Showing papers on "Cell culture published in 1989"
TL;DR: A growth factor for vascular endothelial cells identified in the media conditioned by bovine pituitary follicular cells and purified to homogeneity by a combination of ammonium sulfate precipitation, heparin-sepharose affinity chromatography and two reversed phase HPLC steps is proposed to be named VGF on the basis of its apparent target cell selectivity.
2,334 citations
TL;DR: Endothelial leukocyte adhesion molecule-1 (ELAM-1), a cell surface glycoprotein expressed by cytokine-activated endothelium, mediates the adhesion of blood neutrophils and may be a member of a nascent gene family of cell surface molecules involved in the regulation of inflammatory and immunological events at the interface of vessel wall and blood.
Abstract: Focal adhesion of leukocytes to the blood vessel lining is a key step in inflammation and certain vascular disease processes. Endothelial leukocyte adhesion molecule-1 (ELAM-1), a cell surface glycoprotein expressed by cytokine-activated endothelium, mediates the adhesion of blood neutrophils. A full-length complementary DNA (cDNA) for ELAM-1 has now been isolated by transient expression in COS cells. Cells transfected with the ELAM-1 clone express a surface structure recognized by two ELAM-1 specific monoclonal antibodies (H4/18 and H18/7) and support the adhesion of isolated human neutrophils and the promyelocytic cell line HL-60. Expression of ELAM-1 transcripts in cultured human endothelial cells is induced by cytokines, reaching a maximum at 2 to 4 hours and decaying by 24 hours; cell surface expression of ELAM-1 protein parallels that of the mRNA. The primary sequence of ELAM-1 predicts an amino-terminal lectin-like domain, an EGF domain, and six tandem repetitive motifs (about 60 amino acids each) related to those found in complement regulatory proteins. A similar domain structure is also found in the MEL-14 lymphocyte cell surface homing receptor, and in granule-membrane protein 140, a membrane glycoprotein of platelet and endothelial secretory granules that can be rapidly mobilized (less than 5 minutes) to the cell surface by thrombin and other stimuli. Thus, ELAM-1 may be a member of a nascent gene family of cell surface molecules involved in the regulation of inflammatory and immunological events at the interface of vessel wall and blood.
2,114 citations
TL;DR: Histological thin sections of the regressing tumor showed that anti-APO-1 was able to induce apoptosis in vivo, suggesting induction of apoptosis as a consequence of a signal mediated through cell surface molecules like APO- 1 may be a useful therapeutic approach in treatment of malignancy.
Abstract: To characterize cell surface molecules involved in control of growth of malignant lymphocytes, monoclonal antibodies were raised against the human B lymphoblast cell line SKW6.4. One monoclonal antibody, anti-APO-1, reacted with a 52-kilodalton antigen (APO-1) on a set of activated human lymphocytes, on malignant human lymphocyte lines, and on some patient-derived leukemic cells. Nanogram quantities of anti-APO-1 completely blocked proliferation of cells bearing APO-1 in vitro in a manner characteristic of a process called programmed cell death or apoptosis. Cell death was preceded by changes in cell morphology and fragmentation of DNA. This process was distinct from antibody- and complement-dependent cell lysis and was mediated by the antibody alone. A single intravenous injection of anti-APO-1 into nu/nu mice carrying a xenotransplant of a human B cell tumor induced regression of this tumor within a few days. Histological thin sections of the regressing tumor showed that anti-APO-1 was able to induce apoptosis in vivo. Thus, induction of apoptosis as a consequence of a signal mediated through cell surface molecules like APO-1 may be a useful therapeutic approach in treatment of malignancy.
1,823 citations
TL;DR: It is suggested that the cell-killing activity of TNF is mediated by Fas antigen associated with the TNF-R, an mAb specific for a human cell surface component (termed anti-Fas mAb).
