Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein.

Members of the bcl-2 protein family share regions of sequence similarity, the bcl-2 homology (BH) domains. Bcl-2, the most studied member of this family, has four BH domains, BH1-4, and has a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2-interacting factors should provide a critical advance in understanding the different functions of bcl-2. Here, we characterized bcl-2 interactome by mass spectrometry in human lung adenocarcinoma cells. In silico functional analysis associated most part of the identified proteins to mitochondrial functions. Among them we identified SRA stem-loop interacting RNA-binding protein, SLIRP, a mitochondrial protein with a relevant role in regulating mitochondrial messenger RNA (mRNA) homeostasis. We validated bcl-2/SLIRP interaction by immunoprecipitation and immunofluorescence experiments in cancer cell lines from different histotypes. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein and regulates mitochondrial mRNA levels. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding.

The thiazole derivative CPTH6 impairs autophagy.

We have previously demonstrated that the thiazole derivative 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) induces apoptosis and cell cycle arrest in human leukemia cells. The aim of this study was to evaluate whether CPTH6 is able to affect autophagy. By using several human tumor cell lines with different origins we demonstrated that CPTH6 treatment induced, in a dose-dependent manner, a significant increase in autophagic features, as imaged by electron microscopy, immunoblotting analysis of membrane-bound form of microtubule-associated protein 1 light chain 3 (LC3B-II) levels and by appearance of typical LC3B-II-associated autophagosomal puncta. To gain insights into the molecular mechanisms of elevated markers of autophagy induced by CPTH6 treatment, we silenced the expression of several proteins acting at different steps of autophagy. We found that the effect of CPTH6 on autophagy developed through a noncanonical mechanism that did not require beclin-1-dependent nucleation, but involved Atg-7-mediated elongation of autophagosomal membranes. Strikingly, a combined treatment of CPTH6 with late-stage autophagy inhibitors, such as chloroquine and bafilomycin A1, demonstrates that under basal condition CPTH6 reduces autophagosome turnover through an impairment of their degradation pathway, rather than enhancing autophagosome formation, as confirmed by immunofluorescence experiments. According to these results, CPTH6-induced enhancement of autophagy substrate p62 and NBR1 protein levels confirms a blockage of autophagic cargo degradation. In addition, CPTH6 inhibited autophagosome maturation and compounds having high structural similarities with CPTH6 produced similar effects on the autophagic pathway. Finally, the evidence that CPTH6 treatment decreased α-tubulin acetylation and failed to increase autophagic markers in cells in which acetyltransferase ATAT1 expression was silenced indicates a possible role of α-tubulin acetylation in CPTH6-induced alteration in autophagy. Overall, CPTH6 could be a valuable agent for the treatment of cancer and should be further studied as a possible antineoplastic agent.

Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells.

In addition to act as an antiapoptotic protein, B-cell lymphoma (bcl)-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia-inducible factor-1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes, such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally, we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2.

[The Outpatient Health Care Package for the very premature infant: application of the program to follow-up services for the Neonatal Intensive Care, Pediatrics Department, Università "La Sapienza" I Facoltà, Roma (June 2008-March 2010)]. / II Pacchetto Ambulatoriale Complesso (PAC) del neonato altamente pretermine: applicazione del programma presso il servizio di follow-up della Terapia Intensiva Neonatale, Dipartimento di pediatria, Università "La Sapienza" I Facoltà, Roma (Giugno 2008-Marzo 2010).

Fifty-nine children were enrolled in the Outpatient Health Care Package (OHCP) from 01/06/2008 to 31/03/2010. All children, except two, attended entirely the follow-up appointments; a satisfactory result, considering also that 30% of family were living outside the urban area and more than a third of the families was originated in a foreign country. At 3 months corrected age(CA) Haemoglobin mean values of 47 infants, all in iron treatment, were: 12.26 (10.1-14-1) g/dL; 25% had values between 10.1 and 12 g/dL. Mean values for Calcium were 10.75 (9.50-15.26) mg/dL Mean values for ALP were 393 (179-1075) UI/L, values >1000 UI/L were found in two infants who suspended Vitamin D treatment. At 3 months CA 50 infants performed ABR, 12 of these showing abnormalities. To date 9 infants repeated ABR at 6-9 months CA, 4 of these showed again abnormal results. Overall were found 4 ABR abnormalities among 47 children (8.5%). Outcome of 23 children at 12 months CA: no moderate or severe neurologic abnormalities were found, 4 children (17.4%) presented mild abnormalities, 2 were referred for rehabilitation. No QSM <80% was found (mean QSM 93.7%) in 10 children evaluated. One child presented growth retardation <5 degrees; 2 underwent laser treatment for ROP with normal vision, 7 (30.4%)had sistolic BP > or = 95 degrees; 6 (26%) were rehospitalized. This experience was positive: OHCP promoted a better compliance and standardization of follow-up. It would be desirable to prolong OHCP until school-age, including renal and cardiac functions monitoring.

