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Biomedical subjects

R Gambari

Publications and source records attributed to R Gambari.

At least 73 records · Page 4Linked to original sources
PubMed1994

Interferon-gamma enhances monoclonal antibody 17-1A-dependent neutrophil cytotoxicity toward colorectal carcinoma cell line SW11-16.

17-1A is a murine monoclonal antibody (MAb) specific for the tumor-associated antigen CO17-1A on colorectal carcinoma cells. One of the tumor cell destruction mechanisms induced by in vivo immunotherapy with MAb17-1A has been claimed to be antibody-dependent cellular cytotoxicity (ADCC) by monocytes and NK cells. In the present study we investigated whether human neutrophils (PMN) could be involved in colorectal carcinoma cell lysis and whether IFN-gamma influences this function. We showed that neutrophils are capable of tumor lysis mediated by MAb17-1A, although to a lesser extent than are the mononuclear cells (PBMC). Neutrophil ADCC was, however, markedly increased in the presence of IFN-gamma. Enhancement by IFN-gamma was also observed for PBMC. ADCC by PMN required the binding of MAb17-1A to Fc gamma RIII (CD16) since anti-Fc gamma RIII MAbs efficiently blocked tumor cell lysis. In contrast, in the presence of IFN-gamma the neutralization of Fc gamma RIII did not affect MAb17-1A-mediated cytotoxicity, suggesting that receptors other than Fc gamma RIII were involved in the process. PBMC cytotoxicity was also inhibited by anti-CD16 antibodies but IFN-gamma did not overcome this effect. Finally, the scavenger enzymes superoxide dismutase and catalase did not block ADCC by PMN or PBMC, indicating that oxidants are not key factors in MAb17-1A-mediated lysis; however, in IFN-gamma-activated PMN the oxygen-dependent mechanism was in part involved in tumor lysis.

Animals↗
PubMed1994

A chromatographic procedure for fully automated isolation of DNA from human whole blood.

Isolation of DNA from peripheral blood constitutes a fundamental step in the molecular diagnosis of genetic disorders, analysis of mutations of oncogenes and detection of nucleic-acid sequences of pathogenic organisms in clinical samples. We investigated whether dextran sedimentation of blood followed by the chromatographic isolation of DNA could be applied to the Biomek-1000 automated laboratory workstation in order to develop a fully automated method of DNA isolation from whole blood. Under our experimental conditions, the Biomek-1000 could perform fully automated DNA isolation from seven human whole blood samples in less than 1.5 h. Our data indicate that the DNA isolated from human blood in this way is suitable for polymerase chain reaction.

Autoanalysis↗
PubMed1994

Polymerase-chain reaction as a tool for investigations on sequence-selectivity of DNA-drugs interactions.

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we performed polymerase-chain reaction (PCR) experiments to study the in vitro effects of distamycin, daunomycin, chromomycin and mithramycin. As model systems we employed the human estrogen receptor (ER) gene and the Harvey-ras (Ha-ras) oncogene, in order to obtain PCR products significantly differing for the A + T/G + C frequency ratio. Distamycin, daunomycin, chromomycin and mithramycin are indeed known to differentially bind to different DNA regions depending upon the DNA sequences recognized. The main conclusion of our experiments is that distamycin, daunomycin, chromomycin and mithramycin inhibit polymerase-chain reaction in a sequence-dependent manner. Distamycin inhibits indeed PCR mediated amplification of AT-rich regions of the human estrogen receptor gene, displaying no inhibitory effects on PCR-mediated amplification of GC-rich sequences of Ha-ras oncogene. By contrast daunomycin, chromomycin and mithramycin were found to inhibit PCR-mediated amplification of the Ha-ras GC-rich oncogene sequences. We propose that polymerase-chain reaction technique could be applied to study the in vivo interactions of DNA-binding drugs to specific genes in intact cells.

