Evolutionary conservation of a regulative pathway of erythropoiesis in Poikilothermic vertebrates.

Apoptosis, programmed cell death, plays a central role in haematopoiesis. Mature erythrocytes of non-mammalian vertebrates maintain a permanent nucleus; these cells can undergo apoptosis (eryptosis), as do other somatic cells of a given non-mammalian vertebrate. In this study, we have investigated the expression and subcellular distribution of Bcl-2, Bcl-XL and Bax proteins in the maturation phases and after X-ray irradiation of nucleated erythrocytes of Torpedo marmorata and Caretta caretta and the effect of X-ray irradiation on nucleated circulating erythrocytes of Torpedo marmorata. The cellular distribution of proteins was detected in erythrocytes by using immunocytochemistry at light microscopy and immunoelectron microscopy. The electrophoretic separation and immunoblotting of pro- and anti-apoptotic proteins of immature and mature erythroid cells was performed too, after X-ray irradiation of torpedoes. The results of the immunocytochemical analyses show an increase, in the expression level of Bax in mature as compared to young erythrocytes and a corresponding decrease of Bcl-2 and Bcl-XL. This maturation pattern of Bax, Bcl-2 and Bcl-XL was abrogated in X-ray irradiated torpedo erythrocytes. On the basis of these observations, Bax, Bcl-2 and Bcl-XL seems to play a role in the erythropoiesis of Torpedo marmorata Risso and in Caretta caretta. In conclusion, the same apoptotic proteins of somatic cells appear to be conserved in circulating nucleated erythrocytes thus suggesting to play a role in the maturation of these cells.

Hematopoietic stem cells debut in embryonic lymphomyeloid tissues of elasmobranchs.

The evolutionary initiation of the appearance in lymphomyeloid tissue of the hemopoietic stem cell in the earliest (most primitive) vertebrate model, i.e. the elasmobranch (chondroichthyan) Torpedo marmorata Risso, has been studied. The three consecutive developmental stages of torpedo embryos were obtained by cesarean section from a total of six pregnant torpedoes. Lymphomyeloid tissue was identified in the Leydig organ and epigonal tissue. The sections were treated with monoclonal anti-CD34 and anti-CD38 antibodies to detect hematopoietic stem cells. At stage I (2-cm-long embryos with external gills) and at stage II (3-4 cm-long embryos with a discoidal shape and internal gills), some lymphoid-like cells that do not demonstrate any immunolabeling for these antibodies are present. Neither CD34+ nor CD38+ cells are identifiable in lymphomyeloid tissue of stage I and stage II embryos, while a CD34+CD38- cell was identified in the external yolk sac of stage II embryo. The stage III (10-11-cm-long embryos), the lymphomyeloid tissue contained four cell populations, respectively CD34+CD38-, CD34+CD38+, CD34-CD38+, and CD34-CD38- cells. The spleen and lymphomyeloid tissue are the principal sites for the development of hematopoietic progenitors in embryonic Torpedo marmorata Risso. The results demonstrated that the CD34 expression on hematopoietic progenitor cells and its extraembryonic origin is conserved throughout the vertebrate evolutionary scale.

Evolutionary intraembryonic origin of vertebrate hematopoietic stem cells in the elasmobranch spleen.

The electric ray (Torpedo Marmorata Risso) provides an animal model for the detection of early intraembryonic hemopoietic stem cells in sea vertebrates. The spleen of this bone-marrowless vertebrate appears to be the major site of hemopoietic stem cell differentiation during development and in adulthood. Splenic development in this species was investigated and hemopoietic stem cells were detected in this organ by immunocytochemistry utilizing CD34 and CD38 antibodies. At stage I (2-cm-long embryos with external gills), the spleen contains only mesenchymal cells. At stage II (3-4 cm-long embryos with a discoidal shape and internal gills), an initial red pulp was observed in the spleen, without immunostained cells. At stage III (10-11-cm-long embryos), the spleen contained well-developed white pulp, red pulp and ellipsoids. Image analysis at stage III showed four cell populations, i.e. CD34+/CD38-, CD34+/CD38+, CD34-/CD38+, and CD34-/CD38- cells. The present findings, obtained from an elasmobranch, indicate that the CD34 and CD38 phenotypes are conserved through vertebrate evolution.

Multidisciplinary approach to chest wall resection and reconstruction for chest wall tumors, a single center experience.

BACKGROUND: Chest wall resection and reconstruction (CWRR) is quite challenging in surgery, due to evolution in techniques. Neoplasms of the chest wall, primary or secondary, have been considered inoperable for a long time. Thanks to evolving surgical techniques, reconstruction after extensive chest wall resection is possible with good functional and aesthetic results. METHODS: In our single-center experience, seven cases of extensive CWRR for tumors were performed with a multidisciplinary approach by both thoracic and plastic surgeons. Patients have been retrospective analyzed. RESULTS: Acceptable clinical and aesthetical results have been recorded, with a smooth post-operative course and a low rate of post-surgical complications. Two early complications and one late complication (asymptomatic bone allograft fracture on the site of the bar implant) were recorded. Neither postoperative deaths nor local recurrences were registered after a median follow-up period of 13 months. CONCLUSIONS: Surgical planning is most effective when it is tailored to the patient. Specifically, in the treatment of selected chest wall tumors, the multidisciplinary approach is considered mandatory when an extensive demolition is required. Indeed, here, the radical wide en-bloc resection can lead to good results provided that the extent of resection is not influenced by any anticipated problem in reconstruction.