Umbilical cord interleukin-6 levels are elevated in term neonates with perinatal asphyxia.

BACKGROUND: A correlation between elevation of pro-inflammatory cytokines and white matter injury or abnormal neurologic outcome has been established in the preterm infant. In the full-term neonate, few studies exist linking elevation of cytokines with encephalopathy and poor neurodevelopmental outcome. Our aims were to investigate if serum interleukin-6 concentrations in delivering mothers and their offspring at birth are associated with perinatal asphyxia, and to examine the relation of interleukin-6 levels to the severity of hypoxic-ischemic encephalopathy and to the neurological outcome. DESIGN AND METHODS: Serum interleukin-6 levels were measured at birth, 24 and 48 h of life in 50 consecutive term uninfected newborns with perinatal asphyxia and 113 randomly selected healthy term newborns, and at delivery in their mothers. RESULTS: The median cord interleukin-6 concentrations in the infants who developed hypoxic-ischemic encephalopathy was 376-fold as high as the values in the normal infants (P < 0.0001) and 5.5-fold as high as those in the infants with asphyxia who did not develop hypoxic-ischemic encephalopathy (P < 0.05). There was also a significant relationship between interleukin-6 and the degree of hypoxic-ischemic encephalopathy, and between interleukin-6 and neurodevelopmental outcome at 2 years of age. Regardless of outcome, in the asphyxiated infants the interleukin-6 values were significantly lower at both 24 and 48 h of life than at birth, with a significant decline from 24 to 48 h of life. Among mothers of the asphyxiated neonates, there were no significant differences in interleukin-6 concentrations between those delivering neonates with and without hypoxic-ischemic encephalopathy. CONCLUSIONS: Measurement of IL-6 concentrations in the umbilical cord of neonates with perinatal asphyxia may be useful to identify early, and in a relatively simple way, those who are most likely to have subsequent brain injury and adverse outcome.

[Transplant of livers from living relatives: selection of recipients and donors]. / Il trapianto di fegato da familiare vivente: selezione dei riceventi e donatori.

Living relative liver transplantation is a valid alternative to cadaver transplantation especially at a time when the availability of organs cannot meet the requests of long waiting lists. This procedure was initially introduced in response to the shortage of organs for pediatric cases, but the rapid growth of demand for liver transplantation has led to its extension to the adult population. The procedure raises a number of ethical, logistic and technical questions. The ethical aspect has been widely debated and in order to be acceptable, the procedure must comply with three critical points: the need for innovation, an acceptable risk-benefit ratio and adequate informed consent. The technical aspect is essential for the success of the procedure. It calls for an extensive experience and know-how of hepatobiliary surgery on one hand, and the use of high-resolution vision on the other, an aspect which is crucial for the success of vascular anastomoses. The indications for living relative transplantation are the same as for standard transplants. The sole exception is for adult patients with 2A status who present advanced hepatic imbalance caused by chronic liver disease, thereby reducing the probability of success, above all because a living donor graft is always smaller compared to the ideal dimensions for the recipient. In view of the severe shortage of organs, living relative transplantation is an important alternative for both pediatric and adult patients. The challenge over the coming decades will be to extend living relative transplantation to a growing number of patients, without jeopardizing the health of the donor.

Bone marrow transplantation in Hunter syndrome (mucopolysaccharidosis type II): two-year follow-up of the first Italian patient and review of the literature.

