Interaction between Otx2 and Gbx2 defines the organizing center for the optic tectum.

Otx2 is expressed in the mesencephalon and prosencephalon, and Gbx2 is expressed in the rhombencephalon around stage 10. Loss-of-function studies of these genes in mice have revealed that Otx2 is indispensable for the development of the anterior brain segment, and that Gbx2 is required for the development of the isthmus. We carried out gain-of-function experiments of these genes in chick embryos with a newly developed gene transfer system, in ovo electroporation. When Otx2 was ectopically expressed caudally beyond the midbrain-hindbrain boundary (MHB), the alar plate of the metencephalon differentiated into the optic tectum instead of differentiating into the cerebellum. On the other hand, when Gbx2 was ectopically expressed at the mesencephalon, the caudal limit of the tectum shifted rostrally. We looked at the effects of misexpression on the isthmus- and tectum-related molecules. Otx2 and Gbx2 interacted to repress each other's expression. Ectopic Otx2 and Gbx2 repressed endogenous expression of Fgf8 in the isthmus, but induced Fgf8 expression at the interface between Otx2 and Gbx2 expression. Thus, it is suggested that interaction between Otx2 and Gbx2 determines the site of Fgf8 expression and the posterior limit of the tectum.

A proof of glutathione S-transferase-pi-related multidrug resistance by transfer of antisense gene to cancer cells and sense gene to bone marrow stem cell.

In order to directly prove the involvement of GST-pi in drug resistance, it's antisense gene was transduced into human colorectal cancer cell line which has been shown to express high level of GST-pi and the sensitivity of this cell line to anticancer drugs were assessed. The transfectant showed higher sensitivity to adriamycin (3.3-fold), Cisplatnum (2.3-fold), Melphalan (2.2-fold), Etoposode (2.2-fold) than the parental cell, while the sensitivity to vincristine, mitomicin C, 5-fluorouracil was unchanged by transfection. When the transfectant and parental cells were innoculated in nude mice and treated with adriamycin, a significant suppression of tumor growth was observed with the transfectant as compared to the parental cell. On the basis of this observation, we then transduced sense GST-pi gene into human bone marrow stem cells (CD34+ cells) to protect them from toxicity of anticancer drug. The gene transduced CD34+ cells formed more CFU-GM than nontransduced CD34+ cell in the presence of adriamycin (30 ng/ml). Thus, the autotransplantation of GST-pi gene transduced cell into cancer patients to protect the bone marrow from subsequent highdose chemotherapy is considered to be a new strategy for cancer gene therapy.

[Glutathione levels in human colon cancer cell line M7609 following culture in a low sulfur amino acid medium and its sensitivity to various anticancer drugs].

Glutathione levels in human colon cancer cell line M7609 and its sensitivity to anticancer drugs were investigated in a complete medium RPMI-1640 (medium A) and an incomplete medium (medium B), which was prepared by removing glutathione and sulfur amino acids from medium A. Four different medium conditions were prepared by combining a medium A and a medium B; a medium of 100% medium A (Control), a 50:50 mixture medium of medium A and medium B (Condition 2), a 20:80 mixture medium of medium A and medium B (Condition 3), and a 10:90 mixture medium of medium A and medium B (Condition 4). The cells were cultured in each medium for 14 days, and intracellular levels of glutathione were determined to estimate the cell sensitivity to anticancer drugs. There were no significant differences in glutathione levels among the cancer agents in condition 2, as compared to those in the control. In condition 3, the reduced glutathione levels were decreased to 23.1%, where CDDP, ADM, MMC and melphalane showed 2.5, 2.2, 1.8 and 1.5 times greater antitumor activity than in the control, respectively. In condition 4, cell proliferation was too low to collect adequate cells for glutathione determination. These results demonstrated that the decrease in cellular glutathione concentration with this method might enhance the cytotoxic effects of anticancer drugs.

Involvement of beta 2-glycoprotein I and anticardiolipin antibodies in oxidatively modified low-density lipoprotein uptake by macrophages.

