Verapamil enhances the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin and antitransferrin receptor with Pseudomonas exotoxin

Verapamil, a clinically important calcium channel blocker, has been found to cause a 40-fold enhancement of killing of the human KB cell line by a cytotoxic conjugate of epidermal growth factor with Pseudomonas exotoxin (EGF-PE). Synergistic effects of verapamil and EGF-PE are also seen on HeLa D98 cells and a human epidermal carcinoma cell line, A431. Verapamil also potentiates the effect of a toxic conjugate formed between Pseudomonas exotoxin and a monoclonal antibody to the human transferrin receptor (anti-TFR-PE) and enhances the effect of Pseudomonas exotoxin (PE) alone. Two other calcium antagonists were tested. Diltiazem enhances the cytotoxic effect of EGF-PE, but nifedipine does not. Verapamil does not affect the binding and uptake of 125I-EGF by KB cells, but it significantly delays the disappearance of internalized 125I-EGF from the cells. Density gradient fractionation studies using cell homogenates suggest that 125I-EGF accumulates in an undegraded form in lysosomes when cells are treated with verapamil. By immunofluorescence microscopy using an antibody to PE, EGF-PE was found to accumulate in lysosomes; by electron microscopy the lysosomes had an abnormal appearance. The effects of verapamil on toxicity of EGF-PE and lysosomal function appear to be related. However, it is not known whether the enhanced toxicity of EGF-PE in the presence of verapamil is due to its delayed degradation in lysosomes or some more general effect of verapamil on membrane permeability.     

A Mutation in the Drosophila Profilin Homolog Gene Affects Gametogenesis and Bristle Development in Both Sexes

We have isolated a Drosophila melanogaster mutant, allelic to the profilin gene reported as chickadee . We named the allele chickadee^bin , in which the oogenesis and the spermatogenesis are disrupted, and the bristles are malformed. In the mutant nurse cells, cytoplasmic actin filaments fail to polymerize, and nuclei are displaced. The flow of cytoplasm from nurse cells to the oocyte is abortive. These ovarian phenotypes are principally the same as those reported in chickadee^{wc57 and WF57} (2). In addition, the egg chamber of chickadee^bin contains a reduced number of cystocytes that are binucleafed. In some egg chambers, the oocyte fails to differentiate. All cystocytes in such an egg chamber are morphologically similar to nurse cells with polyploid nuclei. Mutant male flies have defective testes in which the spermatocyst is deficient or reduced in number. Mutant adults have shortened and forked bristles. We discuss the function of profilin in the gametogenesis and bristle development.     

Cultivation and utilization of Undaria pinnatifida (wakame) as food

A brief review is presented concerning wakame, Undaria pinnatifida, one of the most popular seaweeds used for food in Japan. Although it has been cultivated since about 1940, full-scale cultivation occurred after 1955. As methods for providing ‘seed stock’ and of processing the harvested sporophytes progressed, the yield increased rapidly. The main areas of cultivation are in Japan (e.g. Sanriku, Naruto), Korea and China, while ‘wild’ U. pinnatifida has been introduced into France, New Zealand and Australia. The total world yield of wakame exceeds 500 000 t fresh weight. Cultivated and harvested Undaria is boiled and salted (thus becoming green) and refrigerated; in the factory, it is removed its foreign matter and salt and dried. After checking for quality, the product is packaged in forms convenient for cooking and eating.     

Anti-integrin antibodies induce type IV collagenase expression in keratinocytes

During wound healing, pericellular proteolysis is thought to be essential for the detachment of keratinocytes from basement membrane and in their migration into the wound bed. We have characterized integrin-type cell adhesion/migration receptors in human mucosal keratinocytes and examined their function in the regulation of type IV collagenase gene expression. Two major integrins of the β1 class, α2β1 and αβ1, were found to function as collagen and fibronectin receptors, respectively. Antibodies against β1 and α3 integrin subunits were found to stimulate the expression of the 92 kDa type IV collagenase severalfold in a dosedependent manner. Keratinocytes expressed also the 72 kDa type IV collagenase, the synthesis of which remained, however, unchanged in keratinocytes treated with anti-integrin antibodies. Stimulation of 92 kDa enzyme was found to be caused directly by antibody binding to integrins, since Fab-fragments of anti-β1 antibodies alone were able to induce collagenase expression in the absence of secondary, clustering antibodies. Antibodies against α2β1 integrin caused no stimulation. Keratinocytes seeded on different substrata (plastic, collagen, fibronectin, laminin, or vitronectin) showed equal induction of type IV collagenase expression. Expression of 92 kDa type IV collagenase could not be induced by peptides (GRGDS, GRGES), proteins (fibronectin, laminin, fibrinogen., albumin), or antibodies to fibronectin. We suggest that proteolytic processes around keratinocytes can be regulated by extracellular factors signalling through integrin-type receptors. © 1993 Wiley-Liss, Inc.     

