Platinum levels in various tissues of a patient who died 181 days after cisplatin overdosing determined by electrospray ionization mass spectrometry

Platinum (Pt) levels were determined in various tissues and body fluids obtained from a patient who died 181 days after cisplatin overdosing. The symptoms of cisplatin overdose, however, might have almost disappeared by day 40, and the patient’s death was ascribed to the recurrence of malignant lymphoma. Determination of Pt derived from cisplatin was performed by electrospray ionization mass spectrometry (ESI-MS) using silver (Ag) as internal standard. Pt and Ag complexed with diethyldithiocarbamate (DDC) in wetashed blood, and tissue solutions were extracted into isoamyl alcohol, and then acidified with oxalic acid. By injecting an aliquot of the isoamyl alcohol layer into a mass spectrometer in the direct flow injection mode, the quantitation was performed using the signals of Pt(DDC)₃ ⁺ and Ag(DDC)₂ ⁺ at m/z 639 and 403, respectively. The Pt levels ranged from 25ng/ml in blood to 2050ng/g wet weight in the liver of the patient, indicating that Pt remained at high levels in tissues, even after a period as long as 181 days after cisplatin overdosing.     

Triad of thymoma,myasthenia gravis and pure red cell aplasia combined with Sjögren’s syndrome

A 36-year-old woman complained of cough and high fever. Computed tomographic scans demonstrated a mediastinal mass. A couple of months later, she developed dryness in her eyes and mouth. Biopsy of the lip confirmed the diagnosis of Sjögren’s syndrome. She underwent thymo-thymomectomy. Pathological findings of the mass revealed thymoma. At two months after surgery, she developed ptosis and dysphagia that were compatible with myasthenia gravis. The clinical symptoms were adequately controlled with prednisolone. At eleven months after surgery, she presented with severe anemia, which led to the diagnosis of pure red cell aplasia. The following treatment with cyclosporin caused hemoglobin concentration to rise. However, she continues to suffer from dryness of her eyes and mouth. The case is the first to be reported with Sjögren’s syndrome and the triad of thymoma, myasthenia gravis and pure red cell aplasia, and is compared with previously reported cases of the three conditions.     

Activation of phospholipase D by platelet-derived growth factor (PDGF) in rat C6 glioma cells: possible role in mitogenic signal transduction.

The effects of platelet-derived growth factor (PDGF) on phospholipase D (PLD) activity and deoxyribonucleic acid (DNA) synthesis in rat C6 glioma cells have been investigated. Pretreatment of serum-starved C6 cells with PDGF results in enhanced choline production and the phosphatidylethanol (PEt) formation in the presence of ethanol, indicating the activation of PLD acting on phosphatidylcholine (PC). The dose-response curve for choline generation and DNA synthesis were comparable. In addition, the effects of PDGF on both PEt formation and [3H]thymidine incorporation into acid-precipitable material was blocked by the potent protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) but not by N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA1004), a relatively weak inhibitor of PKC, suggesting that PDGF plays an important role as a positive regulator of glioma cell growth via a PLD-mediated mitogenic signal transduction cascades, which depends largely on the activation of PKC.     

Mouse monoclonal antibodies which recognize a human (beta 1-4)galactosyl-transferase associated with tumor in body fluids.

Mouse monoclonal antibodies against human (beta 1-4)galactosyl-transferase (GalT) purified from human ovarian tumor effusion fluids were prepared and characterized. GalT purified from normal human plasma showed a single diffused band in nondenaturing polyacrylamide gel electrophoresis, but GalT purified from human ovarian tumor effusion fluids showed several oligomeric bands and a monomeric band in nondenaturing polyacrylamide gel electrophoresis. These oligomeric bands were dissociated into monomer by urea treatment and polymerized by a 2-mercaptoethanol treatment. Nine monoclonal antibodies (MAb) were prepared by immunization of purified GalT from human ovarian tumor effusion fluids and classified into three groups. Type I MAbs (MAb8611, MAb8913, and MAb8919) reacted only to the GalT monomer. Type II MAbs (MAb4880, MAb8507, and MAb8628) reacted to both the GalT monomer and the GalT polymer. Type III MAbs (MAb7907, MAb8513, and MAb8677) reacted only to the GalT polymer. These MAbs except MAb7907 could recover GalT enzyme activity from effusion fluids by immunoprecipitation. A fraction passed through MAb8513 affinity chromatography still showed reactivity to MAb8919, demonstrating that an epitope of MAb8513 resides on a minor part of GalT. A sandwich immunoassay (MAb8513-MAb8628HRP) was developed, and serum samples from ovarian cancer patients and benign ovarian patients were tested. The levels of sandwich immunoassay of serum samples from cancer were elevated significantly compared to those from benign and did not necessarily correlate to total GalT enzyme activity in serum samples. These results suggested that MAb8513 (Type III) might recognize a unique GalT associated with tumor (GAT).     

Phosphoinositide turnover and Ca2+ mobilization during phagocytosis in cultured chick retinal pigment epithelial cells

Ca2+ mobilization and phosphoinositide turnover were examined during phagocytosis of latex particles in cultured chick retinal pigment epithelial cells (RPE cells). Ca2+ influx into cells and Ca2+ efflux from the cells were enhanced by about 1.5-fold and 3-fold compared to control cells, respectively. A high content of Ca2+ in RPE cells has been reported. Therefore, the Ca2+ efflux observed here may be a reflection of Ca2+ release into cytosol from the intracellular storage site(s). In [3H] inositol-prelabeled RPE cells, addition of latex particles elicited decreases in phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol with the concomitant formation of inositol phosphates including inositol trisphosphate, indicating the hydrolysis of phosphoinositides by phospholipase C. These results suggest that phosphoinositide turnover may be closely coupled with Ca2+ mobilization during phagocytosis in RPE cells.     

