Isoenzyme Activities during the Early Stages of Allium Radicle Germination

As a continuation of a study of the molecular events surrounding the establishment of a mature root apex, changes in the pattern and specific activity of the isoenzymes of aminopeptidase (AmP), esterase (EST), glucose-6-phosphate de-hydrogenase (G6PD) and glutamic-oxaloacetic transaminase (GOT) were investigated during the early germinative growth of the Allium radicle tip. Polyacrylamide disc gel electrophoresis revealed a total of 9 EST fractions. A shift in pattern and reactivity characteristic of pre-emergence to those characteristic of post-emergence was demonstrated. G6PD electropherograms displayed 5 isoenzyme bands. Relative specific activity analyses indicated that this enzyme may be more important during the very early stages of germination. Only quantitative differences were noted in the 3 AmP isoenzymes detected. The progressive increase in AmP isoenzyme activity during the period of maximal protein body degradation suggested a role for these exopeptidases in the degradation of reserve proteins. The 3 isoenzyme bands of GOT exhibited only quantitative changes during germination. Parallel increases in the quantity of soluble protein and GOT specific activity suggest that this isoenzyme is involved in the onion's nitrogen metabolism during germination.     

An extra species of lysine transfer ribonucleic acid in polyoma virus-transformed cells in tissue culture

Isoaccepting tRNAs from various mouse cells were fractionated on columns of benzoylated DEAE cellulose. Lysine tRNA from mouse embryo, adult mouse liver and kidney, primary mouse embryo cells in tissue culture, and an established tissue culture line of mouse fibroblasts (3T3) has two peaks of isoaccepting tRNA; lysine tRNA from two established lines of polyoma virus-transformed cells contains an additional peak of lysine tRNA. The extra peak in transformed cells comprises about 25% of the acceptor capacity for lysine. It is stable to denaturation and renaturation and can be chromatographed, stripped of lysine, recharged, and rechromatographed. The extra peak is present in tRNA from transformed cells and absent in tRNA from normal cells regardless of whether the lysyl-tRNA ligase used for aminoacylation is from normal or transformed cells. Isoaccepting tRNAs for arginine, leucine, serine, valine, histidine, and tyrosine reveal similar profiles for the various tRNAs from normal and transformed cells.     

Polypeptide maps of cells infected with murine type C leukemia or sarcoma oncovirus

The polypeptide composition of murine fibroblast cells and the effect of infection by RNA sarcoma and leukemia viruses were analyzed by two-dimensional gel electrophoresis and tryptic peptide mapping. The polypeptide maps of NIH Swiss mouse embryo fibroblasts (NIH/3T3) and BALB/c mouse embryo fibroblasts (BALB/3T3) were very similar except for two major polypeptides of about 65,000 and 75,000 daltons which were not detected in BALB/3T3 cells. NIH/3T3 cells infected with either Rauscher or Gross oncoviruses and outbred Swiss mouse embryo fibroblasts (3T3 FL) showed two major polypeptrides of about 73,000 and 80,000 daltons not found in uninfected NIH/3T3 cells. The 3T3 FL cells, although uninfected, were also found to contain a high concentration of envelope glycoprotein of an endogenous oncovirus. 3T3 FL cells transformed by Moloney sarcoma virus showed changes in many polypeptides, including several major components: the disappearance or modification of a component of 60,000 daltons, an increased concentration and shift in pl of a glycoprotein of 48,000 daltons, and the apparent loss of several smaller polypeptides. None of the major changes of the transformed cells were associated with cell surface proteins labeled by lactoperoxidase-catalyzed iodination.     