Animals↗
PubMed1994

Capillary electrophoresis: detection of hybridization between synthetic oligonucleotides and HIV-1 genomic DNA amplified by polymerase-chain reaction.

The polymerase chain reaction (PCR) is one of the most efficient techniques for measuring the viral load of HIV-infected samples. Determination of the specificity of PCR products is usually based on Southern blotting and hybridization of the amplified DNA to radioactive oligonucleotide probes specific for sequences comprised between the PCR primers. The recent introduction of capillary electrophoresis (CE) for identification of HIV-1 and HTLV-I PCR products appears interesting in light of its reproducibility, sensitivity and because it is fast and suitable for detection of DNA/DNA and DNA/RNA hybrids. We demonstrate that specific hybridization of a HIV-1 oligonucleotide probe to single-stranded DNA obtained by unbalanced PCR is detectable by capillary electrophoresis. This enabled us the application of a one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR. This procedure is simple, reproducible and is suggested as an integral part of automated diagnostic systems based on the use of laboratory work stations for DNA isolation, preparation of PCR reactions and analysis of PCR products.

Base Sequence↗
PubMed1994

First-trimester human trophoblast is class II major histocompatibility complex mRNA+/antigen.

Lack of expression of the polymorphic class I and class II MHC antigens in the cytotrophoblast is one of the major factors determining the privileged immunologic status of the placenta. In this report, we show that first-trimester human placental cytotrophoblast cells display moderate to strong expression of class II MHC (HLA-DR alpha and -DR beta) and Ii chain transcripts, apparently in absence of detectable class II antigens and Ii chain. In addition, DR alpha, DR beta, and Ii mRNAs, but not antigens, are consistently upregulated by IFN-gamma. Constitutive expression and upregulation of mRNAs are detectable in trophoblast cells kept in short term as well as prolonged (2-3 weeks) culture. These results are reminiscent of an analogous mRNA+/antigen- dissociation occurring, in the case of class I MHC gene products, in a subpopulation of first-trimester cytotrophoblast cells. Thus, analogous mechanisms prevent the expression of potentially hazardous class I and II allodeterminants at early stages of semiallogeneic pregnancy.

Blotting, Northern↗
PubMed1994

A set of Alu-free frequent decamers from mammalian genomes enriched in transcription factor signals.

We have recently reported that the statistical analysis of the frequency distribution of short oligonucleotides within mammalian and viral genomes allows the production of sets of DNA sequences enriched in signals for transcription factors. Such statistical approaches could facilitate the identification of new promoter regions playing a role in the transcriptional regulation of gene expression. In the case of mammalian oligonucleotides, we found that the published set of frequent decamers enriched in transcriptional motifs is not suitable for studies on genes of Homo sapiens and evolutionarily related genomes, because it contains decameric sequences belonging to genomic repeats. We report here that most of the decameric sequences of DNA repeats belong to Alu repeats. Accordingly, we produced a subset of Alu-free frequent decamers. In addition, we eliminated from the subset of Alu-free frequent decamers those that are frequently present within other common human repeats, including (GT)n, (AT)n, (CA)n, (ATT)n, (CAA)n and (GTT)n. The Alu-free (repeats-free) subset of frequent mammalian decamers is enriched in signals for transcription factors and allows the identification of putative signals in genes, such as those coding for plasminogen activator, adenosine deaminase and p53, that contain a large number of Alu-like repeats interspersed within our genomic sequences. The newly generated compilation of frequent decamers described here might be used to locate genomic regions playing functional roles in the expression of genes of Homo sapiens and related primates.

Animals↗
PubMed1994

Differential inhibition of DNA/protein interactions by aromatic amidines with 2, 3 and 4 benzamidine residues.