Anticardiolipin antibodies (aCL) in the sera of patients with antiphospholipid syndrome (APS) recognize an altered structure of beta 2-glycoprotein I (beta 2-GPI) interacting with solid-phase negatively charged phospholipids. beta 2-GPI bound to Cu2+-oxidized plasma lipoproteins, i.e. oxidized very low-density lipoprotein (oxVLDL), oxidized low-density lipoprotein (oxLDL), or oxidized high-density lipoprotein (oxHDL). beta 2-GPI inhibited in vitro uptake, i.e. cell surface binding, cellular association, and proteolytic degradation of oxLDL by murine macrophage J774A.1 cells. The binding of oxLDL to the macrophages was inhibited by the addition of polyinosinic acid (poly (I)), a competitor of the scavenger receptor, but not by another polyanionic acid, polycytidylic acid (poly (C)). Conversely, the binding of oxLDL was significantly increased by the simultaneous addition of human beta 2-GPI and monoclonal aCL derived from NZW x BXSB F1 (WB F1) mice, an animal model of APS, or anti-beta 2-GPI antibodies from BALB/c mice immunized with human beta 2-GPI. These findings indicate that beta 2-GPI may be an antiatherogenic protein and that the autoimmune response against beta 2-GPI may have a role in the development of atherosclerosis in APS.

Transfection of glutathione S-transferase (GST)-pi antisense complementary DNA increases the sensitivity of a colon cancer cell line to adriamycin, cisplatin, melphalan, and etoposide.

The goal of this study was to demonstrate that glutathione S-transferase (GST)-pi is directly involved in the intrinsic and acquired resistance of cancer cells to anticancer drugs. To this end, GST-pi antisense cDNA was transfected into the cultured human colon cancer cell line M7609, which expresses an innately high level of GST-pi and shows intrinsic drug resistance, and into an M7609 strain with acquired resistance to Adriamycin (ADR;i.e., M7609/ADR cells). The changes in the sensitivity of these transfectants to various anticancer drugs were investigated. The intracellular concentrations of GST-pi in M7609/anti-1 cells and M7609/anti-2 cells, two clones that were established by transfection of GST-pi antisense cDNA into M7609 cells, were decreased to approximately half of those detected in the parent cells (M7609) and in the control cells transfected with vector alone (M7609/pLJ). The sensitivities of the antisense transfectants in relation to ADR, cisplatin, melphalan, and etoposide were increased -3.3-fold, 2.3-fold, 2.2-fold, and 2.1-fold, respectively, compared with those of M7609 and M7609/pLJ. On the other hand, the sensitivities of the antisense transfectants to Taxol, vincristine, 5-fluorouracil, and mitomycin C were not significantly changed. Similarly, the transfection of antisense cDNA into M7609/ADR cells resulted in the reduction of intracellular GST-pi concentration (by about half) and an increased sensitivity to ADR (4.4-fold), but no increase in 5-fluorouracil sensitivity. Thus, GST-pi is considered to be a multidrug resistance factor that is responsible for both the intrinsic and acquired resistance of cancer cells to anticancer drugs such as ADR, cisplatin, melphalan, and etoposide.

Characterization of a specific monoclonal antibody 9F5-3a and the development of assay system for oxidized HDL.

We obtained a monoclonal antibody 9F5-3a against oxidative low-density lipoprotein (LDL) modified with CuSO4 and established a sandwich ELISA for detection of oxidized high-density lipoprotein (oxHDL). The 9F5-3a was reacted strongly with oxHDL and to a lesser degree with oxLDL and LDL. In contrast, little or no reactivity was found with HDL. When the generation of oxHDL was limited by the addition of alpha-tocopherol and catalase, reactivity to 9F5-3a was reduced. Incubation of oxHDL with excess lyso-phosphatidylcholine (lyso-PC) also reduced immunoreactivity, but not by only lyso-PC. These results suggested that the epitope is possibly associated with oxHDL-linked lyso-PC induced mainly by the hydroxyl radical.

[Repeated blackout in a patient with accelerated idioventricular rhythm].