Opposite and selective effects of epidermal growth factor and human platelet transforming growth factor-beta on the production of secreted proteins by murine 3T3 cells and human fibroblasts

Growth regulators such as epidermal growth factor (EGF) and type beta transforming growth factor (TGF-beta) regulate the synthesis and secretion of certain proteins by cells in culture. The secretion pattern of each cell line and the effect of growth regulators on the secretion pattern are unique. EGF increased the secreted and intracellular levels of mitogen-regulated protein (MRP) and major excreted protein (MEP) by Swiss 3T3 cells. MRP is related by sequence to prolactin. MEP is a thiol protease located intracellularly in the lysosomes. EGF also selectively induced a 52,000-dalton mitogen-induced protein (MIP 52) secreted by human fibroblasts. Two types of TGF-betas were tested for their effects on the expression of secreted proteins in mouse and human fibroblasts: TGF-beta from human platelets and a growth inhibitor (GI/TGF-beta) secreted by BSC-1 cells. Each selectively decreased the levels of the two secreted proteins induced by growth factors in mouse embryo 3T3 cells and one secreted protein induced by growth factors in human fibroblasts. Platelet TGF-beta and GI/TGF-beta also induced one 48,000-dalton protein secreted by human fibroblasts. Synthesis of DNA and the incorporation of [35S]methionine into total protein in Swiss 3T3 cells were not affected by platelet TGF-beta or GI/TGF-beta. Thus, the inhibitory effect of platelet TGF-beta on the synthesis and secretion of these three proteins is due to a specific effect of platelet TGF-beta on the regulation of MRP and MEP that does not interfere with the ability of EGF to stimulate DNA or protein synthesis.     

Swiss 3T3 mouse embryo fibroblasts transfected with a human prepro-GRP gene synthesize and secrete pro-GRP rather than GRP

A prepro-gastrin-releasing peptide (GRP) gene was introduced into Swiss 3T3 mouse embryo fibroblasts by DNA transfection in an attempt to establish autocrine growth stimulation. Clonal transfectants expressed varying amounts of GRP encoding mRNA. They synthesized and secreted a ～ 15-kd pro-GRP hormone but not fully processed 2.8-kd GRP. Accordingly, no changes in growth properties were associated with GRP gene expression. We postulate that Swiss 3T3 fibroblasts lack the enzymes necessary to process significantly pro-GRP into biologically active peptides and that this deficiency may be responsible for the failure to establish autocrine growth stimulation in the transfected cells.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.     

Serum rapidly mobilizes calcium from an intracellular pool in quiescent fibroblastic cells

Addition of dialysed fetal bovine serum to quiescent cultures of Swiss 3T3 cells loaded with 45Ca2+ causes a very rapid increase in the rate of 45Ca2+ efflux from an intracellular pool. Exposure to serum for 2 min leads to a fall of 0.59 nmol Ca2+/mg protein in the intracellular Ca2+ content of the cells. Inhibitors of mitochondrial function prevent the stimulation of 45Ca2+ efflux by serum. The stimulation of 45Ca2+ efflux by serum is also observed in quiescent cultures of Rat-1, Swiss 3T6 and BHK cells and in secondary cultures of whole mouse embryo fibroblasts.     

An acidic esterase as a biochemical marker for somatic embryogenesis in orchardgrass (Dactylis glomerata L.) suspension cultures

 The isoenzyme pattern of esterases (EC 3.1.1.2) secreted into the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension cultures during defined stages of somatic embryogenesis was compared with that of non-embryogenic suspension cultures during unorganised cell proliferation. Isoelectric focusing revealed the presence of 7–14 predominantly acidic isoforms. Comparison with the corresponding cell-wall isoenzyme pattern showed minor, mainly quantitative differences. The pattern of intracellular soluble esterases did not change markedly during somatic embryo development. A unique esterase whose migration in two-dimensional gel electrophoresis corresponds to an apparent molecular mass of 36 kDa and pI=3.8 was detected only in embryogenic cultures at very early stages of development. Since this isoform appeared long before morphological changes had taken place, it could possibly be used as a biochemical marker for embryogenic potential in D. glomerata L. suspension cultures. Received: 6 June 2000 / Revision received: 17 July 2000 / Accepted: 17 July 2000     

