Alessandra Pica

Occurrence of A-kinase anchor protein and associated cAMP-dependent protein kinase in the inner compartment of mammalian mitochondria.

Evidence showing the existence in the inner compartment of rat‐heart mitochondria of AKAP121 and associated PKA is presented. Immunoblotting analysis and trypsin digestion pattern show that 90% or more of mitochondrial C‐PKA, R‐PKA and AKAP121 is localized in the inner mitochondrial compartment, when prepared both from isolated mitochondria or cardiomyocyte cultures. This localization is verified by measurement of the specific catalytic activity of PKA, radiolabelling of R‐PKA by 32P‐phosphorylated C‐PKA and of AKAP by 32P‐phosphorylated R‐PKA and electron microscopy of mitochondria exposed to gold‐conjugated AKAP121 antibody.

Tumor suppressive activity of a variant isoform of manganese superoxide dismutase released by a human liposarcoma cell line

A cell line derived from a pleiomorphic liposarcoma, named LSA, was previously reported to secrete (a) factor(s) exhibiting oncotoxic properties. The present article describes the isolation, purification and sequence analysis of a protein released by LSA cells into conditioned culture medium. This protein proved to be a variant isoform of manganese superoxide dismutase (MnSOD), hence its designation as LSA‐type‐MnSOD. This LSA‐type‐SOD differed from conventional SODs in its secretion by producer cells, contrasting with the normal localization of SODs in the mitochondrial matrix. Interestingly, during the protein purification process, LSA‐type‐SOD cosegregated with a cytotoxic activity directed against a number of tumor cell lines, as determined under in vitro conditions. This cytopathic effect was most likely due to LSA‐type‐SOD, since it could be fully reproduced using recombinant SOD that was expressed from cDNA clones isolated from LSA cells mRNA preparations and henceforth designated L‐rSOD. In addition to its manifestation in cell lines kept in tissue culture, the oncotoxicity of LSA‐type‐SOD was further reflected in a remarkable capacity of this protein for suppression of mammary tumors in Balb‐C‐FRIII mice. Animals subcutaneously injected with L‐rSOD in the tumor area showed a complete disruption of established mammary carcinomas, as monitored by nuclear magnetic resonance (NMR) scanning. Moreover, metastatic spreading, which was readily detected in the control group, was suppressed in the treated animals. Altogether these data suggest that LSA‐type‐SOD interferes with survival and spreading of neoplastically transformed cells and deserves to be future validated as a therapeutic agent against cancer, either alone or in combination with conventional treatments.

Morphological and biochemical characterization of mitochondria in Torpedo red blood cells

A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.

Co-induction of nitric oxide synthase, Bcl-2 and growth-associated protein-43 in spinal motoneurons during axon regeneration in the lizard tail

In lizards, tail loss transects spinal nerves and the cut axons elongate in the regrowing tail, providing a natural paradigm of robust regenerative response of injured spinal motoneurons. We previously ascertained that these events involve nitric oxide synthase induction in the axotomized motoneurons, suggesting a correlation of this enzyme with regeneration-associated gene expression. Here we investigated, in lizards, whether the cell death repressor Bcl-2 protein and growth-associated protein-43 (GAP-43) were also induced in motoneurons that innervate the regenerated tail in the first month post-caudotomy. Single and multiple immunocytochemical techniques, and quantitative image analysis, were performed. Nitric oxide synthase, GAP-43 or Bcl-2 immunoreactivity was very low or absent in spinal motoneurons of control lizards with intact tail. Nitric oxide synthase and GAP-43 were induced during the first month post-caudotomy in more than 75% of motoneurons which innnervate the regenerate. Bcl-2 was induced in approximately 95% of these motoneurons at five and 15days, and in about 35% at one month. The intensity of Bcl-2 and GAP-43 immunostaining peaked at five days, and nitric oxide synthase at 15days; immunoreactivity to these proteins was still significantly high at one month. Immunofluorescence revealed co-localization of nitric oxide synthase, GAP-43 and Bcl-2 in the vast majority of motoneurons at five and 15days post-caudotomy. These findings demonstrate that co-induction of nitric oxide synthase, Bcl-2 and GAP-43 may be part of the molecular repertoire of injured motoneurons committed to survival and axon regeneration, and strongly favor a role of nitric oxide synthase in motoneuron plasticity.

