James E. Niedel

Distinct patterns of expression of different protein kinase C mRNAs in rat tissues

The identification of multiple protein kinase C (PKC) cDNA sequences from rat, bovine, and human tissues has led to the discovery of a family of PKC genes. Using probes complementary to three cDNA sequences from rat brain, designated PKC-I, -II, and -III, the distribution of PKC transcripts in rat tissues was studied by in situ hybridization histochemistry. In brain, PKC-II and PKC-III transcripts colocalized except in the brain stem and spinal cord, where some cells appeared to contain only PKC-III message. PKC-I mRNA had a different, but partially overlapping, pattern of expression. In spleen, PKC-II and PKC-III, but not PKC-I, transcripts were concentrated in the marginal zone of white pulp, the major B-lymphocyte compartment of rat spleen. The distinct distributions of rat PKC mRNAs suggest that different members of the PKC family have specialized functions.

Constitutive production of macrophage colony-stimulating factor by human ovarian and breast cancer cell lines.

Many nonhematologic tumors produce growth factors that may influence cellular proliferation either by autocrine or by paracrine mechanisms. In the current study, human tumor cell lines were investigated for the constitutive production of macrophage colony-stimulating factor (M-CSF). Culture supernatants obtained from cell lines were analyzed using a radioimmunoassay and a radioreceptor assay specific for M-CSF. Among the various cell types analyzed, all the ovarian cell lines and a majority of the breast cancer cell lines secreted significant amount of an M-CSF-like factor. Treatment of mouse bone marrow cultures with culture supernatants from ovarian cancer cells stimulated the production of macrophage colonies. Analysis of total cellular RNA obtained from the ovarian cell lines by Northern blot showed multiple sizes of M-CSF transcripts with an abundance of a 4.2-kb message. The relative amount of M-CSF transcripts correlated with the level of immunoreactive material seen in the culture supernatants.

Quantitative measurement of sn-1,2-diacylglycerols.

Publisher Summary The hypothesis that sn-l, 2-diacylglycerols function as the intracellular second messengers of growth factors, hormones, and neurotransmitters has gained wide acceptance. This method has been used successfully to measure basal and stimulated diacylglycerol levels in a variety of tissues and cells, including human platelets, rat hepatocytes, sis- and ras-transformed Normal Rat Kidney (NRK) cells, a human leukemia cell line (HL60), and human neutrophils. sn-l, 2-Dioleoylglycerol is prepared from sn-l,2-dioleoyl-glycerol-3-phosphocholine (Avanti) by phospholipase digestion, purified by extraction in ether, and quantitated by ester analysis using cholesteryl acetate as a standard. Bovine heart cardiolipin in chloroform is from Avanti and should be stored under N 2 after opening. This chapter discusses the preparation of membranes containing diacylglycerol kinase. With the recent explosion of interest in diacylglycerols as the in vitro activator of the Ca 2+ -activated phospholipid dependent protein kinase (protein kinase C) and the implication that diacylglycerols function as intracellular second messengers, this assay method provides a useful tool to explore the role of diacylglycerols as the second messengers of neurotransmitters, hormones, and growth factors in a number of biological settings.

Localization of collagenase mRNA in rheumatoid arthritis synovium byin situ hybridization histochemistry

Collagenase has been implicated as playing an important role in the connective tissue destruction that is a major feature of rheumatoid arthritis. Numerous cell types in the hyperplastic rheumatoid synovium are capable of synthesizing collagenase. Past studies have used predominately synovial fibroblasts in culture as a model system for the regulation of collagenase production, but the major cellular source of the enzymein vivo has not been determined. Using the techniques ofin situ hybridization histochemistry and indirect immunofluorescence, we determined the cellular source of collagenase in frozen sections of human synovium. Collagenase mRNA production was localized to cells along the synovial lining layer in rheumatoid arthritis. These were identified as the macrophage-like Type A synovial lining cells by immunofluorescence with antibody LeuM3. Endothelial cells, fibroblasts, and T and B lymphocytes were devoid of detectable collagenase mRNA. Synovial tissue sections from patients with osteoarthritis and trauma did not contain detectable collagenase mRNA. These data identify the Type A macrophage-like synovial lining cell as the primary source of collagenase mRNAin vivo in the rheumatoid arthritis synovium and, potentially, as a major effector cell in the tissue destruction of the disease.

