Nuclear nonhistone proteins in mouse teratocarcinomas

Summary The nonhistone protein pattern of four murine teratocarcinomas with different capacities for differentiation were compared: a multidifferentiated teratocarcinoma OTT2289, a nondifferentiated teratocarcinoma OTT2158, a teratocarcinoma-derived rhabdomyosarcoma TDR114, and a teratocarcinoma-derived neuroblastoma TDN2151. Their nonhistone proteins (NHP) were separated by differential salt extraction and hydroxyapatite chromatography into three fractions, NHP-I, NHP-II and NHP-III. Comparison of the NHP fractions by twodimensional gel electrophoresis in combination with a sensitive silver staining method reveals that there are several tumour line specific proteins in each NHP fraction. We suggest that specific NHP, which can be used as biochemical markers for each of the four investigated tumour lines, may be involved in cell lineage specific control of gene expression.     

Changes in cell surface proteins during differentiation of mouse embryonal carcinoma cells

The cell surface proteins of teratocarcinoma-derived embryonal carcinoma cells (ECC), of parietal endoderm (Pys-2 and F9-AC cl 9), and of fibroblasts (OTT6050f) were radioiodinated by a lactoperoxidase method and analyzed by two-dimensional gel electrophoresis. The combined electrophoretic profiles of proteins from a number of ECC lines allowed the determination of eight ECC-unique polypeptides. Parietal endoderm and fibroblast expressed their own unique polypeptides. The two parietal endoderm-specific polypeptides are identical to the subunits of laminin. Retinoic acid-induced differentiation of one ECC line (F9) resulted in the disappearance of polypeptides specific for ECC and the appearance of those specific for the parietal endoderm.     

Karyotypic normalcy and quasi-normalcy of developmentally totipotent mouse teratocarcinoma cells

Malignant mouse teratocarcinoma cells are, in some cases, able to undergo normal, complete differentiation after injection into blastocysts. Thus far, only three lines—of unrelated origin—have been found (all in this laboratory) to be developmentally totipotent in blastocyst tests. The karyotypes of these lines, and their somatic- and germ-cell derivatives, were investigated by G-banding methods, as a possible clue to their developmental superiority. The first, OTT 6050 (129 strain), is an embryo-derived induced tumor maintained as an ascites transplant line. Its stem cells (from embryoid body “cores”) have 40 chromosomes in the modal class, which comprises two subclasses: one all normal and one with a metacentric chromosome (isochromosome-8). However, mosaic animals from injected blastocysts have only the normal subclass in their teratocarcinomaderived cells; all are of $\text{X}\text{Y}$ male sex chromosome type. Presence of the Y chromosome was verified after transmission through the germ line of two fertile mosaic males, in their F₁ male progeny. The second teratocarcinoma line, 72484-395 (LT strain), is a spontaneous ovarian solid tumor maintained by subcutaneous transplantation. Karyotypes of cells from the tumor, and also of teratocarcinoma-derived cells in mosaic animals, were normal and of $\text{X}\text{X}$ female sex chromosome type. Karyotypes of the F₁ progeny, from tumor-strain germ cells of a fertile mosaic female, were also normal. The third line, NG 2 (129 strain), is a mutant clonal in vitro line deficient in hypoxanthine phosphoribosyltransferase. It originated from an embryo-derived experimental tumor (OTT 5568) that was established in culture (PSA1 line); the culture was then mutagenized and selected for 6-thioguanine resistance. The NG 2 line proved to be quasi-normal, with only two karyotypic anomalies: trisomy of chromosome 6 and $\text{X}\text{O}$ female sex chromosome constitution. Thus, developmental totipotency in all three lines, including one maintained in vitro, is accompanied by karyotypic normalcy or near-normalcy. Other culture lines reported to be aneuploid have not yet given evidence of totipotency. Karyotypic normalcy may therefore have predictive value useful in choosing teratocarcinoma lines with relatively high developmental prospects. This is of importance in identifying those mutant lines that would be promising candidates for introduction, via blastocyst injection, of specific mutant genes into mice.     