A patient with Hunter syndrome, or mucopolysaccharidosis type II (MPS-osis II), was subjected to bone marrow transplantation (BMT), at the age of 2 9/12 years. A two-year follow-up ensued to the purpose of comparing clinical, biochemical, neuropsychologic status pre- and post-BMT. From the clinical standpoint, a complete normalization of hepatosplenomegaly was observed. In addition the skin decreased in thickness and joint mobility improved. The echocardiography showed normalization of left ventricle size. With the exception of verbal capabilities, there was no further deterioration of the neuropsychologic profile. The ultrastructural examination of the liver showed an almost total disappearance of storage material. Normal iduronate sulfatase levels in leukocytes and lymphoblasts were constantly found after BMT. A qualitative and quantitative improvement in urinary glycosaminoglycan (GAG) excretion was also found. The effectiveness of the BMT in our patient is also assessed in the context of the few cases of MPS-osis II that have been reported to date. A final evaluation of the efficacy of BMT in MPS-osis II will be possible only when a higher number of patients, diagnosed as early as possible and transplanted within the first months of life, can be followed-up for more extended periods of time.

Italian multicentre study on very low-birth-weight babies. Neonatal mortality and two-year outcome.

The Italian multicentre study on very low-birth-weight babies is the first collaborative project in Italy on the health status of newborns weighing 500-1499 g at birth: 634 such babies were admitted in 1987-88 to eight Italian NICUs; 424 infants survived and were followed until two years of age, corrected for prematurity. Logistic regression analysis of pre-admission risk factors of in-hospital mortality identified eight statistically significant variables: birth weight, gestational age, sex, antepartum steroids, 1-min Apgar score and, on admission to the NICU, body temperature, pH and absence of spontaneous respiration. Using the equation derived from the logistic model, a theoretical mortality rate was calculated for each centre, predicted on the basis of the local incidence of preadmission risk factors. In no case was the predicted mortality significantly different from the observed one. At two years of age, 8 children were blind and 48 had motor disability. Of these, 46 had cerebral palsy: based on a functional evaluation score 14 had severe (degree 4), 20 intermediate (degree 3) and 12 mild cerebral palsy (degree 2). Among 25 variables entered in a logistic regression as risk factors for cerebral palsy, only periventricular leukomalacia and acidosis were significantly associated with the outcome.

Preliminary molecular analysis of a case of feline mucopolysaccharidosis VI.

Deficiency of the lysosomal enzyme arylsulphatase B (ASB) causes, in man, the Maroteaux-Lamy disease (mucopolysaccharidosis type VI, MPS VI). MPS VI has been described also in Siamese cats. Isolation and characterization of the human and feline cDNAs encoding ASB has been reported as well as the assignment of the feline ASB gene to feline chromosome A1. The present paper describes the Southern and Northern blot analyses on DNA and RNA from an MPS VI affected cat using the human arylsulphatase B probe (ASB2). Our data suggest that a gross deletion/rearrangement of the ASB gene is present in the affected animal.

Biochemical diagnosis of mucopolysaccharidoses: experience of 297 diagnoses in a 15-year period (1977-1991).

We report the results over 15 years (1977-1991) for biochemical diagnoses of patients referred from throughout Italy and suspected of having a mucopolysaccharidosis. Of these, 147 patients were diagnosed as being homozygous or hemizygous for a specific lysosomal enzyme deficiency; 74 pregnancies at risk were monitored in their families; 76 heterozygote diagnoses were performed on their relatives, with a total of 48 positive diagnoses. We also report the analysis of genomic DNA from 11 unrelated Italian Hunter patients, using pc2S15 probe. DNA from two patients, digested with Pst-I, showed a variant pattern of hybridization caused by deletion or rearrangement of the gene.

Coumarin anticoagulation during pregnancy in patients with mechanical valve prostheses.

Between January 1987 and December 1989, 20 female patients with one mechanical valve prosthesis (MVP) for at least 1 year postoperatively were studied while on coumarin therapy for the full length of pregnancy. In each case, caesarean section was scheduled for the 38th week. Patients were selected according to the following criteria: (1) prothrombin ratios remaining within the therapeutic range for more than 85% of their total estimations in the previous 12 months with mean daily doses of warfarin less than 5 mg; (2) stable cardiac status; (3) no previous obstetric diseases and (4) full acceptance of the risks involved in the protocol. The patients were in NYHA functional class I or II. Their ages ranged from 23 to 31 years (mean 26 +/- 3). Ten patients had a mitral prosthesis and 10 an aortic prosthesis. Among the 20 mechanical valve prostheses, 10 were Sorin, 6 Starr-Edwards, 2 Bjørk-Shiley, and 2 Lillehei-Kaster. Eighteen patients were in sinus rythm, 1 in chronic atrial fibrillation, and 1 had a permanent endocardial pacemaker. Nineteen were delivered by caesarean section: warfarin was withdrawn 48 h before surgery and resumed 24 h thereafter. One patient had premature spontaneous delivery at 36 weeks. The mean prothrombin ratio measured weekly in the 20 patients was 2.06 +/- 0.45 INR, using a mean daily warfarin dose of 4.1 mg +/- 1.63. The mean value of the prothrombin ratio during caesarean section for the 19 patients was 1.23 +/- 0.38 INR. In the 20 live births, the mean birth weight was 2.9 kg +/- 0.40.(ABSTRACT TRUNCATED AT 250 WORDS)

