Activity of the cytosolic isozyme of thymidine kinase in human primary lung tumors with reference to malignancy.

Activity increase of the cytosolic isozyme of thymidine kinase (TK) in resected specimens of lung tumor patients would be a useful marker for tumor malignancy and prognosis. In 24 resected cases of malignant lung tumors, the whole enzyme extracts of the tumorous part of the specimens showed that the activities of TK, thymidylate synthetase, and ribonucleotide reductase increased at an average of 469 (P less than 0.001), 208 (not significant), and 193% (P less than 0.02) of the corresponding enzymes in the tumor-uninvolved lung parts, respectively. Two TK isozymes, cytosolic and mitochondrial TKs, were separated better by means of p-aminophenyl 3'-TMP:CH-Sepharose gel affinity column chromatography for precise quantitation of the activity than by polyacrylamide disc gel electrophoresis. These separated isozymes from the tumorous part of the specimens were characteristically very similar to the isozymes of cytosolic and mitochondrial fractions of the xenograft (CPX-101) of human lung tumor transplanted in athymic nude mice, respectively. The cytosolic isozyme activity isolated by this method from the tumorous part was remarkably higher and more varied than that of the tumor-uninvolved part, while that of the mitochondrial isozyme was lower and less agitated. The tumor doubling time showed a good inverse correlation to the activity of the cytosolic isozyme of TK when compared logarithmically (r = -0.798, P less than 0.01). Poorly differentiated tumors exhibited significantly higher activities of the TK cytosolic isozyme than did well-to-moderately differentiated tumors (766.0 +/- 379.1 and 308.1 +/- 119.5 pmol/mg of protein/h, mean +/- SE, respectively), a phenomenon also seen in the activities of the tumors with versus without recurrences within 12 mo after resection (803.6 +/- 278.7 and 124.1 +/- 42.1 pmol/mg of protein/h, respectively). The levels of these relationships using the cytosolic TK activity provided a clearer indication of prognosis and the state of the malignancy than those using the whole extract TK activity.

Co-purification of thymidylate kinase and cytosolic thymidine kinase from human term placenta by affinity chromatography.

It was revealed that thymidylate kinase was purified together with cytosolic thymidine kinase from human term placenta by p-aminophenyl thymidine-3'-phosphate-CH-Sepharose affinity column chromatography, which has been commonly used for purification of thymidine kinase. In addition, it was noted that mitochondrial thymidine kinase and nucleoside diphosphate kinase were concurrently eliminated. In the presence of ATP, cytosolic thymidine kinase and thymidylate kinase could be separated from each other by Ultrogel AcA 34 filtration, and their molecular weights were estimated to be 70,000 and 50,000, respectively. On SDS-polyacrylamide gel electrophoresis, thymidine kinase protein exhibited a band of 26,000, which was compatible with the molecular weight of the enzyme subunit calculated from its cDNA, while thymidylate kinase protein showed 24,000. Thymidylate kinase could utilize either ATP or dATP as an efficient phosphate donor, and showed substrate specificity for dTMP.

Isovolumetric relaxation flow in patients with ischemic heart disease.

OBJECTIVES: The purpose of this study was to clarify the characteristics of flow during the isovolumetric relaxation period and to analyze the relation between these flow patterns and standard hemodynamic indexes. BACKGROUND: Outward motion of the left ventricle during the isovolumetric relaxation period has been observed by cineangiography. However, there is little information about blood flow during this period. METHODS: Seventy-nine patients with ischemic heart disease were examined by pulsed Doppler echocardiography and cardiac catheterization. All patients were classified into three groups according to the observed patterns of isovolumetric relaxation flow: group I (n = 41), flow directed toward the apex; group II (n = 21), flow directed toward the base, and group III (n = 17), low velocity flow (< 12 cm/s) without a dominant direction. Patients in group I were further classified into group Ia (n = 19) with normal ventriculograms and group Ib (n = 22) with asynergy. RESULTS: Left ventricular ejection fraction and negative first derivative of left ventricular pressure were significantly lower in group II (49 +/- 9% and 1,274 +/- 212 mm Hg/s, respectively) and group III (38 +/- 8% and 1,147 +/- 280 mm Hg/s, respectively) than in group Ia (68 +/- 7% and 1,727 +/- 358 mm Hg/s), each p < 0.01. Time constant was significantly prolonged in group II (49 +/- 6 ms) and group III (48 +/- 6 ms) compared with that in group Ia (41 +/- 6 ms), p < 0.05. On left ventriculography, patterns of outward wall motion during the isovolumetric relaxation period were associated with the patterns of isovolumetric relaxation flow. CONCLUSIONS: Changes in left ventricular relaxation are accompanied by alterations in isovolumetric relaxation flow. It is therefore useful to evaluate isovolumetric relaxation flow when investigating early diastolic ventricular function.