Abstract: We have prepared an mAb specific for a human cell surface component (termed anti-Fas mAb). Anti-Fas shows cell-killing activity that is indistinguishable from the cytolytic activity of TNF. Fas antigen was characterized by western blotting, indicating that Fas antigen is a cell surface protein with a molecular weight of 200,000, which is different from the molecular weight of TNF-R. Fas antigen, however, is co-downregulated with the TNF-R when cells sensitive to the cytolytic activity of TNF are incubated with either TNF or anti-Fas. In contrast, Fas antigen on cells insensitive to TNF is not co-downregulated with the TNF-R. We suggest that the cell-killing activity of TNF is mediated by Fas antigen associated with the TNF-R.
1,511 citations
TL;DR: The complete structure of this enzyme is presented along with the evidence that it is identical to the 92-kDa metalloprotease secreted by normal human alveolar macrophages, phorbol ester-differentiated monocytic leukemia U937 cells, fibrosarcoma HT1080 cells, and cultured human keratinocytes.
1,114 citations
TL;DR: The restricted expression of GMP-140 in secretory granules of platelets and endothelium suggests that it has a specific function in the vascular system rather than a general role related to inducible secretion.
Abstract: We used an immunoperoxidase procedure to examine the tissue distribution of the platelet alpha-granule membrane protein, GMP-140. In addition to its presence in megakaryocytes and platelets, GMP-140 antigen was found in vascular endothelial cells of diverse human organs, but it was not detected in other types of secretory cells. [35S]Cysteine-labeled human umbilical vein endothelial cells synthesized a GMP-140 molecule containing complex N-linked oligosaccharides similar to those previously demonstrated in platelets and the megakaryocytic HEL cell line. Using an immunogold procedure on frozen thin sections of endothelial cells, we found GMP-140 antigen to be localized to membranes of electron-dense storage granules. In double-label experiments there was colocalization of GMP-140 with vWf, indicating that these granules are Weibel-Palade bodies. When endothelial cells were stimulated with histamine, GMP-140 rapidly redistributed to the plasma membrane. Immunoassays of cell lysates indicated that, relative to total cell protein, less GMP-140 is present in human umbilical vein endothelial cells than in platelets. The restricted expression of GMP-140 in secretory granules of platelets and endothelium suggests that it has a specific function in the vascular system rather than a general role related to inducible secretion.
1,028 citations
TL;DR: Glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.
966 citations
TL;DR: It is shown that nontransformed Madin-Darby canine kidney epithelial cells acquire invasive properties when intercellular adhesion is specifically inhibited by the addition of antibodies against the cell adhesion molecule uvomorulin; the separated cells then invade collagen gels and embryonal heart tissue.
Abstract: The generation of invasiveness in transformed cells represents an essential step of tumor progression. We show here, first, that nontransformed Madin-Darby canine kidney (MDCK) epithelial cells acquire invasive properties when intercellular adhesion is specifically inhibited by the addition of antibodies against the cell adhesion molecule uvomorulin; the separated cells then invade collagen gels and embryonal heart tissue. Second, MDCK cells transformed with Harvey and Moloney sarcoma viruses are constitutively invasive, and they were found not to express uvomorulin at their cell surface. These data suggest that the loss of adhesive function of uvomorulin (which is identical to E-cadherin and homologous to L-CAM) is a critical step in the promotion of epithelial cells to a more malignant, i.e., invasive, phenotype. Similar modulation of intercellular adhesion might also occur during invasion of carcinoma cells in vivo.
959 citations
TL;DR: This study developed a key methodological tool, the mean graph, which allowed the transformation of the numerical cell line response data into graphic patterns that were particularly expressive of differential cell growth inhibition.
