Autoradiographic localization of 3H-histamine accumulation by the visual system of the locust

Summary The uptake of [³H]-histamine into the retina and optic lobe of the locust, Schistocerca americana gregaria was studied by means of autoradiography at the light- and electron-microscopic levels. Light-microscopic autoradiography showed a significant accumulation of [³H]-histamine in several regions of the optic lobe. Dense accumulations of silver grains were concentrated along the medial border of the medullary neuropil and around the entire periphery of the lobula. No significant accumulations of grains were present within the retina or the neuropil zones of the lamina, medulla or lobula.Electron-microscopic autoradiography showed histamine-accumulating cells along the border of the medulla to exhibit electron density and morphology typical of glial cells. Labelled histamine was present within both glial cell bodies and their processes. In the region surrounding the neuropil of the lobula, [³H]-histamine was concentrated within fine glial processes wrapped around neuronal cell bodies and their axons. No neuronal cell bodies or axons showed accumulation of silver grains above background.These results are consistent with previous studies showing the glial uptake of amino acid and biogenic amine putative neurotransmitters. However, the lack of a demonstration of a specific uptake of histamine in neuropil zones makes it difficult to assess the role of histamine uptake in the inactivation of neurally released histamine in the locust visual system.     

RADIOAUTOGRAPHIC DEMONSTRATION OF 5-HYDROXYTRYPTAMINE-3H UPTAKE BY PULMONARY ENDOTHELIAL CELLS

The lung is able to rapidly remove 5-hydroxytryptamme (5-HT) from the circulation by a Na⁺-dependent transport mechanism. In order to identify the sites of uptake, radioautographic studies were done on rat lungs which had been isolated and perfused with 5-HT-³H and 0 5 mM iproniazid, a monoamine oxidase inhibitor. In control experiments 10^-4 M imipramine was added to the perfusate to inhibit the membrane transport of 5-HT At the light microscope level, silver grains were seen concentrated near capillaries and in the endothelium of large vessels From electron microscope radioautographs a semiquantitative grain count was made and 90% of the silver grains were observed over capillary endothelial cells. The grains were found over the nucleus and cytoplasm of the cell and shewed no preferential association with any particular cytoplasmic inclusion bodies, organelles, or vesicles Other cell types were unlabeled except for a few mast cells, certain vascular smooth muscle cells, and one nerve ending. This radioautographic demonstration of the cell type responsible for the rapid removal of 5-HT from the lung circulation clearly establishes the existence of a new metabolic role for pulmonary endothelial cells.     

Phenylalanine hydroxylation and tyrosine requirement of cultured cells : Evidence of phenylalanine hydroxylation in mastocytoma cells in culture

Growth of cells in tyrosine-free medium can be used to detect and select cells able to convert phenylalanine to tyrosine. Nine different cell types have been tested: fibroblastic cells derived from human skin, human amniotic fluid cells, continuous human lymphoid cells, rat hepatoma, Vero (monkey kidney), HeLa, mouse neuroblastoma, mouse melanoma, and mouse mastocytoma. Two cell lines, mastocytoma and hepatoma, grew well in tyrosine-free medium, and subsequent enzyme assay showed substantial phenylalanine hydroxylation. This enzyme activity has already been documented in hepatoma [1], but the demonstration of phenylalanine hydroxylation by a cell line of non-hepatic origin is new. Fibroblastic cells survived but did not grow without tyrosine. No conversion of [¹⁴C]phenylalanine to tyrosine was demonstrated in these cells.     

In vitro translation of rat liver and Novikoff hepatoma cytokeratin mRNAs

Summary Cytoplasmic poly(A)⁺ RNA was isolated from normal rat liver and Novikoff ascites hepatoma cells, translated in vitro using rabbit reticulocyte lysate system and the translational products were assayed by immunoprecipitation with antibodies specific for Novikoff hepatoma principal cytokeratins p39, p49 (a group of hepatic cytokeratins C, D, and E) and p56. The identity of the precipitated antigens was further confirmed by two-dimensional polyacrylamide gel electrophoresis. Only the Novikoff hepatoma poly(A)⁺ RNA contained translatable mRNA coding for the p39 cytokeratin while the p49 and p56 cytokeratins were translated from both the normal rat liver and Novikoff hepatoma poly(A)⁺ RNAs. Immunoprecipitations employing monoclonal antibody specific for p39 also recovered significant quantities of p56 and 49K cytokeratins, presumably due to oligomeric associations of these proteins with p39 immediately after in vitro synthesis. Similar results were observed after experiments with anti-p56 monoclonal antibody in which p39, not reactive with this antibody, was recovered in immunoprecipitates. Overall, the two-dimensional gel fluorograms of cytokeratins synthesized in vitro from NAH or liver poly(A)⁺ RNA are quite similar to isolated antigenic and cytokeratin profiles reported previously. These results suggest that overt posttranslational processing is not likely responsible for the diversity of cytokeratins observed in the liver.Abbreviations NAH Novikoff ascites hepatoma - HEPES N-2hydroxyethylpiperazine-N-2-ethane sulfonic acid - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Cell surface proteoglycans as a negative modulator in concanavalin a-mediated agglutination of hepatoma cells

