Activation of matrix metalloproteinase-2 in human breast cancer cells overexpressing cyclooxygenase-1 or -2.

Human breast cancer cell line Hs578T was stably transfected with cDNA for cyclooxygenase-1 or -2. When the cells overexpressing cyclooxygenase-1 or -2 were stimulated with concanavalin A, the processing of matrix metalloproteinase-2 was observed with the aid of gelatin zymography. This processing was not seen in mock-transfected and original cells which did not express detectable cyclooxygenase activity. Furthermore, Northern blotting showed 8-13 fold induction of membrane-type 1 matrix metalloproteinase which processed matrix metalloproteinase-2 in the cells expressing cyclooxygenases. These findings suggest that both isoforms of cyclooxygenase mediate the processing of matrix metalloproteinase-2 through induction of membrane-type I metalloproteinase in breast cancer cells.

Diagnosis of familial hypercholesterolemia by measurement of sterol synthesis in cultured skin fibroblasts.

The incorporation of radioactive acetate into the digitonin precipitable fraction (cholesterol) was measured in monolayers of primary cultures of skin fibroblasts. Mean incorporation was increased approximately 20-fold in 4 subjects homozygous for familial hypercholesterolemia (FH) and 4-fold in 6 heterozygotes derived from the immediate family of homozygotes. Incorporation was normal in 4 subjects with Type IV and V hyperlipoproteinemia. In cells that had been preincubated in lipid free medium, incorporation by cells from homozygotes was equal to controls, denoting a derangement in the feedback inhibition of cholesterol synthesis by medium lipids in paralleled the values obtained for sterol synthesis. The assay described could be useful in making an "etiologic" diagnosis of familial hypercholesterolemia and could possible identify variants of monogenic hyperbetalipoproteinemia.

Balloon pump-induced pulsatile perfusion during cardiopulmonary bypass does not improve brain oxygenation.

BACKGROUND: Whether pulsatile flow offers substantial advantages for brain protection during cardiopulmonary bypass is controversial. The purpose of this study is to determine whether differences exist between pulsatile and nonpulsatile bypass concerning the effects on internal jugular venous saturation and on the state of regional cerebral oxygenation during normothermia. METHODS: Twenty-two patients undergoing elective coronary artery bypass grafting were randomly divided into 2 groups: group 1 (n = 11) received nonpulsatile perfusion during cardiopulmonary bypass and group 2 (n = 11) received pulsatile perfusion during bypass. We used an intra-aortic balloon pump to generate pulsatility. A spectrophotometric probe (INVOS 3100R, Somanetics, Troy, Mich) was used to assess the state of regional cerebral oxygenation. A 4F fiberoptic oximetry oxygen saturation catheter was inserted into the right jugular bulb to monitor jugular venous oxygen saturation. Hemodynamic variables, arterial and jugular venous blood gases, and regional cerebral oxygenation were measured at 7 times points. RESULTS: In both groups, jugular venous oxygen saturation decreased at the early stage of the cardiopulmonary bypass (P =.03). Five patients in group 1 and 6 in group 2 had a jugular venous oxygen saturation of less than 50%. In both groups, the regional cerebral oxygenation value decreased during cardiopulmonary bypass (P =.04). CONCLUSIONS: The present results showed that pulsatility generated through the use of intra-aortic balloon pumping did not produce any beneficial effects on jugular venous oxygen saturation and regional cerebral oxygenation at normothermia.

Influence of mild to moderate obesity on left ventricular stress filling pattern in hypertension.

