Abstract: Interferons are a family of proteins first identified by their ability to induce cellular resistance to infection by many viruses. In addition to the antiviral properties it shares with the α- and β-interferons, γ-interferon (IFN-γ), a lymphokine secreted by activated T cells, activates macrophages, stimulates B cells, increases fibroblast and endothelial cell resistance to many non-viral intracellular parasites and modulates cell-surface proteins central to immune cell regulation1–13. To identify molecules involved in the IFN-γ response and characterize their modulation, we have isolated genes that are induced following recombinant IFN-γ treatment of U937 cells, a histiocytic lymphoma cell line with monocytic characteristics14,15. We report here the molecular cloning and characterization of a gene regulated by rIFN-γ in U937 cells as well as in human mononuclear cells, fibroblasts and endothelial cells. Messenger RNA from this gene is induced within 30 min of rIFN-γ treatment and demonstrates maximal (>30-fold) accumulation within 5 h. Increased transcription is partly responsible for this accumulation. This gene encodes a protein of relative molecular mass (Mr) 12,378 which has significant amino-acid homology to platelet factor-4 and β-thromboglobulin, two chemo-tatic proteins released by platelets on degranulation. This IFN-γ-inducible protein may be a member of a family of proteins involved in the inflammatory process.

Abstract: As many as 40 distinct oncogenes of viral and cellular origin have been identified to date Many of these genes can be grouped into functional classes on the basis of their effects on cellular phenotype These groupings suggest a small number of mechanisms of action of the oncogene-encoded proteins Some data suggest that, in the cytoplasm, these proteins may regulate levels of critical second messenger molecules; in the nucleus, these proteins may modulate the activity of the cell's transcriptional machinery Many of the gene products can also be related to a signaling pathway that determines the cell's response to growth-stimulating factors Because some of these genes are expressed in nongrowing, differentiated cells, the encoded proteins may in certain tissues mediate functions that are unrelated to cellular growth control

Abstract: Several lines of evidence suggest a role for the myc oncogene in cell proliferation. Most recently, mitogenic stimulation of quiescent lymphoid, fibroblast and epithelial cells has been demonstrated to lead to a sharp increase in c-myc RNA levels. To determine how c-myc expression is linked to the cell proliferative cycle, we have used centrifugal elutriation to enrich for populations of avian and human cells at different stages of the cell cycle. Centrifugal elutriation is a counterflow centrifugation method that separates cells on the basis of volume, a parameter correlating well with progression through the cell cycle. Using myc-specific anti-peptide antibodies, we show here that the synthesis, half-life and modification of c-myc proteins are constant throughout the cell cycle of normal and transformed cells.

Abstract: A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation. The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity. Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin. Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen. There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar. The molecular weight of the receptor was 50,000 and pI was 4.2. Eukaryotic laminin receptors were visualized by means of the binding of S. aureus in the presence of laminin. Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.

Abstract: Publisher Summary Primary cultures of perinatal mouse or rat brain consist of many cell types, such as astroblasts, oligodendroblasts, ependymal cells, capillary endothelial cells, phagocytic cells, and mesenchymal cells, but are devoid of neurons In spite of cellular heterogeneity, one cell type—the astroblast—appears to dominate quantitatively Most of these cells have been identified immunocyto-chemically by using antibodies against cellular markers, in most cases, cell-type specific proteins From the size of most of the hormonal stimulations, it has been concluded that the action must be on the most abundant cells—the astroblasts The fact that a hormone listed as inhibitory interferes with the action of a stimulating hormone has been interpreted as evidence for the copresence of the corresponding receptors in the same cell surface Thus, the interference of one group of hormones with the action of another group of hormones has been used to allocate a set of receptors to one cell type within a heterogeneous population of cells

Abstract: We have investigated the genetic and physiological control of meiosis in fission yeast. Nutritionally depleted h+/h− diploid cells become irreversibly commited to meiosis immediately prior to the initiation of premeiotic S phase. Premeiotic DNA synthesis requires matP+, matM+, mei2+ and mei3+ but not the mitotic cell cycle control gene, cdc2+, ran1+ is an essential gene, loss of which provokes sexual conjugation, premeiotic DNA synthesis, pseudo-meiosis and the sporulation of haploid cells. Our experiments suggest that sexual differentiation is achieved physiologically by the inhibition of ran1+ activity in a two-step process. In the first step, partial inhibition of ran1+ in starved haploid cells, leads to cell cycle arrest in G1 followed by sexual conjugation. In the second step, a pathway requiring the matP+, matM+ and mei3+ genes of the newly-formed zygote, further inhibits ran1+ and thereby commits the cell to meiosis. mei2+ is required for meiotic commitment after full inhibition of ran1+. ran1+ is normally essential for vegetative cell reproduction but is inessential in cells which have abnormally high levels of cAMP-dependent protein kinase. We propose that the ran1+ gene encodes a highly controlled protein kinase which shares key substrates with cAMP-dependent protein kinase.