Proteoglycan (heparan sulfate-protein conjugate) was solubilized with 8 M urea from rat liver plasma membranes after enzymic (RNAase, neuraminidase) treatments and extensively purified by chromatography and gel filtration. The final products gave an average ratio of hexuronate to protein (weight) of approx. 1.5, contained hexosamine equimolar to hexuronate and were sensitive to β-elimination (the molecular weight being reduced from 20 · 10⁴ to 3 · 10⁴ (gel filtration)).The proteoglycan fraction, when added to trypsinized and untrypsinized ascites hepatoma (AH-130F(N)) cells, inhibited the concanavalin A-mediated agglutination of the cells. However, the alkali-treated proteoglycan (β-elimination) or acid mucopolysaccharide fraction prepared from liver plasma membranes by papain digestion were less effective, and a reference preparation of heparan sulfate was almost ineffective. It was confirmed that significant amounts of proteoglycan labelled with ³⁵SO₄^2? were firmly bound to or taken up by the trypsinized ascites hepatoma cells.These results together with the sensitization of lectin-mediated agglutination by mild protease treatment of cells suggest that cell surface proteoglycans may act as a negative modulator in the lectin-mediated agglutination of cells.     

Citric acid-sonication method for the isolation of nucleoli

A simple method has been developed for the isolation of nucleoli of Novikoff hepatoma ascites cells from nuclei prepared by the citric acid procedure. The purity of the nucleoli is equivalent to that of nucleoli isolated by the sucrose-Ca²⁺ method [3, 16]. Chemical compositions and RNA profiles were virtually identical to those reported earlier [3, 10, 16]. In addition, the nucleoli isolated by this method had no ribonuclease activity. The average yield of 64% is higher than that of the method employing sucrose and Ca²⁺. This method has been successful both for cells obtained directly from ascites fluid and for cells incubated in minimal Eagle's medium for 16 h. For the latter condition, the sucrose-Ca²⁺ method did not yield satisfactory results [13].     

Linkage of the evolutionarily-related serum albumin and alpha-fetoprotein genes within q11-22 of human chromosome 4

Albumin and alpha-fetoprotein are structurally related serum proteins, having a similar gene structure and, conceivably, a common evolutionary origin. To test their relative arrangement in the human genome, the serum albumin and alpha-fetoprotein genes were mapped by in situ hybridization of cloned human albumin or alpha-fetoprotein cDNA to human mitotic chromosome preparations. Analysis of cells hybridized with the serum albumin probe showed that 39% of cells exhibited grains on the proximal portion of the long arm of chromosome 4 (bands q11-22), with these grains comprising 30% of all labeled sites throughout these mitoses. Similarly, in cells hybridized with the alpha-fetoprotein probe, 39% of cells were observed to contain silver grains on 4q11-22, these grains constituting 20% of all labeled sites in these cells. These results demonstrate chromosomal localization and linkage of the serum albumin and alpha-fetoprotein genes within bands q11-22 of the long arm of human chromosome 4.     

