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Biomedical subjects

L Brambilla

Publications and source records attributed to L Brambilla.

At least 37 records · Page 2Linked to original sources
PubMed1999

NADH-Linked substrate-mediated enhancement of mitochondrial calcium accumulation and DNA single-strand breakage elicited by tert-butylhydroperoxide: the source of the cation is a ryanodine-sensitive calcium store.

We previously found that the membrane-permeant NADH-linked substrates pyruvate and beta-hydroxybutyrate enhance the formation of DNA single-strand breaks induced by tert-butylhydroperoxide (tB-OOH) in intact U937 cells. This effect is mediated by a process involving enforced mitochondrial calcium accumulation in the absence of discernible elevation in the cytosolic concentration of free calcium ions. We now show that the intracellular source of the cation is a ryanodine-sensitive calcium store. A high concentration of ryanodine, which suppressed the caffeine-mediated mobilization of calcium ions, also abolished the effects of the NADH-linked substrates on the mitochondrial accumulation of the cation as well as on the tB-OOH-induced genotoxic response. These data constitute a novel demonstration of a physiological mechanism with important pathological implications.

Caffeine↗
PubMed1999

Human immunodeficiency virus negative Kaposi sarcoma and lymphoproliferative disorders.

BACKGROUND: The concomitant occurrence of more than one primary neoplasm in the same individual has led researchers to seek possible common etiopathogenetic factors. Kaposi sarcoma (KS) is a multicentric neoplasm of vascular origin and perhaps viral etiology. Four forms of KS are known: classic or Mediterranean, endemic or African, posttransplant, and epidemic or acquired immunodeficiency syndrome-associated KS. In its classic form KS mainly affects elderly people and often has a long and indolent course that occasionally allows other malignancies to appear. Previous studies of the possible association between human immunodeficiency virus (HIV) negative KS and lymphoproliferative disorders (LDs) have produced discordant results. METHODS: To verify a possibly significant association between HIV negative KS and LDs, data relating to 250 evaluable Italian patients with HIV negative KS were evaluated retrospectively. RESULTS: Of the 250 KS patients, only 6 (2.4%) were found to have had an LD: 2 with Hodgkin lymphoma, 1 with non-Hodgkin lymphoma, 1 with cutaneous T-cell lymphoma, 1 with acute promyelocytic leukemia, and 1 with B-chronic lymphocytic leukemia. CONCLUSIONS: No significant association was found between HIV negative KS and LDs in the patient population in the current study. The authors believe that age, LD, or therapy-related immunodepression played a role in the cases in which KS appeared after the LD by determining the passing to the lytic phase of the herpes-virus HHV8 already present in anatomic sites of latency/persistence.

Adult↗
PubMed1999

NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess.

Introduction of the Lactobacillus casei lactate dehydrogenase (LDH) gene into Saccharomyces cerevisiae under the control of the TPI1 promoter yielded high LDH levels in batch and chemostat cultures. LDH expression did not affect the dilution rate above which respiro-fermentative metabolism occurred (Dc) in aerobic, glucose-limited chemostats. Above Dc, the LDH-expressing strain produced both ethanol and lactate, but its overall fermentation rate was the same as in wild-type cultures. Exposure of respiring, LDH-expressing cultures to glucose excess triggered simultaneous ethanol and lactate production. However, the specific glucose consumption rate was not affected, indicating that NADH reoxidation does not control glycolytic flux under these conditions.

Aerobiosis↗
PubMed1999

Hepatitis C infection in an Italian population not selected for risk factors.

AIMS/BACKGROUND: This study estimated the prevalence of HCV infection and relationship with viremia in a general population. The inhabitants of Albavilla town were personally invited to participate. METHODS: Out of 3997 inhabitants falling within the age range 18-85 years, 2403 (participation rate 60.1%) were examined for transaminases, HCVAb, HCVRNA, genotype and immunoblot assay. The following information was collected: sex, age, blood transfusions, surgery, use of glass syringes, drug addiction, alcohol consumption, tattoos and body mass index. RESULTS: 115 (4.8%) were HCVAb+, the prevalence being 1.2% under 40 years. Transfusion in the past was the only risk factor for HCV infection. Among the HCVAb+ subjects, 71 (61.7%) were HCVRNA+. 40.8% of the HCVAb+/HCVRNA+ group had normal ALT, compared with 68% of those with HCVAb+/HCVRNA-. The HCV genotypes in the 71 HCVRNA+ subjects were: 2a/2c in 58 (81.7%), 40% of them with normal ALT;1b in 11 (15.5%), none with normal ALT; genotype 3 in two (2.8%). CONCLUSION: The prevalence of HCVAb in this general population was 4.8%. About 3% were HCVRNA positive and of these genotype 2a/ 2c was present in 81.6%.