Specific recognition of cytosolic thymidine kinase in the human lung tumor by monoclonal antibodies raised against recombinant human thymidine kinase.

Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells. Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer. The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK. Only one clone of the mAbs inhibited the catalytic activity of TK. By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues. Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells. These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.

Late diastolic mitral regurgitation studied by pulsed Doppler echocardiography.

To evaluate the usefulness of pulsed Doppler echocardiography in assessing late diastolic mitral regurgitation (MR) and to clarify the pathophysiology of MR, 226 consecutive patients who had undergone left ventriculography were studied. By investigating blood flow patterns at the left atrial outflow tract, late diastolic disturbed flow suggesting MR was detected in 15 patients (7%), including 10 (4%) with positive left ventriculographic findings. Among these 15 patients, 14 (93%) had atrial fibrillation and had late diastolic MR in the cardiac cycle with prolonged RR interval. The limitation in number of cardiac cycles that could be analyzed and the rapid heart rate eliminating appearance of the beat with prolonged RR interval may be the reasons for the paucity of late diastolic MR by left ventriculography. Ten patients (66%) with late diastolic MR, including 1 with sinus rhythm, had aortic regurgitation, 3 had high-grade systolic MR and 2 had atrial septal defect. Simultaneous recording of pulmonary artery wedge pressure and left ventricular pressure in 3 patients showed a reversal of pressure gradient in late diastole when the RR interval was prolonged. In conclusion, pulsed Doppler echocardiography was useful for detecting late diastolic MR and in reducing overestimation of systolic MR in left ventriculography induced by erroneous addition of late diastolic MR. The difference of the incidence of this flow between left ventriculography and Doppler examination indicated that this flow depends primarily on heart rate and may come and go in a patient.

Regulation of the activity and polymerization status of recombinant human cytosolic thymidine kinase by thiols and ATP.

The cDNA clone encoding human thymidine kinase (hTK), was expressed in E. coli using a prokaryotic expression vector, pKK 223-3. The kinetics of the recombinant hTK (rhTK) were similar to those of cytosolic TK but not of mitochondrial TK. rhTK was highly purified in the presence of either ATP or dithiothreitol (DTT). The specific activity of rhTK purified in the presence of ATP [rhTK(ATP)] was lower than that of rhTK purified in the presence of DTT [rhTK(DTT)]. Activity of the purified rhTK(ATP) was enhanced by addition of thiols including DTT, cysteine, homocysteine and beta-mercaptoethanol but inhibited by various sulfhydryl reagents such as 5,5'-dithio-bis(2-nitrobenzoic acid). Hence, it was suggested that rhTK is a thiol-type enzyme. Apparent Mr of purified rhTK(ATP) was 100 kDa, which corresponds to the size of a tetramer (25 kDa subunit), while that of purified rhTK(DTT) was 50 kDa, the size of a dimer. The tetramer form of rhTK(ATP) was converted to the dimer by replacement of ATP by DTT. On the other hand, the dimer form of rhTK(DTT) was converted to the tetramer by addition of ATP. Thus, the catalytic activity of human cytosolic TK might be regulated by thiols as well as ATP via its polymerization status.

Initial medical management of patients severely irradiated in the Tokai-mura criticality accident.

