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M J Tocci

Publications and source records attributed to M J Tocci.

30 records · Page 2Linked to original sources
PubMed1988

Demonstration by in situ hybridization of dissimilar IL-1 beta gene expression in human alveolar macrophages and blood monocytes in response to lipopolysaccharide.

IL-1 beta is the major form of IL-1 produced by mononuclear phagocytes. To evaluate the possible mechanisms underlying the observation that mature populations of human mononuclear phagocytes as alveolar macrophages are relatively poor IL-1 producers compared with blood monocytes, the expression of the IL-1 beta gene mRNA transcripts was evaluated in LPS-stimulated normal autologous blood monocytes and alveolar macrophages by using a IL-1 beta cDNA probe. Although Northern analysis demonstrated that stimulated monocytes and alveolar macrophages both express 1.8-kb IL-1 beta mRNA transcripts, cytoplasmic dot blot analysis showed that the total IL-1 beta mRNA content in alveolar macrophages was only 38 +/- 5% of that in blood monocytes. In situ hybridization with antisense and sense IL-1 beta RNA probes demonstrated that whereas most of stimulated blood monocytes contained IL-1 beta mRNA transcripts, a significant proportion of autologous alveolar macrophages stimulated in an identical fashion did not express the IL-1 beta gene. Within 4 h after LPS stimulation, IL-1 beta mRNA transcripts were detected in 81 +/- 6% monocytes, whereas only 16 +/- 9% of alveolar macrophages were positive, and by 18 h this had increased only to 43 +/- 15%. Quantification of the size distribution of the IL-1 beta mRNA expressing mononuclear phagocytes demonstrated that, among the population of alveolar macrophages, the cells expressing this gene were not confined to those that were "monocyte-like." These observations demonstrate that there is a heterogeneity among population of mononuclear phagocytes in their ability to express the gene for IL-1 beta, which could explain the differences observed in IL-1 production.

Gene Expression Regulation↗
PubMed1988

Identification of a high-affinity receptor for interleukin 1 alpha and interleukin 1 beta on cultured human rheumatoid synovial cells.

In this report the binding of recombinant human interleukins 1 alpha and 1 beta (rIL-1 alpha and rIL-1 beta) to primary cultures of human rheumatoid synovial cells is measured and compared to the concentrations of these mediators required for stimulation of PGE2 production by these same cells. The average concentration of IL-1 alpha required for half-maximal stimulation of PGE2 was 4.6 +/- 1.5 pM (+/- SEM) (n = 6), whereas for IL-1 beta half-maximal stimulation was observed at a concentration of 1.3 +/- 0.24 pM (n = 6). Both direct and competitive binding experiments were performed. In direct binding experiments, IL-1 alpha bound with a Kd of 66 pM (n = 1), while IL-1 beta bound with a Kd of 4 pM (n = 2). In competitive binding experiments, IL-1 alpha inhibited binding of 125I-IL-1 alpha with a Ki of 33-36 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 51-63 pM (n = 2). IL-1 beta inhibited binding of 125I-IL-1 alpha with a Ki of 2-3 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 7 pM (n = 2). The binding data were best fit by a model specifying a single class of receptors with homogeneous affinity for either IL-1 alpha or IL-1 beta and with an abundance of 3,000-14,000 sites per cell. Autoradiography showed that the vast majority of the synoviocytes within the cultures possessed IL-1 receptors. Comparison of biological response curves with the binding curves indicates that the observed receptors exhibit sufficiently high affinity to mediate the response of human synoviocytes to low picomolar concentrations of IL-1 alpha and IL-1 beta.

Arthritis, Rheumatoid↗
PubMed1987

Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta.

Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes. To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E. coli strain JM105. rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC). Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained. Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta. Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments. Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained. Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC. Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay. Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts. These observations indicate that E. coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.

Amino Acid Sequence↗
PubMed1987

Evaluation of alveolar macrophages in normals and individuals with active pulmonary sarcoidosis for the spontaneous expression of the interleukin-1 beta gene.

We have evaluated the hypothesis that the presence of large numbers of activated helper/inducer T lymphocytes in the lungs of individuals with active pulmonary sarcoidosis is associated with the exaggerated release of interleukin-1 (IL-1) by alveolar macrophages. Evaluation of media from unstimulated cultured sarcoid alveolar macrophages failed to detect IL-1 activity. When parallel cultures of sarcoid and normal alveolar macrophages were stimulated with lipopolysaccharide (LPS), they released similar amounts of IL-1 activity. Using a highly specific polyclonal anti-IL-1 beta antibody and flow cytometry to evaluate cell-associated IL-1 beta, analysis of fresh alveolar macrophages from patients with active sarcoidosis and normal individuals revealed no detectable cell-associated IL-1 beta, but IL-1 beta was present when macrophages from sarcoid patients and normals were stimulated with LPS. Similar observations were made using immunoblot analysis of cell lysates of the same unstimulated and stimulated macrophages. Finally, Northern analysis of alveolar macrophages for IL-1 beta mRNA transcripts demonstrated minimal, but equivalent, amounts of IL-1 beta in both normal and sarcoid macrophages, as compared to the much larger quantities present in LPS-stimulated alveolar macrophages. Thus, while alveolar macrophages of individuals with sarcoidosis are clearly capable of expressing the IL-1 beta gene, these findings suggest that altered expression of the IL-1 beta gene by alveolar macrophages does not play a central role in the exaggerated lung T-cell activation characteristic of sarcoidosis.

Gene Expression Regulation↗
PubMed1986

Organization and expression of the major genes from the long inverted repeat of the human cytomegalovirus genome.

The long inverted repeat (TRL:IRL) of the human cytomegalovirus (CMV) genome is a major transcription unit in productively infected human fibroblasts. Cloned DNA fragments of the CMV IRL and complementary DNA (cDNA) copies of RNAs transcribed from the TRL:IRL were used as probes in RNA filter hybridization experiments to characterize and map the RNAs transcribed from this region of the virus genome. In human fibroblasts, three poly A+ RNAs of 2.7, 2.0, and 1.2 kb were detected during the early (E) and late (L) phases of virus gene expression. Analysis of cloned cDNAs and RNA mapping studies indicate that the TRL:IRL can be divided into three transcriptionally active regions. The most highly transcribed region lies between 0.805 and 0.816 map units and encodes a major abundant poly A+ RNA of 2.7 kb that is expressed at E and L times postinfection (p.i.). The second region spans map coordinates 0.792-0.797 and encodes a 1.2-kb poly A+ RNA that is relatively low in abundance at E times p.i. but achieves nearly the same abundance as the 2.7-kb transcript at L times p.i. The third region encompasses map units 0.796-0.804 and encodes a less abundant poly A+ RNA of 2.0 kb that attains maximum expression at E times. The 1.2- and 2.7-kb RNAs are transcribed in the same direction, while the 2.0-kb RNA is transcribed in the opposite direction. The 2.7-, 2.0-, and 1.2-kb RNAs, as well as 5.7- and 1.8-kb transcripts were detected at immediate early times p.i. when human fibroblasts were treated with cycloheximide, but not in cells treated with anisiomycin.

Anisomycin↗
PubMed1986

Characterization of a major early gene from the human cytomegalovirus long inverted repeat; predicted amino acid sequence of a 30-kDa protein encoded by the 1.2-kb mRNA.

The human cytomegalovirus (CMV) long inverted repeat (TRL:IRL) encodes three major early mRNAs. One of these RNAs is a 1.2-kb transcript that maps between 0.792 and 0.797 map units on the human CMV genome. Primer extension experiments, in addition to nucleotide sequence analyses of cloned cDNA transcripts and human CMV IRL DNA fragments, demonstrated that the 1.2-kb mRNA was not spliced. A single major open reading frame of 254 amino acids was identified, encoding a basic polypeptide of approximately 30 kDa. This polypeptide contains 19% Arg, Lys, and His residues, and would have a net positive charge of 31 at neutral pH. Examination of the deduced amino acid sequence revealed several potential phosphorylation sites and a hydrophobic carboxy terminus which resembles a membrane anchor sequence. In vitro translation of human CMV infected cell RNA, hybrid selected with either cloned cDNA or human CMV IRL DNA fragments specific for the 1.2-kb mRNA, resulted in a unique translation product that migrated on SDS-polyacrylamide gels with an apparent molecular mass of 37 kDa. Potential transcriptional regulatory sequences were also identified upstream of the gene encoding the 30-kDa protein.

Amino Acid Sequence↗
PubMed1984

Effects of the nucleoside analog 2'-nor-2'-deoxyguanosine on human cytomegalovirus replication.