Adolescent↗
PubMed1999

Replacement of a metabolic pathway for large-scale production of lactic acid from engineered yeasts.

Interest in the production of L-(+)-lactic acid is presently growing in relation to its applications in the synthesis of biodegradable polymer materials. With the aim of obtaining efficient production and high productivity, we introduced the bovine L-lactate dehydrogenase gene (LDH) into a wild-type Kluyveromyces lactis yeast strain. The observed lactic acid production was not satisfactory due to the continued coproduction of ethanol. A further restructuring of the cellular metabolism was obtained by introducing the LDH gene into a K. lactis strain in which the unique pyruvate decarboxylase gene had been deleted. With this modified strain, in which lactic fermentation substituted completely for the pathway leading to the production of ethanol, we obtained concentrations, productivities, and yields of lactic acid as high as 109 g liter(-1), 0.91 g liter(-1) h(-1), and 1.19 mol per mole of glucose consumed, respectively. The organic acid was also produced at pH levels lower than those usual for bacterial processes.

Animals↗
PubMed1998

Protection of U937 cells against oxidative injury by a novel series of iron chelators.

A new series of iron chelators designed to protect tissues against iron-catalysed oxidative damage is described. These compounds are aminocarboxylate derivatives bearing pendant aromatic groups. They were designed to have a relatively low affinity for both ferrous and ferric iron and to be site-specifically oxidizable by hydrogen peroxide through intramolecular aromatic hydroxylation into species with strong iron binding capacity which do not catalyse hydroxyl radical formation. Thus, at the cellular level, oxidative injury is used to convert weak iron chelators into strong iron chelators in order to promote cell survival. The purpose of this local activation process is to minimise toxicity compared to strong iron chelators which may interfere with normal iron metabolism. Compounds within this series were evaluated in vitro in view of their capacity to undergo intramolecular hydroxylation and to protect cultured cells against oxidative injury. Results show that the intramolecular aromatic hydroxylation capacity is critically dependent upon the amino carboxylate chelating moieties and the substituents of the aromatic rings. Cell protection against oxidative injury is only observed with compounds possessing sufficient lipophilicity. The monohydroxylation product of N,N'-dibenzylethylenediamine N,N'-diacetic acid, protects cells against both H2O2 and tBuOOH toxicity with IC50's of 12 and 60 microM, respectively, in agreement with the oxidative activation concept. These results represent the first step toward the development of a new strategy to safe iron chelation for the prevention of oxidative damage.

Ascorbic Acid↗
PubMed1998

Mitochondrial formation of hydrogen peroxide is causally linked to the antimycin A-mediated prevention of tert-butylhydroperoxide-induced U937 cell death.

Antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), both of which bind to the same site of complex III, prevented U937 cell killing promoted by tert-butylhydroperoxide (tB-OOH). This cytoprotection was not directly caused by inhibition of electron transport or reduced formation of tB-OOH-derived toxic species, but rather appeared to be the consequence of a mechanism involving mitochondrial formation of hydrogen peroxide. Ubisemiquinone was most likely the electron donor allowing the formation of superoxides and, as a consequence, of hydrogen peroxide.

Anti-Bacterial Agents↗
PubMed1998

Electron transport-mediated wasteful consumption of NADH promotes the lethal response of U937 cells to tert-butylhydroperoxide.

The toxicity of a short-term exposure to tert-butylhydroperoxide in U937 cells was markedly reduced by chemically or experimentally induced respiratory deficiency. Rotenone mitigated the lethal effects of the hydroperoxide over the same concentration-range in which the complex I inhibitor inhibited oxygen utilization. U937 cells that were made respiration deficient by growing them in the presence of either chloramphenicol or ethidium bromide, were in both circumstances highly resistant to tert-butylhydroperoxide. The improved survival was not a direct consequence of the absence of electron transport, but rather was attributable to the large amounts of NADH which accumulate in the mitochondria of chemically hypoxic or respiration-deficient cells. Indeed, the toxicity elicited by tert-butylhydroperoxide was also abolished by supplementation with either of two different NADH-linked substrates, namely pyruvate or beta-hydroxybutyrate. Accumulation of intramitochondrial NADH, and the resulting cytoprotective effects, was associated with prevention of the loss of nonprotein sulphydryls and mitochondrial membrane potential. Neither rotenone nor pyruvate reduced the toxicity of tert-butylhydroperoxide in thiol-depleted cells. Taken together, these results indicate that depletion of mitochondrial NADH is a critical event in the cytotoxic response to tert-butylhydroperoxide since this pyridine nucleotide prevents mitochondrial dysfunction and cell death caused by the hydroperoxide. As a consequence, in hydroperoxide-treated cells electron transport is highly detrimental since it consumes mitochondrial NADH.

