MDR1, cholesterol certification and cell growth: a comparative study in normal and multidrug-resistant KB cell lines

The product of the MDR1 gene (P-gp) has been implicated in the transport of cholesterol from plasma membrane to endoplasmic reticulum for esterification. In previous studies on leukemia cell lines, we suggested that cholesterol esterification may regulate the rate of cell growth and that the MDR1 gene might be involved in this process by modulating intracellular cholesterol esters levels. To further investigate this matter, the rate of cell growth, cholesterol metabolism, expression of the MDR1 gene, and P-gp activity were compared in KB cell lines displaying differences in expression and function of P-gp (drug-sensitive phenotype versus MDR phenotype). The rate of cell growth correlated with cholesterol esterification in all KB cell lines, whereas the over-expression of MDR1 observed in the MDR cell lines was not always associated with an increased capacity of cells to esterify cholesterol. Two known inhibitors of P-gp activity, progesterone and verapamil, strongly inhibited both cholesterol esterification and cell proliferation in all KB cell lines, but they affected intracellular accumulation of labeled vinblastine only in MDR cell lines. These results further support a role for cholesterol esters in the regulation of cell growth and suggest that the P-gp expressed in MDR KB cells is not involved in the general process leading to cholesterol esterification. Received 14 February 2000; received after revision 10 April 2000; accepted 8 May 2000     

Protein O-GlcNAcylation regulates Drosophila growth through the insulin signaling pathway

Modification of nuclear and cytosolic proteins by O-linked N-acetylglucosamine (O-GlcNAcylation) is ubiquitous in cells. The in vivo function of the protein O-GlcNAcylation, however, is not well understood. Here, we manipulated the cellular O-GlcNAcylation level in Drosophila and found that it promotes developmental growth by enhancing insulin signaling. This increase in growth is due mainly to cell growth and not to cell proliferation. Our data suggest that the increase in the insulin signaling activity is mediated, at least in part, through O-GlcNAcylation of Akt. These results indicate that O-GlcNAcylation is one of the crucial mechanisms involved in control of insulin signaling during Drosophila development.     

Mean cytoplasmic protein concentration of host erythrocytes and the reticulocyte response inPlasmodium berghei infected mice

Summary the decreasing mean cytoplasmic protein concentration (MPC) ofP. berghei host cells is paralled by an increasing parasitemia and percent reticulocyte response. The reticulocyte response parallels the percent parasitemia except during a period of accelerated reticulocytosis noted during the midpoint of the infection at which time the percent reticulocytes increases at a rate more than double the rate of increase of percent parasitemia. Although the reticulocyte population and the host cell population are usually equivalent, the disparity noted suggests the existence of unique characteristics in the reticulocyte response ofP. berghei infected mice.     

EGF-induced programmed cell death of human mammary carcinoma MDA-MB-468 cells is preceded by activation of AP-1

MDA-MB-468 is a human mammary adenocarcinoma cell line that overexpresses the epidermal growth factor (EGF) receptor and undergoes programmed cell death (apoptosis) in response to EGF treatment. Programmed cell death was shown to be greatly enhanced when cells were growth-arrested prior to EGF treatment. Apoptosis was characterized by an initial rounding up and detachment of the cells from their substrate starting about 12 h after EGF treatment, followed by chromatin condensation, nuclear fragmentation and oligonucleosomal fragmentation of the DNA at about 24 to 48 h. Cell death was dependent on de novo protein synthesis. We found a rapid induction of c-fos, c-jun and junB at the mRNA level after about 30 min of EGF treatment and a more delayed upregulation of fosB and fra-1. The junD gene was expressed in the absence of EGF, and it was moderately induced within 30 min of growth factor addition. The increase of the different fos and jun mRNAs were paralleled by an increase of activator protein-1 (AP-1) DNA binding activity. A characterization of the AP-1 complex revealed similar levels of several Fos and Jun proteins. Based on the kinetics of AP-1 accumulation and cell death, it seems likely that AP-1 contributes to the apoptotic cell death of EGF receptor-overexpressing MDA-MB-468 cells. Received 21 July 1997; received after revision 6 November 1997; accepted 6 November 1997     

Thrombospondin co-localises with TGFβ and IGF-I in the extracellular matrix of human osteoblast-like cells and is modulated by 17β estradiol

