Adriano Antonucci

HIF-1alpha cytoplasmic accumulation is associated with cell death in old rat cerebral cortex exposed to intermittent hypoxia.

Intermittent hypoxia, followed by reoxygenation, determines the production of reactive oxygen species (ROS), which may lead to accelerated aging and to the appearance of age‐related diseases. The rise in ROS levels might constitute a stress‐stimulus activating specific redox–sensitive signalling pathways, so inducing either damaging or protective functions. Here, we report that in old rat cerebral cortex exposed to hypoxia, the accumulation in the cytoplasm of hypoxic inducible factor 1α (HIF‐1α) – the master regulator of oxygen homeostasis – concomitant with p66Shc activation and reduced IkBα phosphorylation is associated with tissue apoptosis or necrosis. In young cerebral cortex, we hypothesize that the hypoxic damage may be reversible, based on our demonstration of elevated HIF‐1α levels, combined with a low level of IkBα phosphorylation, a decrease in IAP‐1 and a lack of major change in Bcl2 family proteins. These observations are associated with a low level of cell death induced by hypoxia, suggesting that HIF‐1α activation in cortical neurons may produce rescue proteins in response to intermittent hypoxia.

Complex translocations of the Ph chromosome and Ph negative CML arise from similar mechanisms, as evidenced by FISH analysis

The authors report on 13 patients with chronic myeloid leukemia (CML) studied by serial karyotyping and fluorescence in situ hybridization (FISH) of their bone marrow cells. Ten patients had complex translocations of the Ph chromosome while the remaining three were Ph negative. FISH analysis revealed in all 13 patients the translocation of the ABL protooncogene into chromosome 22 at band q11. Moreover, in all complex translocations but one, FISH with a chromosome 22 painting probe demonstrated on one chromosome 9 at band q34 the presence of material from chromosome 22, in addition to signals on the third chromosome involved in complex changes. Therefore, in this study complex translocations appeared as secondary changes resulting from two consecutive translocations with a total of at least four breaks. The first translocation gave rise to the standard t(9;22)(q34;q11). The second one included a break distal to the original breakpoint at band 9q34 and another one on a third chromosome. Furthermore FISH using S1 and S15 probes, mapped at band 22q11.2 or 22q12, gave evidence that in complex translocations the secondary breakpoint on der(9) was in the translocated segment 22q11-qter between bands q11 and q12. FISH analysis also disclosed the presence of material from chromosome 22 on one chromosome 9 in the three patients with Ph negative CML, demonstrating that in these cases a retranslocation between chromosomes 9q+ and 22q- had occurred. Consequently, the four-break mechanism could also be invoked for the three Ph negative CML patients.

Age-related death-survival balance in myocardium: an immunohistochemical and biochemical study.

During ageing, the occurrence of apoptosis is due to a progressive impairment of normal functions, leading to eliminate redundant, damaged or infected cells. Here we report that also in myocardial tissue, ageing, besides reduction of the number of myocytes and of specialized conduction tissue cells, reduction in Ca(++) transport across the membrane, includes the establishment of apoptosis. In particular, the occurrence of this process, which is less represented than we would have expected, is mediated by the balance between the well known Bcl-2 protein family members, Bad, Bax and Bcl-2, related to the pathway PI-3-kinase/AKT-1, which is known to deliver a survival signal. In fact, aged myocardial cells disclose a suboptimal response, which underlines the possibility that they can become more sensitive to damaging factors or diseases, more frequently occurring during ageing, probably due to an exploited molecular control of apoptosis.

p53 and p66 proteins compete for hypoxia-inducible factor 1 alpha stabilization in young and old rat hearts exposed to intermittent hypoxia.