In resting condition, obese subjects are described as having impaired diastolic filling. To examine the effect of mild to moderate obesity on left ventricular diastolic performance during stress in hypertension, we determined the filling responses to dynamic submaximal exercise in 19 obese hypertensive patients (body mass index, 26 to 30 kg/m2) with a normal left ventricular structure, 19 age- and sex-matched, non-obese hypertensive patients, and 19 age- and sex-matched, non-obese normotensive controls (mean age, 55+/-3 yr). Doppler echocardiographic studies were performed at baseline and 1 min after exercise on a supine ergometer bicycle. At rest, systolic function and filling indices, peak velocities of early (E) and late (A) filling, and their ratio (E/A), were similar in the two hypertensive groups, while normotensive controls had higher peak velocities of E and E/A. At a maximum workload of 75 W, blood pressure and heart rate increased similarly in the two hypertensive groups. Peak velocities of E and A increased significantly after exercise. The percentage change in the peak velocity of E was greater in obese hypertensive patients than in non-obese hypertensive patients and normotensive controls (23+/-4 vs. 12+/-3 and 14+/-3%, p < 0.05). Percentage changes in A and E/A were similar in the three groups. Our study suggests that mild to moderate obesity does not further worsen left ventricular diastolic filling at rest and mitigates diastolic filling abnormalities after exercise in hypertensive patients.

Effects of hypothermic and normothermic cardiopulmonary bypass on brain oxygenation.

BACKGROUND: In this study, we assessed the effects of normothermia and hypothermia during cardiopulmonary bypass (CPB) both on internal jugular venous oxygen saturation (SjvO2) and the regional cerebral oxygenation state (rSO2) estimated by near infrared spectroscopy (NIRS). METHODS: Thirty patients scheduled for elective coronary artery bypass graft surgery (CABG) were randomly divided into two groups. Group 1 (n = 15) underwent surgery for normothermic (> 35 degrees C) CPB, and group 2 (n = 15) underwent surgery for hypothermic (30 degrees C) CPB, and alpha-stat regulation was applied. A 4.0-French fiberoptic oximetry oxygen saturation catheter was inserted into the right jugular bulb to continuously monitor the SjvO2 value. To estimate the rSO2 state, a spectrophotometer probe was attached to the mid-forehead. SjvO2 and rSO2 values were then collected simultaneously using a computer. RESULTS: Neither the cerebral desaturation time (duration during SjvO2 value below 50%), nor the ratio of the cerebral desaturation time to the total CPB time significantly differed (normothermic group: 18+/-6 min, 15+/-6%; hypothermic group: 17+/-6 min, 13+/-6%, respectively). The rSO2 value in the normothermic group decreased during the CPB period compared with the pre-CPB period. The rSO2 value in the hypothermic group did not change throughout the perioperative period. CONCLUSIONS: These findings suggest that near infrared spectroscopy might be sensitive enough to detect subtle changes in regional cerebral oxygenation.

Primary culture of postnatal rat suprachiasmatic neurons in serum-free supplemented medium.

We have previously reported that postnatal hypothalamic neurons can be maintained in low density culture using astrocyte conditioned medium. The present study was designed to establish a method for the culture of postnatal hypothalamic neurons in a chemically defined medium. Neurons were dissociated from the suprachiasmatic nucleus (SCN) of the hypothalamus of 21-day-old rats and plated on plastic dishes. First, the effects of several factors which have been known to exert trophic effects on neuronal cells were examined in culture medium containing 10% fetal bovine serum. We have found that platelet-derived growth factor, interleukin-1 beta and vitronectin in combination markedly increased the number of surviving neurons bearing processes. Next we tested such effects in serum-free minimum essential medium. When these factors were added together the SCN neurons could be maintained in culture for up to 3 weeks without medium change. In this supplemented medium, SCN neurons gradually extended processes from 3-5 days after plating, and the cell number with processes reached maximal at days 8-11. The cells were identified as SCN neurons by the immunocytochemical staining for microtubule-associated protein 2 (MAP2) and vasoactive intestinal polypeptide. This culture method may be valuable for investigating the electrophysiological properties and the mechanisms of regeneration of mature central neurons.

Inhibition by docosahexaenoic acid of receptor-mediated Ca(2+) influx in rat vascular smooth muscle cells stimulated with 5-hydroxytryptamine.

