ATP- and EGF-stimulated phosphatidulinositol synthesis by two different pathways, phospholipase D and diacylglycerol kinase, in A-431 epidermoid carcinoma cells.

The [(3)H]inositol incorporation into the membrane fraction of A-431 human epidermoid carcinoma cells was markedly increased by stimulation of the cells with either epidermal growth factor (EGF), ATP, bradykinin, or a calcium ionophore A23187 in the presence of 1 mM extracellular calcium ions; most incorporated [(3)H]inositol was found to have accumulated as phosphatidylinositol (PI). The EGF- and ATP-stimulated PI synthesis was inhibited by two protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and an intracellular calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), but not by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Pretreatment of cells with pertussis toxin (IAP, islet-activating protein) inhibited the PI synthesis, [Ca(2+)]i elevation, and inositol trisphosphate (IP(3)) production by ATP, suggesting that the phospholipase C(PLC) system coupled with IAP-sensitive G protein is involved in the ATP-stimulated PI synthesis. On the other hand, the ATP stimulation increased the release of [(3)H]choline and [(32)P)phosphatidic acid (PA) from radiolabeled cells, and such release was not inhibited by IAP. In the presence of n-butyl alcohol, which prevents the production of PA by generation of phosphatidylbutanol, the ATP-stimulated PI synthesis was reduced. Because n-butyl alcohol did not inhibit IP(3) production and [Ca(2+)]i elevation, this fact suggests that the lAP-insensitive PLD system is involved in the ATP-stimulated PI synthesis. In A-431 cells, the stimulation of P(2)-purinergic receptors appears to activate the IAP-sensitive PLC system and IAP-insensitive PLD system, both of which are essential for the stimulation of PI synthesis. The present results imply the general prospect that ligand stimulation, which mobilizes second messengers and consumes their precursors, simultaneously provokes the pathway to synthesize and salvage the second messenger precursors as well.

Spherical gradient-index polymer lens with low spherical aberration.

A sphere lens with a spherical gradient index (GRIN) was prepared by the modified suspension polymerization technique. GRIN spheres with quadratic- and linear-index distributions were obtained by two different methods to confirm the effect of the GRIN profile on the focusing property of the sphere lens. It was confirmed in both theory and experiment that the spherical aberration of such GRIN spheres was remarkably decreased compared with that of a homogeneous sphere.

Intussusception caused by a carcinoma of the cecum during pregnancy: report of a case and review of the literature.

A case of intussusception due to a carcinoma of the cecum during pregnancy is reported. A 27-year-old pregnant female was admitted to Shimodate Municipal Hospital because of abdominal pain, nausea and vomiting. Her abdomen was distended, and a relatively hard mass was palpable in the right hypochondrium. Following a diagnosis of intussusception by ultrasonography, a laparotomy was performed. The lesion causing the intussusception was found to be a carcinoma of the cecum, and thus a right hemicolectomy with lymph node dissection was carried out. Histological examination revealed that the tumor was a well-differentiated adenocarcinoma which had invaded the muscularis propria but was superficial to the subserosa. None of the lymph nodes were cancerous. The incidence of colonic cancer above the peritoneal reflection during pregnancy is very low. Only 24 cases have been previously reported; our patient is only the 25th case, as well as being the first case demonstrating Dukes' A. Due to the intussusception, ultrasonography was effective for diagnosis and the patient was able to undergo a curative operation at an earlier stage than other patients.

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells.

Upon stimulation with 10(-6) -10(-3) M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.

Involvement of cellular calcium incorporated from the medium in production of inositol trisphosphate in A-431 cells.