We have recently reported that aromatic polyamidines are powerful inhibitors of in vitro proliferation of tumour cell lines and in vivo tumorigenicity of melanoma cells xenografted into nude mice. Interestingly, we have found that tetrabenzamidines are able to bind DNA, and to inhibit the interaction between transacting factors and specific target DNA sequences. In order to obtain more detailed information on structure-activity relationships, we have analysed the effects of different aromatic polyamidines on the binding of a recombinant protein, the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), to the target sequence of EBV DNA, containing the 12 bp palindromic consensus TAGCATATGCTA. The results obtained suggest that aromatic polyamidines inhibit the interactions between DNA-binding proteins and target DNA sequences with different efficiency, depending (i) on the number of amidine residues and (ii) on the presence of halogen substitutions (Cl, Br or I) on the benzene rings of tetra-benzamidine molecules.

Amidines↗
PubMed1993

Methylation state of the human HLA-DRA gene in T-lymphocytes and B-lymphocytes of transgenic mice. Lack of methylation at one 5'-GCGC site is not required for gene expression.

A consistent lack of DNA methylation at one or both of two GCGC (Hha I) restriction sites in the 5' region of the HLA-DRA gene has been previously documented by the use of methyl-sensitive restriction enzymes in human cells and tissues, irrespectively of their expression of DR alpha products. Evidence presently available, however, does not exclude that a lack of methylation in this region, although not sufficient, might be necessary for gene expression. In this report, we show that only one of the 5'-GCGC sites is protected, although less efficiently than in man, from CG-->mCG modifications in tissues and cells of transgenic mice carrying an expressed single copy of the HLA-DRA gene/diploid genome. We demonstrate that the two 5' GCGC sites of the HLA-DRA transgene are fully methylated in DR alpha- splenocytes (more than 80% T-lymphocytes), while one of them (the most 5' site) is not methylated in a fraction of DR alpha+ splenocytes (more than 95% B-lymphocytes). These results provide evidence that absence of DNA methylation in the 5' region is not necessary for, but might be associated with and possibly secondary to the expression of the DRA gene.

Animals↗
PubMed1993

Sequencing of an RNA transcript of the human estrogen receptor gene: evidence for a new transcriptional event.

Two transcripts of the human estrogen receptor (ER) gene have been described, ER mRNA 1 and mRNA 2, different in their 5' untranslated region. By performing reverse transcriptase-polymerase chain reaction with oligonucleotides specific for the 5' genomic region of the human ER gene we have identified a new ER RNA transcript. The sequence analysis of cDNA from MCF7 breast cancer cells and endometrial human tissues demonstrates that this transcript originates further upstream of the initiation transcription sites so far proposed. Primer extension analysis on RNA from MCF7 cells reveals in the upstream region a possible transcription start site at -3090. In agreement with this result, Northern blot analysis shows, in addition to the canonical 6.3 kb ER mRNA, an ER RNA transcript of approx. 7.4 kb in size. The presence of the additional ER mRNA suggests the existence of a new upstream 5' promoter directing transcription of the human ER gene.

Base Sequence↗
PubMed1993

A new automated method for isolation of genomic DNA from eukaryotic cells.

In this study we determine whether the BIOMEK-1000 Laboratory Workstation (Beckman Instruments, Fullerton, CA) can be used to isolate genomic DNA from eukaryotic cells. The results obtained demonstrate that DNA isolated by BIOMEK-1000 is suitable for amplification of genomic sequences by PCR. Our data prove that automated chromatographic DNA isolation employing the BIOMEK-1000 could be used in molecular diagnosis of human pathologies.

Automation↗
PubMed1993

Use of an automated laboratory workstation for isolation of genomic DNA suitable for PCR and allele-specific hybridization.

In this paper we describe the isolation of genomic DNA by using anion exchange chromatography performed on a Biomek 1000 Automated Laboratory Workstation. This procedure allows the automated isolation of DNA suitable for most molecular analyses employed in diagnosis of genetic pathologies and infectious diseases. The genomic DNA isolated by using the Biomek 1000 was indeed found to be suitable for polymerase chain reaction and allele-specific hybridization.

