New cell line. Epithelial cell line and subline established from premalignant mouse mammary tissue.

A cell line and subline with epithelial characteristics were established from mouse mammary hyperplastic alveolar nodules (HAN). The cells do not grow in suspension cultures in vitro or form tumors in vivo. The cells do produce significant amounts of C-type and A-type virus and low amounts of plasminogen activator.     

Changes in the antitumor resistance of hamsters after experimental removal of non-fixed macrophages

It has been demonstrated that tumours do not appear after subcutaneous transplantation of small amounts (10(2), 2 X 10(2] of AD-12 sarcoma cells to intact hamsters, whereas tumours do appear in 53-72% of hamsters inoculated with starch suspension prior to transplantation of the same amounts of tumour cells (tumour growth stimulation phenomenon). The results show that tumour growth stimulation in hamsters pre-inoculated with starch suspension is connected with distraction of macrophages from the region of tumour cell localization to the abdominal area.     

Alveolar macrophages have greater amounts of the enzyme 5-lipoxygenase than do monocytes.

Alveolar macrophages release greater amounts of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) after A23187 stimulation than do blood monocytes. The mechanisms for this enhanced 5-lipoxygenase activity in alveolar macrophages are unknown. In these studies, we determined whether alveolar macrophages have greater amounts of the enzyme 5-lipoxygenase than do blood monocytes. We confirmed that alveolar macrophages released greater amounts of LTB4 after A23187 stimulation than did equivalent numbers of blood monocytes. In both the presence and absence of A23187, alveolar macrophages had greater amounts of immunoreactive 5-lipoxygenase, determined by Western analysis, on a per cell and a per protein basis than did blood monocytes. The amounts of 5-lipoxygenase enzyme in the cells roughly correlated with the amounts of LTB4 released by both types of cells. These observations suggest that A23187 stimulates alveolar macrophages to release greater amounts of LTB4 and 5-HETE than blood monocytes, in part, due to the greater amounts of 5-lipoxygenase.     

Phylogenetic Distribution of Neuron-Specific Enolase

: Neurons and neuroendocrine cells contain a unique isoenzyme of the glycolytic enzyme enolase which is not found in other cells. This acidic enolase isoenzyme has been designated neuron specific enolase or NSE and is easily identified by its elution on DEAE sephadex. The present study shows that brain tissue from species such as yeast, fish and frog do not contain appreciable amounts of acidic “NSE-like” enolase suggesting that lower species do not have this neuronal isoenzyme.     

Primary anti-trinitrophenyl memory cells investigated by plaque-forming cells and ELISA.

Primary anti-trinitrophenyl antibody production was investigated from spleen cells of mice immunized with trinitrophenylated-keyhole limpet hemocyanin, using the plaque-forming cell method and ELISA. Cells taken 5 days after antigen injection do not produce IgE, but do produce IgM and IgG1 anti-trinitrophenyl antibodies as demonstrated by plaque-forming cells. Substantial increase of IgM, IgG1, and IgE antibody production was seen from cells taken 7 days after immunization, followed by a rapid decline. By ELISA it was seen that cells taken 3 days after immunization already produce small amounts of anti-trinitrophenyl antibodies. Presence of antigen from the start of the cultures did not increase antibody production from cells taken 3 days after immunization, but potentiated antibody secretions from cells taken 5 days or later after immunization. This potentiation was interpreted as recruitment of antibody-forming cells from early memory B cells. The presence of IL-4 from the start of the cultures had no appreciable effect. Cell sorting with specific antibody-coated magnetic beads showed that plaque-forming cells from nonsorted cells, membrane IgE+ or membrane IgE- cells secreted similar amounts of anti-trinitrophenyl IgG1 and IgE antibodies. No difference in anti-trinitrophenyl IgM, IgG1, or IgE production was found in controls; cells sorted negatively or positively for CD23. The data show that memory B cells can be demonstrated already on Day 5 after immunization, and their antigen-induced antibody secretion is IL-4 dependent.     

Metallothionein accumulation may account for intracellular copper retention in Menkes' disease

Cultured lymphoblasts derived from infants with Menkes' disease exhibit the same increased avidity for copper as do fibroblasts and most extrahepatic tissues from these patients. The Menkes' cells preferentially take up not only copper but also, on exposure to elevated metal concentrations, the other metallothionein-binding metals, zinc and cadmium. Menkes' lymphoblasts contain larger amounts of metallothionein than normal cells following exposure to each of these metals; the amount bound to this protein quantitatively accounted for the total cellular increment in metal in Menkes' cells. Induction of metallothionein synthesis caused both normal and Menkes' cells to subsequently take up increased amounts of 67Cu. These observations suggest that an enhanced capacity of Menkes' cells to accumulate metallothionein may be responsible for their increased uptake and retention of copper.     

Differences in DNA-binding proteins isolated from normal and transformed human cells

When the DNA-binding proteins (DBPs) of WI38 normal human fibroblasts and their SV40-transformed counterpart were compared, two DBPs were present in greater amounts in the transformed cells. These two DBPs, P5a and P6b, were also present in greater amounts in HeLa cells versus WI38 cells and in chemically transformed human liver cells versus normal liver cells. Therefore, these DBP differences do not appear to be specific for transformation by SV40. Increased amounts of P5a were present in 7 of 9 transformed cell lines examined. The two tumor cell lines lacking the P5a change were sensitive to density-dependent inhibition of replication, whereas the other seven cell lines were not. This correlation suggests that the increase in P5a may play a role in the release from density-dependent inhibition of replication observed in most transformed cells.     

Queuine in plants and plant tRNAs: Differences between embryonic tissue and mature leaves

In eubacterial and eukaryotic tRNAs specific for Asn, Asp, His and Tyr the modified deazaguanosinederivative queuosine occurs in position 34, the first position of the anticodon. Analysis of unfractionated tRNAs from wheat and from tobacco leaves shows that these tRNAs contain high amounts of guanosine (G) in place of queuosine (Q). This was measured by the exchange of G34 for [³H]guanine catalysed by the specific tRNA guanine transglycosylase from E. coli. Upon gel electrophoretic separation of the labeled tRNAs, seven Q-deficient tRNA species including isoacceptors are detectable. Two are identified as cytoplasmic tRNAs^Tyr and tRNA^Asp and two represent chloroplast tRNA^Tyr isoacceptors. In contrast to leaf cytoplasm and chloroplasts, wheat germ has low amounts of tRNAs with G34 in place of Q.A new enzymatic assay is described for quantitation of free queuine in cells and tissues. Analysis of queuine in plant tissues shows that wheat germ contains about 200 ng queuine per g wet weight. In wheat and tobacco leaves queuine is present, if at all, in amounts lower than 10 ng/g wet weight. The absence of Q in tRNAs from plant leaves is therefore caused by a deficiency of queuine. Tobacco cells cultivated in a synthetic medium without added queuine do not contain Q in tRNA, indicating that these rapidly growing cells do not synthesize queuine de novo.     

Formation of nascent DNA molecules during inhibition of replicon initiation in mammalian cells.

X-irradiation of mammalian cells with moderate doses (100-1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analysed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant component of mammalian DNA.     

Control by the extracellular environment of differentiation pathways in 1003 embryonal carcinoma cells: study at the level of specific intermediate filaments.

1003 is a multipotential embryonal carcinoma (EC) clonal cell line which can be induced to follow different developmental pathways by altering the composition of the culture medium. When grown in serum-containing medium the great majority of 1003 cells remain undifferentiated; they express the ECMA 7 cell-surface embryonic antigen and very low amounts of vimentin. In serum-free medium, most 1003 cells differentiate into neuroepithelial cells. The majority of these cells are still labelled with ECMA 7 antibodies. They contain higher amounts of vimentin than EC cells, but no neurofilament proteins. Neuroepithelial cells then differentiate into neurons through a stage of preneurons containing both vimentin and the 70-K neurofilament protein. Fully differentiated neurons contain 70-K neurofilament protein but no vimentin. The 200-K neurofilament protein is detected later in the neurons. Mesenchymal cells (induced by re-adding serum) express high amounts of vimentin organized in networks. Preneurons , neurons, and mesenchymal cells do not express ECMA 7 antigen.     

Studies of energy linked reactions: a cofactor function for unsaturated fatty acids in oxidative phosphorylation; studies with a yeast auxotroph.

Mitochondria from the yeast unsaturated fatty acid auxotroph KD115 when grown with a high unsaturated fatty acid supplement catalyse normal oxidative phosphorylation and dihydrolipate-dependent ATP synthesis in the absence of added cofactors. Mitochondria from unsaturated fatty acid depleted KD115 cells have half the unsaturated fatty acid content of supplemented cells and do not catalyse oxidative phosphorylation and dihydrolipoate dependent ATP synthesis. Dihydrolipoate-dependent ATP synthesis can be restored specifically by addition of cofactor amounts of oleic acid and oleoyl CoA. The results provide further evidence for a cofactor role for an unsaturated fatty acid in oxidative phosphorylation.     

Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture

We have used antibodies to identify Schwann cells and oligodendrocytes and to study the expression of myelin-specific glycolipids and proteins in these cells isolated from perinatal rats. Our findings suggest that only Schwann cells which have been induced to myelinate make detectable amounts of galactocerebroside (GC), sulfatide, myelin basic protein (BP), or the major peripheral myelin glycoprotein (P0). When rat Schwann cells were cultured, they stopped making detectable amounts of these myelin molecules, even when the cells were associated with neurites in short-term explant cultures of dorsal root ganglion. In contrast, oligodendrocytes in dissociated cell cultures of neonatal optic nerve, corpus callosum, or cerebellum continued to make GC, sulfatide and BP for many weeks, even in the absence of neurons. These findings suggest that while rat Schwann cells require a continuing signal from appropriate axons to make detectable amounts of myelin- specific glycolipids and proteins, oligodendrocytes do not. Schwann cells and oligodendrocytes also displayed very different morphologies in vitro which appeared to reflect their known differences in myelinating properties in vivo. Since these characteristic morphologies are maintained when Schwann cells and oligodendrocytes were grown together in mixed cultures and in the absence of neurons, we concluded that they are intrinsic properties of these two different myelin- forming cells.     

Distribution of murine type B and type C viral nucleic acid sequences in template active and template inactive chromatin.

Template active chromatin and template inactive chromatin have been fractionated from mouse cells infected with the Moloney strain of murine leukemia virus. In vivo the cells produce abundant Rna homologous to Moloney leukemia virus, but do not produce either globin mRNA or RNA homologous to type B mouse mammary tumor virus. The DNA extracted from the template active chromatin or template inactive chromatin contained equal amounts of sequences homologous to Moloney type C virus, to type B virus, or to globin mRNA. The results are discussed with regard to the in vivo structure of chromatin and the difficulties in fractionating chromatin in vitro.     

Dermal fibroblasts from Down''s syndrome patients share a cycloheximide-induced deficiency in collagen adhesion responses with normal aging cells

Human skin fibroblasts from three different Down's syndrome patients (trisomy 21) of very different ages have been tested for their adhesion responses on tissue culture substrata coated with type I collagen, fibronectin (FN), and their combination after or during treatment of cells with cycloheximide to evaluate limitations in specific responses. It was shown previously that in vitro-aged papillary and reticular dermal fibroblasts from normal individuals do not generate F-actin stress fibers when pretreated with cycloheximide on collagen substrata but do so on FN substrata, a deficiency linked to limiting amounts/function of collagen-specific receptors in aging cells. In these studies, all three Down's fibroblast populations demonstrated a similar deficiency in stress fiber formation, evaluated by rhodamine-phalloidin staining, upon cycloheximide treatment at all passage levels. They remained competent for stress fiber formation on FN substrata and for reorganization of microtubule and intermediate filament networks on all substrata, demonstrating the specificity for the collagen matrix and for the F-actin cytoskeleton in this deficiency. The cycloheximide-induced deficiency could be readily reversed in all three cell populations by further incubation of cells in drug-free medium and, in some cases, by prior growth of cells in ascorbate-supplemented medium to stimulate collagen and possibly collagen receptor production. However, several pieces of evidence indicate that reduced amounts of FN and collagen synthesized by fibroblasts do not contribute to the cycloheximide-induced deficiency, including the inability to reverse the effect by treatment of cells with TGF beta. Several conclusions are suggested from these studies: (a) Down's dermal fibroblasts become deficient in collagen-specific receptor(s) upon cycloheximide treatment, which leads to altered transmembrane signaling and inability to reorganize F-actin into stress fibers; (b) Down's dermal fibroblasts at all passage levels have matrix adhesive phenotypes similar to those of aging fibroblasts from normal individuals; and (c) these studies provide further support for cells from Down's patients as a genetic model of aging in normal populations.     

B2 but not B1 cells can contribute to CD4+ T-cell-mediated clearance of rotavirus in SCID mice

Studies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4+ T cells, and CD8+ T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected alphabeta T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of B1 cells suggested that B1 cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding. We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and leads to the production of high levels of RV-specific intestinal IgA. In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA. Cotransfer of mixtures of purified B1 cells and B1-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (B1-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA. To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4+ T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4+ T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4+ T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4+ T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.     

Electrical stimulation of RNA and protein synthesis in the frog erythrocyte

Mature nucleated amphibian erythrocytes can be stimulated to undergo morphological changes resembling de-differentiation when exposed to small amounts of electric current. Puromycin and cyclohexamide will inhibit these morphological changes, and cytochemical staining indicates that RNA synthesis begins in concert with the changes in morphology. Autoradiographic studies show that the erythrocytes exposed to electric current synthesize RNA and protein whereas the erythrocytes not exposed to current do not make appreciable amounts of macromolecules. Electrophoretic separation of proteins from stimulated cells exhibit a band pattern different from that of unstimulated erythrocytes, and scintillation counting shows that some of the new proteins have been synthesized after electrical stimulation.     

Correlation between cell killing and massive second-round superinfection by members of some subgroups of avian leukosis virus.

Avian leukosis viruses of subgroups B, D, and F are cytopathic for chicken cells, whereas viruses of subgroups A, C, and E are not. The amounts of unintegrated linear viral DNA in cells at different times after infection with cytopathic or noncytopathic viruses were determined by hybridization and transfection assays. Shortly after infection, there is a transient accumulation of unintegrated linear viral DNA in cells infected with cytopathic avian leukosis viruses. By 10 days after infection, the majority of this unintegrated viral DNA is not present in the infected cells. The transient cytopathic effect seen in these infected cells also disappears by this time. Low amounts of unintegrated linear viral DNA persist in these cells. Cells infected with noncytopathic viruses do not show this transient accumulation of unintegrated viral DNA. Cells infected with cytopathic viruses and subsequently grown in the presence of neutralizing antibody do not show the transient accumulation of unintegrated viral DNA or cytopathic effects. These results demonstrate a correlation between envelope subgroup, transient accumulation of unintegrated linear viral DNA, and transient cell killing by avian leukosis viruses. The cell killing appears to be the result of massive second-round superinfection by the cytopathic avian leukosis viruses.     

Acid hydrolases and their release in food vacuole-less mutants of Tetrahymena thermophila.

Mutants (NP1 and PSJ5) of Tetrahymena thermophila strains B and D 1968 exist that are unable to construct a functional oral apparatus and form food vacuoles at 37 C but which do so normally at 30 C. Food vacuole-less cells starved in dilute salt solution released similar amounts of acid phosphatase, beta-N-acetyl-glucosaminidase and alpha-glucosidase activity into the medium as wildtype cells during an 8-h period. Actively growing, food vacuole-less cells had approximately 50% less total protein, acid phosphatase, beta-N-acetyl-glucosaminidase, and alpha-glucosidase per cell than wildtype cells after 72-h growth. During this time food vacuole-less cells released significant amounts of the 3 acid hydrolases into the growth medium. For each hydrolase, the total activity released from growing, food vacuole-less cells was less, on a per cell basis, tahn the amount released from food vacuole formers. The proportion of the total activity secreted by the mutant and the wildtype cells was the same for acid phosphatase and beta-N-acetyl-glucosaminidase and somewhat lower for alpha-glucosidase. It is concluded that the release of a significant amount of acid hydrolase activity from Tetrahymena is independent of food vacuole formation and may be analogous to the secretory activity of other nonphagocytic eukaryotic cells.     

The cytochemical localization of adenyl cyclase activity in rat sublingual gland.

Adenyl cyclase activity in mucous acinar cells and serous demilune cells of the rat sublingual gland was localized cytochemically. After incubation with adenylyl-imidodiphosphate (AMP-PNP) as substrate, deposits of reaction product are found along the cell membranes bordering the secretory surfaces of serous demilune cells. These are the membranes which participate directly in secretion by fusing with the granule membranes. The granule membranes of the demilune cells do not reveal reaction product, but the membranes of the granules which are fused with and become part of the cell membrane do show deposits. Thus, it appears that the cell membranes which fuse with granule membranes during secretion are associated with a high level of adenyl cyclase activity. In support of this, the luminal membranes of the mucous acinar cells which do not fuse with granule membranes during secretion are not associated with detectable amounts of adenyl cyclase activity.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.