Studies on the release of barley aleurone cell proteins: Autoradiography

Summary Both uptake and incorporation of radioactivity from [³H]l-leucine into gibberellic-acid (GA₃)-treated aleurone layers of barley (Hordeum vulgare L.) was enhanced by pretreatment with 5 mM potassium bromate. The effect of 5 mM KBrO₃ on amino-acid incorporation was quantitative rather than qualitative and could be partly reversed by the addition of neutralized casein hydrolysate at 10 mg/ml. Autoradiographs of GA₃-treated aleurone cells pulsed with [³H]leucine showed distribution of silver grains predominantly over the endoplasmic reticulum (ER) and aleurone grains. After chasing with carrier l-leucine for 60 min, fewer silver grains were associated with the ER and aleurone grains while nearly half of the silver was associated with the ground cytoplasm of the cell. Autoradiographs were prepared from aleurone cells previously stratified by ultracentrifugation. After a 10-min pulse of label, the silver grains were found over the central ER zone of centrifuged cells; however, with an increase in duration of the chase, label was found distributed throughout the aleurone grain and spherosome region of the cell. The silver grains which were located over the central zone of centrifuged cells at the end of the pulse were almost exclusively associated with the ER. There is no evidence for association of label with dictyosomes or with vesicles derived from dictyosomes. The experimental evidence indicates that labelled amino acids are incorporated into aleurone cells on the ER and are released from these cells without the participation of a membrane-bound vesicle.     

Enzymatic modification of the L and M antigens in LK and HK sheep erythrocytes and their membranes

Summary This paper reports on the effect of two hydrolytic enzymes, neuraminidase and trypsin, on the interaction of blood group L-positive low-potassium-type (LK) and blood group M-positive high-potassium-type (HK) sheep red cells with their respective isoimmune antisera. It was found that treatment of LK and HK red cells with neuraminidase did not change the interaction of these cells with their homologous antibodies as measured by K⁺-pump flux, complement-mediated immune hemolysis and absorption of antibody. Similarly, trypsin pretreatment of LK and HK red cells did not interfere with the hemolytic action of anti-L and anti-M antibodies, respectively. In striking contrast, however, it was observed that pretreatment of LK cells with trypsin rendered these cells insensitive to the K⁺-pump stimulating antibody present in the anti-L serum.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.     

Metaphase figures of rat chromosomes incorporated into mouse cells

Summary Rat chromosomes isolated from ascites hepatoma cells were supplied to the medium of mouse diploid cells culturing in vitro. One, two and four days after cultivation, the rat chromosomes were observed to be incorporated into the metaphase nuclei of the mouse cells at the rate of 0.3, 1.2 and 0.2 per cent, respectively.This investigation was supported by a research grant in Aid for Fundamental Scientific Research from the Ministry of Education, Japan (No. 9001).     

A luminometric assay for peroxisomal beta-oxidation. Effects of fasting and streptozotocin-diabetes on peroxisomal beta-oxidation.

Rat hepatoma cells grown intraperitoneally as an ascites tumour were analysed with respect to their contents of cytosolic glutathione transferases. In contrast with normal liver tissue, the hepatoma cells were dominated by the class Pi glutathione transferase 7-7. All the major hepatic enzyme forms were down-regulated to almost undetectable concentrations. Livers of rats bearing ascites-hepatoma cells expressed low, but significant, amounts of protein which, by electrophoretic and immunochemical properties, appeared identical with transferase 7-7. This enzyme is not detectable in normal hepatocytes. Treatment of rats with trans-stilbene oxide induced the expression of transferase 7-7 in the livers of normal rats as well as in hepatoma-cell-bearing animals. In addition, a 2-fold induction of transferase 7-7 was measured in the hepatoma ascites cells. No significant elevation of any other enzyme forms in the hepatoma cells was noted.     

Ultrastructural relationship between monoamine- and TRH-containing axons in the rat median eminence as revealed by combined autoradiography and immunocytochemistry in the same tissue section

Summary The correlation of dopamine (DA)-, noradrenaline (NA)- or serotonin (5HT)-containing neurons and thyrotropin releasing hormone (TRH)-containing neurons in the median eminence of the rat, as well as the coexistence of monoamines (MA) and TRH in the neurons, were examined by subjecting ultrathin sections to a technique that combines MA autoradiography and TRH immunocytochemistry. The distribution and localization of silver grains after ³H-MA injection were examined by application of circle analysis on the autoradiographs.TRH-like immunoreactive nerve terminals containing the immunoreactive dense granular vesicles were found to have an intimate contact with monoaminergic terminals labeled after ³H-DA, ³H-NA or ³H-5HT infusion in the vicinity of the primary portal capillaries in the median eminence. Synapses between TRH-like immunoreactive axons and MA axons labeled with silver grains, however, have not been observed to date. Findings suggesting the coexistence of TRH and MA in the same nerve terminals or the uptake of ³H-MA into TRH-like immunoreactive nerve terminals, where silver grains after ³H-MA injection were concurrently localized in TRH-like immunoreactive nerve terminals, were rarely observed in the median eminence. Percentages of the nerve terminals containing both immunoreactive granular vesicles and silver grains after ³H-MA injection to total nerve terminals labeled after ³H-MA infusion silver grains were equally very low in ³H-DA, ³H-NA or ³H-5HT, amounting to less than 6.1%.This work was supported in part by grant-in-aid for scientific research from the Japan Ministry of Education (No. 557018).     