Glycoproteins from Teratocarcinomas and Organs of Adult Mice: Analysis by Affinity Chromatography on Lectin-conjugated Agarose1

Glycoproteins were isolated from particulate fraction of four teratccarcinomas and several organs of adult mice by affinity chromatography on lectins conjugated with agarose [concanavalin A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA) and Dolichos biflorus agglutinin (DBA)] and were compared by SDS gel electrophoresis. Glycoprotein components were found to be very similar in three lines of solid teratocarcinoma, namely F9, STT-2 and OTT-10A. Teratocarcinoma OTT6050, which is an ascitic form called embryoid bodies, also gave glycoprotein profiles somewhat similar to those of other teratocarcinomas. However, glycoprotein profiles of most adult organs were significantly different from those of teratocarcinomas. The following points were of special interest. 1) RCA receptors from the four teratocarcinomas gave a strong band with an apparent molecular weight 145,000 daltons, which was either weak or absent in the receptors from adult organs. 2) The WGA receptors of all adult organs except muscle and small intestine gave more intense bands than those of the teratocarcinomas. 3) Glycoproteins with molecular weights of more than 240,000 daltons were present in WGA receptors, RCA receptors and PNA receptors of teratocarcinoma OTT 6050, but not in the receptors of other teratocarcinomas.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

Activation of teratocarcinoma-derived hemoglobin genes in teratocarcinoma-Friend cell hybrids.

Hybrid cells formed by the fusion of murine teratocarcinoma and Friend erythroleukemia cells synthesize hemoglobin in the presence of chemical inducers such as dimethylsulfoxide (DMSO). By making use of the fact that the parental teratocarcinoma and Friend cells carried different alleles at the locus coding for the alpha chain of hemoglobin, it was possible to demonstrate that the teratocarcinoma-derived genes for the globin alpha chains are genetically active in hemoglobin-synthesizing hybrid cells. In addition, evidence is presented suggesting that the teratocarcinoma-derived genes for the beta-globin chains may also be expressed in the hybrids. Apparently the teratocarcinoma-derived genome has become reprogrammed to express erythroid functions following fusion of the teratocarcinoma cell to the Friend cell.     

Embryonal carcinoma cells (and their somatic cell hybrids) are resistant to infection by the murine parvovirus MVM, which does infect other teratocarcinoma-derived cell lines

Minute virus of mice (MVM), a non-defective parvovirus, has been shown to infect cultures of non-pluripotent differentiated teratocarcinoma-derived cells, but pluripotent (and "nullipotent") embryonal carcinoma cells derived from the same teratocarcinoma resist MVN infection. Somatic cell hybrids between an embryonal carcinoma line and Friend erythroblastic leukemia cells are also resistant to MVM, even though Friend cells are susceptible. Among three blastocyst-derived lines tested, only one, a parietal yolk sac cell line, resists MVM infection. These results suggest that teratocarcinoma cultures may provide useful systems in which to study the cellular factors which mediate susceptibility to this teratogenic and oncolytic virus.     

A Comparison of Two Alternatively Spliced Forms of a Metabotropic Glutamate Receptor Coupled to Phosphoinositide Turnover

Abstract: A comparison of the pharmacological and physiological properties of the metabotropic glutamate 1α and 1β receptors (mGluR1α and mGluR1β) expressed in baby hamster kidney (BHK 570) cells was performed. The mGluR1β receptor is an alternatively spliced form of mGluR1α with a modified carboxy terminus. Immunoblots of membranes from the two cell lines probed with receptor-specific antipeptide antibodies showed that mGluRIa migrated with an M_r= 154, 000, whereas mGluR1β migrated with an M_r= 96, 000. Immunofluorescence imaging of receptors expressed in BHK 570 cells revealed that the mGluR1α receptor was localized to patches along the plasmalemma and on intracellular membranes surrounding the nucleus, whereas mGluR1β was distributed diffusely throughout the cell. Agonist activation of the mGluR1α and the mGluR1β receptors stimulated phosphoinositide hydrolysis. At both receptors, glutamate, quisqualate, and ibotenate were full agonists, whereas trans -(+)-1-aminocyclopentane-1, 3-dicarboxylate appeared to act as a partial agonist. The stimulation of phosphoinositide hydrolysis by mGluR1α showed pertussis toxin-sensitive and insensitive components, whereas the mGluR1β response displayed only the toxin-insensitive component. The mGluR1α and mGluR1β receptors also increased intracellular calcium levels by inducing release from intracellular stores. These results indicate that the different carboxy terminal sequences of the two receptors directly influences G protein coupling and subcellular deposition of the receptor polypeptides and suggest that the two receptors may subserve different roles in the nervous system.     

Effects of β-Mercaptoethanol-Containing Medium and Feeder Cells on the Differentiation of Embryonal Carcinoma Cells

Pluripotent, feeder-dependent teratocarcinoma cell lines were cultured without a feeder layer in a medium containing 10 ^−;4 M β-mercaptoethanol (β-medium) and compared for the development of early markers with cells cultured with a feeder layer. The cells cultured in β-medium lost the PNA (peanut agglutinin) receptor typically at low density. This change was accompanied with enhanced secretion of plasminogen activator and the loss of sensitivity to anti-F9 serum, indicating the stem cell differentiation. In contrast, the cells cultured on a feeder layer did not show any marker changes, thus indicating the lack of differentiation. These results indicate that while the presence of feeder cells inhibits cell differentiation, cultivation in β–medium permits the differentiation of pluripotent teratocarcinoma cells.     

A new glycoprotein antigen common to teratocarcinoma, visceral endoderm, and renal tubular brush border

Glycoproteins were purified from teratocarcinoma OTT6050 by affinity chromatography on Dolichos biflorus agglutinin-agarose. The rabbit antiserum raised against the glycoproteins defined a new antigenic marker after absorption with sheep erythrocytes and particulate fraction of the mouse liver. The antigen was detected on the visceral endoderm of 7-day-old embryos, on the tubular brush border of the kidney in adult mice and on certain endodermal cells of teratocarcinomas. Upon sodium dodecyl sulfate-gel electrophoresis, the antigens from teratocarcinoma OTT6050 and from the kidney migrated as band(s) of molecular weight around 500,000. Although the antigens from the two sources were immunologically identical, they should be different in carbohydrate sequence as judged from their behavior upon affinity chromatography on lectin-agarose.     

Tumour cell inhibition of macrophage cytokine activity

Abstract Macrophages elaborate both effector and regulatory immune functions. It was hypothesised that tumours can exert a local alteration of macrophage function. Murine peritoneal macrophage-derived cytokines were assayed in the presence and absence of cells, cytosol fractions or conditioned media (TCCM) from established murine tumour lines. Interleukin-1β, interleukin-6 and tumour necrosis factor-α activities were significantly inhibited by tumour cells or their products, as were the corresponding recombinant human cytokines. Intracellular protein kinase C activation was also measured and was significantly inhibited by murine TCCM, thus suggesting one possible site of inhibitor action. Data analyses indicate that the inhibitory factor(s) is probably not an already well-characterised macrophage inhibitor.     

Initial analysis of the molecular image of pamlin, a sea urchin cell adhesion protein, by transmission electron microscopy

Pamlin, an important extracellular protein required early for sea urchin embryogenesis, is readily isolated from the embryos of Hemicentrotus pulcherrimus . A molecular image analysis of pamlin was conducted using immuno-electron microscopy, rotary shadowing and negative staining technique-applied electron microscopy. The electron microscopy showed that a monoclonal antibody to the pamlin α-subunit bound to a position 13.5 nm from one end of a purified 255 kDa pamlin molecule, which is a 132 nm long and 6.8 nm wide linear structure. The pamlin structure is composed of three subunits, a 47 nm long 52 kDa α-subunit that attaches to one end of a 105 nm long 180 kDa β-subunit, and a 15.6 nm diameter globular 23 kDa γ-subunit that binds to the middle of the β-subunit. The α- and β-subunits together form a 125–140 nm linear structure. Intermolecular aggregation frequently occurred between the free end of two β-subunits of the αβγ pamlin molecule, leaving the entire α-subunit surface free. Occasionally associations between the ends of α-subunits, or between an α-subunit and the middle of a β-subunit also occurred, but no aggregations of pamlin formed through the γ-subunit. These homophilic molecular aggregations of pamlin formed a large supramolecular network. In addition, the single pamlin molecule rounded at one end under high calcium ion concentration to form a 'loop', suggesting the presence of a calcium sensitive region in the molecule.     

Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane-Bound Polysomes

Abstract: Free and membrane-bound polysomes were prepared from rat forebrain and added to a cell-free system containing rabbit reticulocyte factors and L-[³⁵S]methionine. The translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (α¹, α², β¹, and α²) that are found in rat forebrain cytoplasm. The membrane-bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two-dimensional gel. MB has a molecular weight and isoelectric point similar to α-tubulin. Only trace amounts of α- and β-tubulin and actin were synthesized by the membrane-bound polysomes. MB co-purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane-bound polysomes) and α- and β-tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of α- and β-tubulin; MB yielded peptides not found in tubulin. We conclude that membrane-bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane-bound polysomes and is similar, but not identical, to α-tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.     

Two New Saponins from Lysimachia capillipes Hemsl.

To investigate the saponins from whole plants of Lysimachia capillipes Hemsl., two new saponins, named capilliposide E (1) and capilliposide F (2), were isolated. The structures of the new sa ponins were elucidated as 3 β, 22α-dihydroxy- 16α-acetat-28→ 13 -lactone-oleanane-3 -O- [β-D-glucopyranosyl(1→2)-α-L-arabinpyranoyl]-22-O-β-D-glucopyranoside (1) and 3 β, 22α-dihydroxy- 16α-acetat-28→ 13-1actone-oleanane-3-O- { [β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)]-α-L-arabinpyranoyl }-22-O-βD-glucopyranoside (2). The structures of these compounds were determined by 1D- and 2D-NMR, MS techniques, and chemical methods.     

Comparative Study on the Ability of Various Teratocarcinomas to Form Chimeric Mouse Embryos

Various embryonal carcinoma cells of different origins were compared as to the ability to form chimeric blastocysts by means of aggregating with normal 8-cell stage mouse embryos. The teratocarcinoma lines examined were OTT6050 and five newly established ones including a spontaneous testicular teratocarcinoma STT-2. The present results have revealed that distinct differences existed in the ability of colonizing blastocysts among teratocarcinomas and also among embryonal carcinoma cell lines.
Since STT-2 stem cells were found to be incorporated into blastocysts most efficiently, further development of the blastocysts were examined in utero. It was found that STT-2 stem cells could be incorporated into the fetuses up to the 7-to 28-somite stages. This is the first case to demonstrate that testicular teratocarcinoma cells with the male germ cell origin have the developmental potency to participate into mouse embryogenesis.     

Characterization of antisera against mouse teratocarcinoma OTT6050: Molecular species recognized on embryoid bodies,preimplantation embryos,and sperm

Antisera raised in rabbits by hyperimmunization with small embryoid bodies of the transplantable teratocarcinoma OTT6050 recognize several distinct antigenic protein species on the surfaces of cells of the immunogen. Some of these antigens were found on the cells of preimplantation mouse embryos, on cells from parietal yolk sac carcinoma, and on mouse sperm. These antigens have been distinguished by polyacrylamide electrophoresis of the immune precipitates from detergent extracts of lactoperoxidase-iodinated cells. The intact embryoid bodies from the ascitic form of the OTT6050 teratocarcinoma exhibited five major protein bands (approximate MW 150K, 115K, 82K, 48K, and 12K), one band that ran at the dye front of the gels (R_f ≥1) and one minor band (approximate MW 22K). Two different rabbit antisera recognized an essentially identical pattern of antigens which, however, varied on the different cell types tested. Antisera were also elicited in syngeneic male mice using glutaraldehyde-fixed or irradiated OTT6050 embryoid bodies. The isoantisera had very poor titers in comparison to the absorbed xenoantisera, as assessed by complement-mediated cytotoxic activity against the immunizing cell types. Complement-mediated cytotoxicity could also be demonstrated using parietal yolk sac carcinoma cells, preimplantation mouse embryos from all cleavage stages, blastocysts, and immunosurgically isolated inner cell masses, as targets. The complexity of the antisera generated by intact embryoid bodies described here indicates that these structures bear multiple antigenic specificities not present on adult somatic cells, some of which are stage-specific embryonic polypeptides.     

The α- and β-Tubulin Genes of Euplotes octocarinatus

ABSTRACT. The α- and the β-tubulin genes of the hypotrichous ciliate Euplotes octocarinatus were isolated from a size-selected macronuclear DNA library. The α-tubulin gene is located on a 1,587 bp macronuclear DNA molecule and the β-tubulin gene on a 1,524 bp macronuclear DNA molecule. Sequencing revealed that all the cysteine residues of the two genes are encoded by the common cysteine codons UGU and UGC and none by an UGA codon. This is in contrast to the genes of E. octocarinatus sequenced so far, where some of the cysteines are encoded by the opal codon UGA. The tubulin genes end like other Euplotes genes with a TAA. They do not contain introns. The last codon for an amino acid in the α-tubulin gene is a GAA which codes for glutamic acid. This is in contrast to what has been reported for most α-tubulin genes, but it supports findings for other hypotrichous ciliates. No evidence for the existence of more than one type of α- and one type of β-tubulin genes could be obtained.     

β-Synuclein occurs in vivo in lipid-associated oligomers and forms hetero-oligomers with α-synuclein

α-synuclein (αS) and β-synuclein (βS) are homologous proteins implicated in Parkinson's disease and related synucleinopathies. While αS is neurotoxic and its aggregation and deposition in Lewy bodies is related to neurodegeneration, βS is considered as a potent inhibitor of αS aggregation and toxicity. No mechanism for the neuroprotective role of βS has been described before. Here, we report that similar to αS, βS normally occurs in lipid-associated, soluble oligomers in wild-type (WT) mouse brains. We partially purified βS and αS proteins from whole mouse brain by size exclusion followed by ion exchange chromatography and found highly similar elution profiles. Using this technique, we were able to partially separate βS from αS and further separate βS monomer from its own oligomers. Importantly, we show that although αS and βS share high degree of similarities, βS oligomerization is not affected by increasing cellular levels of polyunsaturated fatty acids (PUFAs), while αS oligomerization is dramatically enhanced by PUFA. We show the in vivo occurrence of hetero-oligomers of αS and βS and suggest that βS expression inhibits PUFA-enhanced αS oligomerization by forming hetero-oligomers up to a quatramer that do not further propagate.     

Regulation of major efflux transporters under inflammatory conditions at the blood-brain barrier in vitro

ATP-driven efflux transport proteins at the blood-brain barrier protect the healthy brain but impede pharmacotherapy of the disordered CNS. To investigate the question how ATP-binding cassette (ABC)-transporters are regulated during inflammation or infection we analysed the effects of the cytokines tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) on the expression of brain multidrug resistance proteins in primary cultures of porcine brain capillary endothelial cells. We found that TNF-α and IL-1β rapidly decrease Abcg2 ( BMDP/BCRP ) mRNA expression within 6 h. After 24 and 48 h the mRNA level came back to control values. The mRNA reduction at 6 h was counter-regulated by the anti-inflammatory glucocorticoid hydrocortisone. Abcg2 protein levels were suppressed at prolonged stimulations but not after 6 h of stimulation which correlates with Abcg2 specific substrate uptake measurements. Abcb1 (p-glycoprotein) protein expression was transiently increased after TNF-α addition within 6 h of incubation followed by a reduction after 24 and 48 h whereas the Abcb1 mRNA levels were not changed. IL-1β caused a continuous decrease in protein expression of both ABC-transporters. Long-term treatment with an assumed TNF-α-downstream agent, the vasoconstrictor endothelin-1, induced Abcg2 protein expression but suppressed Abcb1. Other efflux pumps like multidrug resistance-associated proteins/Abcc were rarely affected. The present results imply a complex regulation of the two most abundant ABC-transporters at the blood-brain barrier during early inflammation stages suggesting that Abcb1 (p-glycoprotein) is an early target of TNF-α-signalling counterbalanced by Abcg2.