Abstract: The objective of this study was to develop and investigate an approach to optimally detect, rank, display, and analyze patterns of differential growth inhibition among cultured cell lines. Such patterns of cellular responsiveness are produced by substances tested in vitro against disease-oriented panels of human tumor cell lines in a new anticancer screening model under development by the National Cancer Institute. In the first phase of the study, we developed a key methodological tool, the mean graph, which allowed the transformation of the numerical cell line response data into graphic patterns. These patterns were particularly expressive of differential cell growth inhibition and were conveniently amenable to further analyses by an algorithm we devised and implemented in the COMPARE computer program.
928 citations
TL;DR: The cell cycle-dependent phosphorylation of Rb is demonstrated and a model to explain how Rb may suppress cell growth by acting as a cell cycle regulatory element is proposed.
903 citations
TL;DR: The release of this growth factor by human embryonic fibroblasts raises the possibility that KGF may play a role in mesenchymal stimulation of normal epithelial cell proliferation, and Lack of mitogenic activity on either fibro Blasts or endothelial cells indicated that K GF possessed a target cell specificity distinct from any previously characterized growth factor.
Abstract: A growth factor specific for epithelial cells was identified in conditioned medium of a human embryonic lung fibroblast cell line. The factor, provisionally termed keratinocyte growth factor (KGF) because of its predominant activity on this cell type, was purified to homogeneity by a combination of ultrafiltration, heparin-Sepharose affinity chromatography, and hydrophobic chromatography on a C4 reversed-phase HPLC column. KGF was both acid and heat labile and consisted of a single polypeptide chain of approximately 28 kDa. Purified KGF was a potent mitogen for epithelial cells, capable of stimulating DNA synthesis in quiescent BALB/MK epidermal keratinocytes by greater than 500-fold with activity detectable at 0.1 nM and maximal at 1.0 nM. Lack of mitogenic activity on either fibroblasts or endothelial cells indicated that KGF possessed a target cell specificity distinct from any previously characterized growth factor. Microsequencing revealed an amino-terminal sequence containing no significant homology to any known protein. The release of this growth factor by human embryonic fibroblasts raises the possibility that KGF may play a role in mesenchymal stimulation of normal epithelial cell proliferation.
TL;DR: TF‐1 is a cell line of immature erythroid origin that requires GM‐CSF, IL‐3, or EPO for its growth and that has the ability to undergo differentiation into either more mature erystroid cells or into macrophage‐like cells.
Abstract: We have established a novel cell line, designated as TF-1, from a patient with erythroleukemia, which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1, but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factorβ and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion, and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I, the morphological and cytochemical features, and the constitutive expression of globin genes, indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate, TF-1 entered two different pathways. Specifically, hemin and delta-arninolevulinic acid induced hemoglobin synthesis, whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary, TF-1 is a cell lineof immature erythroid origin that requires GM-CSF, IL-3, or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is auseful tool for analyzing the human receptors for IL-3, GM-CSF, and EPO or the signal transduction of these hemopoietic growth factors.
TL;DR: It is shown that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2-transformed NIH 3T3 cells from forming colonies in soft agar.
Abstract: The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.
TL;DR: A potent, non-cell autonomous, anti-tumor effect of IL-4 which is effective against a wide range of tumor cell types in vivo is identified and seems to be mediated by an inflammatory infiltrate composed of eosinophils and macrophages.
TL;DR: A general and efficient transfection procedure, based on compacted lipopolyamine-coated plasmids, has been developed on endocrine cells of the intermediate lobe of the pituitary as a general tool for physiological work on primary cells.
Abstract: A general and efficient transfection procedure, based on compacted lipopolyamine-coated plasmids, has been developed. The active species is obtained by simple addition of excess synthetic lipospermine solution to the DNA and binds within minutes to the cell membrane. This technique has been developed on endocrine cells of the intermediate lobe of the pituitary as a general tool for physiological work on primary cells; it is not toxic and does not interfere with physiological regulations in melanotrope cells. A variety of eukaryotic cell cultures also have been transfected with success for transient and stable expression.
