Biomedical subjects
Publications and source records attributed to M S Bianchi.
We have characterized the mtDNA in normal and breast cancer tissues from seven patients. Both types of tissue showed point mutation heteroplasmies and extensive deletions of the mtDNA. The analysis of these polymorphisms allowed us to infer the mtDNA composition of the breast cell that underwent malignant transformation.
beta-endorphin (beta-EP) levels increase in maternal plasma starting from the 10th to the 12th week of pregnancy and reach a peak during labor and at delivery. Respiratory autogenous training (RAT) has been acclaimed as one of the most effective non-pharmacological methods of obtaining hypoalgesia and relaxing perineal muscles during labor. In order to study the effects of the RAT method in both maternal and fetal beta-EP levels during labor, 28 pregnant women were enrolled in this study. Fourteen attended a RAT program, while the others (control group) did not. beta-EP levels in both groups were measured in maternal plasma at early and late labor, at delivery and on the 4th day of the puerperium and in umbilical cord blood at birth. From late labor on, beta-EP maternal plasma levels increased significantly less in the RAT group than in the control group. The gap in the beta-EP levels between both groups was still significant at puerperium and in umbilical cord blood. In conclusion, the RAT method favorably modulates the secretion of beta-EP during labor and at delivery. This result supports the effectiveness of the RAT method in reducing both maternal and fetal stress during labor.
The sex determining region Y gene (Sry) is the strongest candidate to be the testis determining factor gene (Tdy). Several South-American Akodon species have two varieties of Y chromosome. One type transmitted via male specimens induces testis development. The second Y variety fails to induce male gonadal differentiation and gives rise to fully fertile XY females. These variant females test positive for Sry. Moreover, sequencing of a partial open reading frame of the conserved region of Sry from males and XY females shows no sequence difference. Sry is two- to sixfold amplified in six of eight akodont species tested. Since Sry amplification was found in species having and not having XY females, amplification apparently does not in itself play a primary role in the origin of sex reversal. The development of fully fertile ovaries in XY Akodon females is not due to a deletion of Sry or to mutations in the Sry segment analyzed in this report. Sex reversal may be due to abnormal expression of this gene at the stage of gonadal differentiation. Alternatively, other genes in the sex-determining pathway may be involved. Several of the Akodon species showing Sry amplification also have amplification of Zfy, which may map to the same region of the Akodon Y chromosome.
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Zinc finger-Y (Zfy) and zinc finger-X (Zfx) genes were analyzed by Southern blotting in male and female specimens of 10 species belonging to the oryzomyne-akodontine stock of Cricetidae rodents. DNA fragments were used as characters to construct a parsimony tree of the genes. Zfx and Zfy trees in general coincide with the evolutionary history of the taxa. Both trees show Oryzomys longicaudatus genes as the outgroup whereas Akodon xanthorrhinus genes are also distant from those of the other species. Oxymycterus rufus and Bolomys obscurus share related sequences, while genes from the other six Akodon species form a group of their own. It was found that 9 out of the 10 species analyzed show Zfy amplification in a range varying from 2 to 24 copies and with a pattern that is clade specific. The estimation of the average changes per character strongly suggests that Zfy has evolved more rapidly than Zfx; our estimates of the rate of nucleotide substitution are 4.6 times higher for Zfy than for Zfx.
The superoxide dismutase (SOD) activities in normal and tumor breast tissues from 14 human females were determined by the epinephrine autoxidation assay. SOD levels showed a marked interindividual variability in normal and malignant cells. However, each donor had a higher SOD activity in cancer than in normal tissue samples. In three cases in which Mn- and CuZnSOD activities were determined, it was found that tumoral increases in SOD were due to increases in both enzymatic forms. Therefore, it seems reasonable to assume a similar situation for all cases in our series. The level of DNA methylation in the SOD-1 gene was assessed in the first four donors. The four cases exhibited full methylation of SOD-1 genes corresponding to normal as well as to cancer cells. It is concluded that the variability in CuZnSOD activities is not related with the state of methylation of the SOD-1 gene. MspI restriction fragment polymorphisms between DNA samples from normal and malignant cells were detected in the four DNA donors. This phenomenon may be due to point mutations changing the frequency of MspI sites or to methylation of the external C in CCGG sequences.
The activity of the antioxidant enzymes catalase (CAT), peroxidases (POD), and superoxide dismutases (SOD) in whole blood and different blood fractions was analyzed in 20 normal human beings and correlated with the chromosomal sensitivity of lymphocytes to bleomycin (BLM) (measured as frequency of dicentric chromosomes per BLM dose). Our results demonstrate that both the physiologic activities of the enzymes and the chromosomal sensitivity to BLM exhibit an ample and significant interindividual variability. An inverse and linear correlation between chromosomal sensitivity to BLM and the concentration of 1) CAT and POD in plasma and 2) SOD in whole blood, erythrocytes, and plasma was found. On the other hand, the chromosomal sensitivity to BLM showed a direct correlation with the concentration of SOD and POD in mononuclear leukocytes. It is suggested that a determination of antioxidant enzyme (AOE) activities in a given cell population may serve to predict the chromosomal sensitivity to BLM.
Out of 1172 females recruited in a screening program for genital infections, 144 (12.28%) were cervical and/or urethral positive for Chlamydia trachomatis (Ct) by enzyme immunoassay (EIA). Patients positive for Ct showed no significant differences in terms of demography, sexual practices and clinical evidence as compared to a control group formed by Ct-negative females randomly selected. Historical data showed a higher frequency of previous pelvic inflammatory disease (PID) in patients as compared to controls. 11 of the 94 patients' partners tested were sperm positive for Ct. Of the 63 patients for whom therapeutic data were available, 38 were treated with josamycin, 16 with tetracycline and the others with different drugs. After treatment, EIA for Ct was negative for 92.1% of the patients treated with josamycin and for 68.7% of those treated with tetracycline. The results of this study confirm a high prevalence of asymptomatic Ct infection which may be correctly diagnosed by EIA performed on cervical and urethral samples. They also indicate that negative test results can be obtained by an appropriate antibiotic treatment.
The dependence syndrome concept, developed for alcohol and extended to other abused substances, postulates that its elements form a continuum of severity. Dependence severity should predict treatment success if this concept has predictive validity. Because most studies that examined this aspect of validity used alcoholic populations, we undertook the present 1-year follow-up of 48 opiate addicts to investigate whether dependence severity predicted program retention, treatment compliance, and psychological functioning. We also controlled demography, withdrawal severity, and substance use frequency. The best predictor of program retention was the addicts' rating of substance use frequency. Severity of opiate dependence predicted program retention in White addicts only. For non-White opiate addicts, concurrent depression and cocaine abuse had much greater association with program retention compared to dependence severity.
COLO320DM and COLO320HSR are cell lines derived from a human colon carcinoma. Both lines have an amplification of the MYC oncogene: COLO320DM in the form of double minute chromosomes, COLO320HSR as a large marker chromosome with homogeneously staining regions. We have used pulsed field gel electrophoresis (PFGE) to analyze undigested DNA from both COLO320 cell lines. Upon blotting and hybridization with a MYC probe, the autoradiograms showed the existence of discontinuities in the amplified DNA domains. Additional evidence indicating a preferential concentration of DNA breaks in the region containing the MYC amplicons was obtained with the alkaline unwinding technique. We propose that amplicons are connected by hairpin formation or Hoogsteen pairing between free DNA ends.
We have used a modification of the Southern hybridization method to analyze the removal of UV-induced pyrimidine cyclobutane dimers from the domain of the c-myc oncogene. The study was performed in human COLO320HSR cells, which exhibit a 30- to 40-fold amplification of c-myc that is maintained in a marker chromosome as a homogeneously staining region. Intron 2 and the region upstream from the gene showed better dimer removal than intron 1 or the region downstream from the c-myc gene. Regions showing less repair coincide with regions that are hotspots for mutations and chromosome translocations. Therefore, it is proposed that the inefficiency of DNA repair may play an important role in the origin of c-myc rearrangements.
Growing monolayer COLO320 human cells having a 30- to 40-fold amplification of a MYC domain were pulse treated for 15 min with increasing doses of bleomycin (BLM). Cellular DNA was extracted at the end of the BLM treatment or after an interval of 30 min, during which the cells were allowed to repair the DNA damage at 37 degrees C in culture medium without BLM. Damage and repair in total cellular DNA was assessed by alkaline unwinding and by neutral and alkaline gel electrophoresis. The response to BLM in the domain of MYC oncogene was evaluated by Southern blotting of EcoRI-digested DNAs separated by neutral or alkaline gel electrophoresis. We found that MYC domains from COLO320HSR showed a higher frequency of double-strand DNA breaks than MYC domains from COLO320DM cells. At the level of total cellular DNA, both cell lines showed the same frequency of double-strand nicks. No repair of double-strand breaks was observed. Total DNA from COLO320HSR cells was more sensitive to single-strand breakage than DNA from COLO320DM. At the gene level, the frequency of single-strand scissions was higher in COLO320DM than in COLO320HSR MYC domains. Both cell lines had good capability to close single-strand scissions. However, at high BLM doses the damage was more efficiently repaired by COLO320DM cells. We propose that chromatin organization in total cellular DNA and in the amplified MYC domain probably plays an important role in the DNA response to BLM.
The karyotype of moose (2n = 68) is characterized by very large C-bands close to the centromeres of most chromosomes. The C-banded material represents 40% of the genome. For further characterization of the heterochromatin chromosome spreads were treated with restriction endonucleases and the restriction enzyme (Re) banding pattern was analyzed. HaeIII, AluI, MboI, RsaI and HinfI produced informative Re-bands. DdeI induced an even digestion with no banding. Staining with chromomycin A3 produced bright fluorescence in regions corresponding to C-bands. Labeling with BrdUrd during late S phase differentiates four regions in the C banded area. The sequence of these regions from centromere to telomere are: late, early, late and early replicating. The authors propose the existence of five satellite DNA families with distinctive characteristics of G-C and A-Trichness and different replication timing, and point out the different clusters for the endonucleases detailed above and their varying location in the chromosomes examined.
The baseline sister-chromatid exchange (SCE) frequencies of human plasma lymphocyte cultures (PLC), but not pig PLC, were nearly twice as high as those of whole-blood cultures (WBC). Addition of human red blood cells (RBCs) to human PLC decreased the SCE frequency in proportion to the RBC-leukocyte co-incubation interval. When the period of RBC-leukocyte co-incubation was equivalent to the total length of the culture period (72 h), the SCE frequency was similar to that observed in WBC. Shorter co-incubation periods yielded SCE frequencies intermediate between those of PLC and WBC. Regardless of the species, cell proliferation was slower in PLC than in WBC. Experiments where RBCs were added to PLC showed that the time sequence of RBC incorporation also affects the cell-cycle progression of human and pig lymphocytes. When either human or pig RBCs were added immediately after PLC stimulation, the cell-cycle kinetics was similar to that of WBC. Shorter co-incubation periods made cell-cycle progression intermediate between PLC and WBC values. Thus, PBCs modulate the baseline frequency of SCEs in human PLC and the cell-cycle progression of both human and pig lymphocytes in a time-dependent manner. Two possible hypotheses for the heightened frequency of SCEs of human lymphocytes in RBC-free cultures were assessed. The loss of RBC-to-lymphocyte cellular contact in PLC did not influence the SCE frequencies of lymphocytes. Finally, the increase of SCEs in human PLC could not be related to differences in the generation time of lymphocytes in culture.
Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes in removing UV damage from constant kappa and mu genes.
COLO320DM and COLO320HSR are two human cell lines with 30-40-fold c-myc amplification. Half of the c-myc gene copies in COLO320DM are truncated and expressed as the predominant mRNA, while half are normal. Most c-myc copies in COLO320HSR are normal and expressed. Truncated c-myc genes are fully methylated, while the normal ones are fully demethylated irrespective of their stage of expression. The normal transcriptionally active c-myc from fibroblast cells is fully methylated, while c-myc from granulocytes (probably downregulated) is almost fully demethylated. These results indicate a lack of correlation between expression and the state of methylation for human c-myc oncogenes. Furthermore, exons 1 and 2 and intron 1 of c-myc are CpG-rich islands. Since these islands are constitutively demethylated, it is assumed that demethylation is the constitutive state and methylation the facultative state of the c-myc oncogene.
The concentration of the anti-oxidant enzymes catalase (CAT), peroxidases (POD) and superoxide dismutases (SOD) in different blood fractions, and the chromosomal sensitivity of lymphocytes to bleomycin-induced free radicals (expressed as frequency of dicentrics per bleomycin dose) were analyzed in 10 normal human donors. Our results demonstrate that the physiological concentration of the enzymes as well as the chromosomal sensitivity exhibited a wide interindividual variability. An inverse correlation between chromosomal sensitivity to bleomycin and SOD concentration in whole blood, plasma and red cells was found. On the other hand, no correlation between the yield of bleomycin-induced dicentrics and the concentration of CAT or POD was detected in any of the blood fractions analyzed. These findings suggest that the concentration of SOD may play an important role in the cellular susceptibility to DNA damage by free radicals.
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