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Biomedical subjects

M Marino

Publications and source records attributed to M Marino.

At least 19 recordsLinked to original sources
PubMed1996

Changes in the levels of glycerophosphoinositols during differentiation of hepatic and neuronal cells.

Glycerophosphoinositols are metabolites formed by a phosholipase A2 and a lysolipase specifically acting on membrane phosphoinositol lipids. High levels of these compounds characterize epithelial cells and fibroblasts transformed by ras and other cellular oncogenes. Here we have analyzed the glycerophosphoinositol levels in cells that are considered models of cell differentiation. Using rat hepatocytes at different stages of liver development we have shown that the glycerophosphoinositol basal levels of fetal cells were up to fourfold higher than in adult hepatocytes. No changes in glycerophosphoinositol were observed in regenerating rat liver, a model of differentiated cells proliferating in a synchronous manner, where only glycerophosphoinositol 4-phosphate increased by 80%. Similarly to fetal hepatocytes, a modest but significant increase (30%) in the levels of glycerophosphoinositols was observed in undifferentiated NG-108-15 cells as compared to the same cells induced to differentiate by cAMP. In a different neuronal cell line, PC12 cells, increased glycerophosphoinositol levels characterized the differentiated cells. Based on these observations we suggest that high glycerophosphoinositol levels characterize cellular phenomena associated with the activation of ras/mitogen-activated protein kinase pathways.

Animals↗
PubMed1996

Cloning and chromosomal localization of a cDNA encoding a mitochondrial porin from Drosophila melanogaster.

We have raised polyclonal antibodies against purified the Drosphila melanogaster mitochondrial porin. They showed high titre and specificity and were thus used as a tool for screening an expression library. The isolated clone 1T1 showed 74% sequence identity in the last 19 residues at the C-terminus of human porin. A subclone of 1T1, containing the porin-like sequence, was thus used as a probe for re-screening a cDNA library and several positive clones were plaque-purified. We present here the sequence of a 1363 bp cDNA encoding a protein of 279 amino acids. Its identity with porin was also confirmed by N-terminal Edman degradation of the purified protein. The D. melanogaster porin shows an overall 51.8% identity with human porin isoform 1 (porin 31HL or HVDAC1) and an overall 55.7% identity with human porin isoform 2 (HVDAC2). Hydrophobicity plots and secondary structure predictions showed a very high similarity with data obtained from known porin sequences. The D. melanogaster porin cDNA was used as a probe for in situ hybridization to polytenic salivar gland chromosomes. It hybridizes with different intensities in two sites, in chromosome 2L, at region 31E and in chromosome 3L at region 79D. Thus, also in Drosophila melanogaster porin polypeptide(s) belong(s) to a multigene family.

Amino Acid Sequence↗
PubMed1996

Activation of Sulfolobus solfataricus alcohol dehydrogenase by modification of cysteine residue 38 with iodoacetic acid.

Reaction of thermostable NAD(+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus with iodoacetate at pH 9.0 and 37 degrees C significantly increases the oxidation rate of aliphatic and aromatic alcohols and decreases the reduction rate of aromatic aldehydes. The archaeal ADH is chemically modified and activated in a Michaelis-Menten-type reaction, where one molecule of the reagent binds per active site. NAD+ in micromolar concentration protects the enzyme against the inhibitor in an uncompetitive manner, while imidazole significantly increases the extent of the activation. Carboxymethylation selectively modifies one out of five cysteine residues per subunit, namely, Cys 38, located in the catalytic site, as determined by peptide and sequence analysis, and enhances by up to 25-fold the oxidation rate of benzyl alcohol. Carboxymethylated SsADH is less thermostable and shows a temperature optimum 30 degrees C lower than that of the native enzyme. The carboxymethylated enzyme exhibits a lower affinity toward the oxidized and reduced coenzyme. The dissociation constants for NAD+ and NADH determined at 25 degrees C and pH 8.8 are 60- and 200-fold higher, respectively, compared to the native enzyme. The significant isotope effect in alcohol oxidation suggests that hydride transfer partially limits the turnover rate of the reaction catalyzed by the modified enzyme, whereas the rate-limiting step for the native enzyme is NADH dissociation. Carboxymethylated enzyme probably gives higher maximum velocities of oxidation because of the faster dissociation of the modified enzyme-coenzyme complex.