Diffusible factor(s) controlling density inhibition of 3T3 cell growth: a new approach

In 3T3 Swiss mouse fibroblasts, incorporation of phosphate into cells and phosphorylation of small organic compounds were increased by shaking dense cultures. This response was not obtained with SV40 transformed Swiss 3T3 cells (SV-3T3). It appeared likely that these results could be accounted for by an inhibitor released from 3T3 cells but not from SV-3T3 cells. Our new method of co-incubation of sparse and dense cultures allowed us to demonstrate inhibition of growth and phosphate metabolism in sparse 3T3 cultures which were shaken in the presence of dense cultures. The inhibition was much less when the cultures were co-cultivated but not shaken. The inhibition of phosphate incorporation in acid-soluble and acid-insoluble fractions of sparse cultures was observed as early as 20 minutes of co-incubation in the presence of dense cultures, so this inhibition is not the result of depletion of growth factors in the medium. Our experiments suggest that an inhibitor(s) was released from dense cultures of 3T3 cells.     

Stimulation of sugar uptake in cultured fibroblasts by epidermal growth factor (EGF) and EGF-binding arginine esterase.

Mouse epidermal growth factor causes a rapid increase in 2-deoxyglucose uptake in stationary phase mouse (3T3) cells or human fibroblasts. Maximum effect is approximately two fold over control levels for 3T3 cells and about 50% over controls for human fibroblasts. Maximum effect on 3T3 cells is seen about two hours after addition of 10 ng/ml EGF to the culture medium. Stimulation is easily measureable within the first fifteen minutes after addition of the hormone and may be detected at hormone concentrations as low as 0.1 ng/ml. The EGF-binding arginine esterase found associated with EGF in the mouse submaxillary gland causes an enhancement of the EGF effect. In serum-free medium, the EGF effect is still readily observed, but no enhancement by the esterase is seen. SV40 virus-transformed 3T3 cells show no effect on deoxyglucose uptake after addition of 10 ng/ml EGF to the culture medium, but a response may be demonstrated after these cells are incubated for 12 hours or more in serumless medium. EFG stimulates transport of 3-O-methylglucose in stationary phase 3T3 and human fibroblasts but no EGF stimulation of alpha-amino-isobutyrate uptake in 3T3 cells is seen under conditions is reproted to inhibit intracellular degradation of human EGF by human fibroblasts, does not diminish the EGF effect on deoxyglucose uptake in human fibroblasts.     

Dihydrocytochalasin B disorganizes actin cytoarchitecture and inhibits initiation of DNA synthesis in 3T3 cells

Dihydrocytochalasin B (H2CB) disrupts the actin structure of Swiss/3T3 mouse fibroblasts and inhibits the ability of serum growth factors to stimulate DNA synthesis in quiescent cultures. Low doses of H2CB (2-10 X 10(-7) M) added to serum-arrested cells reversibly block initiation of DNA synthesis by serum; by epidermal growth factor and insulin; or by epidermal growth factor, fibroblast growth factor and insulin. H2CB is effective only when added to cells within 8-10 hr after stimulation. Low doses of H2CB cause cell rounding and a loss of actin microfilament bundles, but they do not interfere with glucose or thymidine transport. These results suggest that stimulation of 3T3 cells involves at least one obligatory actin-mediated step. Transformed cells appear to obviate this step, for H2CB does not inhibit the entry into S phase of SV40-transformed or Moloney murine sarcoma virus-transformed 3T3 cells synchronized by mitotic shake-off.     

Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.     

Murine Sarcoma and Leukemia Viruses: Assay Using Clonal Lines of Contact-Inhibited Mouse Cells

A continuous cell line of highly contact-inhibited cells (NIH/3T3) has been developed from NIH Swiss mouse embryo cultures. Its growth properties are similar to those of 3T3 and BALB/3T3. Although 3T3 is relatively insensitive to focus formation by murine sarcoma viruses, cloned lines of both NIH/3T3 and BALB/3T3 have been isolated that are highly sensitive to sarcoma virus focus formation and leukemia virus growth. The sensitivity and specificity are comparable to those found with primary embryo cells. MSV-transformed lines of NIH/3T3 have been obtained.     

