Search PubMed⌕ Search

Biomedical subjects

J J Lucas

Publications and source records attributed to J J Lucas.

At least 91 records · Page 5Linked to original sources
PubMed1983

Programmed macromolecular synthesis in regenerating karyoplasts.

Conditions for the preparation, purification, and maintenance of karyoplasts which could regenerate to reform whole viable cells were defined. Results of biochemical analyses of such karyoplasts at various times during regeneration indicated that a reproducible biosynthetic program was followed. Thus, an examination of the polypeptides made during regeneration by two-dimensional gel electrophoresis showed that the pattern of radiolabeled polypeptides synthesized at each time studied was specific and was significantly different from that observed at other times during regeneration. Polypeptides associated with three major cellular fractions--nuclear, cytoskeletal-microtrabecular, and soluble--were among the most dramatically regulated molecules. Other polypeptides, such as the major components of microfilaments and intermediate filaments, were synthesized at relatively constant rates and were assembled into structures throughout regeneration. Likewise, microtubules appeared to be reformed throughout regeneration, even in the absence of identifiable centriole-associated organizing centers. Finally, analysis of DNA synthesis by autoradiography showed that, even when prepared from whole cells synchronized at the G1/S interface, karyoplasts could not begin making DNA until they had regenerated an almost complete complement of cytoplasm.

Animals↗
PubMed1982

Estrogen-induced changes in chick oviduct membrane glycoproteins.

Membrane protein and glycoprotein changes during estrogen-induced differentiation of the chick oviduct were studied. Membrane preparations from chicks treated with diethylstilbestrol for 3, 6, 10, and 20 days; from chicks treated with hormone for 10 days and then withdrawn for 2 days; and from mature hens were incubated with either GDP-[14C]Man or GDP-[3H]Man. Double label analysis of proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicates that labeling of proteins with apparent molecular weights of 91,000 and 57,000 is enhanced upon diethylstilbestrol treatment. The effect is reversed upon hormone withdrawal. Analysis of [14C]Man-proteins by SDS-PAGE and fluorography reveals at least 18 labeled proteins, confirms the double label gel results, and shows changes in three additional proteins. Estrogen-induced changes in membrane proteins and glycoproteins were also studied by Coomassie blue staining, periodic acid-Schiff staining, and autoradiography of 125I-concanavalin A binding following SDS-PAGE. Changes are observed in at least 9 Coomassie blue, 7 periodic acid-Schiff and 13 125I-concanavalin A bands. Some proteins are enhanced by estrogen treatment and are diminished upon withdrawal, whereas other proteins are diminished by estrogen treatment and are enhanced by withdrawal. Hen oviduct membrane preparations were labeled with combinations of UDP-GlcNAc, GDP-Man, and UDP-Glc. Tunicamycin inhibits incorporation of [3H]Man and [14C]GlcNAc into protein by 84% and 65%, respectively. SDS-PAGE reveals that transfer of GlcNAc to a 36,000 protein is the least sensitive to tunicamycin. SDS-PAGE of proteins labeled by incubation with GDP-[14C]Man and UDP-[3H]Glc indicate that the presence of Glc does not alter the typical Man-protein-labeling pattern and processing of Glc from individual acceptors uniform.

Acetylglucosamine↗
PubMed1981

Increased transfer of oligosaccharide from oligosaccharide pyrophosphoryl dolichol to protein acceptors upon estrogen-induced chick oviduct differentiation.

In an earlier report (Lucas, J. J., and Levin, E. (1977 J. Biol. Chem. 252, 4330-4336), we showed that immature chick oviduct membranes could not transfer [14C]mannose from GDP-[14C]Man to endogenous oligosaccharide pyrophosphoryl dolichol or protein. Estrogen treatment of chicks however, stimulated [14C]mannose transfer to those acceptors. In order to determine whether the oligosaccharide transfer responsible for N-glycosylation of oviduct proteins is present in immature oviduct membranes exogenous carboxymethylated alpha-lactalbumin and radiolabeled oligosaccharide-lipid were added to oviduct membranes. Because purity of the oligosaccharide-lipid is essential for consistent transfer of oligosaccharide to carboxymethylated alpha-lactalbumin a high pressure liquid chromatography procedure has been developed for oligosaccharide-lipid purification. The data obtained indicate that immature chick oviduct membranes do have an enzyme capable of oligosaccharide transfer and that the activity is enhanced 3- to 4-fold by estrogen treatment of chicks. A Glc-oligosaccharide is transferred approximately 3-fold more effectively than the nonglycosylated Man-oligosaccharide by membrane preparations from immature chicks, estrogen-treated chicks, and mature hens.

Animals↗
PubMed1981

Enhanced chick oviduct dolichol kinase activity during estrogen-induced differentiation.

Crude microsomal preparations from hen oviduct catalyze the transfer of [32P]phosphate from [gamma-32P]CTP or [gamma-32P]dCTP to endogenous dolichol, forming dolichyl [32P]monophosphate. The oviduct kinase activity assayed with [gamma-32P]CTP is stimulated by divalent cations and exogenous dolichol. The enzymatic formation of dolichyl [32P]monophosphate is inhibited by dCDP and CDP, but not CMP, ADP, GDP, or UDP. The hen oviduct kinase is inhibited 50% by the addition of 38 microM CDP, but 101 microM dCDP is required for 50% inhibition. The amount of dolichol kinase activity in chick oviduct microsomes increases 3.7-fold within 10 days of estrogen administration. The hormone-induced increase in kinase activity is also observed when membranes from untreated and estrogen-treated chicks are assayed in the presence of saturating levels of exogenous dolichol. The microsomal preparations from oviducts of untreated chicks and fully induced birds both exhibit an apparent Km value of 7.1 microM for CTP. An apparent Km of 14 microM has been determined for dCTP. Thus, the developmental change in dolichol kinase activity does not appear to be the result of a difference in the amount of available endogenous dolichol or an alteration in the reactive site for the nucleoside triphosphate substrate, but is probably due to an increased level of the enzyme.

Animals↗
PubMed1981

A staining procedure for identifying viable cell hybrids constructed by somatic cell fusion, cybridization, or nuclear transplantation.

A general procedure for identifying viable hybrid cells was developed. One cell type was labeled by a brief incubation in the Kodak laser dye rhodamine 123, which accumulates in the mitochondria; a second cell type was labeled by a brief incubation in the Hoechst fluorochrome 33258, which binds to chromatin. The substances which are eventually lost from the organelles, appeared to be nontoxic; the plating efficiencies of numerous cell lines tested was unaffected. Either whole cells or cytoplasts labeled with rhodomine 123 were fused, using inactivated Sendai virus, to whole cells or karyoplasts labeled with Hoechst 33258. When living cells were illuminated with ultraviolet light, individual whole cell hybrids, cybrids or cytoplasmic- nuclear hybrid cells could be rapidly identified by the appropriate staining pattern.

Animals↗
PubMed1981

Heterogeneous binding of high mobility group chromosomal proteins to nuclei.

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.

Animals↗
PubMed1981

Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed.

Animals↗
PubMed1980

A novel N-acetylglucosaminyl polyprene in hen oviduct.

Incubation of hen oviduct membranes with UDP-[14C]N-acetylglucosamine (GlcNAc) leads to the formation of four endogenously labeled lipids. One of these lipids has not previously been characterized. Based on its chromatographic behavior on SG-81 paper and DEAE-cellulose, it is a monophosphate diester. The oviduct lipid is extremely sensitive to mild acid hydrolysis and, based on a comparison with alpha-GlcNAc-phosphoryl-undecaprenol from Bacillus cereus, the oviduct lipid contains GlcNAc linked to a polyisoprene as the beta anomer, the polyisoprene containing an alpha-unsaturated isoprene unit. Molecular sieve chromatography indicates that the molecular weight of the lipid is on the order of mannosyl-phosphoryl-dolichol and larger than alpha-GlcNAc-phosphoryl-undecaprenol. Formation of the GlcNAc-lipid is insensitive to tunicamycin. Dolichyl phosphate stimulates formation of a GlcNAc-lipid with similar chromatographic properties.

Animals↗
PubMed1980

Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adiopose tissue.

Microsomal preparations from rat adipose tissue catalyse the transfer of [(14)C]mannose from GDP-[(14)C]mannose to an endogenous acceptor forming a [(14)C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate beta-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [(14)C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate. The kinetics of transfer of [(14)C]mannose from GDP-[(14)C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The K(m) and V(max.) values for transfer from GDP-mannose were virtually indistinguishable in the four preparations.In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [(14)C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose. Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.

Adipose Tissue↗
PubMed1980

Construction of viable mouse-human hybrid cells by nuclear transplantation.

Viable interspecies cytoplasmic-nuclear hybrid cells were constructed by fusion of karyoplasts prepared from the highly tumorigenic A9 mouse fibroblast cell line and cytoplasts prepared from the Detroit 532 normal human diploid cell strain. The identity of the hybrid cells was ascertained using a variety of morphological, immunological, and genetic criteria, including: nuclear pattern of staining with the fluorochrome Hoechst 33258, appearance of the actin-myosin containing cytoskeleton, presence of fibronectin, and resistance to azaguanine and diphtheria toxin. About 90% of the hybrid cells were viable, that is, capable of division. Changes in the morphology of the hybrid cells, apparently nuclear directed, were observed before cell division occurred. Using the techniques described here, large numbers of interspecies hybrid cells suitable for many types of biochemical analyses can be routinely produced.

Animals↗
PubMed1979

Effect of progesterone, estrogen withdrawal and secondary estrogen treatment of mannosylphosphoryldolichol synthesis in chick oviduct membranes.

Oviduct membranes from chicks treated with diethylstilbestrol have a fully induced level of an enzyme that transfers mannose from GDP-Man to form mannosylphosphoryldolichol (Lucas, J.J. and Levin, E. (1977) J. Biol. Chem.252, 4330--4336). Withdrawal of diethylstilbestrol for 5 days causes a decrease in oviduct weight, lysozyme, and 60% of the mannosyltransferase activity. Chicks withdrawn from treatment for 10 days followed by secondary stimulation with diethylstilbestrol exhibit a more rapid increase in the mannosyltransferase activity than chicks that have not been previously treated with diethylstilbestrol. Further experiments indicate that the decrease in mannosylphosphoryldolichol synthesis after hormonal withdrawal may be the result of decreased levels of endogenous dolichyphosphate in the membrane preparations.

Animals↗
PubMed1979

Separation of cytoplasts and whole cells using density gradients of renografin.

Cytoplasts prepared from L929 or Hepa-2 cells were separated from whole cells using density gradients of renografin. Using this technique, cytoplasts can be isolated from cell lines which cannot be routinely enucleated with an efficiency of 100%. The purified cytoplasts excluded the vital dye trypan blue and were utilized in nuclear transplantation experiments to reconstruct whole viable cells capable of division. In addition, the renografin gradient technique was used to separate the newly reconstructed cells from any contaminating "non-renucleated" cytoplasts. This will permit immediate biochemical characterization of cytoplasmic-nuclear hybrid cells without interference from contaminating cytoplasts.

Animals↗
PubMed1979

Purification and characterization of regenerating mouse L929 karyoplasts.

Within 72-96 hr after preparation, about 10% of the karyoplasts made from mouse L929 cells regenerated to reform whole viable cells. As soon as 30 hr after preparation, however, nearly all of the remaining 90% of karyoplasts were dead. By separating living and dead karyoplasts at 30 hr, therefore, that fraction destined to complete regeneration was effectively purified. Complete separation was accomplished by sedimentation through Ficoll-paque (Pharmacia), a patented preparation originally developed for the separation of monocytes from whole blood. With the addition of this technique to the previously reported purification scheme for karyoplasts, various biochemical and morphological studies were attempted. Of particular importance are results indicating that karyoplasts that regenerate do not initially contain any more cytoplasm than the average karyoplasts in a preparation--that is, about 10% of the cytoplasm within a whole cell. Electron microscopy of karyoplasts immediately after preparation indicated an unequal partitioning of cytoplasmic organelles at the time of enucleation. For example, karyoplasts initially contain about 11.4% of the mitochondrial volume of whole cells, but only 2.9% of the Golgi apparatus. The size of the karyoplasts and the volume occupied by a variety of organelles was followed throughout the process of regeneration. Although there was an approximately linear increase in the diameter of regenerating karyoplasts, there appeared not to be a simple concordant increase in the volume occupied by all cellular organelles. An extensive investigation was performed to determine whether or not karyoplasts contained centrioles. Immediately after enucleation, 15,000 random thin sections through karyoplasts, which represented about 100 complete bodies, were examined for the presence or absence of centrioles. No centrioles were observed. Examination of the cytoplasts revealed that they contained a sufficient number of centrioles to account for all of the centrioles that were present in the whole cells before enucleation. Centrioles were first detected in karyoplasts in 24 hr after preparation, about the same time that karyoplasts regained the ability to adhere to the surface of tissue culture dishes. At this time, however, the average karyoplast had less than one centriole. By 72 hr, the regenerated karyoplasts had approximately the same number of centrioles as whole cells.

Animals↗