Thrombospondin (TSP) is a multifunctional glycoprotein which is synthesised by several cell types including osteoblasts, and incorporated into the extracellular matrix (ECM) of these cells. The function and regulation of TSP in bone is not clear. In this study, using a long term culture model of human osteoblast-like cells, we examined the distribution of TSP in the ECM and its modulation by added estradiol. In this model the osteoblast-like cells form a regular multilayer which continues to increase in depth up to 50 days post confluence. In the ECM of these cultures and in 19-week fetal bone, the bone markers osteocalcin and alkaline phosphatase were diffusely distributed in the matrix. In contrast, labelling for TSP was concentrated, confined to the banded collagen and its immediately adjacent ECM. This pattern of labelling resembled that of the growth factors transforming growth factor-I (TGF), and insulin-like growth factor-I (IGF-I), with which TSP label co-localised. Labelling intensities were comparable between fetal bone and the in vitro material for TSP, TGF and IGF-I. TSP label was present by 10 days post confluence, reached a maximum by 20 days, and declined slowly thereafter, a time course which was similar to that of IGF-I. Incubation of osteoblast-like cell cultures with 17 estradiol resulted in an increase in multilayer depth and a maximal 3-fold increase in TSP labeling at 30 days as well as approximately 2-fold increases for TGF and IGF-I. The dose-response relationship for these responses to estradiol treatment was biphasic with maximal increases at 10^–10 M–10^–11 M of added estradiol. Treatment with 17 estradiol produced labelling intensities that were not significantly different from controls. Studies with other cell types have suggested that TSP may be involved in modulation of growth factor activity. The similarities between TSP, TGF and IGF-I, in terms of their distribution and regulation by 17 estradiol treatment, may indicate a role for TSP in modulating bone cell proliferation and function through interaction with local growth factors.     

Ethylene production and cell wall formation in mesophyll protoplasts

Summary The regulation of ethylene synthesis, in relation to the presence of a new cell wall, has been investigated forNicotiana sylvestris leaf protoplasts. It is clear that the production of ethylene is not controlled by the new wall, which has no action on the ethylene formation. The addition of dichlorobenzonitrile to cultivated protoplasts causes the inhibition of wall formation, without any other apparent deleterious effects.     

Zur leukotaktischen Wirkung von Aminosäuren in Liquor cerebrospinalis

Summary Intercisternal injections of-aminobutyric acid,l-asparagin,l-glutamine,l-leucine andl-norleucine exert a strong leukotactic action and in dogs result after a short time in a maximum cell increase of 76,000/3 cells.     

Life behind cell walls: paradigm lost, paradigm regained

This review of the living cell wall [1] and its protein components is in two parts. The first is anecdotal. A personal account spanning over 40 years research may perhaps be an antidote to one stereotypical view of scientists as detached and humorless. The second part deals with the meaning of function, particularly as it applies to hydroxyproline-rich glycoproteins. Function is a difficult word to define objectively. However, with help from such luminaries as Humpty Dumpty: "A word means what I want it to mean, neither more nor less," and Wittgenstein: "Giving examples of usage ... is the only way to talk about meaning," it is possible to construct a ziggurat representing increasingly complex levels of organization from molecular structure to ecology. Forty years ago I suggested that hydroxyproline-rich structural proteins played a key role in cell wall functioning. But because the bulk of the wall is carbohydrate, there has been an understandable resistance to paradigm change. Expansins, paradoxically, contribute greatly to this resistance because their modus operandi as cell-wall-loosening proteins is based on the idea that they break hydrogen bonds between polysaccharide chains allowing slippage. However, this view is not consistent with the recent discovery [Grobe et al. (1999) Eur. J. Biochem 263: 33-40] that β-expansins may be proteases, as it implies that the extensin network is not a straightjacket but a substrate for expansin in muro. Such a direct role for extensins in both negative and positive regulation of cell expansion and elongation may constitute a major morphogenetic mechanism operating at all levels of plant growth and development.     

Dynamics of increase in muscle fibers in fishes in relation to size and growth

Summary Dynamics of increase of white myotomal muscle fibers of four species of freshwater teleosts (Salmo gairdneri, Pimephales notatus, Esox masquinongy andE. americanus vermiculatus) from three families (Salmonidae, Cyprinidae, Esocidae) representing a variety of maximum attainable sizes and growth rates, have been investigated. There are at least three major differences in these dynamics, and there appears to be an association between the ability of a fish species to attain large size (and grow fast) and its ability to recruit new fibers into this predominant tissue of the myotomal mass.     

The growth of Atlantic salmon (Salmo salar L.) myosatellite cells in culture at two different temperatures

Temperature is known to affect fish growth, and in Atlantic salmon there is an influence on muscle cellularity. Primary muscle cell culture makes it possible to investigate direct effects of temperature on myogenic cells. Salmon myosatellite cells were cultured for the first time in this study. The cells were cultured at either 5°C or 11°C. Increased temperature led to an increase in differentiation rate and especially hypertrophic growth (Q₁₀=4.0). No nuclear proliferation was evident in the satellite cell population isolated at either temperature. This may be due to the presence of different subpopulations of myogenic cells at different developmental ages or the presence of indirect factors in vivo.     

Growth and the average duration of larval life in the southern hemisphere lamprey,Geotria australis Gray

Summary The average duration of larval life in the anadromous lamprey,Geotria australis (the sole representative of the Geotriidae) is estimated as 41/4 years. Compared with other lampreys, the ammocoetes ofG. australis have a slow growth rate, increase in length during the year preceding metamorphosis and typically enter metamorphosis at a small mean length (<100 mm) and weight (<1.2 g).     

Circadian rhythm in the subcommissural organ of the frog,Rana arvalis Nilsson,under natural conditions

Summary A circadian activity of SCO ependymal cells, judged by changes in the nuclear volume, has been found in juvenile frogs (Rana arvalis) under natural summer conditions. The nuclear volume reaches its maximum at 12.00 h and a minimum at 24.00 h. A significant increase in activity occurs between 06.00 and 09.00 h and a gradual decrease is observed from 12.00 to 24.00 h.     