Variable Susceptibility to Apoptosis Induced by Calcium Ionophore in Hybridomas between HL-60 Promyelocytic and CEM T-Lymphoblastic Leukemia Cell Lines: Relationship to Constitutive Mg-Dependent Endonuclease

We recently reported that treatment with calcium ionophore, A23187, induces apoptosis in human myclogenous leukemia cells but causes necrotic cell death in T-lymphoblastic leukemia cells. To better understand the underlying mechanisms of such different modes of cell death, we established hybridomas between HL-60 promyelocytic and CEM T-lymphoblastic leukemia cells. The resulting hybridomas were divided into three groups in terms of their susceptibility to apoptosis following exposure to A23187: (1) hybridomas highly sensitive to apoptosis, (2) hybridomas with intermediate sensitivity to apoptosis which occurs later and to a lesser extent, and (3) hybridomas resistant to apoptosis. However, growth inhibition after 72 h of incubation and an initial rise in intracellular free calcium concentrations induced by A23187 were similar in the three groups. Expression of Ca²⁺-independent/Mg²⁺-dependent endonuclease, which had an optimal pH of 7.5-8.5 and was inhibited by Zn²⁺, was correlated with the susceptibility of the hybridomas to A23187-induced apoptosis. Thus, this endonuclease may play, at least in part, an important role in the induction of apoptosis in leukemia cell lines. Analysis of hybridomas between apoptosis-sensitive and apoptosis-resistant cells is useful in the elucidation of genetic factors which regulate cell death.     

Liquid chromatographic determination of myocardial interstitial epinephrine

This study describes a high-performance liquid chromatographic method with electrochemical detection (HPLC–ED) for monitoring of epinephrine (Epi) in the myocardial interstitial space. The in vitro detection limit for Epi was 200 fg in a 50-μl injection. Using a cardiac dialysis technique, 60-μl dialysates were sampled from the myocardial interstitial space (6-min fractions). After an alumina procedure, the dialysate Epi concentration was measured using the HPLC–ED system. Although the basal Epi concentration was undetectable, local administration of desipramine increased Epi concentration of the dialysate to 38.1±18.5 pg/ml. This system affords a new possibility for estimating myocardial interstitial Epi level.     

An Active Efflux System for Heavy Metals in Cisplatin-Resistant Human KB Carcinoma Cells

The mechanism for cisplatin resistance in cisplatin-resistant KCP-4 cells was studied. Although multidrug resistance-associated protein (MRP) was not detected in KCP-4 cells, the cells were more resistant to heavy metals than multidrug-resistant C-A120 cells that overexpressed MRP. KCP-4 cells expressed metallothionein, but it was scarcely involved in cisplatin resistance in these cells. KCP-4 cells did not express canalicular multispecific organic anion transporter (cMOAT). The glutathione(GSH) level was 4.7-fold higher in KCP-4 cells than in KB-3-1 cells. When the GSH level in KCP-4 cells was decreased by treating the cells with buthionine sulfoximine and nitrofurantoin, the accumulation of and sensitivity to cispaltin in the cells were increased. C-A120 cells were only 3.0-fold more resistant to cisplatin than KB-3-1 cells and this resistance was not affected by the increased glutathione level. The accumulation of platinum in C-A120 and KCP-4 cells was 68.5 and 20.4% of that in KB-3-1 cells, respectively, while the intracellular levels of antimony potassium tartrate in C-A120 and KCP-4 cells were 13.2 and 9.9% of that in KB-3-1 cells, respectively. The ATP-dependent efflux of antimony was enhanced in both C-A120 and KCP-4 cells. These results, taken together, suggest an efflux pump for heavy metals different from MRP and cMOAT is involved in cisplatin resistance in KCP-4 cells.     

Activator Protein-1 Binding Activities in Discrete Regions of Rat Brain After Acute and Chronic Administration of Methamphetamine

Abstract: The activator protein-1 (AP-1) binding activities increased in three brain regions (striatum, nucleus accumbens, and cingulate cortex) after a single methamphetamine (METH) injection to rats. Pretreatment with SCH 23390, but not (−)-eticlopride, significantly inhibited the enhanced AP-1 binding activities induced by acute METH administration. The magnitude of enhancement of AP-1 binding activities 3 h after the last dose of chronic METH administration (4 mg/kg once daily for 14 days) was significantly attenuated as compared with those 3 h after a single METH administration. The AP-1 binding activities after a 1-, but not 4-, week abstinence from chronic administration of METH were still significantly higher than those of the saline-treated controls. A METH challenge after a 4-week abstinence period induced significantly lower AP-1 binding activities in rats chronically injected with METH than in rats chronically injected with saline. The supershift assay revealed that the levels of Jun family protein, but not Fos-related antigen, increased significantly in the striatum and nucleus accumbens of chronically METH-treated rats after a 1-week abstinence. These results suggest that chronic METH administration leads to delayed decay of the induced AP-1 binding activities and Jun component levels after abstinence for up to 1 week but results in no change in or decreases these activities and attenuates METH challenge-induced AP-1 binding activities after abstinence for 4 weeks.     

A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization.

A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), Sephadex G-100, single-stranded DNA cellulose and hydroxyapatite. The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.