A nuclear criticality accident occurred in Japan on September 30, 1999, which resulted in severe exposure of three victims to mixed flux of neutrons and gamma-rays. Estimated average doses for the three victims were 5.4 Gy of neutrons and 8.5 Gy of gamma-rays for Patient A, 2.9 Gy of neutrons and 4.5 Gy of gamma-rays for Patient B, and 0.81 Gy of neutrons and 1.3 Gy of gamma-rays for Patient C. They then suffered the consequences of the effects of ionizing radiation resulting in acute radiation syndrome. In Patients A and B, bone marrow failure was so severe that they received haematopoietic stem cell transplantation. The graft initially took successfully in both patients, although in Patient B it was later taken over by his own haematopoietic cells. They also suffered from severe skin lesions, later exhibited gastrointestinal bleeding and eventually died of multiple organ failure 82 and 210 days after the accident, respectively. The survival of these patients beyond the period of agranulocytosis means that bone marrow failure per se caused by exposure to ionizing radiation may now be overcome. Patient C also developed bone marrow failure and was treated with granulocyte colony-stimulating factor as well as supportive care. He recovered without major complications and is now under periodical follow-up. Remarkably, during the prodromal phase, all the patients exhibited hypoxaemia, two of whom also showed interstitial oedema of the lungs. In Patient C these manifestations improved within a week. The circumstances of the accident and the initial medical treatment of the victims are described.

Initial symptoms of acute radiation syndrome in the JCO criticality accident in Tokai-mura.

A criticality accident occurred on September 30, 1999, at the uranium conversion plant in Tokai-mura (Tokai-village), Ibaraki Prefecture, Japan. When the criticality occurred, three workers saw a "blue-white glow," and a radiation monitor alarm was sounded. They were severely exposed to neutron and gamma-ray irradiation, and subsequently developed acute radiation syndrome (ARS). One worker reported vomiting within minutes and loss of consciousness for 10-20 seconds. This worker also had diarrhea an hour after the exposure. The other worker started to vomit almost an hour after the exposure. The three workers, including their supervisor, who had no symptoms at the time, were brought to the National Mito Hospital by ambulance. Because of the detection of gamma-rays from their body surface by preliminary surveys and decreased numbers of lymphocytes in peripheral blood, they were transferred to the National Institute of Radiological Sciences (NIRS), which has been designated as a hospital responsible for radiation emergencies. Dose estimations for the three workers were performed by prodromal symptoms, serial changes of lymphocyte numbers, chromosomal analysis, and 24Na activity. The results obtained from these methods were fairly consistent. Most of the data, such as the dose rate of radiation, its distribution, and the quality needed to evaluate the average dose, were not available when the decision for hematopoitic stem cell transplantation had to be made. Therefore, prodromal symptoms may be important in making decisions for therapeutic strategies, such as stem-cell transplantation in heavily exposed victims.

Characterization of clofibrate-induced retrograde Golgi membrane movement to the endoplasmic reticulum: clofibrate distinguishes the Golgi from the trans Golgi network.

Clofibrate-induced retrograde Golgi membrane movement was blocked or retarded when NRK cells were treated with sodium azide/2-deoxyglucose, nocodazole, taxol, and destruxin B, indicating that it depends on energy, and the dynamic state of microtubules, and being acidic or vacuolar-type ATPase function. PDMP and phospholipase A2 inhibitors also blocked it. These characteristics are similar to those of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA), inducers of retrograde Golgi membrane movement. However, clofibrate was distinguished from BFA in that BFA action was insensitive to phospholipase A2 inhibitors and from NDGA in that NDGA stabilized microtubules against nocodazole and its action was almost insensitive to taxol. The trans Golgi network (TGN) was resistant to clofibrate, while BFA and NDGA dispersed it. To our knowledge, clofibrate is the first drug to show such different effects on the Golgi and TGN and, therefore, is expected to be a useful tool to distinguish their architecture and/or membrane dynamics.

Glucosylceramide synthesis inhibitors block pharmacologically induced dispersal of the Golgi and anterograde membrane flow from the endoplasmic reticulum: implication of sphingolipid metabolism in maintenance of the Golgi architecture and anterograde membrane flow.

PDMP (D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and PPMP (D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol), inhibitors of glucosylceramide synthesis, blocked brefeldin A (BFA)- and nordihydroguaiaretic acid-induced dispersal of the Golgi and trans Golgi network, and Golgi-derived vesicles were retained in the juxtanuclear region. PDMP and PPMP did not stabilize microtubules but blocked nocodazole-induced extensive fragmentation and dispersal of the Golgi, and large Golgi vesicles were retained in the juxtanuclear region. PPMP is a stronger inhibitor of glucosylceramide synthesis than PDMP, but PDMP showed a stronger activity against BFA-induced retrograde membrane flow. However, PPMP showed a stronger activity for Golgi disruption and inhibition of anterograde trafficking from the endoplasmic reticulum, and rebuilding of the Golgi architecture. Cumulatively, these results suggest that sphingolipid metabolism is implicated in maintenance of the Golgi architecture and anterograde membrane flow from the endoplasmic reticulum but not in Golgi dispersal induced by BFA.