PSF and CMF,autocrine factors that regulate gene expression during growth and early development ofDictyostelium

Throughout growth and development,Dictyostelium cells secrete autocrine factors that accumulate in proportion to cell density. At sufficient concentration, these factors cause changes in gene expression. VegetativeDictyostelium cells continuously secrete prestarvation factor (PSF). The bacteria upon which the cells feed inhibit their response to PSF, allowing the cells to monitor their own density in relation to that of their food supply. At high PSF/bacteria ratios, which occur during late exponential growth, PSF induces the expression of several genes whose products are needed for cell aggregation. When the food supply has been depleted, PSF production declines, and a second density-sensing pathway is activated. Starving cells secrete conditioned medium factor (CMF), a glycoprotein of Mr 80 kDa that is essential for the development of differentiated cell types. Antisense mutagenesis has shown that cells lacking CMF cannot aggregate, and preliminary data suggest that CMF regulates cAMP signal transduction. Calculations indicate that a mechanism of simultaneously secreting and recognizing a signal molecule, as used byDictyostelium to monitor cell density, could also be used to determine the total number of cells in a tissue.     

Analysis of <Emphasis Type="Italic">Dictyostelium</Emphasis> TACC reveals differential interactions with CP224 and unusual dynamics of <Emphasis Type="Italic">Dictyostelium</Emphasis> microtubules

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-α-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.     

Relationship of prodigiosin condensing enzyme activity to the biosynthesis of prodigiosin and its precursors inSerratia marcescens

Summary Prodigiosin condensing enzyme (PCE) activities were present inSerratia marcescens wild type 08, mutants OF, WF and 9-3-3. Their specific activities exhibited different maxima and at different times during the late log phase or the early stationary phase of cell growth. The levels of prodigiosin and its precursors also showed a significant increase at this period. The results support that prodigiosin and/or its precursors are secondary metabolites. The ubiquity of the PCE activity in mutants deficient in prodigiosin biosynthesis suggest that this particular enzyme may also be present in non-pigmented clinical isolates.     

Inverse regulation of spore germination and growth by cyclic AMP inStreptomyces hygroscopicus

Summary The cyclic AMP level in germinating spores ofStreptomyces hygroscopicus rises to a maximum at outgrowth of germ tubes. Exogenous cyclic AMP results in an inverse effect on germination speed and growth.     

Purification and properties of a heat-resistant exotoxin produced byMacrophomina phaseolina (Tassi) Goid in culture

Summary A partially purified preparation of a water-soluble, heat-resistant, nonspecific exotoxin produced by a strain ofMacrophomina phaseolina, isolated fromPhaseolus mungo L. could reduce Cu⁺⁺ ions and produced a red colour with 2,4-dinitrophenyl hydrazine reagent. It caused inhibition of seed germination, wilting of cut seedlings, stunted growth of young seedlings and loss of permeability of the cell membrane. Seedlings ofP. mungo, grown in presence of the toxin showed a slight increase in the contents of protein and total RNA over control, but a significant increase in the specific activities of F-1, 6-BP aldolase and G-6-P isomerase.     

A new cell line (XTY) from a tumor ofXenopus laevis

A new cell line (XTY) was derived from a tumor of a femaleXenopus laevis. This cell line has been proliferating in standard amphibian culture medium for more than 4 years (470 generations) and has a doubling time of 75.5 h at 25°C. The cells aggregate into large groups, and their stellate morphology and the expression of desmin demonstrated by immunocytochemistry suggest that their origin is not epithelial.     

Sur l'action de l'acide 2,4-dichlorophénoxyacétique sur la croissance des racines deZea mays et dePisum cultivéesin vitro

Summary (1) A response ofZea mays andPisum roots cultured aseptically to 2,4-dichlorophenoxyacetic acid has been observed.Depending on the concentrationZea mays shows an increase of the growth rate (optimal concentration 10^–11 mol) which turns over to an inhibition (above 10^–7 mol). The curve is similar to that obtained by 3-indoleacetic acid, which proves the phytohormonal character of 2,4-D.(2) ThePisum root is more sensitive than theZea mays root. A concentration of 10^–7 molinhibitsPisum in a high degree, whileZea mays is no more inhibited, thus demonstrating the selective herbicidal action of 2,4-D against isolated roots of Dicotyledons culturedin vitro.     

Effects of weak electromagnetic fields onPhysarum polycephalum: Mitotic delay in heterokaryons and decreased respiration

Summary Continuous exposure ofPhysarum polycephalum to a 75 Hz, 2.0 G, and 0.7 V/m electromagnetic field results in a depressed respiration rate and a lengthening of the mitotic cell cycle. If unexposedPhysarum are mixed with exposedPhysarum the onset of synchronous mitosis in the mixed culture is delayed, occurring at a time between those of the 2 parent cultures.Acknowledgment. This work was supported by the Naval Electronics Systems Command through an Office of Naval Research Contract and a grant from Parkside's Center for the Application of Computers.