Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport.

Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin. Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes. Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes. Addition of insulin enhanced Na+-dependent uptake of a system A amino acid (alpha-amino isobutyric acid; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake. From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed.

Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH2Cl: synergistic role of phosphotyrosine and intracellular Ca2+ concentration.

The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not the non-membrane-permeating chloramine, taurine-NHCl) induced detachment of fetal mouse cardiac myocytes and other cell types (fibroblasts, epithelial cells, and endothelial cells) from the culture dish, concomitant with cell shrinkage ("peeling off"). Stimulated human neutrophils also induced peeling off of cultured mouse cardiac myocytes when the latter were pretreated with inhibitors of .OH and elastase. Immunofluorescence microscopy revealed that the NH2Cl-induced peeling off of WI-38 fibroblasts is accompanied by disorganization of integrin alpha 5 beta 1, vinculin, stress fibers, and phosphotyrosine (p-Tyr)-containing proteins. Decrease in the content of the p-Tyr-containing proteins of the NH2Cl-treated cells was analyzed by immunoblotting techniques. Coating of fibronectin on the culture dish prevented both NH2Cl-induced peeling off and a decrease in p-Tyr content. Preincubation with a protein-tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), also prevented NH2Cl-induced peeling off, suggesting that dephosphorylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling off was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiac myocytes and WI-38 fibroblasts. The absence of extracellular Ca2+ prevented both NH2Cl-induced peeling off and increased [Ca2+]i, both of which did occur on subsequent incubation of the cells in Ca2+-containing medium. These observations suggest that an increase in [Ca2+]i is also necessary for peeling off. Depletion of microsomal and cytosolic Ca2+ by incubation with the microsomal Ca2+-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ) plus EGTA prevented both NH2Cl-induced increases in [Ca2+]i and peeling off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl-induced [Ca2+]i increment. A combination of p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinase) and an increase in [Ca2+]i by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphorylation and increased [Ca2+]i in NH2Cl-induced peeling off.

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion.

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.

Partial purification of Na+-Ca2+ antiporter from plasma membrane of chick heart.

To study Na+-Ca2+ exchange, proteins of membrane vesicles from chick hearts were solubilized with cholate in the presence of phospholipids and the cholate extract was treated with pronase. These purified proteoliposomes, reconstituted by subsequent dilution and centrifugation to eliminate the cholate, catalyzed Ca2+ uptake depending on the intraliposomal Na+ (Nai+) concentration. The maximal amount of Ca2+ accumulating in the liposomes was 140 nmol/mg protein and the initial rates of Nai+-dependent Ca2+ uptake were routinely 20 to 40 nmol/mg per 3 s at 25 degrees C, but only 2 to 4 nmol/mg per 3 s for the crude proteoliposomes from the cholate extract not treated with pronase. Thus the pronase treatment resulted in 10-fold purification. Nai+-dependent Ca2+ uptake by purified proteoliposomes was 30- to 50-fold higher than that by the initial membrane vesicles. The fundamental properties of Nai+-dependent Ca2+ uptake in purified proteoliposomes such as Km for Ca2+, the sensitivity for Na+ and pH dependency, were nearly equal to those in membrane vesicles and crude proteoliposomes. Thus, pronase treatment was very useful for obtaining reconstituted liposomes containing highly enriched Na+-Ca2+ antiporters which were functionally intact.

Comparison of kinetic characteristics of Na+-Ca2+ exchange in sarcolemma vesicles and cultured cells from chick heart.

The kinetic characteristics of Na+-Ca2+ exchange in isolated sarcolemma vesicles from new-borne chick heart, which contain about 70% of right-side-out vesicles, were compared with those of cultured embryonic chick heart cells. Na+-Ca2+ exchange was monitored as Nai-dependent Ca2+ uptake. Increase in the internal concentration of Na+ ([Na+]i) in these two preparations caused increase in both the initial rate and the saturation-level of Ca2+ uptake. Plots of the rate of Ca2+ uptake against [Na+]i showed similar saturation-kinetics in these two preparations. The apparent Michaelis constant (Km) (0.35 mM) for Ca2+ uptake by the intact cells was much higher than that (0.031 mM) for Ca2+ uptake by the vesicles. The degree of inhibition by Mg2+ was also higher in the cells than in the vesicles. Some possible reasons (age of the chicks used, membrane potential, etc.), for these differences were examined and are discussed.