The effect of docosahexaenoic acid treatment on intracellular Ca(2+) dynamics in rat vascular smooth muscle cells stimulated with 5-hydroxytryptamine (5-HT) has been investigated in order to elucidate one of the mechanisms for its beneficial effect on cardiovascular disorders. The treatment of cells with 30 microM docosahexaenoic acid for 2 days inhibited an increase in intracellular Ca(2+) concentration induced by 5-HT (10 microM) and a depolarizing concentration of KCl (80 mM). Docosahexaenoic acid treatment significantly inhibited divalent cation influx stimulated by 5-HT and KCl, as measured by Mn(2+) quenching method, whereas had no effect on 5-HT-induced Ca(2+) release from the internal stores. Docosahexaenoic acid treatment also significantly inhibited 5-HT receptor-mediated Ca(2+) influx through Ni(2+)-insensitive channels that were distinct from store-operated channels. These results suggest that the specific inhibition of intracellular Ca(2+) dynamics in vascular smooth muscle cells may contribute to the beneficial properties of docosahexaenoic acid on cardiovascular disorders.

5-hydroxytryptamine induces transient Ca2+ influx through Ni2+-insensitive Ca2+ channels in rat vascular smooth muscle cells.

The effects of Ni2+, a non-selective cation channel inhibitor, on 5-hydroxytryptamine (5-HT)- and angiotensin II (Ang II)-induced intracellular Ca2+ dynamics in rat aortic smooth muscle cells were investigated. Ni2+ (1 mM) significantly inhibited the transient increase in intracellular Ca2+ concentration ([Ca2+]i) induced by Ang II (100 nM) in aortic smooth muscle cells, as measured using fura-2. However, Ni2+ did not suppress the transient increase in Ca2+ influx induced by 5-HT (10 microM), while significantly suppressed the sustained increase. Ca2+ influx evoked by high KCl (80 mM), thapsigargin (TG) (1 microM) or depletion of intracellular Ca2+ store was almost completely suppressed by Ni2+. Ni2+ had no effect on 5-HT-induced inositol triphosphate production and Ca2+ release from the intracellular store(s). These results suggest that 5-HT, but not Ang II, induces transient Ca2+ influx through Ni2+-insensitive Ca2+ channels, which are distinguishable from the voltage-dependent or store-operated Ca2+ channels.

Pharmacological characteristics of GABAA responses in postnatal suprachiasmatic neurons in culture.

The suprachiasmatic nucleus (SCN) is considered to be an endogenous circadian pacemaker. Previous studies have suggested functional roles of gamma-aminobutyric acid (GABA) in the control of circadian rhythms. In this study, the responses to applied GABA in cultured SCN neurons dissociated from postnatal rat hypothalamus were investigated using whole-cell voltage-clamp techniques. GABA and muscimol induced a large current response (EC50 values 5.3 and 1.6 microM, respectively), which was blocked by the GABAA antagonist bicuculline. This current response was also blocked by Zn2+ (0.5-50 microM) in a concentration-dependent manner, but was not potentiated by diazepam (10 microM) or ethanol (21 mM). These characteristics seem to correspond to those of GABAA receptors that lack gamma-type subunits.

The expression of E-cadherin as a prognostic factor in breast cancer.

We examined the clinical significance of E-cadherin expression in 98 patients with breast cancer. E-cadherin expression significantly correlated with histologic grade (p=0.0323) and tended to correlate with the absence of axillary lymph node metastases (p=0.0646). The expression of E-cadherin was associated significantly with the recurrence rate (p=0.0334) and marginally associated with disease-free (p=0.0597) but not overall survival, in univariate analysis. The association between E-cadherin expression and disease-free survival was lost in multivariate analysis. The prognostic value of E-cadherin expression in breast cancer appears to be limited. A larger, prospective study with long-term follow-up is needed.

Effect of docosahexaenoic acid on smooth muscle cell functions.

