Sensitivity of the cell cycle to TGFβ1 does not correlate with transformation of a rat liver epithelial cell line

The effects of TGF1 on cell cycle events in a rat liver derived epithelial cell line (BL9) and in two in vitro transformants of this line were studied by flow cytometry. Using either ethidium bromide staining or the incorporation of bromodeoxyuridine to evaluate DNA synthesis it was shown that TGF1 prevented the entry of G0/G1 phase BL9 cells into S phase. TGF1 did not exert its inhibitory effect(s) on DNA synthesis by the modulation of early events in the cell cycle. The tumorigenic transformed BL9 cell lines gave contrasting responses to the effects of TGF1. DNA synthesis in a BL9 cell line derived by transfection with an active N-ras oncogene was unaffected by TFG1 and thus appeared refractory to its growth controlling effects. On the other hand cells from a BL9 cell line derived by in vitro transformation with activated aflatoxin B₁ retained their sensitivity to the effects of TGF1. Thus the loss of the inhibitory effect of TGF1 on DNA synthesis is not obligatory for the malignant transformation of rat liver epithelial cells.Abbreviations TGF1 transforming growth factor 1 - BSA bovine serum albumin - FBS foetal bovine serum - BrdUrd bromodeoxyuridine - PI propidium iodide - PBS phosphate buffered saline     

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.     

Growth and activities of enzymes of primary metabolism in batch cultures of Catharanthus roseus cell suspension under different pCO2 conditions

In vitro enzyme activities of glycolysis, pentose-phosphate pathway and dark CO₂ fixation were assayed in batch cultures of heterotrophic Catharanthus roseus cells under various gassing rates and partial pressures of carbon dioxide. Detrimental effects of low pCO₂ culture conditions on the growth characteristics could be linked to marked changes in levels of enzymes of primary metabolism during growth. The enzyme levels observed during the early stages of growth were found to be more stable when a constant pCO₂ (20 mbar) was maintained and enabled exponential growth to be reached more rapidly.The importance of carbon dioxide as a conditioning factor of the culture medium is discussed.     

Comparative effects of four surfactants on growth,contraction and adhesion of cultured human fibroblasts

A range of surfactants, including the anionic sodium dodecyl sulfate, the cationic cetyltrimethylammonium bromide and the nonionics octylphenoxy polyethoxyethanol (Triton X100) and polyoxyethylene 20 sorbitan monoleate (Tween 80) was studied for effects on proliferation, contractibility and attachment of cultured human fibroblasts. Only ionic surfactants exhibited a stimulatory effect on fibroblast proliferation, whereas all the surfactants tested increased the contraction of collagen gels containing fibroblasts, with the greatest effect from the non-ionic surfactants. This activity was not correlated with an increase of cell population or cell attachment within the collagenous matrix. The activity of the surfactants was seen only at levels close to their LD₅₀ values and in a narrow range of concentrations. Thus, we consider that they are the result of the so-called hormesis phenomenon.Abbreviations CTAB cetyltrimethyl-ammonium bromide - FCS fetal calf serum - LD₁₀₀ dose lethal to 100% of exposed - MCD maximal contraction dose - PDL population doubling level - SDS sodium dodecyl sulfate     

A free-radical hypothesis for the instability and evolution of genotype and phenotypein vitro

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IAl or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P4501Al and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P4501A1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.Some of the data presented here were taken from a thesis submitted by D.K.H. in partial fulfillment of the requirements for the Ph.D. degree in the University of Texas Graduate School of Biomedical Sciences.     

Human and mouse S-protein mRNA detected in Northern blot experiments and evidence for the gene encoding S-protein in mammals by Southern blot analysis

Summary Human S-protein is a serum glycoprotein that binds and inhibits the activated complement complex, mediates coagulation through interaction with antithrombin III and plasminogen activator inhibitor I, and also functions as a cell adhesion protein through interactions with extracellular matrix and cell plasma membranes. A full length cDNA clone for human S-protein was isolated from a lambda gt11 cDNA library of mRNA from the HepG2 hepatocellular carcinoma cell line using mixed oligonucleotide sequences predicted from the amino-terminal amino acid sequence of human S-protein. The cDNA clone in lambda was subcloned into pUC18 for Southern and Northern blot experiments. Hybridization with radiolabeled human S-protein cDNA revealed a single copy gene encoding S-protein in human and mouse genomic DNA. In addition, the S-protein gene was detected in monkey, rat, dog, cow and rabbit genomic DNA. A 1.7 Kb mRNA for S-protein was detected in RNA from human liver and from the PLC/PRF5 human hepatoma cell line. No S-protein mRNA was detected in mRNA from human lung, placenta, or leukocytes or in total RNA from cultured human embryonal rhabdomyosarcoma (RD cell line) or cultured human fibroblasts from embryonic lung (IMR90 cell line) and neonatal foreskin. A 1.6 Kb mRNA for S-protein was detected in mRNA from mouse liver and brain. No S-protein mRNA was detected in mRNA from mouse skeletal muscle, kidney, heart or testis.     

An interplexiform cell in the goldfish retina: light-evked response pattern and intracellular staining with horseradish peroxidase

Summary The light-evoked response pattern and morphology of one interplexiform cell were studied in the goldfish retina by intracellular recording and staining. The membrane potential of the cell spontaneously oscillated in the dark. In response to a brief light stimulus, the membrane potential initially gave a slow transient depolarization. During maintained light, the oscillations showed a tendency to be suppressed; the response of the cell to the offset of the stimulus was not so prominent. The perikaryon of the interplexiform cell was positioned at the proximal boundary of the inner nuclear layer. The cell had two broad layers of dendrites; one was diffuse in the inner plexiform layer, the other was more sparse in the outer plexiform layer. The morphological and electrophysiological characteristics of the cell are discussed in relation to dopaminergic interplexiform cells and the light-evoked release pattern of dopamine in the teleost retina.