and baseline haemodynamic characteristics were similar for the two groups. 4. Ejection fraction (EF) increased without changes of heart rate and end-diastolic dimension after ACE inhibitor in group I as well as group II. The change of EF was not different for the two groups. No correlation was found between changes in EF and blood pressure in group I patients. 5. This study indicates that ACE inhibitor might directly influence left ventricular function independent of systemic haemodynamic changes.