There is increasing evidence that fish oil-enriched diets attenuate the progression of several types of human and experimental renal, intestinal and cardiovascular disorders, including hypertension. Docosahexaenoic acid (DHA), may be one of the active biological component. We previously reported that dietary DHA suppressed the progression of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). The purpose of this study is to clarify the in vitro effect of DHA on cultured smooth muscle cell functions such as cell growth, hypertrophy, NO release, and intracellular Ca2+ metabolism, which are involved in the regulatory mechanisms of vascular tone. Addition of DHA to the culture medium of aortic smooth muscle cells isolated from SHRSP and normotensive Wistar Kyoto rats (WKY) had no significant effects on cell growth or on cell hypertrophy induced by angiotensin II as measured by flow cytometry. DHA had no stimulatory effect on interleukin-1beta (10 ng/ml)-induced nitric oxide release from smooth muscle cells of SHRSP, but rather slightly inhibited it. However, the treatment of smooth muscle cells with DHA (30 microM) for 2 days significantly suppressed the increase in intracellular Ca2+ concentration induced by angiotensin II, but not by thapsigargin. This was due to the suppression of Ca2+ influx, as determined by Mn2+ influx experiment. These results indicate that DHA specifically suppresses Ca2+ mobilization into smooth muscle cells. This may be one of the mechanisms by which dietary DHA prevents the development of hypertension in SHRSP.

Conversion of progesterone-1,2-3-H to 5beta-pregnane-3,20-dione by brain tissue.

The presence of a 5beta-reductase acting to convert progesterone to 5beta-pregnane-3,20-dione is described in the soluble 105,000 x g fraction of a preparation of dog cerebral cortex. The function of this enzymatic activity is obscure but may be important in regulation of sensorium. 5beta-pregnane compounds are potent depressors of the central nervous system.

Cyclothialidine analogs, novel DNA gyrase inhibitors.

DNA gyrase inhibitors, cyclothialidines B, C, D and E were isolated from four Streptomycete strains (NR 0659, NR 0660, NR 0661 and NR 0662). Their structures have been elucidated based on the amino acid analysis of the hydrolysates, NMR and HRFAB-MS experiments and shown to be cyclothialidine analogs. The absolute stereochemistry has been determined by the chiral HPLC analysis of the hydrolysates. Cyclothialidines B, D and E are novel and potent inhibitors of DNA gyrase.

Adjuvant chemotherapy with a combination of mitoxantrone, methotrexate, 5-fluorouracil for node-positive breast cancer: phase II pilot study.

A phase II pilot study was carried out on 30 patients to ascertain the toxicity and efficacy of combination chemotherapy with mitoxantrone, methotrexate, 5-fluorouracil (NMF) in the adjuvant setting for axillary lymph node-positive breast cancer. The NMF regimen was mitoxantrone 10 mg/m2, methotrexate 40 mg/m2, and 5-fluorouracil 600 mg/m2 administered i.v. on day 1, repeated every 3-4 weeks for 6 cycles. The median nadir WBC count was 2,000/microl; grade 4 leukocytopenia occurred only in 1 patient. Nausea and vomiting appeared as grade 0 and 1 severity in 26/30 patients. Alopecia was extremely mild, appeared as grade 0 and 1 in 29/30 patients. The overall and relapse-free survival rates were 67.8% and 68.4% at the 82-month follow-up, respectively. The overall survival rate in premenopausal patients was significantly better than that in postmenopausal patients (P<0.05). NMF is a well-tolerated combination regimen, suitable as adjuvant chemotherapy for node-positive breast cancer.

Left ventricular aneurysm without coronary artery disease, incidence and clinical features: clinical analysis of 11 cases.

OBJECTIVE: To examine the incidence, underlying disease and clinical features of left ventricular aneurysm (LVA) not related to coronary artery occlusion. METHODS: Retrospective review of consecutive patients who underwent both left ventriculography and coronary angiography. PATIENTS: LVA was confirmed in 11 of 2,348 consecutive patients (0.47%). RESULTS: The location of LVA was mainly in the apical region (81.8%). In five of the 11 patients (45.5%), the underlying heart disease was hypertrophic cardiomyopathy (HCM), including 4 patients of dilated phase and one patient of midventricular type. The serial ECG changes from left ventricular hypertrophy to abnormal Q wave and endomyocardial biopsy were useful for the differential diagnosis of these cases against myocardial infarction. The underlying disease of the remaining patients was: myocarditis (2 patients), arrhythmogenic right ventricular dysplasia (1 patient), Chagas' disease (1 patient), glycogen storage disease (1 patient), and sarcoidosis (1 patient). Ventricular tachycardia appeared in 9 of 11 cases (81.8%) including 2 patients with sustained ventricular tachycardia. CONCLUSION: LVA formation without coronary artery disease was a rare phenomenon. The underlying disease was varied but the incidence of hypertrophic cardiomyopathy in the dilated phase was comparatively high. Ventricular tachycardia was a significant complication in these patients.