Kinetic studies on sodium-dependent calcium uptake by myocardial cells and neuroblastoma cells in culture.

Kinetic analyses were made on intracellular Na+-dependent Ca2+ uptake by myocardial cells and neuroblastoma cells (N-18 strain) in culture. Cells loaded with various concentrations of Na+ could be prepared by incubating them in Ca2+-free medium containing various concentrations of Na+. Cells pre-loaded with various concentrations of Na+ were incubated in medium containing Ca2+ and 45Ca. The resulting 45Ca uptake by the two types of cell depended greatly on the initial intracellular concentrations of Na+. Lineweaver-Burk plots of the initial rate of Ca2+ uptake against the external concentration of Ca2+ fitted well to straight lines obtained by linear regression (r greater than 0.95). This result shows that Ca2+ uptake by the two types of cell was achieved by a carrier-mediated transport system. This Na+-dependent Ca2+ uptake was accompanied by Na+ release and the ratio of Na+ release to Ca2+ uptake was close to 3 : 1. A comparison of the kinetic data between myocardial cells and N-18 cells suggested that N-18 cells possess a carrier showing the same properties as that of myocardial cells, i.e.: (1) a similar dependency on the intracellular concentration of Na+; (2) the coincidence of the apparent Michaelis constants for Ca2+ (0.1 mM); (3) the similarities of the Ki values for Co2+, Sr2+ and Mg2+ (Co2+ less than Sr2+ less than Mg2+) and (4) a similar dependency on pH. However, the maximal initial rate, V, of N-18 cells was about 1/100 that of myocardial cells. The rate of Na+-dependent Ca2+ uptake by non-excitable cells was much lower than that by myocardial cells.

A novel ubiquinone reductase activity in rat cytosol.

Ubiquinone (UQ) reductase activity which reduces UQ to ubiquinol (UQH2) in rat tissues was roughly proportional to the UQH2/total UQ ratio in respective tissues. The highest activity was found in the liver, showing the highest UQH2/total UQ ratio. A greater part of liver UQ reductase activity was located in the cytosol. Within a week, the liver UQ reductase activity decreased by 80% even at -20 degrees C. The DT-diaphorase activity was stable. UQ reductase required NADPH as the hydrogen donor and was not inhibited by a less than 1 microM concentration of dicoumarol. There was no stimulation of UQ reductase in the presence of bovine serum albumin nor in Triton X-100. Yet, both stimulated DT-diaphorase. As a result, UQ reductase appeared to be a novel NADPH-UQ oxidoreductase and responsible for the UQ redox state in liver.

Polymorphonuclear leukocytes-induced injury in hypoxic cardiac myocytes.

Growing evidence suggests that free radicals derived from polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia-reperfusion injury. To elucidate the cellular mechanism by which activated PMNs exacerbate ischemic myocardial damage, we investigated the extent of cell injury, assessed by the morphological deterioration, free radical generation, and lipid peroxidation in mouse embryo myocardial cells coincubated with activated PMNs. The generation of PMN-derived free radicals was related to the extent of myocardial cell injury. When myocardial cell sheets were subjected to hypoxia and glucose-free media, myocardial cells were injured (cristalysis in the mitochondria and disruption of the sarcolemma) after adding various PMN activators, and the injury extended to the adjacent cells. Chemiluminescent emission and production of thiobarbituric acid-reactive substances in the coincubated cells increased markedly compared with myocardial cells or PMNs alone. The augmented lipid peroxidation coincided with the progression of myocardial cell injury. Catalase inhibited the myocardial cell injury by 52%, the chemiluminescence by 46%, and lipid peroxidation by 50%, whereas superoxide dismutase exhibited less pronounced inhibition. These results indicate that a chain reaction of lipid peroxidation in myocardial cells induced by PMN-derived free radicals closely correlates with membrane damage and contributes to the propagation of irreversible myocardial cell damage.