Plastic changes and nitric oxide synthase induction in neurons that innervate the regenerated tail of the lizard Gekko gecko: I. Response of spinal motoneurons to tail amputation and regeneration.

The lizard tail regenerates after autotomy or amputation. After horseradish peroxidase injections in the regenerate, motoneurons were retrogradely labeled only in the three spinal segments rostral to the amputation, whose spinal nerves are severed by tail loss. The changes in these motoneurons, compared to those of lizards with original intact tails, were investigated 5, 15, and 30 days after caudotomy and at 8 months in lizards with mature regenerates. Morphometric analysis of Nissl‐stained motoneurons rostral to the amputation revealed marked hypertrophy, peaking at 15 days, when chromatolysis and nuclear eccentricity were also evident; motoneuron perikarya remained significantly larger than in controls after tail regeneration. The dUTP nick‐end labeling (TUNEL) stain for apoptotic neurons did not reveal labeled cells in the spinal cord 5 and 15 days after caudotomy. Nitric oxide synthase (NOS) expression was studied with nicotinamide adenine‐dinucleotide phosphate (NADPH)‐diaphorase histochemistry and evaluated quantitatively with densitometry. A few caudal spinal motoneurons were lightly stained in lizards with intact tails. Induction of NADPH‐diaphorase positivity was evident in the vast majority of these cells 5 days after caudotomy and was very marked at 15 and 30 days, during tail regrowth. These data were confirmed by neuronal NOS immunohistochemistry. After tail regeneration, histochemical positivity was markedly down‐regulated in the tail spinal motoneurons but persisted in the majority of these cells. The findings show that in the lizard caudotomy elicits in axotomized caudal spinal motoneurons NOS induction associated with plasticity phenomena and in particular with vigorous regeneration of axons that innervate the regrowing tail. J. Comp. Neurol. 417:60–72, 2000. ©2000 Wiley‐Liss, Inc.

Biophysical and biochemical characterization of a liposarcoma-derived recombinant MnSOD protein acting as an anticancer agent.

A recombinant MnSOD (rMnSOD) synthesized by specific cDNA clones derived from a liposarcoma cell line was shown to have the same sequence as the wild‐type MnSOD expressed in the human myeloid leukaemia cell line U937, except for the presence of the leader peptide at the N‐terminus. These results were fully confirmed by the molecular mass of rMnSOD as evaluated by ES/MS analysis (26662.7 Da) and the nucleotide sequence of the MnSOD cDNA. The role of the leader peptide in rMnSOD was investigated using a fluorescent and/or 68Gallium‐labelled synthetic peptide. The labelled peptide permeated MCF‐7 cells and uptake could be inhibited in the presence of an excess of oestrogen. In vivo it was taken up by the tumour, suggesting that the molecule can be used for both therapy and diagnosis. The in vitro and in vivo pharmacology tests confirmed that rMnSOD is only oncotoxic for tumour cells expressing oestrogen receptors. Pharmacokinetic studies in animals performed with 125I‐ and 131I‐labelled proteins confirmed that, when administered systemically, rMnSOD selectively reached the tumour, where its presence was unambiguously demonstrated by scintigraphic and PET scans. PCR analysis revealed that Bax gene expression was increased and the Bcl2 gene was down regulated in MCF7 cells treated with rMnSOD, which suggests that the protein induces a pro‐apoptotic mechanism.

Plastic changes and nitric oxide synthase induction in neurons which innervate the regenerated tail of the lizard Gekko gecko: II. The response of dorsal root ganglion cells to tail amputation and regeneration.