Background: During development and aging, as well as under hypoxia, many cells can adapt to a stressful environment, while others are damaged and die by apoptosis. In particular, intermittent hypoxia, i.e., hypoxia followed by reoxygenation, determines different responses in young and adult myocardia. Objective: In the rat myocardium exposed to hypoxia, the roles played by p53 and p66 Shc proteins in matching, in an age-dependent mode, in stabilizing hypoxia-inducible factor-1α (HIF-1α), and in preventing its biological activity, which usually induces synthesis of rescue proteins against this stress, were investigated. Methods: Five animals from three groups, each consisting of 10 male Wistar rats, 8 days and 3 and 24 months old, were kept under physiological conditions; 5 young and 5 old rats were exposed to intermittent hypoxic challenges (12 h at 10% O2 followed by 12 h at 21% O2) for 8 days. Pregnant rats were kept for 3 days under hypoxic conditions before delivery, and 5 neonate rats were kept in intermittent hypoxia for 8 days. Left ventricles were excised and processed for TUNEL and Western blotting analyses. Results: HIF-1α stabilization by p53 along with decline in Bcl2, substantial caspase-3 expression, and a large number of apoptotic events make the hypoxic young myocardium the most damaged when compared to the neonatal one, in which HIF-1α is not stabilized. Moreover, high expression and activation of p66 in hypoxic young and in normoxic old myocardia suggests a pathological increase of the response to oxidative stress in the former and a physiological progressive increase in the latter. Conclusions: The different responses to hypoxic challenge during life show that the young seem the most reactive and damaged, as is well documented by p53-mediated HIF-1α stabilization. The neonate, not showing any modification in terms of HIF-1α expression and activation, seems ‘adapted’ to such an environment, since it was maintained in hypoxia 3 days before and 8 days after birth. In the old, increasing p66 expression and tyrosine phosphorylation, probably exerting a slight HIF-1α stabilization in the two experimental conditions, provide evidence of longevity and oxidative stress resistance, as suggested by the low number of apoptotic events seen upon hypoxic challenge, and this fact could be due to impairment of oxygen-sensing mechanisms or to adaptation of the cells to apoptosis.

Molecular and morphological modifications occurring in rat heart exposed to intermittent hypoxia: role for protein kinase C α

Abstract Exposure of rats to intermittent hypoxia determines different responses at tissue and cell level. Heart mainly undergoes the effects of hypoxic injury and its response is determined both by the relationship between oxygen supply and demand and by its functional state. Since molecular mechanisms mediate cells sensing and response to low O2 concentration, here we explore the role played by Protein Kinase C α (PKC α) in the signal transduction mechanisms leading to the occurrence of morphological responses in rat neonatal, young and old heart subjected to intermittent hypoxia. Along with a key role for hypoxia inducible factor and vascular endothelial growth factor in the occurrence of continuous state of dynamic adaptation of vasculature, PKC α presumably phosphorylates IkBα in rat normoxic and hypoxic neonatal hearts, supporting the hypothesis of a rescue strategy carried out against hypoxia, together with an hypertrophic response. In hypoxic young heart PKC α activation, paralleled by sustained Bax homodimerization and caspase-3 activation, along with reduced p-IKBα and Inhibiting Apoptosis Protein (IAP) expression, suggests that the young early and deeply undergoes the effects of lowered oxygen tension. In addition, since no modifications concerning PKC alpha driven signalling system are evidenced in both the experimental conditions, we suggest an oxygen impaired sensing during ageing.

Detection of Apoptosis in Peripheral Blood Cells of 31 Subjects Affected by Down Syndrome Before and After Zinc Therapy

Thirty-one patients affected by Down syndrome (DS) were investigated to study the presence of apoptosis in peripheral blood cells in relation to the plasma levels of zinc. Twelve patients had undergone therapy with ZnSO4, while the remaining 19 were untreated. The presence of programmed cell death was evaluated by means of electron microscopy, in situ nick translation (NT), and agarose gel electrophoresis of DNA. These approaches evidenced the presence of apoptosis in peripheral blood cells of patients before therapy with ZnSO4, while after zinc supplementation there was a reduction in the number of apoptotic cells. These results suggest that the process of programmed cell death in peripheral blood cells of patients with Down syndrome is related to the plasma levels of zinc ion.

Cytogenetic survey of 80 patients with acute nonlymphocytic leukemia

The authors report a cytogenetic survey of 80 patients with acute nonlymphocytic leukemia. The prognostic value of chromosome aberrations has been evaluated with three methods. The first one showed that patients with NN or AN bone marrow cellularity have a significantly better prognosis than those with AA cellularity; the second method confirmed the relatively good prognosis for patients with t(8;21) and abnormal 16 and a poor one for those with rearrangements of chromosomes 5 and/or 7. The authors also noted, surprisingly, that patients with hyperdiploidy had a significantly poorer prognosis than those with hypodiploidy and especially pseudodiploidy. The third method showed that patients with very complex karyotypes and a worse outcome than those with simple changes. Finally, they discuss the prognostic value of unusual and/or undeciphered chromosome changes detected in 18 patients, with a mean survival of 9.6 months, showing that these changes have a negative prognostic significance.

A woman with an apparent non‐mosaic 45,X delivered a 46,X,der(X) liveborn female

A liveborn female with a phenotype suggestive of Down syndrome is reported. Cytogenetic lymphocyte analysis showed a 46,X der(X) karyo‐type. Fluorescence in situ hybridization (FISH) with a biotinylated probe specific for chromosome 21 showed no signal on the der(X). This marker was homogeneously painted using a specific probe for X chromosome. In addition, FISH analysis detected telomeres on the rearranged X. Therefore, the probands karyotype was revaluated as 46,X,del(X) (pter p22.2::p11.3 qter). Cytogenetic analysis of 150 lymphocytes in the mother disclosed a homogeneous 45,X karyotype. FISH analysis of interphase nuclei using the X chromosome painting probe showed two domains of different sizes in 0.8% of cells. This led us to study further metaphases in the mother. In one out of 450 metaphases scored, after FISH with the X chromosome painting probe, the del(X) was observed, confirming that the rearranged X chromosome found in the newborn had segregated from a 45,X/46,X,del(X) mother.

Detection of minimal residual disease by polymerase chain reaction in patients with different hematologic diseases treated by bone marrow transplantation

Thirteen male patients affected by different hematologic diseases who underwent bone marrow transplantation (BMT) with female donors were investigated by cytogenetic analysis and polymerase chain reaction (PCR) amplification of a DNA sequence specific for the Y chromosome. In six of these patients, PCR showed the presence of the Y chromosome-related sequence; in only three of these did cytogenetic analysis confirm the presence of mixed chimerism. In the remaining three patients, the results of the PCR were confirmed by in situ hybridization on cell nuclei with a probe for the alpha-satellite of the Y chromosome. We compare results obtained with the two methods and discuss the meaning of the minimal residual disease detected by PCR in patients submitted to BMT.

PKC α-mediated CREB activation is oxygen and age-dependent in rat myocardial tissue

Both hypoxia and aging affect the morphology and the function of rat myocardial tissue. Moreover the heart tries to counteract the impaired function by activating specific signalling cascades. Here we report the involvement of CREB protein in “in vivo” response to hypoxic challenge and during aging in rat hearts. CREB is activated in parallel to HIF-1α nuclear translocation in the young after hypoxia exposure followed by reoxygenation, while this kind of response is not so dramatic in the old, neither in terms of CREB activation, neither in terms of HIF-1α expression and translocation, suggesting in the old the existence of an impaired oxygen-sensing mechanism or an adaptation of the cells to hypoxia. Moreover in the young a PKC α/Erk pathway seems to be involved in the activation of HIF-1α along with CREB, suggesting an attempt of the young to counteract the damage evoked by hypoxia, while in the old a PKC α/p38 MAPK/CREB pathway could determine the occurrence of both aging and aged cell hypoxia response.