Alcohol Dehydrogenase↗
PubMed1996

Evidence of induced non-tolerance in HLA-identical twins with hemoglobinopathy after in utero fetal transplantation.

Fetus-to-fetus transplantation has been suggested for the treatment of hemoglobinopathies in utero. However, dissimilar results have to date been obtained by different groups. We describe a case in which fetus-to-fetus transplantation in HLA-identical twins was performed at the 19th week of gestation by infusion of 0.8 ml of fetal blood from normal to beta-thalassemia affected fetus with the main aim of inducing tolerance. No evidence of engraftment, determined by KM19 polymorphism, was present after 2 years of the procedure. Moreover, an alloreactive cytotoxic T lymphocyte precursor (CTLp) study of affected fetus vs donor and other different stimulators showed that immunization vs tolerance was the real effect of the procedure.

Adolescent↗
PubMed1995

The great heterogeneity of thalassemia molecular defects in Sicily.

This paper reports the results of 1428 beta-thalassemia chromosomes studied in Sicily during a hemoglobinopathy control program starting in 1983. Molecular screening was performed by direct restriction enzyme analysis, allele specific oligonucleotide (ASO) hybridization, reverse dot blot analysis (RDB) and, for the rare or new mutations, by direct sequencing of polymerase chain reaction (PCR) products. Using these approaches 1410 (98.7%) out of 1428 beta-globin gene defects were characterized, involving 22 different beta-thalassemia mutations. Three of these were present at high frequency (beta(0)39, IVS1, 110 and IVS1,6); the other beta-globin gene defects were found at lower frequency. In the latter, we found a smaller group of mutations at a frequency lower than 10% (IVS1, 1, IVS2, 745, beta S) and a larger one at a frequency lower than 2% [-87, IVS1,2, IVS2,1, fr 6, fr 8 (-AA), fr 44, fr 76, -101, IVS1, 116, IVS1, 3'end G-C, IVS1,5 G-A, IVS1,5 G-C, cod 30, Lepore, delta beta, beta C]. The possible origin of this very large number of mutations is discussed, taking into account the historical point of view. Moreover, this approach has made a first trimester prenatal diagnosis program possible in our region in practically all cases, with a great improvement in general thalassemia management.

DNA↗
PubMed1995

Identification of two forms (31-33 and 48 kD) of the urinary soluble p55 tumor necrosis factor receptor that are differentially N- and O-glycosylated.

The structure and the activity of urinary soluble TNF receptor type 1 (sTNF-R1), isolated from the urine of normal individuals, has been characterized and compared with that of recombinant sTNF-R1 expressed in CHO cells and with that of a nonglycosylated form expressed in Escherichia coli. Urinary sTNF-R1 was resolved in a major band of 31-33 kD and in a 48 kD band (less than 5% of total) by reducing SDS-PAGE; CHO sTNF-R1 was resolved in two bands of 29 and 31 kD. All bands were recognized by various anti-sTNF-R1 antibodies as well as by TNF-alpha in western and ligand blotting assays. No cross-reaction was observed with anti-TNF-R2 antibodies. N- and O-glycosylation studies indicated that (1) the 29-31 kD recombinant form as well as the 31-33 kD urinary form are N-glycosylated; (2) the differences between the 29-31 and 31-33 kD recombinant and natural products are mainly related to differences in the N-linked sugar content; and (3) the 48 kD sTNF-R1 isolated from urine also contains O-linked sugars. The urinary sTNF-R1 antigen mixture was able to inhibit TNF-alpha cytotoxicity with a potency comparable to that of nonglycosylated E. coli sTNF-R1. At variance, urinary sTNF-R1 was able to inhibit TNF-beta sevenfold more efficiently than E. coli sTNF-R1. In conclusion, two subtypes of sTNF-R1 have been isolated from urine: a main N-glycosylated form of 31-33 kD and a N- and O-glycosylated form of 48 kD that appears to be a minor constituent of the urinary sTNF-R1 antigen.