Abstract: The Ca2+-dependent cell-cell adhesion system (CDS) is thought to be essential for the formation and maintenance of cell adhesion in a wide variety of tissues. Previous studies suggested that CDS has some cell-type specificity; for example, the monoclonal antibody ECCD-1 selectively recognizes CDS of certain epithelial tissues in mouse embryos but not nervous tissues. In the present study, we have obtained a monoclonal antibody, designated NCD-1, that disrupts connections between brain cells of mouse embryos. A series of experiments suggested that NCD-1 specifically recognizes CDS. We then determined the distribution of the NCD-1 antigen in various mouse tissues. NCD-1 reacted with cells of the following tissues and cell lines: nervous tissues from various sources, lens, striated muscle, cardiac muscle, glioma G26-20, adrenocortical tumor Y1, and melanoma B16. None of these cells reacted with ECCD-1, and the cells reactive with ECCD-1 did not react with NCD-1. There was also a class of cells that did not react with either ECCD-1 or NCD-1. These results suggest that cells in the body can be classified into at least three groups containing CDS of differing specificities. A map of the tissue localization of these different classes of CDS also suggests that the expression of cell-type-specific cell adhesion molecules in each tissue plays a crucial role in adhesion between the same cell types and segregation of different cell types in processes essential for animal morphogenesis.

Abstract: • Proliferative vitreoretinopathy (PVR) is the most common cause of failure in retinal reattachment surgery. Giant retinal tears are associated with a particularly high incidence of PVR. It involves the formation of cellular membranes that contract and cause traction retinal detachments. Retinal pigment epithelial (RPE) cells are a major component of these cellular membranes. Displacement of viable RPE cells from their normal position into the vitreous cavity is necessary for membrane formation to occur. This study demonstrates that cryotherapy, but not diathermy, enhances intravitreal dispersion of viable RPE cells in bovine eyes in vitro. This suggests that in some circumstances, excessive cryotherapy may play an exacerbating role in the initiation of PVR.

Abstract: Axons of dorsal root ganglion neurons express on their surfaces one or more proteins which are mitogenic for Schwann cells (Salzer, J., R. P. Bunge, and L. Glaser, 1980, J. Cell Biol., 84:767-778). Incubation of co-cultures of dorsal root ganglion neurons and Schwann cells with 4-methylumbelliferyl-beta-D-xyloside, an inhibitor of proteoglycan biosynthesis, decreases the mitogenic response of the Schwann cell by over 95%. The effect of the beta-D-xyloside has been localized to the neurons; pretreatment of neurons but not of Schwann cells with the inhibitor causes a marked reduction of the mitogenic response. In addition, Schwann cells treated with beta-D-xyloside are still mitogenically responsive to soluble Schwann cell mitogens (cholera toxin and glial growth factor). Neurons treated with heparitinase and membrane vesicles prepared from heparitinase-treated neurons show diminished mitogenicity for Schwann cells, while other proteoglycan lyases have no effect. We conclude that a cell surface heparan sulfate proteoglycan is a component of the Schwann cell mitogen present on the surface of dorsal root ganglion neurons.

Abstract: To investigate the differentiation-promoting potential of c-fos in embryonal carcinoma cells (EC cells) we have designed various human metallothionein promoter-mouse-c-fos gene constructs containing also the selectable SV40 promoter-driven neo gene. Upon transfection into F9 EC cells and selection for neo resistance, the following results were obtained. (i) With each of the constructs, colonies of morphologically altered and differentiated (i.e., TROMA-1 and TROMA-3 expressing) cells were identified. (ii) Expression of c-fos was required to affect the differentiation state of F9 cells to a significant extent, but a low level was sufficient; no enhancement of differentiation was noticeable even after 100-fold induction of c-fos expression by cadmium. (iii) F9 cell clones were isolated which, in spite of very high levels of exogenous c-fos expression, had stem cell morphology. These cells, however, continuously generated morphologically altered and differentiated cells upon subculturing. (iv) In other EC cell lines, which resemble stem cells more closely than the 'partially differentiated' F9 cells, c-fos expression showed either a less pronounced (P19 cells) or no differentiation-promoting effect at all (PC13 cells). Our results suggest that the c-fos gene product acts in concert with other, probably 'spontaneously' occurring events to promote differentiation of certain EC cell lines.