Inhibition of inward rectifier K+ currents by angiotensin II in rat atrial myocytes: lack of effects in cells from spontaneously hypertensive rats.

We examined the effects of angiotensin II (Ang II) on inward rectifier K+ currents (IK1) in rat atrial myocytes. [125I]Ang II-binding assays revealed the presence of both Ang II type 1 (AT1) and type 2 (AT2) receptors in atrial membrane preparations. Ang II inhibited IK1 in isolated atrial myocytes with an IC50 of 46 nmol/l. This inhibition was abolished by the AT, antagonist RNH6270 but not at all by the AT2 antagonist PD123319. Treatment of cells with pertussis toxin or a synthetic decapeptide corresponding to the carboxyl-terminus of Gialpha-3 abolished the inhibition by Ang II, indicating the role of a Gi-dependent signaling pathway. Accordingly, Ang II failed to inhibit IK1 in the presence of forskolin, dibutyryl-cAMP or protein kinase A catalytic subunits. In spite of the increased binding capacities for [125I]Ang II, Ang II failed to affect IKI in cells from spontaneously hypertensive rats (SHR). AT, immunoprecipitation from atrial extracts revealed decreased amounts of Gialpha-2 and Gialpha-3 proteins associated with this receptor in SHR as compared with controls. The reduced coupling of AT, with Gialpha. proteins may underlie the unresponsiveness of atrial IK1 to Ang II in SHR cells.     

Expression of leukocyte adhesion molecules (ICAM-1/LFA-1) related to clinical behaviour in B cell lymphomas.

We investigated the expression of adhesion molecules of lymphocyte function-associated antigen-1 alpha (LFA-1 alpha) and its ligand intercellular adhesion molecule-1 (ICAM-1) on 74 well-characterized B cell lymphomas. The LFA-1 was expressed on B cell lymphomas (21/74; 28 per cent), but to a lesser degree than ICAM-1 which was highly expressed (48/74 cases; 64 per cent). From the results of bone marrow examination of 39 cases with B cell lymphomas, 13 of 16 cases with a lack of ICAM-1 molecule showed a higher incidence of marrow involvement, but nine of 23 cases with the expression of ICAM-1 molecule showed a lower incidence. These findings suggest that the lack of expression of the ICAM-1 molecule by B cell lymphomas correlates with bone marrow involvement by lymphoma cells (p < 0.05). Expression of the LFA-1 molecule appears not to correlate with marrow involvement (p < 0.05).     

Mass contents of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol in human platelets stimulated with a thromboxane analogue and thrombin.

Mass contents of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) were measured in U46619-stimulated human platelets. 1 microM of U46619 induced maximum responses in aggregation, 5-hydroxytryptamine (5HT) secretion and increase in intracellular free Ca2+ concentration ([Ca2+]i). Aggregation was almost comparable to that induced by maximal dose (1 U/ml) of thrombin, while 5HT release was almost half. The initial [Ca2+]i peak in response to U46619 was about half of thrombin stimulation. Production of IP3 and DG was, however, less than one tenth of that seen in thrombin stimulation. The profile (time course and concentration-dependency) of IP3 formation did not correlate with that of [Ca2+]i, suggesting that U46619 stimulates IP3-dependent and -independent Ca2+ mobilization. DG production was small but sustained for more than 5 min. These findings support the recent hypothesis that aggregation is regulated by a delayed accumulation of DG. The low level of 5HT secretion could be explained by the low production of second messengers, IP3 and DG.     

Involvement of MMP-7 in invasion of pancreatic cancer cells through activation of the EGFR mediated MEK-ERK signal transduction pathway

AIMS: To clarify the involvement of matrix metalloproteinase-7 (MMP-7) in cell dissociation and the subsequent invasion of pancreatic cancer cells.METHODS: Western blotting, in vitro invasion assay, immunocytochemistry, and immunohistochemistry were performed in pancreatic cancer cell lines or pancreatic cancer tissue.RESULTS: The active form of the MMP-7 protein was expressed exclusively in the conditioned medium of dissociated (PC-1.0 and AsPC-1) pancreatic cancer cells, whereas proMMP-7 protein was only detected in the conditioned medium of non-dissociated (PC-1 and Capan-2) cells. Both intracellular and conditioned medium localised MMP-7 was greatly reduced by treatment with the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the mitogen activated protein kinase kinase (MEK) inhibitor U0126 in pancreatic cancer cells. MMP-7 treatment significantly induced the disruption of tight junction (TJ) structures and subsequent cell dissociation, and activation of the EGFR mediated MEK- ERK (extracellular signal regulated protein kinase) signalling pathway in the non-dissociated pancreatic cancer cells. Moreover, the strong in vitro invasiveness of dissociated cells was inhibited by AG1478 and U0126 treatment, whereas the weak invasiveness of non-dissociated cells was apparently induced by MMP-7 treatment. In addition, MMP-7 expression was stronger at the invasive front than at the centre of human pancreatic tumours.CONCLUSION: MMP-7 is involved in cell dissociation and the subsequent invasion of pancreatic cancer cells. It induces the disruption of TJ structures and forms a positive feedback loop with activation of the EGFR mediated MEK-ERK signalling pathway.