The property of intensive 45Ca2+ uptake by A-431 human epidermoidal carcinoma cells was indicated to be an influx, not binding to the cell surface, since the two apparent dissociation constants (Kd) between 45Ca2+ and cells were almost the same when measured in either the presence or absence of 1 mM [ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA); these constants were approximately 5-10 x 10(-6) and 1 x 10(-4) M, respectively, which are much higher than the chelating constant of EGTA for Ca2+ (approximately 10(-11) M). Furthermore, addition of A23187, a calcium ionophore, rapidly released the 45Ca2+ incorporated into cells at both 37 degrees C and 0 degrees C. The 45Ca2+ associated with the cells was slowly released or exchanged when cells were incubated in medium depleted of Ca2+, or in that containing 1 mM non-radioactive Ca2+. The ability of A-431 cells to respond to extracellular ATP by elevating their level of intracellular calcium ions, as well as by producing inositol trisphosphate (InsP3), was suppressed in cells depleted of cellular calcium. These data suggest that calcium ions are extensively incorporated or exchanged with those outside the cells, maintained as stored calcium, and involved in production of InsP3, when A-431 cells are stimulated by ATP to trigger the signal transduction system.

[Effects of L-ascorbic acid and bone metabolism factors on alkaline phosphatase activity of and 45Ca2+ incorporation by ROS 17/2.8 cells].

Among several bioactive substances known as coupling factors, transforming growth factor-beta (TGF-beta), interleukin-1 (IL-1), and prostaglandin (PG) E1 and E2 increased not only the activity of alkaline phosphatase but also the rate of incorporation of 45Ca2+ into ROS 17/2.8 during a 3-day culture: the former two factors are known to be formed at the site where bone is resorbed, while PG's are known as one of the factors involved in bone resorption. Parathyroid hormone, another hormone that affects bone metabolism, elevated the incorporation of 45Ca2+ by and decreased the alkaline phosphatase activity of the cells. The facts indicate the possibility that the osteoblastic cells are involved in the transport of calcium ions when bones are being resorbed. On the other hand, when these osteosarcoma cells were cultured in DMEM containing ascorbate and beta-glycerophosphate, followed by staining with silver nitrate by the procedure of von Kossa, there appeared many groups of cells that were positively stained as dark brown spots. Cells were then cultured under the same conditions in the presence of radioactive calcium, and the radioactivity accumulated was measured. The result showed that the presence of both ascorbate and beta-glycerophosphate in the culture medium dramatically increased the accumulation of 45Ca2+. It appears from these facts that ROS 17/2.8 cells are capable of incorporating and/or accumulating calcium ion if they are cultured under appropriate conditions. These cells will probably be able to produce a calcified matrix in vitro.

A simple and rapid purification procedure for the preparation of tetramethylrhodamine-labeled antibodies.

A conjugate preparation of antibody (Fab) and tetramethylrhodamine (TMR) generally adsorbs free TMR which is very difficult to remove because of its strong hydrophobic binding. On the basis of criteria such as sodium dodecyl sulfate (SDS) gel electrophoresis and staining of the plasma membrane of live cells, we found that simple extraction with n-butyl alcohol or iso-amyl alcohol could remove the contaminating free dye. The procedure is especially useful when one needs to prepare conjugates with low nonspecific binding for the study of lateral diffusion of cell membrane-associated antigens.

Postnatal changes in an alpha subunit isoform, alpha(S), of Na+,K(+)-ATPase in the submandibular gland of rats.

The alpha and alpha(+) isoforms of Na+,K(+)-ATPase were isolated from the kidney and brain of rats and purified. Their antisera were raised to analyze the alpha isoforms in rat tissues. We found that the submandibular gland (SMG) contains a new immunoreactive alpha subunit isoform, designated alpha(S) in this report, in addition to alpha identical with those found in the kidney or brain. The new alpha(S) strongly reacted with anti-alpha-antiserum but to a much lesser extent with anti-alpha(+)-antiserum. The alpha(S) had a slightly lower molecular weight (approximately 90,000) than the brain and kidney alpha isoforms. Various fractions of SMG tissues were added to the SMG microsomes and incubated in order to test whether or not the alpha(S) is formed artificially; no increase of alpha(S) was observed by these treatments, suggesting that the alpha(S) was not the product formed from alpha during the preparation of microsome sample, but was rather a protein originally present in the SMG. The alpha(S) protein was not detected in the SMG of 2- or 5-week-old rats, but it gradually increased in rats older than 8 weeks, reaching the maximum in 30-week-old animals. The Na+,K(+)-ATPase activity in the SMG increased concomitantly with the increase of alpha(S), indicating that Na+,K(+)-ATPase comprising alpha(S) also shows enzyme activity; it is speculated that alpha(S) may have some unique and unknown function(s) in older rats.