A comparative study of plasma membrane Mg2+-ATPase activities in normal,regenerating and malignant cells

Plasma membranes have been prepared from rat normal liver cells, regenerating liver cells and Yoshida ascites hepatoma 66 cells after intact cells were first bound to polylysine-coated polyacrylamide beads, and the membrane-associated Mg²⁺-ATPase activity was assayed directly on beads with membrane attached. With plasma membranes from normal liver cells, K_m for ATP and V were found to be higher than those in regenerating liver cells and hepatoma cells. Vanadate caused a different sensitivity of the activity, without an effect in normal liver cells and with an inhibition in regenerating liver cells and hepatoma cells. The activity in normal and regenerating liver cells decreased with increasing temperature above 24–30°C, while the activity in hepatoma cells continued to increase linearly to 37°C. Unlike the enzyme in normal and regenerating liver cells, the hepatoma enzyme was shown to have a higher phase transition temperature and lower activation energies. In all three kinds of cells the activity was increased by the dephosphorylation of plasma membranes and unaffected by the phosphorylation. By means of histochemical Mg²⁺-ATPase staining applied on polyacrylamide gels, at least three major bands which show the enzymic activity were visible in normal and regenerating liver and a single band was detected in hepatoma cells.     

Rat alpha-fetoprotein: in vitro production of four molecular variants by clonal cell lines.

Mass and six clonal cultures derived from Morris hepatoma 7777 by standard tissue culture techniques synthesize and secrete alpha-fetoprotein $\text{in vitro}$. During serial passage, the alpha-fetoprotein which accumulates in the media of these cultures contains two concanavalin A-affinity molecular variants. Each of the concanavalin A-affinity molecular variants shows two electrophoretic variants. Mass and clonal cell populations of hepatoma cells continue to secrete $\text{in vitro}$four molecular variants of rat alpha-fetoprotein known to occur $\text{in vivo}$. These results demonstrate for the first time that individual hepatoma cells have the potential to synthesize four molecular variants of alpha-fetoprotein and that this phenotypic property is maintained during serial subculture $\text{in vitro}$.     

Catumaxomab: Clinical development and future directions

Catumaxomab, a monoclonal bispecific trifunctional antibody, was approved in the european Union in April 2009 for the intraperitoneal treatment of patients with malignant ascites. The marketing authorization holder Fresenius Biotech GmbH developed catumaxomab (Removab^®) together with its partner TRiOn Pharma GmbH, Germany. it is the first substance worldwide with a regulatory label for the treatment of malignant ascites due to epithelial carcinomas. Since the peritoneum is of mesothelial origin and therefore lacks epCAM expression, the intraperitoneal administration of catumaxomab is an attractive targeted immunotherapeutic approach. Catumaxomab is able to destroy epCAM positive tumor cells in the peritoneal cavity known as the main cause of malignant ascites. in addition, catumaxomab is a potential therapeutic option for several primary tumors since the epCAM molecule is expressed on the majority of epithelial carcinomas. This review focuses on the clinical development of catumaxomab and indicates future directions.Key words: catumaxomab, Removab^®, monoclonal antibody, trifunctional, EpCAM, malignant ascites, peritoneal carcinomatosis, immunotherapy     

Release of glycopeptides and mucopolysaccharides from ascites hepatoma cells by tryptic treatment.

Two types of ascites hepatoma cells, AH 66 and AH 130 FN, were treated with trypsin to observe the release of complex carbohydrates constituting the plasma membranes. From AH 66 cells, mucopolysaccharide (heparan sulfate) was preferentially released. From AH 130 FN cells, N-glycosidic glycopeptides were preferentially released whereas no mucopolysaccharide (chondroitin sulfate A) was released.     

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Lysis of tumor cells by antibody and complement. VI. Enhanced killing of enzyme-pretreated tumor cells.

The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement. Pretreatment of line-10 cells with certain proteolytic enzymes rendered tham susceptible to the killing action of antibody and guinea pig complement. The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors. The effect of the enzyme treatment could reversed by incubating the treated cells at 37 degrees C (but not at 0 degrees C), in the absence of the enzyme. Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens. The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination. Enzyme preparations that proved ineffecitve in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, beta-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase.