Biomedical subjects
N Soares
Publications and source records attributed to N Soares.
Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.
A vaccination strategy designed to enhance the immunogenicity of self-antigens that are overexpressed in tumor cells is to identify and slightly modify immunodominant epitopes that elicit T-cell responses. The resultant T cells, however, must maintain their ability to recognize the native configuration of the peptide-MHC interaction on the tumor cell target. We used a strategy to enhance the immunogenicity of a human CTL epitope directed against a human self-antigen, which involved the modification of individual amino acid residues predicted to interact with the T-cell receptor; this strategy, moreover, required no prior knowledge of these actual specific interactions. Single amino acid substitutions were introduced to the CAP1 peptide (YLSGANLNL), an immunogenic HLA-A2+-binding peptide derived from human carcinoembryonic antigen (CEA). In this study, four amino acid residues that were predicted to potentially interact with the T-cell receptor of CAP1-specific CTLs were systematically replaced. Analogues were tested for binding to HLA-A2 and for recognition by an established CTL line directed against CAP1. This line was obtained from peripheral blood mononuclear cells from an HLA-A2+ individual vaccinated with a vaccinia-CEA recombinant. An analogue peptide was identified that was capable of sensitizing CAP1-specific CTLs 10(2)-10(3) times more efficiently than the native CAP1 peptide. This enhanced recognition was shown not to be due to better binding to HLA-A2. Therefore, the analogue CAP1-6D (YLSGADLNL, Asn at position 6 replaced by Asp) meets the criteria of a CTL enhancer agonist peptide. Both the CAP1-6D and the native CAP1 peptide were compared for the ability to generate specific CTL lines in vitro from unimmunized apparently healthy HLA-A2+ donors. Whereas CAP1 failed to generate CTLs from normal peripheral blood mononuclear cells, the agonist peptide was able to generate CD8+ CTL lines that recognized both the agonist and the native CAP1 sequence. Most importantly, these CTLs were capable of lysing human tumor cells endogenously expressing CEA. The use of enhancer agonist CTL peptides may thus represent a new efficient direction for immunotherapy protocols.
[Prevalence of several cardiovascular risk factors in a population in the municipality of Viseu].
STUDY OBJECTIVE: To assess the prevalence of the main changeable cardiovascular risk factors (hypertension, hypercholesterolemia and smoking) in the population of the Viseu municipality. MATERIAL AND METHODS: The population was obtained through a publicity campaign in local radios and on lighted placards in the town of Viseu. We chose a set of volunteers above 20 years of age, who answered a questionnaire about smoking habits, academic qualifications, profession and residence. After the inquiry, the total cholesterol and blood pressure were determined. We considered hypertension (HBP) values > or = 140/90 mmHg and hypercholesterolemia > 200 mg/dl. RESULTS: 1852 persons were inquired (3.2% of the population of the Viseu municipality) 1173 of which were females. According to the age groups, we verified that the ages between 50 and 69 years represented 47.9% of the total amount of volunteers. In what concerns smoking habits, we found a prevalence of 9.1% (15.9% in males and 5.2% in females). In the study of the prevalence of HBP we found a value of 38.5%, higher in males (42.8%) than in females (35.9%). The prevalence of hypercholesterolemia found was 34.9% (no significant differences between the sexes). CONCLUSION: In comparison with other studies carried out in the Portuguese population, a low prevalence of smoking habits, high hypertension and similar hypercholesterolemia were found.
Chromosomal protein HMG-14. Identification, characterization, and chromosome localization of a functional gene from the large human multigene family.
The human HMG-14 multigene family is one of the largest retropseudogene families known. To identify and isolate a functional human HMG-14 gene, genomic clones, selected with the cDNA, were screened with a set of 6 oligonucleotides. A single genomic clone was isolated suggesting that the human genome contains few, and perhaps only one, functional genes. An 8882-base pair (bp) genomic clone containing the complete, 6804-bp-long human gene together with 850 bp 5' to the start of transcription and 1228 bp 3' to the end of transcription was sequenced. The gene is comprised of 6 exons ranging in size from 30 to 839 bp, two of which code for the entire DNA binding site of the protein, and has several features typical of "housekeeping" genes. Using human-rodent somatic cell hybrids, the HMG-14 gene was localized to human chromosome 21. A restriction fragment length polymorphism, useful for further analysis and mapping, has been detected. The present article, which describes the first isolation and characterization of a gene coding for chromosomal protein HMG-14, indicates that genes coding for HMG-14 and HMG-17 may share several distinctive characteristics. Comparison with the human and chicken HMG-17 genes reveals that all contain 6 exons, that all have exons of similar size, that all have 5' regions highly enriched in GC residues and that all have features typical of housekeeping genes.
Mouse non-histone chromosomal protein HMG-17 cDNA sequence.
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Modulation of the cellular ratio of chromosomal high mobility group proteins 14 to 17 in transfected cells.
The cDNAs coding for human nonhistone chromosomal high mobility group (HMG) proteins 14 and 17 have been introduced into the eukaryotic expression vector pSVL under the transcriptional control of the SV40 late promoter and the constructs used to transfect COS cells. Transfection with plasmid pSVL14s, containing the HMG-14 cDNA in the sense orientation, increased the endogenous levels of HMG-14 mRNA 50-fold and the levels of HMG-14 protein 3-fold. Transfection with pSVL17s, which contains the HMG-17 mRNA in the sense orientation, resulted in a 19-fold increase in mRNA levels and a 3-fold increase in the protein level. Transfection with pSVL17as, containing the HMG-17 in the antisense orientation, resulted in a noticeable decrease in the protein levels. The overproduction of HMG mRNAs does not affect the level of other cellular mRNAs and the increase in the cellular level of either HMG-14 or -17 did not affect the level of the other HMG or that of any other cellular protein examined. The results suggest that COS cells can tolerate large excess of HMG mRNAs and protein, that the relative amounts of HMG-14 and HMG-17 and their mRNAs are not constant, and that neither the transcription nor the translation of the proteins is coordinately regulated.
Cell cycle regulated synthesis of an abundant transcript for human chromosomal protein HMG-17.
The abundance and cell cycle dependent expression of the mRNA for human nonhistone protein HMG-17 were studied in synchronized HeLa cells. Slot blot analysis indicates that the HMG-17 mRNA is a very abundant message, significantly more so than histone or actin mRNA. RNA prepared from tissue culture cells contains higher amounts of HMG-17 transcripts than RNA prepared from liver suggesting a correlation between the rate of cell division and HMG-17 mRNA levels. HMG-17 mRNA is present in the cells throughout the cell cycle however there is a significant increase in the mRNA levels late in S phase suggesting that the protein is deposited on chromatin after nucleosome assembly. Synthesis of the HMG-17 transcript is not coupled to DNA replication suggesting that the cell cycle related expression during late S phase is regulated in a different manner from that of the nucleosomal histones.
Chromosomal protein HMG-17. Complete human cDNA sequence and evidence for a multigene family.
Antibodies elicited against chromosomal protein HMG-17, purified from calf, were used to screen a human lambda gt11 cDNA expression library and isolate the full length cDNA coding for this protein. Sequence analysis reveals that the nucleotide distribution along this cDNA is highly asymmetric. The amino acid sequence, deduced from the reading frame, reveals that the human HMG-17 is, respectively, 96 and 92% homologous with the calf and chicken protein. The amino acid substitution are conservative suggesting evolutionary constraints on the conformation of the protein. The human genome contains 35-50 HMG-17 gene copies which, as revealed by Southern analysis, are distributed at several loci. Northern analysis of total RNA isolated from 3 human cell lines, indicates that each cell contains a single-size mRNA coding for this protein. Nucleotide sequences which cross-hybridize, under stringent conditions, with the human HMG-17 cDNA are present in the genome of rodents and absent from the genomes of sea urchin, Drosophila, and yeast. The availability of a probe for the HMG-17 gene may help elucidate the cellular role of this protein which may confer specific conformations to transcribable regions in the genome.
Differential binding of chromosomal proteins HMG1 and HMG2 to superhelical DNA.
The binding of chromosomal proteins HMG1 and HMG2 to various DNA structures was examined by a nitrocellulose filter binding assay using a 32P labelled supercoiled plasmid. Binding assays and competition experiments indicated that HMG2 has a higher affinity than HMG1 for supercoiled DNA. Studies at various ionic strengths and pH values reveal differences in the interaction of the two proteins with DNA. The results suggest that HMG1 and HMG2 are involved in distinguishable cellular functions.
Antibodies to 5 M urea soluble chromosomal proteins from HeLa cells.
The tissue specificity of a chromosomal protein fraction, extractable from chromatin with 5 M urea at low ionic strength, has been examined in HeLa, A549 and HT 29 cells. Electrophoresis in polyacrylamide gels indicates that each cell type has a different content of 5 M urea soluble proteins which are distinguishable from the histones, from the tight DNA-binding proteins and from the high-mobility-group chromosomal proteins. Antibodies against 5 M urea soluble proteins extracted from HeLa cells were produced in mice. Although each of the mice tested prior to immunization contained a detectable amount of antibodies against both the 5 M urea soluble proteins and tight DNA-binding proteins, immunization elevated the level of the antibodies in the serum over 100-fold. The antibodies do not distinguish between the 5 M urea extracts obtained from different sources because most of the antibodies are directed against antigens shared by the cells studied. Immunofluorescence studies reveal that components which cross-react with 5 M urea soluble chromosomal proteins are also present in the cytoplasm. We conclude the following. (1) 5 M urea extracts from chromatin a group of proteins which differs among cells. (2) Mice contain detectable amounts of autoantibodies against these chromosomal proteins. (3) Immunization with the 5 M urea extractable fraction elicits antibodies against a restricted number of antigenic components which are shared among the cells studied. (4) 5 M urea extractable proteins are found both in the nucleus and cytoplasm; part of these may be cytoskeletal elements. Because the antisera do not react with histones, high-mobility-group proteins and tight DNA-binding proteins, they may be used for various functional studies on the 5 M urea extractable chromosomal protein fraction.
Localization of HMG chromosomal proteins in the nucleus and cytoplasm by microinjection of functional antibody fragments into living fibroblasts.
We have used microinjection and cell fractionation to localize the chromosomal high mobility group proteins (HMG) in human fibroblasts. Electrophoretic analysis of nuclear and cytoplasmic fractions from the fibroblasts indicates that the concentration of HMG-1,2 in the cytoplasm is 2.9 times larger than in the nucleus indicating that the majority of the cellular HMG-1,2 is present in the cytoplasm. In contrast, HMG-17 remains predominant in the nuclear fraction. We conclude that the cellular distribution of HMG-1,2 is significantly different from that of HMG-17. To avoid possible artifacts due to cell fractionation, fluoresceinated HMG-1 and HMG antibodies were microinjected into living fibroblasts. The cellular distribution of the injected proteins was monitored using fluorescent microscopy. Fluoresceinated HMG-1 microinjected into the cytoplasm moves very rapidly into the nucleus and concentrates in the nucleolus of living human fibroblasts. However, some control non-nuclear proteins also migrated into the nucleus raising the possibility that exogenous injected proteins do not always distribute in the same pattern as the endogenous proteins. The localization of microinjected F(ab)2 fragments derived from anti-HMG-1 was compared to that of microinjected F(ab)2 derived from anti-histones. Whereas the anti-histone F(ab)2 when injected into the cytoplasm migrated into the nucleus, the anti-HMG-1 F(ab)2 remained in the cytoplasm. Microinjection of anti-HMG-17 and anti-histone inhibited transcription in living cells, anti-HMG-1,2 did not. We conclude that HMG-1,2 proteins are present in both the nucleus and cytoplasm of living fibroblasts.
Effect of X-radiation on DNA and histone synthesis in ataxia telangiectasia and normal lymphoblastoid cells.
The possibility that the radiosensitivity of lymphoblastoid cell lines from patients with ataxia telangiectasia (A-T) is due to an aberrant content of histones has been examined. The histone pattern of lymphoblastoid cell lines derived from A-T patients was found to be indistinguishable from that obtained from normal individuals. X-ray irradiation led to a greater decrease in cell growth rate in the A-T cells than in the normal cells but was accompanied by a greater decrease of DNA synthesis rate in the normal cells. This difference in radiosensitivity was not reflected in differences in the content or rates of synthesis of histones or of major non-histone proteins in these cells. Reduction in the rate of DNA synthesis was not associated with the appearance of the lysine-rich histone variant H1. We conclude that the hypersensitivity to ionizing radiation in A-T cells is not due to fundamental differences in the composition or synthesis of the major chromosomal proteins.
Antigenic determinants of high mobility group chromosomal proteins 1 and 2.
The antigenic determinants of nonhistone high mobility group chromosomal proteins 1 (HMG-1) and 2 (HMG-2) were studied with rabbit antisera elicited against HMG-1 and against HMG-2 and monoclonal antibodies elicited by HMG-1. The monoclonal antibodies did not distinguish between the two proteins, suggesting that they have specificity toward a shared determinant. Whereas anti-HMG-1 did not, anti-HMG-2 did distinguish between the proteins, suggesting that the anti-HMG-2 serum contains antibodies against peptides which differ between the proteins. Peptides were generated from HMG-1 and HMG-2 by controlled digestion with trypsin and pepsin. Analysis of the digests by ELISA and by sodium dodecyl sulfate electrophoresis followed by diazobenzyloxymethyl transfer, antibody binding and autoradiography revealed that most of the antibodies are against sequential determinants some of which are smaller than 3000 in molecular weight.
Response of human hematopoietic precursor cells (CFUc) to hyperthermia and radiation.
Currently, whole-body and local hyperthermia is being evaluated in clinical studies as a potential method of cancer treatment. Since the hyperthermic sensitivity of normal human bone marrow cells is not known, we have studied the in vitro response of these cells to two anticancer modalities when administered alone or in combination. Cell survival following various treatment schedules was determined by colony formation of bone marrow cells (CFUc) in soft agar suspensions. Within the survival range studied, a thermal tolerant plateau on the cell survival was not observed for temperatures of 42 degrees or less. However, thermotolerance induction could not be ruled out. In addition, when hyperthermia (42.5 degrees for 1 hr) and radiation (100 rads) were sequenced, the human CFUc survival remained the same regardless of whether the radiation was administered before, during, or after the hyperthermic exposure. Under our experimental conditions, we found the human CFUc to be more radiosensitive (D0 = 84 rads) than what has been reported previously. The radiation survival response of human CFUc was similar for cells irradiated either in vitro or in vivo. The possible clinical implications for these data are discussed.
Plasma and cerebrospinal fluid pharmacokinetics of acivicin in Ommaya reservoir-bearing rhesus monkeys.
Acivicin was administered iv to rhesus monkeys bearing Ommaya reservoirs, and serial blood and cerebrospinal fluid (CSF) samples were collected and analyzed to determine the time course of drug concentrations in these body fluids. After iv doses of 4 or 20 mg/kg (50 or 250 mg/m2), acivicin plasma concentrations demonstrated a rapid initial decline (distribution phase), and then declined exponentially with a terminal (elimination phase) half-life of 3--4 hrs. CSF concentrations increased over a period of 2--2.5 hrs, reaching peak values of 2.0--2.7 micrograms/ml at 20 mg/kg and 0.3--0.5 microgram/ml at 4 mg/kg; thereafter, CSF levels declined in parallel with plasma, with a CSF/plasma concentration ratio of 0.10--0.17. A three-compartment pharmacokinetic model gave a close fit of predicted and observed plasma and CSF concentration data. Significant and predictable CSF penetration by iv administered acivicin in monkeys is consistent with observation of CNS side effects in patients in the phase I clinical trial and suggests that acivicin should be evaluated in the treatment of CNS malignancies and metastases.
Specificity of 51Cr-release cytotoxicity of lymphocytes immune to murine sarcoma virus.
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In vitro cytotoxicity studies of murine sarcoma virus-induced immunity in mice.
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Effect of prior vaccination on experimental blastomycosis.
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