Alleles↗
PubMed1992

DNA binding activity and inhibition of DNA-protein interactions. Differential effects of tetra-p-amidino-phenoxyneopentane and its 2'-bromo derivative.

In the present study are reported the differential DNA binding activity of the anti-tumor polyamidine tetra-p-amidinophenoxyneopentane (TAPP-H) and its 2'-halo derivative (TAPP-Br), and their effects on the binding of the recombinant Epstein-Barr virus (EBV) nuclear antigen to a synthetic oligonucleotide mimicking the target DNA sequence present in the EBV genome. In addition, the proliferation kinetics and cell cycle analysis of human leukemia K562 cells treated with TAPP-H and TAPP-Br are reported. The possible in vivo relationship between DNA binding affinity and cytotoxicity is also discussed.

Antigens, Viral↗
PubMed1992

Analysis of upstream sequences of the human estrogen receptor gene.

An upstream region of the human estrogen receptor gene (ER) spanning from -2776 to -38 in relation to a previously described exon 1 was sequenced and analyzed for the possible presence of open reading frames (ORF) and regulatory motifs. We identified a long 459 bp ORF beginning at -2301. Most of the transcriptional regulatory motifs present within the ER upstream sequence are distributed either upstream or downstream of this long ORF which displays a TATA box, a CACC motif and a canonical cap site in the 5' flanking region.

Base Sequence↗
PubMed1992

Enrichment of oligonucleotide sets with transcription control signals. II: Mammalian DNA.

We studied the frequency distribution of oligonucleotides 10 bp long in a sample of 1.6 Mb of mammalian genes, containing 579 sequences from GenBank(R) 55.0, with the aim of detecting transcription control signals. 2216 decamers had a frequency higher than 10 times the mean and were subjected to further statistical analysis. For each of the 2216 decamers (parents), we counted the individual frequencies of the 30 decamers differing from the parent by one base mutation (progeny) and then calculated two variance/mean chi squares for the progeny, with and without the parent. We then studied the distribution of the ratio between the two chi squares. Out of 2216 decamers, 346 had a chi square ratio of 1.9 or larger. In this final set, which corresponds to less than 0.033 per cent of all possible decamers, 18 were found to contain 23 eukaryotic transcription control elements 5-10 bp of length, such as Sp1 and others. Furthermore, when compared to 210 random sets containing 346 decamers, this set contains a highly significant excess of the longer signals.

Algorithms↗
PubMed1992

New isoxazole derivatives of retinoids: synthesis and activity on growth and differentiation of tumor cells.

The effects of several newly synthesized isoxazole analogues of retinoids on differentiation and proliferation of 'in vitro' cultured tumor cell lines are reported. Some of the tested compounds exhibit significative differentiating action, inducing adipogenic conversion of the Chinese hamster FH06T1-1 cell line in a range of 2-10 times the activity of retinoic acid and retinol. In addition, most of the compound tested display antiproliferative activity comparable to that of natural retinoids. The reported data could be of interest for experimental anticancer therapy.

Adipose Tissue↗
PubMed1991

Frequencies of codons in histones, tubulins and fibrinogen: bias due to interference between transcription signals and protein function.

The distribution of codons was studied in 65 proteins: 48 histones, 14 tubulins, and three fibrinogens, With the methodology used, (1) we confirmed that the preterminator state of a codon has no detectable effect on codon bias. (2) The well-known effect of CG suppression was visible. We also found that (3) some codons which are very rare, are equal to parts of known transcription signals. Thus, we advanced that to avoid signal interference, the use of these codons is suppressed when a synonymous codon is available. In addition we found that in the whole series of codons, transcription signals are less frequent than in a random sequence of equal composition. Finally we observed (4) that tryptophan is absent in histones. This absence was related not to the TGG codon itself, but to characteristics of the amino acid. We conclude that the functional constraints of a protein can influence, at least for synonymous codon usage, the evolution of its own coding sequence.

Animals↗