A new hexapeptide from the leader peptide of rMnSOD enters cells through the oestrogen receptor to deliver therapeutic molecules.

A 24-amino acid leader peptide of a new human recombinant manganese superoxide dismutase can enter cells and carry molecules. Here, we demonstrated that six of the 24 amino acids penetrate cells through a particular gate represented by a specific amino acid sequence of the oestrogen receptor (ER). We analysed the internalization of the synthetic hexapeptide and the cytotoxic activity of the hexapeptide conjugated to cisplatin on a cell line panel. In most cell lines, the hexapeptide delivered an amount of cisplatin that was 2 to 8 times greater than that released by cisplatin when the drug was used alone. This increased delivery increases the therapeutic index of cisplatin and reduces side effects caused by a high dosage or long-term treatment times. We may consider this hexapeptide a new molecular carrier to deliver molecules with therapeutic activity into ER(+) cells for diagnostic purposes and clinical or immune therapy.

Poland syndrome: an algorithm to select the appropriate chest wall surgical reconstructive treatment.

AIM: Poland syndrome is a constellation of rare congenital anomalies of the chest wall, with or without alterations to the ipsilateral superior extremity. Actually Foucras' classification is commonly used to choose the most appropriate surgical treatment, but often only a radiological classification proves unsatisfactory in order to achieve the best aesthetic result. MATERIAL AND METHODS: Since November 2006 in our institute have been treated 6 patients (3 M, 3 F) with Poland Syndrome affected by only chest wall and/or breast deformities. RESULTS: We treated 6 patients opting for different surgical procedures, depending on the deformity detected. We experienced only one procedural complication, a fat necrosis with superior migration of the prosthesis, successfully managed. DISCUSSION: Surgical alternative treatments of the Poland's abnormalities of the chest wall are independent from the corrective surgery of the ipsilateral affected forearm ad hand. Surgeons should be able to develop an operative plan to address aesthetic goals while preserving muscular functionality. Indeed surgical techniques should be minimally invasive and possible available in every hospital structure. CONCLUSIONS: This study has been designed to review a series of surgical options of breast reconstruction in patients with Poland Syndrome in order to develop a new flow chart to plan the best surgical choice analyzing only breast/chest wall deformities according to Blondeel's point of view about reconstruction of the new breast and thoracic wall. KEY WORDS: Breast reconstruction, Poland syndrome.

Reconstruction of the loss of substance of the dorsum of the hand: from the conventional techniques to regenerative surgery procedures.

AIM: Our aim is focused on the advantages of new technologies compared with those of traditional methods in the reconstruction of the loss of substance of the dorsum of the hand. MATERIAL OF STUDY: We observed 37 patients from 2007 to 2010 with loss of substance of the dorsum of the hand, also associated with significant comorbidities. In 27 patients we chose surgical reconstruction, in 10 patients we opted for conservative reconstruction with the use of new technologies. RESULTS: After a median follow-up from one to three years, in all cases the skin coverage was reinstated and mobility was restored, thereby adhering to the principles of both morphological and functional reconstruction. DISCUSSION: Concerning reconstruction by means of flaps, the main principles dictate is the new coverage must appear as much as possible, similar to the original tissue. The reconstruction must be in a single surgical time. On the other hand, the advent of advanced dressings and bioengineering has optimized conservative skin repair. CONCLUSIONS: We have seen a considerable broadening of the indications for conservative reconstruction. This method is suitable for reconstruction of the dorsum of the hand with good effectiveness, it makes it possible to obtain a tissue of good quality, which is flexible and smooth on the tendons and is not excessively thick. These methods are achieved with relative ease even in patients with poor general health. Although costly, this procedure will ultimately save the patient from further surgeries and hospitalization expenses, making it advantageous when considering the benefit-cost ratio.

A molecular carrier to transport and deliver cisplatin into endometrial cancer cells.

The leader peptide of a recombinant manganese superoxide dismutase (rMnSOD-Lp) acts as a molecular carrier. Clonogenic tests on normal (MRC-5) and endometrial adenocarcinoma cells (HTB-112) were carried out in the presence of rMnSOD-Lp, cisplatin alone (CC) or cisplatin conjugated to the rMnSOD-Lp (rMnSOD-Lp-CC). The platinum delivered into the cells was measured by atomic spectrophotometric absorbance. The treatments on tumor and normal cells were finally evaluated by LM and TM microscopy. Tumor cell death in the case of 0.5 µM cisplatin on its own was minimal, while in the presence of 0.5 µM rMnSOD-Lp-CC, no tumor cells survived. Atomic absorbance analysis showed that rMnSOD-Lp-CC delivered approximately four times more cisplatin into HTB-112 cells than the amount delivered using cisplatin alone. By LM observation, the cells treated with rMnSOD-Lp-CC showed signs of nuclear and cytoplasmic fragmentation, that is, apoptosis induced by the treatment. The therapeutic effect of rMnSOD-Lp-CC on endometrial cancer cells was significant, while on the normal cells it showed only a minimal toxicity. We believe that rMnSOD-Lp deserves to be considered as a molecular carrier to deliver cisplatin directly into tumor cells, thus transforming its antireplicative activity into a specific and selective antitumor agent.

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 (68)Gallium-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 (125)I- and (131)I-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.

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 (32)P-phosphorylated C-PKA and of AKAP by (32)P-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-FR(III) 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.