TL;DR: Overexpression of MnSOD confers increased resistance to TNF plus cycloheximide on the 293 human embryonic kidney cell line and implicate mitochondrially generated O2- as a key component of TNF-mediated tumor cell killing.
TL;DR: Data demonstrated a paracrine but not autocrine regulation of the growth and differentiation of myeloma cells by IL-6, which is not the autocrine growth factor of these well-documented human myelomatic cell lines.
TL;DR: Transient-transfection experiments demonstrated that the inductive effects of TNF-alpha were due to specific activation of the HIV long terminal repeat, providing evidence that T NF-alpha may play a role in the mechanisms of pathogenesis of HIV infection.
Abstract: Tumor necrosis factor alpha (TNF-alpha), also known as cachectin, was demonstrated to induce the expression of human immunodeficiency virus (HIV) in a chronically infected T-cell clone (ACH-2). Concentrations of recombinant TNF-alpha as low as 50 pg/ml induced a significant increase over background of HIV expression in the ACH-2 cells as determined by supernatant reverse transcriptase activity. The HIV-inducing effects of TNF-alpha could not be explained by toxic effects on the cells. In addition, both the uninfected parental cell line (A3.01) and the infected ACH-2 cells were shown to have high-affinity receptors for TNF-alpha. Transient-transfection experiments demonstrated that the inductive effects of TNF-alpha were due to specific activation of the HIV long terminal repeat. These studies provide evidence that TNF-alpha may play a role in the mechanisms of pathogenesis of HIV infection.
TL;DR: This work has investigated the mechanism by which oligonucleotides enter living cells and identified an 80-kDa surface protein that may mediate transport.
Abstract: Addition of antisense oligonucleotides to cell cultures has been used to specifically inhibit gene expression. We have investigated the mechanism by which oligonucleotides enter living cells. These compounds are taken up by cells in a saturable, size-dependent manner compatible with receptor-mediated endocytosis. Polynucleotides of any length are competitive inhibitors of oligomer transport, providing they possess a 5'-phosphate moiety. Using oligo(dT)-cellulose for affinity purification, we have identified an 80-kDa surface protein that may mediate transport. Knowledge of the oligonucleotide transport mechanism should facilitate the design of more effective synthetic antisense oligomers as potential clinical agents.
TL;DR: Two independent cDNA clones encoding the erythropoietin receptor (EPO-R) were isolated from a pXM expression library made from uninduced murine erythroleukemia (MEL) cells and showed no similarities to known proteins or nucleic acid sequences in the data bases.
TL;DR: Analysis of the kinetics of [3H]thymidine incorporation by cultured fibroblasts demonstrated that the response to IL-1 is delayed approximately 8 hours relative to their response to PDGF, and antibodies toPDGF completely block the mitogenic response toIL-1.
Abstract: Both interleukin-1 (IL-1) and platelet-derived growth factor (PDGF) induce proliferation of cultured fibroblasts and smooth muscle cells. These polypeptide mediators are released by activated macrophages and other cell types in response to injury and are thought to have a role in tissue remodeling and a number of pathologic processes. Analysis of the kinetics of [3H]thymidine incorporation by cultured fibroblasts demonstrated that the response to IL-1 is delayed approximately 8 hours relative to their response to PDGF. IL-1 transiently stimulated expression of the PDGF A-chain gene, with maximum induction after approximately 2 hours. Subsequent synthesis and release of PDGF activity into the medium was detected as early as 4 hours after IL-1 stimulation, and downregulation of the binding site for the PDGF-AA isoform of PDGF followed PDGF-AA secretion. Antibodies to PDGF completely block the mitogenic response to IL-1. Therefore, the mitogenic activity of IL-1 for fibroblasts and smooth muscle cells appears to be indirect and mediated by induction of the PDGF A-chain gene.
TL;DR: The method for cell number measurement in monolayer cultures by crystal violet staining published recently was modified and significantly improved and it is shown that the sensitivity of the assay can be varied and easily controlled by choosing the appropriate pH during the staining procedure.
TL;DR: It is reported that G-CSF and GM- CSF influence the migration and proliferation of human endothelial cells suggesting that these molecules may act as regulatory signals outside the haematopoietic system.
Abstract: Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) belong to a family of glycoprotidic growth factors required for the survival, growth and differentiation of haematopoietic precursors and which affect the function of circulating mature cells. They are produced by resting or stimulated stromal cells of the haematopoietic microenvironment (fibroblasts and endothelium) and by immunocompetent cells (T cells and monocytes/macrophages). The action of these CSF molecules was thought to be restricted to cells of haematopoietic origin. Here, we report that G-CSF and GM-CSF influence the migration and proliferation of human endothelial cells suggesting that these molecules may act as regulatory signals outside the haematopoietic system.
Journal Article•
TL;DR: The inhibitory action of IL-6 on TNF production is consistent with a predominantly antiinflammatory role ofIL-6 in the intact organism and may represent the negative arm of a regulatory circuit.
Abstract: Incubation of the human U937 histiocytic lymphoma cell line with granulocyte-macrophage colony stimulating factor (GM-CSF) rendered the cells responsive to induction of TNF by LPS. Treatment with IL-6 reduced TNF production in GM-CSF-primed U937 cells. The inhibitory effect was most pronounced (approximately equal to 80%) when IL-6 was added either along with GM-CSF or within the first 3 h of GM-CSF treatment. Both GM-CSF or IL-6 inhibited [3H]TdR uptake in U937 cells, and simultaneous treatment with GM-CSF and IL-6 resulted in an additive inhibitory effect on cell proliferation. However, the inhibition of TNF production could not be explained by the inhibitory effect of IL-6 on cell growth, nor was it due to a reduction in cell viability. An inhibition of TNF production by IL-6 was also demonstrated in cultured human peripheral blood monocytes. Treatment with IL-6 also resulted in a dose-dependent reduction of the 17-kDa TNF band revealed by SDS-PAGE after labeling monocytes with [35S]cysteine and immunoprecipitation with anti-TNF mAb. In addition, treatment with IL-6 resulted in a reduction of monocyte in vitro cytotoxicity for tumor target cells. Finally, in mice sensitized by the administration of Bacillus Calmette-Guerin, the injection of IL-6 significantly reduced the levels of TNF found in the serum upon challenge with LPS. Inasmuch as TNF is known to be an inducer of IL-6, the inhibitory action of IL-6 on TNF production may represent the negative arm of a regulatory circuit. The inhibitory action of IL-6 on TNF production is consistent with a predominantly antiinflammatory role of IL-6 in the intact organism.
Journal Article•
TL;DR: It is demonstrated that the B7 gene encodes a unique molecule belonging to the Ig superfamily and that B7 expression is limited to normal activated B cells and noncirculating B cell malignancies.
Abstract: The human B cell restricted activation antigen B7 identifies an in vivo primed subpopulation of B cells that demonstrate an accelerated response to triggers of B cell activation and proliferation. The cDNA encoding B7 was molecularly cloned by cDNA expression. The identity of the B7 cDNA was confirmed by indirect immunofluorescence and immunoprecipitation of a 44/54-kDa protein from B7 transfected COS cells. The sequence of the B7 polypeptide predicts a type I membrane protein of 262 amino acids with eight potential N-linked glycosylation sites in the extracellular region and a short, highly positively charged cytoplasmic tail. The extracellular region is homologous to the Ig gene superfamily and consists of two contiguous Ig-like domains. The first Ig domain has the characteristics of a variable domain and the second that of a constant region domain. B7 mRNA was detected only on anti-Ig activated B lymphocytes and not in other hematopoietic cells. After in vitro activation of B cells with anti-Ig, B7 mRNA was maximally expressed between 4 and 12 h with four RNA transcripts of 1.7, 2.9, 4.2, and 10 kb. The 2.9-kb mRNA predominated in in vitro-activated B cells whereas the 1.7-kb mRNA was most abundant in tumor cells of B cell origin. B7 expression was confined to several histologically defined subgroups of B cell malignancies. The majority of non-Hodgkin's lymphomas were B7+ whereas the B cell leukemias and circulating non-Hodgkin's lymphomas were generally negative. These results demonstrate that the B7 gene encodes a unique molecule belonging to the Ig superfamily and that B7 expression is limited to normal activated B cells and noncirculating B cell malignancies.