Breast conserving treatment versus modified radical mastectomy in Japanese patients with operable breast cancer.

From January, 1988 to October, 1995, 96 patients with operable breast cancer were treated by breast conserving treatment (BCT) including wide excision and axillary dissection followed by breast radiation. During the same period, 188 patients were treated by modified radical mastectomy (MRM) with or without breast reconstruction. In order to compare the survival of BCT and MRM groups, univariate and multivariate analyses were performed in this retrospective study. Univariate analysis revealed that the 5-year survival rates in the BCT and MRM groups were 97% +/- 2% and 87% +/- 3%, respectively (p < 0.05 with the Cox-Mantel test). However, the baseline variables were different between the groups. The adjusted Cox regression analysis revealed that the results of BCT were almost equivalent with those of MRM. Moreover, no breast recurrence was found in the BCT group. Therefore, it is suggested that our technique of BCT is as effective as modified radical mastectomy in treating operable breast cancer in Japanese patients.

[Malfunctioning of cerebral function monitors in three cases of carotid endarterectomy].

In three patients, EEG, jugular venous oxygen saturation (Sjvo2) and near infrared spectroscopy (NIRS) were monitored to detect cerebral ischemia during carotid endarterectomy. In all cases, no changes in Sjvo2 and NIRS were observed during carotid artery occlusion, but in two patients EEG showed changes when carotid artery was clamped. It is important to know the precise mechanism of cerebral monitors to assess the cerebral ischemia in patients with preexisting neurological disorder during carotid endarterectomy.

[The change of oxygen consumption and oxygen delivery in patients undergoing heart surgery during perioperative period].

We examined the changes of oxygen consumption (Vo2), oxygen delivery (Do2) and Vo2/Do2 in 38 patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) during perioperative period. There were no significant correlations between Svo2 and cardiac index. The Vo2/Do2 showed a good level at pre-CPB and 1 hour after CPB, but the value became deteriorated 4 and 12 hours after CPB. These data suggest that we should be careful about the oxygen debt in tissue 4 and 12 hours after CPB.

[Diastolic function in patients with coronary artery disease before and after CABG].

We evaluated diastolic cardiac function in 16 patients with coronary artery disease before and after coronary angioplasty using transesophageal echocardiography. Peak early diastolic and late diastolic filling velocities (E wave and A wave) and velocity time integrals of early and late diastolic filling (VTI-E and VTI-A) were measured by pulsed doppler echocardiography. The patients were divided into two groups: Group A; E/A > or = 1. Group B; E/A < 1. There were statistical differences between the two groups in E-acceleration time and VTI 0-33%/ total VTI at pre-coronary angioplasty period (P < 0.05). There were statistical differences in E-decelaration time and E-decelaration slope of group B before and after coronary angioplasty (p < 0.05). These results suggest that the left ventricular diastolic function in group B has recovered partly at one hour after coronary angioplasty.

[A case of aortic dissection after the termination of cardiopulmonary bypass].

We report, a case of aortic dissection after the termination of cardiopulmonary bypass (CPB), which was diagnosed by transesophageal echocardiography (TEE). A 70-year old male with aortic regurgitation received aortic valve replacement. After the termination of CPB, the aortic dissection was diagnosed by TEE. Furthermore wall motion abnormality was found by TEE, and aorto-coronary bypass was performed after observation by TEE. This case report suggests that TEE is useful not only for diagnosis but also for therapeutic orientation of aortic dissection.