There has been no report that Accelerated Idioventricular rhythm (AIVR) causes a syncope attack. The patient described in this report is very rare because AIVR chronic without any heart diseases has been observed for 13 years, and because it is considered that AIVR is closely associated with his repeated blackouts. A 62-year-old male was admitted to our hospital because of repeated syncopal attacks. He reported that he occasionally felt lightheaded after strenuous lifting and pushing or pulling against resistance. We found that AIVR could produce a remarkable arterial pressure drop partly because of ventricular asynergy and loss of timed atrial contribution. Furthermore, we observed syncopal attacks during Valsalva maneuver and found abnormalities of baroreceptor reflex (Lack of reflex tachycardia and weakened evershoot phenomenon). It is concluded that AIVR is not a benign arrhythmia in this patient because it has been a possible cause of syncope attack due to systemic arterial pressure drop and baroreceptor abnormalities.

[Extent and degree of coronary flow reserve in hypertrophic cardiomyopathy assessed by delta Fract map unfolding coronary flow reserve index].

Patients with hypertrophic cardiomyopathy (HCM) are known to have ischemic events and decreased coronary flow reserve, but the variabilities in the site and degree of fall between patients with this disease have not been clarified. To elucidate these variabilities, we performed exercise myocardial single photon emission computed tomography (SPECT) using double dose method in 30 patients with HCM (6 with obstruction, 17 with non-obstruction, 7 with apical hypertrophy) and 10 normals. Then, the delta Fract (coronary flow reserve index) map was obtained for each subject. Exercise and then rest Tl-201 myocardial scintigraphy were performed after administration of Tl-201. The data were reconstructed, making the circumferential curves from the same level of short-axis imaging during exercise and at rest. By subtracting the values at rest from the values during exercise, which were divided by the values at rest, delta Fract in each frame was obtained, and described on the unfolded map. The extent and degree of coronary flow reserve were visually estimated by this delta Fract map. Patients were categorized into 5 groups: diffuse fall of coronary flow reserve (D-type), 6 cases; localized fall of the septum or lateral wall (L-type), 5 cases; fall of apical region (A-type), 5 cases; mild fall (M-type), 6 cases; and normal pattern (N-type), 8 cases. We concluded that delta Fract map is useful for evaluating the extent and degree of coronary flow reserve in HCM.

[Assessment of transient dilation of the left ventricular cavity in patients with hypertrophic cardiomyopathy by exercise thallium-201 scintigraphy].

Exercise Tl scintigraphy (EX-Tl) provides a noninvasive means of identifying myocardial perfusion abnormalities in patients (pts) with hypertrophic cardiomyopathy (HCM). We have noted that some pts with HCM have a pattern of transient dilation of the left ventricle (LV) on the immediate post exercise images as compared with 3 hour redistribution images. We presumed that left ventricular dilation was caused by subendocardial hypoperfusion. So we studied transient dilation of the LV in 50 pts with HCM and 20 controls (C). Initial and delayed conventional short tomographic images were obtained after reconstruction of 30 projections acquired over 180 degrees. Thirty six radii every 10 degrees were generated from the center of the middle myocardial images of the short axis. An area surrounded by the thirty six points of maximal count on each radius was calculated in initial and delayed images. Transient Dilation Index (TDI) as an index of dilation was determined by dividing an area in initial image by an area in delayed image. TDI in pts with HCM was larger than that in C. Pts with HCM were classified into the two groups, Group A: TDI greater than 1.11 (mean + 2 SD in C), 24 pts, Group B: TDI greater than 1.11, 26 pts. Frequency of pts with history of chest pain in Group A was higher than that in Group B, and frequency of pts with positive exercise ECG in Group A was higher than that in Group B. End diastolic volume in Group B did not change 10 minutes after exercise by radionuclide ventriculography. In conclusion, transient dilation of the LV in pts with HCM by EX-Tl is in appearance, and may reflect subendocardial ischemia.

[Regional myocardial blood flow measurements in resting and dipyridamole induced vasodilative state by Xenon-133 clearance method].