[Effects of various factors involved in bone metabolism on 45Ca2+ incorporation and alkaline phosphatase activity of ROS 17/2.8 cells].

The effects of interleukin 1, transforming growth factor-beta (coupling factors), prostaglandin E1, and prostaglandin E2 on incorporation of 45Ca2+ and on alkaline phosphatase activity were studied using cultured ROS 17/2.8 cells, one of cell lines derived from rat osteosarcoma. We found that all these factors stimulate both the incorporation of 45Ca2+ and alkaline phosphatase activity of these cells. On the other hand, one of the bone resorption hormones, parathyroid hormone (PTH), suppressed the proliferation of cells and decreased the alkaline phosphatase activity at considerably low concentrations (1 X 10(-12)-1 X 10(-11) M). However, the hormone stimulated the incorporation of 45Ca2+ by these cells in a dose-dependent manner; the maximum stimulation on day 3 was observed at 1 X 10(-7) M and it was approximately 3 times the control value. The data suggest therefore, that the osteoblasts incorporated calcium ions and transported them while bone resorption was occurring. Thus the ROS 17/2.8 cell line appears to be an advantageous experimental system for the study of calcium metabolism of osteoblasts in vitro.

Development of enucleated parthenogenones that received pronuclei or nuclei from fertilized mouse eggs.

This study compares the developmental potential of enucleated mouse parthenogenones that received pronuclei from fertilized eggs with those that received nuclei from late two-cell embryos. The proportion of reconstituted parthenogenones, which received pronuclei at the one-cell stage, that developed to blastocysts in vitro and to live fetuses after transfer to recipients was significantly lower than that of reconstituted control eggs. However, the in vitro and in vivo developmental potential of reconstituted parthenogenones that received nuclei at the two-cell stage from fertilized late two-cell embryos was not different from that of reconstituted control eggs. These results were contrary to those reported previously by Mann and Lovell-Badge (1984), who showed that parthenogenetic eggs receiving pronuclei from fertilized eggs developed well both in vitro and in vivo.

[Two cases of obstructive jaundice due to advanced gastric cancer with marked response to the intravenous administration of cisplatinum].

Two cases of obstructive jaundice due to advanced gastric cancer were treated with intravenous administration of cisplatinum. The first case was a 46-year-old female who had undergone gastrojejunostomy 5 months earlier because of Borrmann type 3 gastric cancer. The tumor involved the head of the pancreas and a portion of the duodenum with distant intraperitoneal dissemination (S3N3P3H0). She was admitted to Shimodate Municipal Hospital on June 8 because of abdominal pain and jaundice. Her abdomen was distended with ascites, and there was a fist-sized tumor in the lower portion. CT examination revealed that the jaundice was caused by obstruction due to the main tumor. Histologically, the tumor consisted of poorly differentiated adenocarcinoma. Intravenous administration of CDDP (50 mg/body/week X 4), MMC (4 mg/body/week X 4) and FT (400 mg/body/day for 4 weeks) was carried out. After the chemotherapy, the jaundice, abdominal pain and ascites disappeared, and the abdominal tumor had markedly reduced in size which was regarded as PR. The second case was 66-year-old male who had received subtotal gastrectomy and transverse colectomy 16 months ago because of Borrmann type 3 gastric cancer. The tumor comprised well-differentiated adenocarcinoma and infiltrated to the mesentery of the transverse colon with positive lymphnodes (S3N1P1H0, stage IV). This time he was admitted to the hospital because of general fatigue and jaundice. According to CT examination, the common bile duct was obstructed by metastasized lymphnode around the pancreas. He had elevated serum level of total bilirubin (7.7 mg/gl) and CA 19-9 (23,000 U/ml). After the administration of CDDP (50 mg/body/week X 4) and MMC (4 mg/body/week X 4), his complaints disappeared and the serum total bilirubin level and CA 19-9 level returned within normal range. These data suggest that combination chemotherapy using CDDP was effective in these 2 cases.

Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation.

The exposure of mouse zygotes pre-stained with Hoechst 33342 to u.v. irradiation for 20-30 sec significantly or completely inhibited development to blastocysts in vitro. However, development to the blastocyst stage of enucleated eggs receiving pronuclei from untreated eggs was as good as that of control reconstituted eggs when the cytoplasm originated from eggs exposed to u.v. irradiation for 20-30 sec, but was significantly lower when the cytoplasm was from eggs exposed for 40 sec. The chromosomes at the second metaphase stage could be removed with 15 sec of exposure to u.v. irradiation under a fluorescence microscope. Most eggs enucleated at the second metaphase that received a single inner cell mass nucleus (75%) showed pronuclear formation 6 h after activation; 23% of them developed to morphologically normal 2-cell eggs and 5% developed to blastocysts. These results demonstrate that the cytoplasm of mouse zygotes is more resistant to u.v. irradiation after Hoechst staining. Eggs at the second metaphase, from which chromosomes have been removed under a fluorescence microscope, can therefore be used as cytoplasm recipients for nuclear transplantation of inner cell mass nuclei.

Full-term development of mouse blastomere nuclei transplanted into enucleated two-cell embryos.

The nuclei from four- and eight-cell mouse embryos were transplanted into enucleated two-cell embryos. It was found that such embryos not only developed to the blastocyst stage in vitro (72% and 35%), but also developed to full term (22% and 8%) after transfer to recipient mice. However, development of embryos which contained nuclei from the inner cell mass was not observed. Since the development of enucleated zygotes which contain advanced nuclei is limited (the present study; McGrath and Solter: Science, 226:1317-1319, '84; Robl, Gilligan, Critser, and First: Biol. Reprod., 34:733-739, '86), it appears that cytoplasmic factors are important for the development of nuclei from advanced cells.

Electrofusion for the pronuclear transplantation of mouse eggs.

The present study was undertaken to determine the efficiency of HVJ treatment and electrofusion for pronuclear transplantation in the mouse. The output voltage and duration of the pulses were fixed to 200 microsec at 10 V or to 150 microsec at 15 V for electrofusion, because the maximum rates of blastomere fusion of 2-cell embryos and development of fused embryos in vitro were obtained under these conditions. Although the proportion of eggs with fused karyoplast (78%) and the fused eggs developed to morulae or blastocysts the proportion of pregnant recipients and young obtained after treatment of fused eggs was not significantly different between these two procedures. It is advised that electrofusion can be used as a fusogenic procedure for pronuclear transplantation in the mouse in some cases where HVJ cannot be applied.

Serial observations of colonic carcinogenesis in the rat. Premalignant mucosa binds Ulex europeus agglutinin.

We evaluated certain histochemical tests for their ability to detect premalignant mucosa in the dimethylhydrazine model of colonic carcinogenesis. Biweekly colonoscopic biopsies of the descending colon were performed for 29 wk in control and dimethylhydrazine-treated rats. Biopsy specimens of the splenic flexure, rectum, and any visualized tumors were taken. The specimens were stained with periodic acid-Schiff to detect neutral mucins, high-iron diamine alcian blue to detect sialylated and sulfated mucins, fluoresceinated peanut agglutinin, and fluoresceinated Ulex europeus agglutinin. None of the first three tests consistently detected premalignant mucosa. However, Ulex europeus agglutinin, which bound to only 3% of control biopsy specimens throughout the course of the study, bound to increasing numbers of biopsy specimens in the dimethylhydrazine-treated animals, reaching a maximum of 90% positivity by 13-16 wk. Moreover, Ulex europeus agglutinin bound strongly to all biopsy specimens from tissues adjacent to tumors and to 93% of tumors. Mucosal atrophy and focal dysplasia were present more frequently in specimens taken from the rectum (but not the splenic flexure) of dimethylhydrazine-treated animals than of control animals, but there was no correlation between the histochemical markers and either atrophy or dysplasia. We conclude that Ulex europeus agglutinin binding is a consistent feature of premalignant colonic mucosa in dimethylhydrazine-treated rats.