Abstract: The total cellular content of the fluorescent mitochondrial-specific dye rhodamine 123 (Rh-123) was quantified by butanol extraction as a function of time of exposure and dose for a variety of cell lines. These results were compared with observations made by fluorescence microscopy on dye localization and mitochondrial morphology. There appeared to be two categories of cell types based on Rh-123 uptake: those which progressively accumulate the dye, such as Ehrlich ascites tumor cells, carcinoma-derived lines MCF-7, PaCa-2, EJ, HeLa, and normal fibroblast line CCL 64; and those which appear to equilibrate with the extracellular dye within 1 h of incubation in Rh-123 (1 microgram/ml) with a minimal level of uptake, such as the normal epithelial-derived lines CV-1 and MDCK and the transformed fibroblast line 64F3. Within the first category, the absolute value of uptake per cell correlated with the concentration of Rh-123 in the medium and with the period of exposure to the dye up to a point of apparent cellular saturation. The length of time required for apparent saturation depended on the cell type. In the second category equilibration was very early, and the total uptake was a function of the extracellular concentration of Rh-123. This probably does not represent a saturation level of dye content in the non-accumulating, low uptake cell lines. Fluorescence microscopy revealed that Rh-123 localization was initially mitochondrial-specific for all of the cell lines examined. Over time, alterations in mitochondrial morphology and cytoplasmic fluorescence were observed in the high uptake cell lines but not in the minimal uptake cell lines. Incubation of the high uptake HeLa cell line with the mitochondrial membrane potential inhibitor p-trifluoromethoxyphenylhydrazone substantially decreased Rh-123 uptake. These observations may indicate a transformation-related characteristic of carcinoma cell mitochondria. It may be possible to exploit the mechanism responsible for the progressive accumulation of Rh-123 by carcinoma-derived cell types for chemotherapeutic approaches to certain types of carcinomas.

Abstract: Hematopoietic tissues and some leukemic cell lines express elevated levels of c-myb transcripts. We have separated a subpopulation of chicken embryo yolk sac cells that represents about 5% of the yolk sac hematopoietic cells and appears to contain all of the detectable c-myb transcripts. The level of myb expression in this cell population is higher than previously reported for any normal cell population and is in the range of that found in cells transformed by avian myeloblastosis virus and E26 virus. Since the myb gene probe used also detects full-length viral transcripts as well as the v-myb mRNA, it appears that the level of expression of c-myb in this normal population may exceed that found in some transformed cell populations that depend on v-myb to maintain the transformed phenotype. This c-myb-expressing cell population has been identified as primarily M-CFC, the committed progenitor for the macrophage lineage. As cells differentiate to the promonocyte stage there is an abrupt decrease in c-myb expression of greater than 100 fold. These studies thus describe a normal cell population that expresses c-myb at levels similar to the level of v-myb in cells that depend on v-myb for the maintenance of their transformed phenotype. Furthermore, these studies provide direct evidence for the developmental regulation of c-myb during the process of normal macrophage differentiation.

Abstract: Glycosidases have been demonstrated to be elevated in the interstitial fluid of tumors, sera of animals and patients with tumors, and in some tumor tissue as compared to normal adjacent tissue. Elevations of serum β-N-acetylglucosaminidase and β-glucuronidase most commonly have been found to occur and these enzymes have been shown to be secreted into the extracellular medium by many different tumor cell types in vitro. The mechanism of cellular release of these hydrolytic enzymes probably involves tumor lysosomal exocytosis. Increased tumor glycosidase levels may promote increased tumor cell shedding from primary tumors, local invasion and perhaps be responsible directly, or indirectly for structural changes in tumor cell surface glycoconjugates. These cell surface changes could facilitate tumor cell thrombus formation, secondary site implantation and attachment in the microcirculation to endothelial cells and/or subendothelial basement membrane components.