Myocardial imaging with Xenon-133 and a gamma camera was employed to evaluate the relationship between angiographically demonstrated coronary artery stenosis and regional myocardial blood flow (r-MBF) in 22 patients with ischemic heart disease and 9 control subjects. After a left anterior oblique cineangiogram was obtained, the cinecamera was replaced by the mobile gamma camera in the same plane and location over the pericardium of patients. Then, 370 MBq (10 mCi) of 133Xe was injected into the left coronary artery as a bolus. After the first measurement of r-MBF in resting state, a second injection was made 3 minutes after intravenous injection of 0.56 mg/kg of dipyridamole for 4 minutes. The r-MBF in different regions of the heart was calculated by Kety formula. Coronary flow reserve (CFR) was defined the rMBF ratio between resting and dipyridamole induced vasodilative state. In the resting state, good correlation (r = 0.711) was observed between pressure rate product and the r-MBF at the areas perfused by non stenotic vessels. The r-MBF of the areas perfused by severe stenotic vessels (greater than 90%) was smaller than that of the areas perfused by non stenotic vessels, however there was overlapping of flow. In the dipyridamole induced vasodilative state, the r-MBF of the areas perfused by intact vessels increased approximately 2.4 times of the resting state, and showed good correlation with pressure rate product. In contrast, the r-MBF of the areas perfused by stenotic vessels did not correlate with pressure rate product, but depended on the degrees of coronary stenosis (50-75% stenosis; 1.9 times, greater than 90% stenosis; 1.3 times, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

[Utility of reinjection method in stress Tl-201 myocardial scintigraphy to assess viability of the infarcted myocardium].

To assess viability of the infarcted myocardium, we performed stress Tl-201 myocardial scintigraphy using reinjection method in 37 patients with old myocardial infarction, and in 13 patients of them, Tl myocardial imagings were performed in resting state on the other day within 4 weeks after this examination. In this method, 111 MBq (3 mCi) of thallium was injected at the peak of exercise and initial and delayed images were acquired, then additionally 37 MBq (1 mCi) of thallium was injected after delayed scanning (reinjection) and we obtained post-reinjection images. Delayed images showed redistribution in 15 patients (41%), and no redistribution in 22 patients (59%). In post-reinjection images, 7 (19%) of 15 patients with redistribution and 3 (8%) of 22 patients without redistribution showed improvement of thallium uptake, in total 10 patients (27%) had additional thallium uptake in infarcted regions. And the degree of thallium uptake in post-reinjection images were almost equal to that in resting images. In conclusion, myocardial imaging using thallium reinjection method may be useful for the assessment of viability of the infarcted myocardium.

[Value and limitations of stress thallium-201 scintigraphy in evaluating the viability of infarcted myocardial segments].

The aim of this study was to evaluate the value and limitations of stress thallium-201 scintigraphy in evaluating the viability of infarcted myocardial segments following percutaneous transluminal coronary angioplasty (PTCA) and aortocoronary bypass surgery. The relative thallium activity (RTA: the ratio of the count rate in the infarcted segment to that in the intact segment) obtained from delayed images of stress myocardial scintigraphy was used as an index of myocardial viability and was compared with the following parameters. 1) Thallium-201 myocardial perfusion images were made during transient coronary arterial occlusion (PTCA) in eight patients with old myocardial infarction. The RTA of delayed images after successful PTCA was compared with those of stress thallium-201 scintigraphy. There were no significant differences of the RTA between delayed images following PTCA and those of stress myocardial scintigraphy. We therefore, presumed that incomplete redistribution of the delayed images of stress thallium-201 scintigraphy did not truly reflect the degree of coronary stenosis. 2. Regional ejection fraction of the left ventricle after postextrasystolic potentiation (PESP) using equilibrium radionuclide ventriculography was studied in 18 patients with old myocardial infarction, and was compared with the RTA of delayed images of stress myocardial scintigraphy. A significant correlation was observed between the changes in regional ejection fraction by PESP and the RTA three hours after exercise. 3. The RTAs of early and delayed thallium-201 scintigrams were measured at rest in 30 patients with old myocardial infarction, and were compared with the RTA of delayed thallium 201 scintigrams after exercise.(ABSTRACT TRUNCATED AT 250 WORDS)

[Coronary flow reserve index corrected by exercise load using Tl-201 SPECT: evaluation by PTCA].