Italian Multicenter Study on Very Low Birth Weight Babies.

The Italian Multicenter Study on Very Low Birth Weight babies (IMS-VLBW) is the first collaborative investigation performed in Italy on the health status of newborns weighing less than 1500 g at birth. Eight Neonatal Intensive Care Units (NICUs) participated in the study: Cagliari, Napoli, Padova, Palermo, Roma, Sassari, Trieste, Udine. Data were analyzed in the Laboratorio di Epidemiologia e Biostatistica of the Istituto Superiore di Sanità. The objectives of the study were established in the following: a) to collect accurate descriptive data on neonatal morbidity, mortality and long term outcome of VLBW babies admitted to NICUs; b) to analyze the risk factors of unfavourable outcome (death or handicap) and to analyze, with respect to outcome, the relationships between risk factors, neonatal diseases and therapeutical procedures; c) to test the feasibility of a multicenter follow-up programme based on the use in all participating Centers of the same diagnostic criteria (the results of follow-up will be presented in a forthcoming paper). In the years 1987 and 1988, 634 newborns weighing 500-1499 g at birth were enrolled in the study. In-hospital mortality for the whole group was 33.1% (65.1% in the 500-999 g birth weight class and 19.2% in the 1000-1499 g class). Mortality was not different for inborn vs outborn babies. A high incidence of unfavourable perinatal conditions was observed in these babies, namely birth asphyxia, sub-optimal care during transport, poor clinical conditions on arrival to the NICU. Neonatal diseases, like respiratory distress syndrome and peri-intra ventricular hemorrhage were also frequent and severe. A logistic regression analysis of pre-admission risk factors of in-hospital death identified eight statistically significant variables: birth weight; gestational age; sex; antenatal steroid stimulation of lung maturity; first minute Apgar score; absence of spontaneous respiration, body temperature and pH on arrival to the NICU. Using the equation derived from the logistic regression analysis a theoretical mortality rate, predicted on the basis of the local incidence of pre-admission risk factors, was calculated for each Center. In no case the predicted mortality was statistically different from the observed one, suggesting that in our study differences in observed mortality rates from one Center to another are largely influenced by pre-admission risk factors.

Adherence of bacteria to cardiac valve prostheses.

The adherence of bacterial cells to valvular prostheses has been studied. Bacteria were selected on the basis of their surface features (fimbriae, hydrophobicity and specific receptors). It was found that only strains having fimbriae and high cell surface hydrophobicity adhered to bioprostheses, while they did not adhere to metallic prostheses to any significant extent. Adherence to bioprostheses depended on the exposure time and it was affected by the saline concentration of the suspension medium. Furthermore, different bacterial binding capacity was observed for bioprostheses from different companies.

Behaviour of preterm newborns reaching term without any serious disorder.

We studied the behaviour of 20 preterm infants (average gestational age 33 weeks) brought to term, without any serious disorder. These infants were compared with a group of 21 healthy term infants. To evaluate behaviour we used the 26 items of the Brazelton Neonatal Behavioural Assessment Scale (BNBAS). Our preterm infants had on the whole better scores than those reported in the literature for preterm infants with various disorders. Apart from lower ability to bring hand to mouth and in getting used to visual stimuli (these differences are statistically significant) they had, in the items of orientation a lower score only in ability to follow a voice and a face (not statistically significant). These results show that preterm infants reaching term without any serious disorder do on the whole as well as full-term infants. This correlates with the observations of Dubowitz on behaviour and particularly on visual function of preterm infants and confirms the preliminary report of Daum regarding the influence of the type of neonatal pathology on the ability of orientation at the moment of term.