Changes in the cytoskeleton of 3T3 fibroblasts induced by the phosphatase inhibitor,calyculin-A

Summary Addition of the protein phosphatase inhibitor, calyculin-A, to 3T3 fibroblasts causes a marked change in cell morphology. Initially the cells become rounded, develop surface blebs and then detach from the substratum. In the detached cells an unusual ball-like structure is observed. This study focuses on the cytoskeleton during these calyculin-A-induced morphological changes. Stress fibres disappear as the cells begin to round and aggregates of actin are formed towards the apical surface of the cell. These aggregates condense, in the detached cells, to form the ball structure of approximately 3 m diameter. Between the ball and the nucleus are cables of intermediate filaments that appear to be attached to the surface of the ball and to the nuclear lamina. Using a procedure designed for the isolation of nuclei the nucleus-ball complex can be obtained. Analysis of the nucleus-ball preparation by immunofluorescence and electron microscopy demonstrate that the ball contains actin and that intermediate filaments are located between the ball and the nucleus. In this preparation, the intermediate filaments also appear to attach to the surfaces of the ball and the nucleus. Electrophoretic analysis of the nucleus-ball preparation indicates that, in addition to actin, a major component of the ball is myosin. It is suggested that the formation of the ball is caused by an actin-myosin-based contractile process, initiated by the phosphorylation of myosin. The aggregation of the actomyosin draws together the intermediate filaments into the area between the ball and nucleus. This hypothesis requires that vimentin binds both to the nucleus and to some component of the ball.     

Proliferative cell nuclear antigen expression in follicular tumours of the thyroid with special reference to oxyphilic cell lesions

The expression of proliferative cell nuclear antigen (PCNA) in follicular tumours of the thyroid was examined by immunohistochemistry. Both usual nonoxyphilic cell follicular tumours (non-OCT) and oxyphilic cell tumours (OCT) were subdivided into benign, indeterminate, encapsulated carcinoma, and widely invasive carcinoma types. Among non-OCT the percentages of PCNA-positive cells in benign tumours, encapsulated carcinomas, and widely invasive carcinomas was 2.5%–8.6%, 11.8%–39.1%, and 18.6%–20.0%, respectively. There was a statistically significant difference between benign tumours and encapsulated or widely invasive carcinomas, as in previous studies. A value of 10% was appropriate to distinguish benign from malignant lesions. PCNA-positive cells in indeterminate-type non-OCT were not significantly different from those in benign tumours, ranging from 4.3%–19.6%, and occurring at more than 10% in three of six tuours. Among OCT the positivity was less than 10% in benign tumours (4.5%–7.8%) and more than 10% in malignant tumours (14.1%–35.9%) and all the eight indeterminate tumours (12.5%–27.3%), with a statistically significant differences between the benign tumour and each of the latter types. These results indicate that the examination of PCNA is valuable in diagnosis of thyroid follicular tumours and that the use of similar diagnostic criteria may be warranted in both non-OCT and OCT.     

Ontogeny of NADPH-diaphorase in rat forebrain and midbrain

 This study characterizes the developmental expression of NADPH-diaphorase from embryo to adulthood in the forebrain, midbrain and cerebellum of rat brain via histochemical staining. On embryonic day 12 no neurons stained. Labeling was observed in certain nuclei from E15 through the postnatal period to adulthood. Labeling in neurons increased or maintained a constant level with increased age. The embryo demonstrated substantial labeling in neurons of the caudate putamen, bed nucleus of the stria terminalis, preoptic area, lateral hypothalamic area, paraventricular thalamic nucleus, ventromedial hypothalamic nucleus, magnocellular nucleus posterior commissure, and periaqueductal central gray. Additional neuronal labeling was observed postnatally in the olfactory bulb, cerebral cortex, amygdala, various nuclei of the thalamus, interpeduncular nucleus, linear nucleus of the raphe, pretectal area and superior colliculus. In the cerebellum, labeling appeared only after P14 in cells of the molecular cell layer and granular cell layer. The sizes of labeled neurons developed significantly from P4 to P14 in several nuclei. The distinctive temporal and spatial expression pattern of NADPH-diaphorase implies that the NO/cGMP system may play an important role in physiological and developmental functions. Accepted: 8 September 1997     

Pig kidney xenotransplantation: Progress toward clinical trials

Pig organ xenotransplantation offers a solution to the shortage of deceased human organs for transplantation. The pathobiological response to a pig xenograft is complex, involving antibody, complement, coagulation, inflammatory, and cellular responses. To overcome these barriers, genetic manipulation of the organ‐source pigs has largely been directed to two major aims—(a) deletion of expression of the known carbohydrate xenoantigens against which humans have natural (preformed) antibodies, and (b) transgenic expression of human protective proteins, for example, complement‐ and coagulation‐regulatory proteins. Conventional (FDA‐approved) immunosuppressive therapy is unsuccessful in preventing an adaptive immune response to pig cells, but blockade of the CD40:CD154 costimulation pathway is successful. Survival of genetically engineered pig kidneys in immunosuppressed nonhuman primates can now be measured in months. Non‐immunological aspects, for example, pig renal function, a hypovolemia syndrome, and rapid growth of the pig kidney after transplantation, are briefly discussed. We suggest that patients on the wait‐list for a deceased human kidney graft who are unlikely to receive one due to long waiting times are those for whom kidney xenotransplantation might first be considered. The potential risk of infection, public attitudes to xenotransplantation, and ethical, regulatory, and financial aspects are briefly addressed.     