For simple, non-invasive evaluations of coronary flow reserve during exercise, we investigated the coronary flow reserve index using the ratio of change of fractional distribution in the regional myocardium to oxygen demand. The subjects included 24 patients with effort angina pectoris and 11 normal volunteers. PTCA was performed for 11 patients. Using the biological properties of Tl-201, single photon emission computed tomography (SPECT) was performed at submaximal exercise and at rest by two successive injections of Tl-201 within a short time interval (double dose method). The correlation between the fractional distribution in the myocardium (delta-Fract) and the oxygen demand during exercise and at rest were evaluated. delta-Fract correlated well with the rate of change of the pressure rate product (delta-PRP), and delta-Fract/delta-PRP (CRI) was a good indicator of the coronary flow reserve index. CRI in the ischemic region correlated well with the percent area stenosis of the supplying coronary artery and improved with resolution of percent area stenosis of the target coronary artery after PTCA, but in some cases they were not above the normal range. CRI as obtained by exercise SPECT using the double dose method is useful for non-invasively estimating the coronary flow reserve during exercise, and it can be used for evaluating the effect before and after PTCA.

[An examination of left ventricular peak filling rate and ventricular relaxation rate by simultaneous acquisition of radionuclide ventriculography and left ventricular pressure].

We investigated whether peak filling rate (PFR) obtained from ECG gated radionuclide ventriculography (RNV) reflects left ventricular (LV) relaxation rate or not. Five patients with angina pectoris, 5 patients with old myocardial infarction and 6 controls were studied by simultaneous acquisition of RNV and LV pressure. RNV was performed in modified left anterior oblique position before and during angiotensin II (A II) infusion to elevate their blood pressure about 25 mmHg (A-1) and 50 mmHg (A-2). The data were acquired in a list mode and LV volume curves were reconstructed by forward and backward gating from the R wave of ECG. Actual PFR and corrected PFR by stroke volume (/SV), by end diastolic volume (/EDV), by instantaneous volume (/IV) were calculated from LV volume curve and its first differential curve. LV pressure was simultaneously measured by the catheter-tip micromanometer, and the time constant (T) of assumed exponential decline in LV pressure was calculated as index of LV relaxation rate. Although there was no correlation between actual PFR and T, corrected PFR (/EDV) and PFR (/IV) correlated with T. PFR (/EDV) decreased and T increased during A-1 and A-2. Decrease of PFR (/EDV) corresponded with increase of T. Thus, corrected PFR (/EDV) obtained from RNV reflects the rate of LV relaxation, and was considered to be an useful index to evaluate LV diastolic function.

[An attempt of thallium-201 myocardial perfusion imaging during transient coronary arterial occlusion by PTCA].

To evaluate the myocardial perfusion during transient coronary occlusion, we attempted to obtain the myocardial scintigraphy during percutaneous transluminal coronary angioplasty (PTCA). Tl-201 was injected at the last inflation of angioplastic balloon and occlusion was kept on for 60 sec. Planar images or SPECT were obtained immediately after PTCA. With this protocol, myocardial perfusion defects were observed during PTCA and fully redistributed 3 hours after Tl injection. Extent of ischemic lesions were almost same as that observed during exercise in two cases without collateral vessels. In a case with well visualized collateral vessels, perfusion defect was smaller in PTCA images than that in exercise stressed images. We conclude that intravenous injection of Tl-201 during PTCA is useful to assess the alteration of myocardial perfusion due to transient coronary occlusion without increasing the risk of angioplastic procedure.

[Evaluation of transient dilation of the left ventricle on exercise thallium-201 scintigraphy].

