A practical test for in vitro evaluation of photosensitization: Assessment with hematoporphyrin derivative (HPD)

A simple procedure is described for the determination of the photosensitizing potency of drugs, using three leukemic cell lines, two of lymphocytic origin, L1210 and P388 and one of erythroid type, Friend-745. The procedure allows one to investigate several aspects of the photosensitization properties of tested compounds such as cellular localization and direct (trypan blue exclusion) or delayed (clonogenicity) photomediated toxicities.The method was assessed using crude hematoporphyrin derivative (HPD) as well as dihematoporphyrin ether (DHE) or commercially available Photofrin II. Results were compared to those obtained with normal cells, e.g spleen lymphocytes and erythropoietic stem cells (CFU-e), and discussed in the light of the relative response of normal versus transformed cells.Abbreviations DHE Dihematoporphyrin Ether - FCS Fetal Calf Serum - HPD Hematoporphyrin Derivative - PDT Photodynamic Therapy     

Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers

Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (10⁷–2×10⁷ cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors.     

Ferric cacodylate efficiently stimulates growth of rat renal glomerular epithelial cells in vitro

Rat renal glomerular epithelial cells (SGE1 cell line) can be maintained and grown continuously in serum-free medium supplemented with insulin, iron-saturated transferrin (Tr), selenium, bovine serum albumin (BSA), linoleic acid, and epidermal growth factor (EGF). Of the growth supplements used, Tr is essential for proliferation of the cells. In the present study, we describe the use of a unique iron-chelate complex, ferric cacodylate (Fe-Cac), positively charged molecules in neutral buffer, that could almost replace Tr in serum-free culture. It even stimulated the growth of SGE1 cells more efficiently than ferric chloride (FeCl₃) and other iron-chelate complexes, such as ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-Cit). The growth-stimulatory activity of Fe-Cac was exerted at iron concentrations of more than 0.01 g/ml, whereas a 10-fold excess of iron concentration was required with FeCl₃, Fe-NTA and Fe-Cit. We observed that SGE1 cells grew until confluent, then formed hemicysts (domes) in serum-free medium containing Fe-Cac, suggesting that Fe-Cac did not merely permit cell growth but also supported polarization and organization of the cells into a functional epithelial architecture. Moreover, since the stimulatory activity of Fe-Cac was completely abolished by desferrioxamine, a strong iron chelator, it is suggested that iron is crucial for growth of SGE1 cells. When the cells were treated with suramin, an inhibitor of cellular pinocytosis and endocytosis of a large spectrum of ligands including receptor-bound growth factors, growth-stimulatory activity of Tr was inhibited, whereas the activity of Fe-Cac was not affected. These results, taken together, strongly suggest that the growth-stimulatory activity of Fe-Cac is associated with iron delivery into the cells through the cell membrane by diffusion, which is different from Tr receptor-mediated endocytosis. The use of Fe-Cac for investigating iron-regulated cell proliferation is suggested.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Melanin as a natural germ cell marker for intraspecific transplantation experiments in Ambystoma mexicanum (Urodela,Amphibia)

Summary In urodele amphibians, the lack of a reliable germ cell marker restricts the experimental study of the germ lineage. In the present work, we conducted genetic and histological analyses in order to demonstrate that melanin from oocytes constitutes a germ cell marker available for intraspecific experiments in Ambystoma mexicanum. Then, using this marker, we implanted germ cells from undifferentiated gonads (stage 48) into the blastocoel of host embryos and investigated their fate and determined state. Our results show that, from this stage on, the donor cells do not differentiate into other cell types; therefore, they are restricted in developmental capacity and irreversibly determined as germ cells. On the other hand, exogenous germ cells were found in an isotopic position until the young tail-bud stage, and then were found in an ectopic position; these results suggest that, from the middle tail-bud stage on, an active process contributes to migration of primordial germ cells to the gonadal territory.     

Pattern of cell proliferation in embryogenesis and planula development ofHydractinia echinata predicts the postmetamorphic body pattern

Summary During embryogenesis and planula development of the colonial hydroidHydractinia echinata cell proliferation decreases in a distinct spatio-temporal pattern. Arrest in S-phase activity appears first in cells localized at the posterior and then subsequently at the anterior pole of the elongating embryo. These areas do not resume S-phase activity, even during the metamorphosis of the planula larva into the primary polyp. Tissue containing the quiescent cells gives rise to the terminal structures of the polyp. The posterior area of the larva becomes the hypostome and tentacles, while the anterior part of the larva develops into the basal plate and stolon tips. In mature planulae only a very few cells continue to proliferate. These cells are found in the middle part of the larva. Labelling experiments indicate that the prospective material of the postmetamorphic tentacles and stolon tips originates from cells which have exited from the cell cycle in embryogenesis or early in planula development. Precursor cells of the nematocytes which appear in the tentacles of the polyp following metamorphosis appear to have ceased cycling before the 38th hour of embryonic development. The vast majority of the cells that constitute the stolon tips of the primary polyp leave the cell cycle not later than 58 h after the beginning of development. We also report the identification of a cell type which differentiates in the polyp without passing through a post-metamorphic S-phase. The cell type appears to be neural in origin, based upon the identification of a neuropeptide of the FMRFamide type.     

Binding of azide and thiocyanate ligands to copper(II) model complexes

Summary Binding of azide to a series of copper(II) complexes has been investigated by absorption, CD and EPR spectroscopy. Axial binding of azide to Cu(II) can be differentiated from equatorial binding through the lower intensity and lack of optical activity of the LMCT band. The affinity of azide for Cu(II) increases with the overall positive charge of the complex. The preliminary data on thiocyanate binding to Cu(II) seem to agree with the trends observed for the corresponding azide adducts.     

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.     

A possible contribution by glial cells to neuronal energy production enzyme-histochemical studies in the developing rat cerebellum

Summary Recent reports have revealed that certain neurons do not survive in vitro in the presence of glucose, which is the primary substrate and exclusive source of energy in the brain. But these neurons can survive in the presence of low-molecular-weight agents such as pyruvate, which are supplied by glial cells (Selak et al. 1984). To test whether this result also holds true in vivo, we investigated the distribution of hexokinase, lipoic dehydrogenase, -hydroxybutyrate dehydrogenase, and glucose-6-phosphate dehydrogenase activities in the developing rat cerebellum. Hexokinase activity was relatively higher in glial cells than in neurons. After postnatal day 8, the activity of hexokinase could hardly be detected in Purkinje cells, whereas it was highest in Bergmann glial cells. Purkinje cells were the only type of neuron with high levels of lipoic dehydrogenase at all ages tested. -Hydroxybutylate dehydrogenase activity was also high in Purkinje cells, especially in those from young rats. Relatively high glucose-6-phosphate dehydrogenase activity was demonstrated in basket and stellate cells from adult brain. Thus, it appears that, in vivo, certain neurons utilize relatively little glucose, and it is indeed possible that glial cells may supply some substance(s) other than glucose, for example pyruvate, as the primary source of energy.