Adenosine Triphosphate↗
PubMed1997

Mechanism of the antimycin A-mediated enhancement of t-butylhydroperoxide-induced single-strand breakage in DNA.

Inhibitors of complex III increased the DNA strand scission induced by t-butylhydroperoxide (tB-OOH) and cumene hydroperoxide but did not affect DNA damage induced by H2O2. The hypothesis that these effects are selectively linked to inhibition of the electron transport from cytochrome b to cytochrome c1 is validated by the following observations: (1) two complex III inhibitors, antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, enhanced the tB-OOH-induced DNA cleavage over the same concentration range as that in which inhibition of oxygen consumption was observed; (2) the complex III inhibitor-mediated enhancement of tB-OOH-induced DNA damage was abolished by the complex I inhibitor rotenone or by glucose omission, and (3) the enhancing effects of antimycin A were not observed in respiration-deficient cells. The mechanism whereby the complex III inhibitors potentiate DNA cleavage promoted by tB-OOH was subsequently investigated with intact as well as permeabilized cells. H2O2, produced at the level of mitochondria via a Ca2+-dependent process, was found to account for the enhancing effects of antimycin A.

Antimycin A↗
PubMed1997

Mitochondrial respiratory chain deficiency leads to overexpression of antioxidant enzymes.

U937 cell growth in the presence of either chloramphenicol or ethidium bromide rapidly leads to respiratory deficiency. The novel finding of this report is that this response is paralleled by a specific increase in Se-dependent and independent glutathione peroxidase activities as well as of glutathione peroxidase and heme oxygenase mRNAs. Under the same experimental conditions, catalase activity and catalase mRNA do not show appreciable changes. These results can be explained by an increased formation of H2O2 at the early times of development of respiratory deficiency followed by induction of antioxidant enzymes.

Antioxidants↗
PubMed1997

Stimulation of oxygen consumption promotes mitochondrial calcium accumulation, a process associated with, and causally linked to, enhanced formation of tert-butylhydroperoxide-induced DNA single-strand breaks.

The NADH-linked substrates pyruvate, L-glutamine, and beta-hydroxybutyrate, while enhancing the rate of oxygen consumption, also increased the formation of DNA single-strand breaks induced by tert-butylhydroperoxide in intact U937 cells. A cause-effect relationship between these two parameters was established by showing that: (a) rotenone, an inhibitor of complex I, abolished respiration and prevented the enhancement of the DNA-damaging response under all the above circumstances; (b) the membrane-impermeant, complex I-activating substrate L-malate gave similar results in permeabilized cells; and (c) none of the NADH-linked substrates affected the DNA-damaging response to tert-butylhydroperoxide in respiration-deficient cells. Stimulation of electron transport potentiated the DNA-cleaving ability of tert-butylhydroperoxide via a process involving enforced mitochondrial calcium accumulation in the absence of a discernible elevation in the cytosolic concentration of free Ca2+. Finally, mitochondrial calcium was found to promote the mitochondrial formation of DNA-damaging levels of hydrogen peroxide. In conclusion, the data herein presented define a previously unexpected role of respiratory substrates in the control of the deleterious effects of an organic hydroperoxide at the level of genomic DNA. The enhanced DNA cleavage mediated by NADH-linked substrates in response to tert-butylhydroperoxide would appear to depend on a sequence of events involving stimulation of electron transport, mitochondrial accumulation of Ca2+, and mitochondrial formation of DNA-damaging levels of hydrogen peroxide via a Ca(2+)-dependent process.

3-Hydroxybutyric Acid↗
PubMed1997

Vinorelbine therapy for Kaposi's sarcoma in a kidney transplant patient.

We report the case of a patient with Kaposi's sarcoma after kidney transplantation. Despite the discontinuation of azathioprine and a reduction in the cyclosporin dosage, the disease continued to evolve, and antineoplastic treatment became necessary. After 14 cycles of vinorelbine chemotherapy, there was a 75% regression of the initial lesions, despite the continuation of cyclosporin A.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Low levels of hydrogen peroxide and L-histidine induce DNA double-strand breakage and apoptosis.