We have noted that some patients with ischemic heart disease have a pattern of transient dilation of the left ventricle on the immediate post exercise images as compared with 3 hour redistribution images. We assessed correlation between transient dilation of the left ventricle and coronary arteriographic findings in 32 patients with ischemic heart disease and 15 controls. Initial and delayed conventional short axis tomographic images were obtained after reconstruction of 20 projections acquired over 180 degrees. Thirty six radii by every 10 degrees were generated from the center of the middle myocardial images of the short axis. An area surrounded by thirty six points of maximal count on each radius was calculated in initial and delayed images. Transient Dilation Index (TDI) as an index of dilation was determined by dividing an area in initial image by an area in delayed image. There was no difference in TDI between patients with 1 vessel disease and controls, but TDI in patients with two vessel disease and patients with three vessel disease was larger than that in controls. Thus, transient dilation of the left ventricle on exercise Tl scintigraphy was likely to be related to number of stenotic vessel. We concluded that TDI as a simple quantitative index of transient dilation of the left ventricle was useful for detecting multi vessel stenosis.

[Nuclear cardiological examination for evaluation of hypertrophic cardiomyopathy with midventricular obstruction and apical aneurysm: a case report].

Midventricular hypertrophy (MVO) is a rare and complicated myocardial disease. Its pathophysiology and prognosis remain unknown, and few nuclear cardiological findings for MVO were reported. On a 44-year old man with MVO, thallium-201 (Tl) myocardial scintigraphy and gated blood pool scintigraphy (GPS) were performed to evaluate their usefulness. GPS revealed a characteristic hour-glass deformity of the left ventricular cavity, apical aneurysm and asynchrony due to obliteration of the midventricle. Anteroseptal hypertrophy in midventricle and myocardial damage in apico-inferior region were detected by Tl. Thus, nuclear cardiological studies are proved to be useful and essential in not only diagnosis but also evaluating pathophysiology and observing natural history noninvasively.

[Intraventricular flow dynamics in hypertrophic cardiomyopathy with midventricular obstruction investigated by Doppler echocardiography].

Seven patients with hypertrophic cardiomyopathy having midventricular obstruction (MVO) were examined using two-dimensional, conventional Doppler and color Doppler echocardiography to investigate intraventricular flow conditions. The controls were 35 patients with hypertrophic cardiomyopathy without MVO. All MVO patients had "hour-glass" LV cavities during systole, resulting from either hypertrophy at the midventricular level or hypertrophied papillary muscles, where systolic mosaic signals originated. Systolic peak flow velocities at the midventricle ranged from 2.5 to 4.2 m/s, proving the presence of a pressure gradient between the apex and the base of the LV. In fact, a pressure drop of 15-30 mmHg was demonstrated in four patients who underwent cardiac catheterization. These high velocity jet flows were not detectable at the midventricle in the control subjects. Peak ejection velocities in the outflow tracts were significantly lower in patients with MVO compared to those with hypertrophic cardiomyopathy and subaortic stenosis (129 +/- 29 vs 384 +/- 111 cm/s; p less than 0.001). As midventricular obliteration became severe, systolic jets at the midventricle increased in velocity. Waveforms changed from single- to double-peaked, and lasted until the isovolumic relaxation or the rapid filling phase beyond the second heart sound. Consequently, isovolumic relaxation waveforms at the midventricle using the apical approach changed the direction; from "the base to apex" to "the apex to base". An isovolumic signal away from the transducer was only observed in two patients without MVO. Diastolic color reversal and mosaic signals at the midventricle were also seen in five of the seven patients with MVO. Peak flow velocities in the rapid filling phase were significantly higher at the papillary muscle level than at the mitral valve level, indicating that MVO continues up to early diastole. It was suggested that MVO disturbs intraventricular flow dynamics during both systole and diastole. Color Doppler echocardiography is particularly useful in determining the site of obstruction and allows further evaluation by pulsed and continuous wave Doppler techniques to precisely measure pressure gradients. With routine, careful use of Doppler echocardiography, MVO may prove to be a more common entity than was previously believed.