The nucleoside analog 2'-nor-2'-deoxyguanosine (2'NDG) effectively inhibits the replication of several laboratory and clinical isolates of human cytomegalovirus. These isolates included viruses obtained from congenitally infected infants and patients suffering from acquired immune deficiency syndrome. The dose of 2'NDG that inhibited cytomegalovirus plaque formation ranged from 0.1 to 1.6 micrograms/ml. At 10 micrograms/ml, 2'NDG completely blocked the production of virus progeny but not the expression of immediate early and early virus gene functions. Cytomegalovirus DNA was not detectable in 2'NDG-treated virus-infected human embryo lung cells when assayed by CsCl density gradient centrifugation. In contrast, the guanosine analog acyclovir at 100 micrograms/ml did not inhibit the production of virus or the synthesis of cytomegalovirus DNA. In virus-infected cells, 2'NDG and acyclovir at 10 and 100 micrograms/ml, respectively, inhibited the incorporation of [3H]thymidine and 32Pi into cellular DNA by ca. 50%. Uninfected human embryo lung cells grown in these concentrations of acyclovir or 2'NDG exhibited a slightly transient lag phase but, overall, cell growth was not retarded, and there was no decrease in cell viability. The extended lag in cell division was not due to inactivation or breakdown of the antiviral compounds but may be due in part to a temporary decrease in cellular DNA synthesis.

Acyclovir↗
PubMed1983

Cloning of total mRNA populations from adult and embryonic mice.

Total clone banks of cDNAs synthesized from poly(A)-RNA obtained from three stages of the developing mouse were constructed. The stages chosen were 13-day-old embryo, neonatal, and fully grown adult. To have as complete a bank as possible, large numbers of individual clones were generated approximately 400,000 for the 13th day embryo and neonatal mouse and approximately 610,000 for the adult bank. In each case the clone bank was constructed by inserting double stranded cDNA into the PstI site of pBR322 by the "G-C tailing" method. Sequences cloned in this way could be separated from the plasmid host DNA by treatment of the resultant total chimeric plasmid population with PstI. Aliquots of the cloned cDNA material were labeled with 32P by "nick translation" using Escherichia coli DNA polymerase I for the preparation of hybridization probes. Back-hybridization of these probes to the total clone banks allowed the determination of the sequence diversity among the above three very different developmental stages. The use of such clone banks should allow the identification of developmental stage specific mRNAs.

Age Factors↗
PubMed1980

On the cloning of eukaryotic total poly(A)-RNA populations in Escherichia coli.

A total clone bank of cDNAs synthesized from mouse liver poly(A)-RNA was constructed in Escherichia coli K-12 RR1 using the plasmid pBR322. Sequences of cDNAs were inserted into the PST-I site of pBR322 by the "G-C-tailing" method. Bulk preparations of these cDNA sequences were obtained by treatment of the resultant total hybrid plasmid population with PST-I. Aliquots of this cDNA material were then labeled with 32P by "nick translation" using E. coli DNA polymerase I for the preparation of hybridization probes. Experiments involving the back-hybridization of these probes to the total hybrid-plasmid clone bank population revealed that virtually all of the liver poly(A)-RNA sequences were represented in the total clone bank. The long term stability of these sequences residing in RR1 host cells was then examined through extensive serial passage of the initial total clone cell population culture. The results showed: 1) that the small percentage of natural pBR322 molecules (containing no cDNA inserts) usually present in such total clone bank preparations do not outgrow the respectively hybrid specie of plasmids in these populations: 2) few, if any, cDNA sequences are completely lost; and 3) some redistribution of the abundant and more unique DNA sequences probably does occur. The use of total clone hybrid-plasmid populations (constructed from poly(A)-RNA isolated from different tissues) described here should allow the identification of tissue-specific RNA sequences through the use of cross-hybridizaton techniques.

Animals↗
PubMed1979

Susceptibility of lymphoblastoid cells to infection with human cytomegalovirus.

Human lymphoblastoid cells of B- and T-cell origin were examined for their in vitro susceptibility to infection with human cytomegalovirus (CMV). Results of infectious-center assays, at virus to cell ratios of 10, indicated that in each of the lymphoblastoid cell lines tested less than 1% of the cells produced infectious virus. Under these conditions, CMV specific antigens were undetectable. Infection of lymphoblastoid cells with CMV resulted in atypical virus growth curves similar to those obtained with persistently infected human embryonic kidney cells. Although some variation existed in the relative sensitivity of lymphoblasts, cells of B (Raji, P3J-HR-1, RPMI 8226) and T (CCRF-CEM) origin were susceptible to infection with CMV. Variation in the sensitivity of lymphoblasts to CMV infection did not correlate with differences in virus adsorption or the presence of Epstein-Barr virus deoxyribonucleic acid. These studies suggest that human lymphoblastoid cells could serve as a model to examine persistent CMV infection in lymphoid cells of various origin.

Adsorption↗