The results presented in this study demonstrate that L-histidine triggers a lethal response in U937 cells exposed to nontoxic, albeit growth-inhibitory, levels of H2O2. Treatment for 1 h with the cocktail H2O2/L-histidine promotes the formation of a low level of DNA double-strand breaks that are rapidly rejoined, and this process is followed by secondary DNA fragmentation at about 7 h of post-treatment incubation, at which time cells are still viable. The appearance of oligonucleosomal DNA fragments associated with the detection of morphological changes typical of apoptosis strongly suggests that a portion of the cells was undergoing an apoptotic process. The relative level of cells with fragmented chromatin never exceeded 15-20% throughout the 20 h post-treatment incubation. Treatment with high concentrations of H2O2 in the presence of L-histidine was found to trigger necrotic cell death. The results presented in this paper provide further experimental evidence in support of the notion that DNA double-strand breaks mediate the lethal effects of the cocktail H2O2/L-histidine and suggest that this type of DNA lesion can promote both apoptotic and necrotic cell death, depending on the concentration of the oxidant.

Apoptosis↗
PubMed1996

Pyruvate enhances DNA single-strand break formation while abolishing cytotoxicity in U937 cells exposed to tert-butylhydroperoxide.

tert-Butylhydroperoxide (tB-OOH) induces killing and DNA single strand breaks (SSBs) in cultured U937 cells. Pyruvate while increasing the rate of oxygen consumption also increased the magnitude of the DNA scission produced by tB-OOH. Rotenone, an inhibitor of complex I, abolished both effects but did not, however, affect the DNA SSB-frequency observed after treatment with tB-OOH alone. These results collectively suggest that pyruvate potentiates the DNA-damaging activity of tB-OOH via stimulation of oxygen consumption. Importantly, under the same experimental conditions, pyruvate was found to abolish both the decline in nonprotein sulfhydryls (NPSH) and the cytotoxicity induced by tB-OOH. Thus, cells with energized mitochondria are more sensitive to the DNA-damaging effects of tB-OOH and display resistance against its cytotoxic effects. As a consequence, DNA SSBs promoted by tB-OOH do not appear to be toxic for the cell.

Cell Line↗
PubMed1996

The respiratory-chain poison antimycin A promotes the formation of DNA single-strand breaks and reduces toxicity in U937 cells exposed to t-butylhydroperoxide.

Antimycin A at levels that abolish oxygen consumption had a slight, although statistically significant, inhibitory effect on the toxicity elicited by t-butylhydroperoxide in U937 cells. The protective effect was observed after 6 h of post-treatment incubation, but was no longer apparent after 24 h. Unexpectedly, these events were associated with a marked accumulation of DNA single-strand breaks produced by low levels of t-butylhydroperoxide. Both an oxygen- and a carbon-centred radical were found to arise during treatment with t-butylhydroperoxide, and their formation was significantly lowered by antimycin A. Thus inhibition of electron transport at the level of complex III appears (a) to decrease the formation of toxic species which mediate, at least partially, the lethal effects elicited by t-butylhydroperoxide, and (b) to enhance the formation of DNA-damaging species generated at low concentrations of t-butylhydroperoxide. Rotenone and cyanide, which respectively inhibit complexes I and IV, did not affect DNA damage elicited by t-butylhydroperoxide. These results suggest that DNA single-strand breaks do not mediate the toxicity of t-butylhydroperoxide, and that specific mitochondrial functions might modulate the formation of the toxic and of DNA-damaging species generated by organic hydroperoxides.

Antimycin A↗
PubMed1996

Cytotoxic impact of DNA single vs double strand breaks in oxidatively injured cells.

Hydrogen peroxide is a potent inducer of DNA single strand breaks (SSBs) in cultured mammalian cells. These lesions, however, are efficiently repaired and do not appear to mediate the cytotoxic response. This inference is based on the observations that a) inhibiting the rate of SSB-removal does not result in an increased cytotoxicity; b) using different experimental conditions it is possible to dissociate the formation of DNA SSBs from the cytotoxic response; c) the induction/loss of the oxidant-resistant phenotype in cell variants characterized by different levels of resistance to the lethal effect of the oxidant does not correlate with resistance to DNA SSB-induction; d) a much larger accumulation of DNA SSBs can be observed following treatment with H2O2 at 4 degrees C, as compared to 37 degrees C, although the opposite is true in terms of cytotoxicity. In the presence of micromolar levels of L-Histidine, H2O2 also induces DNA double strand breaks (DSBs), a type of lesion which we suggest may mediate the lethal event. This conclusion finds experimental support in the following observations: a) DNA DSBs are generated at survival-range concentrations, and a linear correlation exists between the level of this lesion and cytotoxicity; b) this correlation curve overlaps with the curves generated under similar experimental conditions using different cell lines with different sensitivity to the oxidant alone, or different clones derived from the same cell line, some of which showed a high degree of resistance to H2O2. Finally, the formation of DNA DSBs appears to enhance both apoptotic and necrotic cell death.

Animals↗