Arachidonyltrifluoromethy ketone, a phospholipase A(2) antagonist, induces dispersal of both Golgi stack- and trans Golgi network-resident proteins throughout the cytoplasm.

Arachidonyltrifluoromethy ketone (AACOCF(3)), a phospholipase A(2) antagonist, reversibly induced dispersal of Golgi stack- and trans Golgi network (TGN)-resident proteins throughout the cytoplasm in NRK cells as followed by immunocytochemical staining of ManII and TGN38, respectively. The action of AACOCF(3) was partly blocked by other PLA(2) antagonists, suggesting it be not caused by a general inhibition of phospholipase A(2). AACOCF(3) neither dissociated beta-COP from membranes nor prevented brefeldin A-induced beta-COP release. Action of AACOCF(3) on the Golgi stack and TGN is different from that of brefeldin A and nordihydroguaiaretic acid. The most prominent difference is that the Golgi stack and TGN showed a similar sensitivity to AACOCF(3), while the TGN was dispersed more slowly than the Golgi stack in brefeldin A- or nordihydroguaiaretic acid-treated NRK cells. This novel action of AACOCF(3) may be used as pharmacological tool and give new insights into vesicle-mediated traffic and Golgi membrane dynamics.

Right ventricular aneurysm due to myocardial fatty infiltration: report of a case.

A case of right ventricular aneurysm due to myocardial fatty infiltration was reported. A 39-year-old man was admitted to our hospital for evaluation of several episodes of syncopal attack presumably induced by malignant arrhythmia. There were frequent observations of ventricular premature beats, which occurred occasionally in couples. Cardiac catheterization disclosed the aneurysm situated at the outflow tract of the right ventricle. Aneurysmectomy was performed and the ventricular premature beats and syncopal attacks were effectively abolished. Etiology of the aneurysm was proved to be a fatty infiltration in the myocardium. To the best of our knowledge, there has been no case report of right ventricular aneurysm due to fatty infiltration in the myocardium.

[Effect of age on left ventricular relaxation: pulsed Doppler echocardiographic evaluation].

There is no noninvasive index to assess the left ventricular relaxation property during the isovolumic relaxation period except for the isovolumic relaxation time, which is derived from M-mode echocardiography or phonocardiography. The intracardiac blood flow can now be precisely observed using pulsed Doppler echocardiography. We evaluated the left ventricular relaxation property and studied the effect of age on the relaxation property using this method. Cardiac catheterization and pulsed Doppler echocardiography were performed for 27 patients with various heart diseases, none of whom had wall motion abnormalities on left ventriculography, to determine the relationships between invasive and noninvasive parameters. The effects of age were examined in 25 normal healthy persons ranging in age from 26 to 69 years. In all cases, the intracardiac blood flow during the isovolumic relaxation period (isovolumic relaxation flow) as obtained by pulsed Doppler echocardiography was laminar, and directed from the base toward the apex of the left ventricle. The duration of this flow (the time interval from the beginning to the end of this flow; IRT), the acceleration time (the time interval from the beginning to the peak velocity; ACT) and the acceleration rate (the slope of the beginning to the peak velocity; AcR) were measured, and IRT, ln AcT (natural logarithm of AcT) and ln AcR (natural logarithm of AcR) were compared with the parameters obtained by cardiac catheterization. The time constant T of the fall in left ventricular pressure during the isovolumic relaxation period, which was proposed as an index of the left ventricular relaxation property, correlated well with IRT (r = 0.82, p less than 0.001), ln AcT (r = 0.69, p less than 0.001) and ln AcR (r = -0.77, p less than 0.001), but there were no significant correlations with maximum blood pressure, left ventricular ejection fraction, maximal positive dp/dt or the left ventricular end-diastolic volume index. Age and these three Doppler indexes in normal healthy persons correlated significantly as follows; IRT (r = 0.80, p less than 0.001), ln AcT (r = 0.60, p less than 0.001) and ln AcR (r = -0.61, p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)