Protective effect of gamma-glutamylcysteinylethyl ester on dysfunction of the selenium-deficient rat heart.

We investigated the protective effect of intracellular GSH against cardiac dysfunction in selenium (Se)-deficient neonatal rats and cultured fetal rat myocytes. A Se-deficient diet with or without daily subcutaneous injections of gamma-glutamylcysteinylethyl ester (gammay-GCE) (a membrane-permeating GSH precursor) was given to rats from gestation day 4 via the dam to postnatal day 14. Se deficiency induced a 62% incidence of electrocardiographic abnormalities such as sinus arrhythmias or extrasystole, a 63% reduction in dP/dt in the left ventricle, and an increase in thiobarbituric acid reacting substances (TBARS), but no ultrastructural cardiac lesions were observed. Administration of gamma-GCE increased the intracellular GSH concentration ([GSH]i) of both neonatal rat hearts and cultured fetal rat cardiac myocytes. gamma-GCE-like sodium selenite prevented the cardiac dysfunction and the TBARS increment. gamma-GCE also prevented H2O2 toxicity in the cultured myocytes. The Vmax, but not the Km, for GSH of Se-dependent GSH peroxidase (Se-Gpx) activity in Se-deficient rat heart homogenates was one-third that of normal rat heart homogenates. Although gamma-GCE did not affect the Se-Gpx Vmax and Km for GSH, it did induce a substantial and significant increase in [GSH]i, which was postulated to increase the velocity of H2O2 decomposition by Se-Gpx activity 1.6-fold. These data suggest that the increase in [GSH]i may have played a role in preventing the TBARS increase and cardiac dysfunction in Se-deficient rats.

Effect of initial tumor stage on patient follow-up after potentially curative surgery for cutaneous melanoma.

The optimal follow-up strategy after completion of therapy for melanoma is not known. We evaluated the effect of TNM stage on the self-reported surveillance strategies employed by practicing plastic surgeons caring for otherwise healthy patients subjected to potentially curative treatment for cutaneous melanoma. Hypothetical patient profiles and a detailed questionnaire based on these profiles were mailed to a random sample (N=3,032) of the 4,320 members of the American Society of Plastic and Reconstructive Surgeons. The effect of TNM stage on the surveillance strategies chosen was analyzed by repeated-measures ANOVA. There were 1,142 responses to the 3,032 surveys; 395 were evaluable. Plastic surgeons often do not provide postoperative follow-up themselves; this was the most frequent reason for non-evaluability. Surveillance of patients after resection of melanoma relies most heavily on office visits, chest X-ray, CBC, and liver function tests. All other surveillance modalities are used infrequently. Most respondents modify their surveillance practices slightly according to the patient's initial TNM stage. Most commonly used modalities are employed significantly more frequently with increasing TNM stage. This effect persists through ten years of follow-up, but the differences across stages are tiny. We conclude that most plastic surgeons performing surveillance after potentially curative surgery in otherwise healthy patients with melanoma use similar follow-up strategies for patients of all TNM stages. These data permit the rational design of a controlled clinical trial of high-intensity vs. low-intensity follow-up.

Reciprocal changes of plasma glucose and ketone bodies in fasted and acutely diabetic rats after CNS stimulation.

To assess the effect of chemical stimulation of the central nervous system (CNS) on ketogenesis, we injected neostigmine (5 x 10(-8)mol) into the third cerebral ventricle in normal rats fasted for 48 h and fed rats with diabetes induced by streptozotocin (STZ, 80 mg/kg). The hepatic venous plasma levels of ketone bodies (3-hydroxybutyrate and acetoacetate), free fatty acids (FFA), and glucose were measured for 120 min after the injection of neostigmine under pentobarbital anesthesia. In the normal rats, plasma glucose levels were significantly increased but neither ketone bodies nor FFA were affected by CNS stimulation with neostigmine. In contrast the plasma levels of ketone bodies and FFA were significantly increased in STZ-diabetic rats, while glucose levels remained unchanged. The intravenous infusion of somatostatin (1.0 microgram/kg/min) suppressed the increase in plasma ketone bodies following CNS stimulation in STZ-diabetic rats. These findings suggest that CNS stimulation with neostigmine may accelerate ketogenesis by promoting the lipolysis, which may be induced by glucagon, in fed diabetic rats but not in normal fasted rats.