Absorption and metabolism of vitamin K in Swiss 3T3 mouse fibroblasts--a model system for study of vitamin K absorption and metabolism

The vitamin K cycle previously described in liver has been demonstrated in Swiss 3T3 mouse fibroblasts. Vitamin K epoxide and gamma-carboxyglutamic acid were isolated from the cells and chemically characterized. Menaquinone (MK4) is also metabolized to its epoxide and vitamin K epoxide is reduced to vitamin K in these cells. Thus Swiss 3T3 mouse fibroblasts provide a useful model system for the study of vitamin K metabolism. Possible functions of the vitamin K-dependent protein(s) in fibroblasts are discussed.     

Biosynthesis in vitro of mono- and di-sialosylgangliosides from gangliotetraosylceramide by cultured cell lines and young rat brain. Structure of the products, and activity and specificity of sialosyltransferase

Incubations in vitro of GA1, labeled with 3H in the terminal D-galactopyranosyl group, with nonradioactive CMP-NeuNAc in the presence of homogenates of C21 rat brain glial cells, NIE mouse neuroblastoma cells, 3T3 mouse fibroblasts, SV 40-transformed 3T3 cells, chick embryo fibroblasts, Rous sarcoma virus-transformed chick embryo fibroblasts, and 9-day old rat brain resulted in all cases in the formation in high yield of GM1b, in which the neuraminidase-labile NeuNAc group is linked at O-3 of the terminal D-galactosyl residue, as shown by permethylation studies. No trace of the naturally occurring neuraminidase-stable GM1a was detected in any case. In addition, with NIE cells, and normal and RSV-transformed chick embryo fibroblasts, a disialosylganglioside (GD1) differing from GD1a and GD1b, and bearing only one substituent at O-3 of the terminal D-galactopyranosyl residue was formed. It was also biosynthesized from GM1b and CMP-NeuNAc by NIE and chick embryo cells but not by C21 cells, or rat brain. However, C21 cells and rat brain were capable of synthesizing GD1a from GM1a. Periodate oxidation degraded both NeuNAc groups in GD1 to a 7-carbon fragm:nt, indicating lack of substitution at O-8. GM1b could not be detected as a natural product in rat brain.     

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.     

Properties of the testicular lactate dehydrogenase isoenzyme.

1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of "heavy mitochondria" and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.     

Cyclic AMP does not mediate inhibition of DNA synthesis by interferon in mouse Swiss 3T3 cells

The purpose of this study was to determine whether cyclic AMP (cAMP) plays any direct or indirect role in the antiproliferative effect of mouse L-cell interferon in Swiss 3T3 cells. Firstly, we found that interferon did not affect intracellular levels of cAMP in these cells in the absence or the presence of cAMP-elevating agents. Secondly, we examined the effect of interferon on the stimulation of DNA synthesis of quiescent 3T3 cells by a range of cyclic AMP-elevating agents, including cholera toxin, cAMP derivatives, and prostaglandin E, added in the presence of insulin or vasopressin. Interferon inhibited cyclic AMP-stimulated DNA synthesis as measured by incorporation of radioactive thymidine into acid-insoluble material and autoradiographic analysis of the fraction of labelled cells. Dose-response curves and kinetics of inhibition were identical to those obtained in cultures stimulated by combinations of growth factors that do not increase the intracellular level of cAMP. The inhibition by interferon of cAMP-stimulated DNA synthesis was also observed in secondary cultures of mouse embryo fibroblasts, where cAMP-elevating agents provide a mitogenic signal in the absence of other added growth factors. These results show that the inhibitory effect of interferon on DNA synthesis in Swiss 3T3 cells is not mediated by cyclic AMP.