TL;DR: It is reported that the 72-kDa type IV procollagenase purified from HRAS-transformed human bronchial epithelial cells, SV40- transformed lung fibroblasts, and normal skin fibro Blasts exists in a stable but noncovalent stoichiometric complex with a 24-k da inhibitor referred to here as "TIMP-2."
Abstract: Simian virus 40 (SV40)-transformed human lung fibroblasts secrete both 72-kDa type IV collagenase and a closely related 92-kDa type IV collagenase that was not detected in the parental cell line. The 92-kDa type IV procollagenase purified from these cells exists in a noncovalent complex with the tissue inhibitor of metalloproteases, TIMP. Here we report that the 72-kDa type IV procollagenase purified from HRAS-transformed human bronchial epithelial cells, SV40-transformed lung fibroblasts, and normal skin fibroblasts exists in a stable but noncovalent stoichiometric complex with a 24-kDa inhibitor referred to here as "TIMP-2." TIMP-2 is closely related to TIMP, as demonstrated by comparison of the partial amino acid sequence of this protein to that of TIMP, although it does not cross-react with TIMP-specific antibody. The TIMP-2 inhibitor interacts with the 72-kDa type IV collagenase in preference to the 92-kDa type IV collagenase that forms a complex exclusively with TIMP. The 72-kDa type IV collagenase-TIMP-2 complex can be activated with organomercurials to yield a catalytically competent enzyme. Activation occurs concomitantly with autoproteolytic cleavage of the amino terminus of the protein and does not require dissociation of the complex. Both activity and activation of the complex can be completely inhibited by further addition of stoichiometric quantities of purified TIMP-2 or recombinant TIMP.
TL;DR: The release of FSdGF by pituitary cells and its target cell specificity raise the possibility thatFSdGF may play a role in angiogenesis.
Abstract: A growth factor with specificity for vascular endothelial cells has been identified in conditioned medium of pituitary-derived folliculo stellate cells. This factor, named folliculo stellate-derived growth factor (FSdGF), was purified to homogeneity by a combination of heparin-Sepharose affinity chromatography, Bio-Gel P-60 exclusion chromatography, Mono S ion-exchange chromatography, and hydrophobic chromatography on a C4 reverse-phase HPLC column. FSdGF was characterized as a homodimer composed of two subunits with a molecular mass of 23 kDa. FSdGF was a potent mitogen for vascular endothelial cells with activity detectable at 25 pg/ml and saturation at 500 pg/ml. It did not stimulate the proliferation of other cell types such as bovine vascular smooth muscle cells, corneal endothelial cells, adrenal cortex cells, granulosa cells, BALB/MK cells, or BHK-21 cells. Microsequencing revealed an N-terminal sequence having no significant homology to any known protein. The release of FSdGF by pituitary cells and its target cell specificity raise the possibility that FSdGF may play a role in angiogenesis.
TL;DR: It is reported here that in contrast to the normal low frequency of γδ-bearing cells in lymphoid tissues, peripheral blood, or normal skin, the frequency is increased five to eightfold in particular granulomatous reactions of leprosy.