Current practice of patient follow-up after potentially curative resection of cutaneous melanoma.

Follow-up care for patients who have undergone potentially curative resection of cutaneous melanoma varies widely among physicians, and the underlying rationale has not been assessed. To quantify current practice patterns and to discern motivation, a custom-designed questionnaire was mailed to U.S. and non-U.S. surgeons, all of whom were members of the American Society of Plastic and Reconstructive Surgeons (ASPRS). Surveys were mailed to 3,032 ASPRS members, chosen randomly from a total of 4,320 members. Of the 1,142 questionnaires that were returned, 395 were evaluable. Nonevaluability was usually due to lack of melanoma patients receiving follow-up in the surgeons' practices. Surveillance of patients after resection of melanoma relies most heavily on office visit, chest x-ray, complete blood count, and liver function tests. There was surprisingly little influence of elective node dissection on follow-up practices. Imaging tests such as computed tomography, magnetic resonance imaging, and position emission tomography scan were rarely employed. Surveillance is motivated by many factors, particularly early detection of recurrence of the index melanoma and second primary melanomas. This survey provides information regarding current follow-up strategies recommended by ASPRS surgeons after potentially curative resection of cutaneous melanoma. There is considerable variation in surveillance intensity and in motivation among practitioners, thus representing a lack of consensus.

Long-term observation of a case of pulmonary arteriovenous fistula with pulmonary hypertension.

A case of pulmonary arteriovenous fistula (PAVF) with pulmonary hypertension (PH) occurring in an adult woman is described. Resection of PAVF was not performed and she has been followed up for 5 years, receiving repeated right cardiac catheterization. We discuss the causal relationship of PH and the development of PAVF.

Effect of ubiquinone-10 on fluctuation of beating rhythm of cultured myocardial cell sheets.

Effects of ubiquinone-10 (UQ-10) on fluctuation of beat time intervals and on beat frequencies were investigated in cultured ventricular myocytes obtained from mouse fetuses and quail embryos. Fluctuation rates of beat time intervals of mouse and quail myocardial cell sheets growing in the culture medium supplemented with 10% fetal bovine serum increased significantly to 1.2-fold and twice, respectively by removing serum from the medium. Beat frequencies of the cell sheets also significantly decreased simultaneously with the increase in the fluctuation rates. However, such changes of beating states were almost completely recovered within 10 min by addition of 100 microM UQ-10 as an isotonic lecithin emulsion to the medium. The addition of UQ-10 also significantly increased intracellular ATP content in the cell sheets concomitantly. These results suggested that UQ-10 circulating in serum may participate in the maintenance of regular and rhythmical beating of myocardial cells, presumably through stimulating intracellular ATP generating systems.

[Acute onset of tuberculous meningoencephalitis presenting with symmetric linear lesions in the bilateral thalamus: a case report].

A 18-year-old woman was admitted to our hospital because of high fever and headache. Nuchal stiffness was present, and a CSF examination showed lymphocyte-domonant pleocytosis and a decreased level of glucose. Although antibiotics, aciclovir and an antimycotic drug were administered, disturbance of consciousness, involuntary movements, and pyramidal tract signs appeared. Soon after the medications were changed to antituberculous medicines, the meningoencephalitis started to subside, and was finally cured. Judging from the clinical findings, the CSF findings, the effectiveness of antituberculous medicines, an elevated ADA level in CSF, and positive conversion in tuberculin tests, the final diagnosis was made as tuberculous meningoencephalitis. At the severest stage of the disease, a brain MRI showed symmetric, linear lesions without the effect of Gd-enhancement in the bilateral thalamus, which thereafter disappeared along with the healing of the illness. From all these things, we conclude that thalamic and other parenchymal lesions should be kept in mind in case of acute tuberculous meningoencephalitis.