Cyclic AMP Levels and Cellular Kinetics During Maturation of Human Promyelocytic Leukemia Cells

Differentiation of human promyelocytic leukemia cells (HL‐60) in response to several classes of inducing agents is characterized by the sequential appearance of granulocytic or monocytic markers. Compounds that increase intracellular cAMP in HL‐60 cells induce a program of maturation in which cells demonstrate early functional phagocytic properties.

Inhibition of 125I-chemotactic peptide uptake by protease inhibitors.

Abstract A chymotrypsin inhibitor, 1-tosylamido-2-phenylethyl chloromethyl ketone and two chymotrypsin substrates, L-tyrosine-benzyl ester-p-tosylate and L-phenylalanine-benzyl ester-p-tosylate, irreversibly inhibited the uptake of formyl-Nle-Leu-Phe-Nle-Tyr( 125 I)-Lys by human neutrophils. The carboxylproteinase inhibitor pepstatin A reversibly inhibited the same process and induced chemotaxis of human neutrophils. For pepstatin A, a close correlation was found between inhibition of formyl peptide uptake and chemotaxis demonstrating that the biological activity of pepstatin A was mediated through the formyl peptide chemotactic receptor. The chymotrypsin inhibitors and substrates were one to two logs more potent as inhibitors of chemotaxis than as inhibitors of formyl peptide uptake demonstrating that the mechanism of inhibition of chemotaxis was independent of inhibition of uptake.

Transcription interruption may be a common mechanism of c-myc regulation during HL-60 differentiation

Human promyelocytic leukemia cells (HL-60) differentiate along a monocytoid pathway in response to recombinant human tumor necrosis factor or recombinant human interferon gamma. Together, these agents act synergistically to induce phenotypic differentiation. Since reduced expression of mRNA for the proto-oncogene c-myc correlates with differentiation of HL-60 cells induced by other agents, we tested the abilities of tumor necrosis factor and interferon gamma to regulate expression of c-myc mRNA. Tumor necrosis factor rapidly and effectively reduced c-myc mRNA levels. In contrast, interferon gamma did not affect c-myc mRNA levels, nor did it show synergy with tumor necrosis factor in reducing c-myc. Transcription run-on studies confirmed that tumor necrosis factor caused interruption of c-myc transcription after exon 1. Phorbol diesters also caused interruption of transcription of c-myc. Thus, interruption of transcription may be a common mode of regulation of c-myc during induced differentiation of HL-60 cells.

Rapid purification of chicken gizzard α-actinin

Abstract A method of purification has been developed which yields highly purified α-actinin and requires less than one day to complete. The α-actinin is extracted from washed chicken gizzard muscle with water at 37°. Actin and a 55,000 dalton protein are quantitatively precipitated from the extract with 20 mM MgCl2. The α-actinin is subsequently precipitated from the extract by 30% (NH4)2SO4 and fractionated on DEAE cellulose. Spontaneous protein aggregation is prevented by adding 10% glycerol.

Affinity labeling of the Fc receptor on human monocytes using bifunctional cross-linking agents☆

To affinity label the Fc receptor on human monocytes, Fc fragments of monoclonal human IgG1 radiolabeled with iodine 125 were covalently bound to the surface of intact monocytes using a variety of bifunctional cross-linking agents including ethylene glycol bis(succinimidyl succinate), dithio-bis-(succinimidyl proprionate), maleimidobenzoyl N-hydroxysuccinimide, glutaraldehyde and dimethyl suberimidate. After cross-linking, cells were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. Each of these cross-linkers caused a portion of cell-bound Fc fragments to form a covalent complex with a monocyte membrane component. This complex migrated on electrophoresis with an apparent molecular weight of 120,000. Deducting the molecular weight of Fc fragments alone (53,000) the molecular weight of the second component of the complex therefore was about 67,000. A similar estimate of receptor size also was obtained after reduction with dithiothreitol. Complex formation was potently inhibited by unlabeled Fc fragments, IgG1 or IgG3, all of which would be expected to compete with Fc fragments for IgG Fc receptor on human monocytes, but was not inhibited by Fab fragments, IgG2 or IgG4, which do not bind avidly to this receptor. By quantitating the amount of complex formed in the presence of varying concentrations of labeled ligand, it could be demonstrated that complex formation was saturable, and that Fc fragments formed complexes with avidity comparable to that with which Fc fragments bound to receptors on intact monocytes. The findings establish the feasibility of using radiolabeled Fc fragments to affinity label the IgG Fc receptors on human leukocytes. Potential advantages of this approach to studying receptor structure are discussed.