The lizard tail regenerates after amputation, which severs the spinal cord and spinal nerves. Dorsal root ganglia (DRGs) do not regenerate in the regrowing tail, which is innervated by DRGs rostral to the amputation. With Nissl staining, NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we investigated NOS expression and its relationship with structural changes in DRG neurons of caudotomized lizards. First, by horseradish peroxidase retrograde tracing we here provided evidence that the sensory innervation of the regenerated tail derives only from the three pairs of DRGs rostral to the amputation plane. These ganglia were then analyzed in control animals with original intact tail, at 5, 15 and 30 days after caudotomy, and at 8 months in lizards with mature regenerates. Caudotomy elicited in DRG neurons marked hypertrophy that persisted after tail regeneration. In control ganglia, most neurons were lightly NADPH-diaphorase-positive, a few were unstained or intensely stained. Tail transection elicited marked staining up-regulation, and an increase in the proportion of intensely positive neurons. The staining intensity peaked in DRG neurons at 15 days and was still significantly increased in respect to controls several months after complete tail regeneration. NOS immunoreactivity in DRGs matched the histochemical findings. NADPH-diaphorase positivity was also enhanced in the dorsal horn superficial laminae of the corresponding spinal segments. We demonstrate that transection of the lizard spinal nerves, provoked by tail loss, elicits in the axotomized primary sensory neurons marked NOS enhancement, which accompanies axon elongation in the regrowing tail and persists after the end of this process.

Hematology of the Mediterranean population of sea turtle (Caretta caretta): comparison of blood values in wild and captive, juvenile and adult animals

In order to establish baseline hematological and biochemical values in loggerhead turtles from the Mediterranean Sea, 84 specimens were sampled, comprising 24 wild turtles in good health at the time of capture and 60 turtles tested after indoor rehabilitation at the Sea Turtle Rescue and Rehabilitation Centre of the Zoological Station Anton Dohrn in Naples, Italy. The following parameters were evaluated: red cell counts (RBC, 488–575u2009×u2009103/μL), white cell counts (WBC, 17–24u2009×u2009103/μL) and thrombocyte counts (TBC, 19–49u2009×u2009102/μL), hemoglobin (Hb, 8–14xa0g/dL), hematocrit (Ht, 23–34%), mean corpuscular volume (MCV, 487–723xa0fL), mean corpuscular hemoglobin (MCH, 170–261xa0pg), mean corpuscular hemoglobin concentration (MCHC, 34–42%), white and red blood cell differential counts, and a panel of hematochemical tests, composed of glucose (97–164xa0mg/dL), cholesterol (74–144xa0mg/dL), blood urea nitrogen (35–200xa0mg/dL), uric acid (1–2.7xa0mg/dL), total bilirubin (0.20–0.40xa0mg/dL), GOT (44–184xa0IU/L) and GPT (6xa0IU/L) transaminases, calcium (6.7–8.7xa0mg/dL), and magnesium (3.6–5.4xa0mEq/L). Comparisons of the statistically analyzed data from the turtles which were divided into groups on the basis of age and/or lifestyle (wild or captive) revealed that erythroid parameters attained higher values in captive turtles. This suggested a positive influence of the rich and complete diet fed in captivity upon the hemopoietic process of the turtle. On the other hand, data suggest a more intense and active hemopoiesis in young turtles, compared to adult specimens.

Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies

The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species.

Haemopoietic Regeneration After Autohaemotransplant in Sublethal X-Irradiated Marbled Electric Rays

Abstract: Haemopoiesis of Elasmobranchs provides a useful model for haematopoietic studies owing to the compartmentalisation of various maturative lineages in spleen (erythropoiesis and lymphocytopoiesis), oesophageal wall and epigonal tissue (myelopoiesis and lymphocytopoiesis). The survival capacity and the recovery of peripheral blood cells and haemopoietic organs were studied in Torpedo marmorata Risso subjected to different single doses of total-body X-ray and following autologous haemotransplantation. Torpedoes were found to be very radioresistant animals from the high dose (100 Gy) of X-ray able to produce complete destruction of haematopoietic tissues but no immediate lethality. Here we report a successful autologous peripheral blood transplantation following 100 Gy X-ray that produces death of the control torpedoes from failure of haemopoiesis. The recovery of the circulating red blood cells after autohaemotransfusion was completed in 28 days, whereas that of the white cells was slower. On day 28 after autohaemotransplant the animals displayed recovery of the haemopoietic tissues, as demonstrated by the BUdR method, but irradiated controls displayed no blood cell or haemopoietic regeneration.