Prevention of postischemic reperfusion injury: The improvement of myocardial tissue blood flow after ischemia by terminal Nicorandil-Mg cardioplegia

We evaluated the preventive effect of postischemic reperfusion injury by Nicorandil-Mg cardioplegia given just prior to reperfusion as terminal cardioplegia. Twenty seven dogs were placed on cardiopulmonary bypass and the aorta was cross-clamped for 90 min under hypothermic (17–19°C) cardioplegic arrest. The canine hearts were divided into three groups: in group A (n=10) the hearts were reperfused without any treatment; in group B (n=9) the hearts received coronary perfusion with Nicorandil-Mg solution (Nic, 8 mg/l; Mg, 20 mEq/l; glucose, 50 g/l) for 2 min just prior to reperfusion; and in group C (n=8) the hearts received coronary perfusion with Nicorandil-Mg free solution (glucose, 50g/l). During and after ischemia, the myocardial tissue PCO₂ (t-PCO₂) was continuously monitored by an ion-sensitive field effective transistor (ISFET) sensor. In addition, the myocardial tissue blood flow (TBF), oxygen consumption, and lactate flux were then calculated at 5, 10, 20, and 40 min of reperfusion. In the initial reperfusion period, Group B showed an improved TBF compared to group A and C (at 5 min, group B was 42.7±11.9; group A was 29.4±11.2, P<0.025; and group C was 33.9±9.2% of the preischemic control level, P<0.05). T-PCO₂ in group B was significantly decreased at 5 min of reperfusion (group B, 127.5±22.5 42.5±9.7; group A, 117.5±23.0 85.2±17.4, P<0.001; group C, 122.3 mmHg 68.2±18.7 mmHg, P<0.01), and group B had a better metabolic recovery. These results suggest that terminal Nicorandil-Mg cardioplegia might reduce the rate of postischemic reperfusion injury.     

CD98 regulates the phosphorylation of HER2 and a bispecific anti-HER2/CD98 antibody inhibits the growth signal of human breast cancer cells

Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.     

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Interleukin-1β-induced, nitric oxide-dependent and -independent inhibition of vascular smooth muscle contraction

Stimulation of vascular smooth muscle by bacterial lipopolysaccharide has been shown to produce interleukin-1β and to induce vasodilation in septic shock. To understand the mechanisms of interleukin-1β-induced relaxation, we examined the effects of interleukin-1β on contractility and cyclic GMP contents of vascular smooth muscle. After treatment of the rat aorta with interleukin-1β (20 ng/ml) for 6 h, the cyclic GMP content increased and the contraction induced by phenylephrine (1 μM) was partially inhibited. An inhibitor of nitric oxide (NO) synthase, N^G-monomethyl-

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-arginine (

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-NMMA, 100 μM), prevented the inhibitory effect of interleukin-1β. After treatment with interleukin-1β for 24 h, the phenylephrine-induced contraction was inhibited more strongly. Neither

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-NMMA (100 μM) nor aminoguanidine (100 μM) reversed the inhibition, whereas methylene blue (10 μM) partially reversed the inhibition. After treatment with interleukin-1β for 12 or 24 h, the cyclic GMP content increased but to a level lower than that obtained with a 6-h treatment. The effects of sodium nitroprusside (1 μM) to inhibit the phenylephrine-induced contraction and to increase the cyclic GMP content were markedly suppressed by the 24-h interleukin-1β treatment. In contrast, the 24-h interleukin-1β treatment did not change the ability of 8-bromo-cGMP to relax the phenylephrine-stimulated aorta. Addition of

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-NMMA (1 mM) during the 24 h treatment prevented NO production and preserved the sodium nitroprusside-induced cGMP generation by interleukin-1β. The 24 h interleukin-1β treatment increased the threshold concentration of KCl needed to induce contraction without changing the maximum contraction. In the presence of 25.4 mM KCl or the non-selective K⁺ channel inhibitor, tetraethylammonium, the inhibitory effect of the 24-h interleukin-1β treatment on phenylephrine-induced contraction was restored. These results suggest that interleukin-1β inhibits vascular smooth muscle contraction by a time-dependent, dual mechanism. After a 6-h treatment with interleukin-1β, the NO/cyclic GMP system is activated. After a 24-h interleukin-1β treatment, in contrast, the NO/cyclic GMP system may be desensitized and the contraction of vascular smooth muscle is inhibited by another mechanism, possibly membrane hyperpolarization.     

Accumulation of L-[2-(F-18)]fluorophenylalanine in peri-infarct area in a patient with acute cerebral infarction

We studied the brain uptake of amino acid in a patient with acute cerebral infarction with L-[2-(F-18)] fluorophenylalanine and positron emission tomography. The increased accumulation of the ligand was specifically found in the peri-infarct area where oxygen metabolism was still maintained but decreased later in the 72-day follow-up period. The kinetic analysis revealed that increased accumulation was not due to increased transport from the blood to the brain but to delayed washout from the brain to the blood. Although the mechanism is still unknown, abnormally high accumulation of L-[F-18]fluorophenylalanine may predict delayed neuronal changes after ischemic insults of the brain.