Abstract: We have investigated the expression of certain cell-cycle-dependent genes in human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). The genes studied had been previously identified as cell-cycle dependent in other cell types from different species and were induced by different mitogens. One of these genes (2F1) and the gene for the interleukin 2 receptor were induced by PHA even in cultures partially depleted of accessory cells where the lymphocytes grew in size but failed to enter S phase. The other genes (c-myc, 4F1, JE-3, and KC-1) were induced only in complete cultures of PBMC stimulated by PHA. These results confirm the dissociation between growth in size and cell DNA replication that can occur during cell-cycle progression. Moreover, the time course of appearance of detectable levels of RNA for these genes suggests that they may be used as markers of cell-cycle progression in the transition of lymphocytes from G0 to S phase.

Abstract: RU 486 is an original multifaceted antihormone. It appears to be a potent progestin and glucocorticoid antagonist while exhibiting no agonistic effect, even at very high doses. Thus, according to the bioassay used, RU 486 administered orally at doses between 3 and 20 mg/kg completely inhibits the effect of exogenous progesterone on the endometrial proliferation in rabbits, on the volume density of uterine gland cell mitochondria, on the deciduomata formation and on the maintenance of pregnancy in ovariectomized rats. Furthermore, it proves to be antinidatory and abortive in rats and mice. In cycling monkeys it induces menstruation when administered during the mid-luteal phase.

Abstract: The expression of enzymes in LLC-PK1 and MDCK cells was used to study the retention of differentiated properties of the renal epithelial cell lines by a biochemical approach. Activities of marker enzymes, for which intracellular and intranephron localization is known, were determined from crude cell homogenates of LLC-PK1 and MDCK monolayer cultures. The activity patterns of the particular enzymes found were then compared with the in vivo distribution of the enzymes along the rat nephron. LLC-PK1 cells exhibit high activities of apical membrane enzymes when compared with MDCK cells, whereas in the latter high activity of Na-K-ATPase could be detected. The activities of lysosomal enzymes, mitochondrial enzymes, and transaminases were higher in LLC-PK1 than in MDCK cells. Glycolytic enzymes, however, displayed identical activity levels in both the LLC-PK1 and MDCK cells, which may be due to the fact that these are continuous cell lines and to the culture conditions used, since glucose is a major energy source in the culture media.

Abstract: We report here that soluble factors from activated mononuclear leukocytes have a dramatic effect on cultured endothelial cells. While human umbilical vein endothelial cells grown under standard conditions show a polygonal, epithelial-like morphology, cells exposed to culture media conditioned by lectin-activated human mononuclear leukocytes become extremely elongated and/or send out numerous cytoplasmic processes, assuming a dendritic configuration. This effect cannot be mimicked by exogenous cyclic AMP, is reversible upon interruption of the treatment, and appears specific for endothelial cells, since it has not been observed so far with other cell types. The shape changes are accompanied by a reorganization of the endothelial cell cytoskeleton: actin microfilament bundles tend to be disposed in parallel arrays, while intermediate filaments and microtubules penetrate up to the extremity of the cytoplasmic processes. Colchicine prevents endothelial cell elongation but only slightly impairs the formation of lateral cell processes ("dendritic configuration"). Purified interleukins were tested for their ability to induce these changes of cell shape. Escherichia coli-recombinant human interleukin 2 had no effect, and gamma-interferon only a slight effect on endothelial cell morphology. Interleukin 1 induced moderate cell elongation, while combined treatment with both interleukin 1 and gamma-interferon resulted in shape changes indistinguishable from those elicited by supernatants of activated mononuclear leukocytes. The possible relevance of the observed endothelial cell changes to the reported angiogenic activity of mononuclear cell products is discussed.

Abstract: An in vitro assay for examining the sublethal effects of chemotherapeutic agents on vascular endothelial integrity is described. Using vascular endothelial cell monolayers, the kinetics of binding of radiolabeled platelets or metastatic tumor cells were altered when endothelial cells were pretreated for 2 hr with low, clinically relevant concentrations of certain drugs. Electron microscopic examination by scanning electron microscopy revealed that these same drugs caused endothelial cell retraction and exposure of subendothelial matrix. Platelets and tumor cells were found bound only to the exposed areas of subendothelial matrix. Some drugs (bleomycin, 1,3-bis(2-chloroethyl)-1-nitrosourea, vincristine) induced rapid endothelial cell retraction and increased platelet and tumor cell binding to exposed subendothelial matrix, while one of the drugs tested (Adriamycin) caused delayed (1 to 3 day after a 2-hr drug treatment) endothelial cell retraction and increased cell binding. Of the drugs tested, only 5′-fluoro-2′-deoxyuridine which interferes with DNA replication failed to induce endothelial cell retraction and increased tumor cell and platelet binding. The results suggest that certain drug effects on the vascular endothelium can be assessed using the vascular endothelial cell monolayer model.