Abstract: THE majority of T cells bear the T-cell receptor (TCR) αβ complex which recognizes foreign antigen peptides only in the context of self major histocompatibility complex (MHC) molecules1. Such T cells function in a variety of effector roles and secrete cytokines that mediate the activation and differentiation of other cells in the immune system. Recently, a small subpopulation T cells was found to bear a distinct TCR composed of γ and δ subunits2. In man, TCR γδ+ cells are distributed as ~5 per cent of the CD3+ cells in all organized lymphoid organs as well as in the skin- and gut-associated lymphoid tissues3. Although a limited number of germ-line genes encode the TCR γ and δ subunits, extensive junctional variation particularly in the δ gene, results in unprecedented diversity for this receptor4,5 The nature of the specificity and immunological functions of these T cells remains enigmatic. We report here that in contrast to the normal low frequency of γδ-bearing cells in lymphoid tissues, peripheral blood, or normal skin, the frequency is increased five to eightfold in particular granulomatous reactions of leprosy. TCR γδ+lymphocyte lines from these leprosy skin lesions proliferate in vitro specifically to mycobacterial antigens. This reactivity to foreign antigens appears to require presentation in the context of self-molecules. Moreover, culture supernatants from activated γδT lymphocytes induce adhesion and aggregation of bone-marrow monocytes in the presence of granulocyte monocyte-colony stimulating factor (CSF), suggesting that products of γδ-bearing T cells may play a role in the immune response, possibly by stimulating granuloma formation.
TL;DR: In vitro results suggest that the Ca2+ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of theCa2+ gradient in vivo.
Abstract: Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.05 mM Ca2+ medium, and terminal differentiation is induced when the Ca2+ concentration is increased to 1 mM. However, only a small fraction of the cells express the markers K1, K10, CE, or filaggrin in the higher Ca2+ medium. To explore the factors required for marker expression, cultured epidermal cells were exposed to intermediate Ca2+ concentrations and extracts were analyzed using specific antibody and nucleic acid probes for the four markers of interest. These studies revealed that marker expression was enhanced at a restricted concentration of Ca2+ in the medium of 0.10-0.16 mM. At this Ca2+ concentration, both protein and mRNA levels for each marker were substantially increased, whereas at higher or lower Ca2+ concentrations they were diminished or undetected. The percentage of cells expressing each marker was increased two- to threefold in the permissive Ca2+ medium as determined by immunofluorescence analysis. This optimal level of Ca2+ was required both to initiate and sustain marker expression. At the permissive Ca2+ concentration, expression of the markers was sequential and similar to the order of appearance in vivo. K1 was expressed within 8-12 h and K10 was expressed in the ensuing 12-24-h period. CE and filaggrin were expressed in the subsequent 24 h. Inhibition of K1 expression by cycloheximide suggested that an inducible protein was involved. Other investigators have determined that a shallow Ca2+ gradient exists in epidermis, where the basal cells and spinous cells are in a Ca2+ environment substantially below serum Ca2+ levels. These in vitro results suggest that the Ca2+ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of the Ca2+ gradient in vivo.
TL;DR: Measurements of calcium uptake and cyclic GMP production by cerebellar granule cells grown in primary culture demonstrated that ethanol preferentially inhibited N‐methyl‐D‐aspartate (NMDA) receptor‐gated cation channel function.
Abstract: Measurements of calcium uptake and cyclic GMP production by cerebellar granule cells grown in primary culture demonstrated that ethanol preferentially inhibited N-methyl-D-aspartate (NMDA) receptor-gated cation channel function. Concentrations of ethanol as low as 10 mM inhibited NMDA-stimulated Ca2+ uptake by greater than 30%, and ethanol also inhibited NMDA-stimulated (Ca2+-dependent) cyclic GMP accumulation in a similar, dose-dependent manner. Responses to kainate were significantly less sensitive to ethanol. Studies using various concentrations of NMDA, as well as phencyclidine (PCP) and glycine, suggested that ethanol affected the "coagonist" binding site of the NMDA receptor-channel complex, rather than the PCP recognition site.