Abstract: Cultured bovine aortic endothelial cells were examined for renin activity by biochemical, immunological, and immunohistochemical techniques. When cell sonicates were incubated with renin substrate, linear generation of angiotensin I was observed (1.12 +/- 0.2 ng angiotensin I/10(6) cells per hr). The effect of pH on this activity was similar to that of bovine renal renin, and renin antibodies inhibited a large portion of the enzymatic activity. Furthermore, immunofluorescence microscopy with antirenin antisera confirmed the presence of renin within these cells. Biosynthetic radiolabeling, followed by immunoprecipitation, demonstrated de novo synthesis of a renin precursor in the endothelial cells, which was processed to a more mature protein. Thus, bovine aortic endothelial cells in culture contain and biosynthesize renin, a key component of the renin-angiotensin system. The expression of renin activity by endothelium may contribute to the local regulation of vascular tone.

Abstract: Erythrocyte skeletal proteins are known to play an important role in determining membrane deformability. In order to see whether transmembrane proteins also influence deformability and, if so, whether this influence is mediated by an interaction with the membrane skeleton, we examined the effect on deformability of ligands specific for transmembrane proteins. We found membrane deformability markedly reduced in erythrocytes that were pretreated with glycophorin A-specific ligands. In contrast, ligands specific for band 3 and A and B blood group antigens had no effect. The increase in membrane rigidity appeared to depend upon a transmembrane event and not upon a rigidity-inducing lattice on the outside surface of the cell in that a monovalent Fab of antiglycophorin IgG caused decreased deformability. We therefore looked for a ligand-induced association of glycophorin and the skeletal proteins and found, in Triton X-100-insoluble residues, a partitioning of glycophorin with the skeletal proteins only after preincubation with a ligand specific for glycophorin. We then studied cells and resealed membranes with skeletal protein abnormalities. In spectrin-deficient and protein 4.1-deficient erythrocytes and in 2,3-diphosphoglycerate-treated resealed membranes, the antiglycophorin IgG was only one-third as effective in decreasing deformability as it was in normal cells. Thus, normal skeletal proteins appear to be essential for liganded glycophorin to affect membrane deformability maximally. Taken together, these observations indicate that there is a ligand-induced interaction between glycophorin A and skeletal proteins and that this interaction can directly influence membrane deformability.

Abstract: Namalva, a human B cell lymphoma line, produced a factor with a molecular weight of approximately 60,000 which enhanced the proliferation of normal activated human B lymphocytes. The factor also enhanced the proliferation of certain B cell lines. It can be distinguished physiologically and biochemically from other lymphokines known to enhance B cell proliferation, namely, interleukin (IL) 1, IL 2, and interferon. The production of B cell growth factor by B cell tumor lines may contribute to their ability to grow autonomously and may reflect an important component of the neoplastic potential of the cell. B cell growth factor produced by tumors may also affect normal cells in vivo.

Abstract: The hemagglutinin (HA) of influenza virus was used to obtain efficient and rapid bulk delivery of antibodies and horseradish peroxidase (HRP) into the cytoplasm of living tissue culture cells. By exploiting HA's efficient cell surface expression, its high affinity for erythrocytes, and its acid-dependent membrane fusion activity, a novel delivery method was developed. The approach is unique in that the mediator of both binding and fusion (the HA) is present on the surfaces of the target cells. A recently developed 3T3 cell line which permanently expresses HA, Madin-Darby canine kidney cells infected with influenza virus, and CV-1 cells infected with a simian virus 40 vector carrying the HA gene were used as recipient cells. Protein-loaded erythrocytes were bound to the HA on the cell surface and a brief drop in pH to 5.0 was used to trigger HA's fusion activity and hence delivery. About 3 to 8 erythrocytes fused per 3T3 and CV-1 cell, respectively, and 75-95% of the cells received IgG or HRP. Quantitative analysis showed that 1.8 X 10(8) molecules of HRP and 1.4 X 10(7) IgG molecules were delivered per CV-1 cell and 6.2 X 10(7) HRP molecules per 3T3 cell. Cell viability, as judged by methionine incorporation into protein and cell growth and division, was not impaired. Electron and fluorescence microscopy showed that the fused erythrocyte membranes remained as discrete domains in the cell's plasma membrane. The method is simple, reliable, and nonlytic. The ability to simultaneously and rapidly deliver impermeable substances into large numbers of cells will permit biochemical analysis of the fate and effect of a variety of delivered molecules.