Animals↗
PubMed1995

Modelling the three-dimensional structure and the electrostatic potential field of two Cu,Zn superoxide dismutase variants from tomato leaves.

The three-dimensional structure of tomato P31 and T10 Cu,Zn superoxide dismutases (SODs) were computer modelled using the structure of the bovine enzyme as a template. The structure-essential residues retain in the models the position occupied in the other Cu,Zn SODs of known 3D structure and the overall packing of the beta-barrel is maintained. Formation of 'aromatic pairs' occurs between newly inserted aromatic residues. The number of total charges changes in the two variants and some charged residues located in the proximity of the active site in most Cu,Zn SODs disappear in tomato enzymes. Calculation of the electrostatic potential field, carried out by numerically solving the Poisson-Boltzmann equation, indicates that in both variants a negative potential field surrounds all the protein surface except the active site areas, characterized by positive potential values, as already observed in the bovine enzyme. This result confirms that coordinated mutations of charged residues have occurred in the evolution of this enzyme giving rise to a peculiar electrostatic potential distribution common to all members of this protein family.

Amino Acid Sequence↗
PubMed1994

Changes in plasma dolichol levels, transport, and hepatic delivery during rat liver regeneration.

During the proliferative process that follows partial hepatectomy in the rat, the dolichol content increases in both plasma and liver. Its transport in the blood by lipoproteins also changes. The difference in the distribution of dolichols of various chain lengths in plasma and in the liver is further enhanced during liver regeneration. The dolichol released by perfused liver shows a homologue distribution more similar to that observable in blood than in the liver, thus confirming the importance of the liver as a regulatory site for the blood dolichol supply.

Animals↗
PubMed1994

Hodgkin's disease: diagnostic accuracy of fine needle aspiration; a report based on 62 consecutive cases.

We report on our series of 62 cases occurring between January 1977 and December 1990, which were diagnosed as Hodgkin's disease by fine needle aspiration (FNA) samples. The overall accuracy of the cytological diagnosis was high, with only four incorrect diagnoses and a positive predictive value of 93.5%. The value of FNA as a first level diagnostic technique in the screening of lymphadenopathies is discussed, as well as the limitations and pitfalls of the cytological diagnosis.

Adolescent↗
PubMed1994

A newborn with ring chromosome 10, aganglionic megacolon, and renal hypoplasia.

A newborn infant is reported who had aganglionic megacolon, renal hypoplasia, severe growth retardation, generalised hypotonia, and various dysmorphic features. Chromosome analysis of lymphocytes and fibroblasts showed a ring chromosome 10 with breakpoints at p13-15 and q26. AluI digestion showed that the ring chromosome was monocentric. FISH with an alpha satellite probe specific for chromosome 10 showed one signal only in about 20% of interphase nuclei. It is suggested that aganglionic megacolon could result from dynamic somatic mosaicism owing to loss of the ring chromosome.

Chromosome Mapping↗
PubMed1993

Identification of a new receptor subtype for tumor necrosis factor-alpha.

Two distinct receptors, which bind both tumor necrosis factor-alpha and tumor necrosis factor-beta (TNF-alpha and TNF-beta), have been previously identified and cloned from transformed cells. The present study identifies a novel receptor subtype in normal human liver which binds TNF-alpha but not TNF-beta. TNF-alpha but not TNF-beta competes for 125I-TNF-alpha binding and incorporation into affinity-labeled complexes in human liver plasma membranes (HLPM). Antisera to the cloned receptors competed for 125I-TNF-alpha binding to plasma membranes isolated from various transformed cell lines but not to HLPM. However, mRNAs corresponding in size to both known TNF receptors were detected in liver RNA, making it likely that post-transcriptional modifications account for the TNF-alpha specificity of HLPM. These observations suggest that the effects of TNF-alpha and TNF-beta on some normal tissues may be more distinct than previously realized.

Antibodies, Monoclonal↗
PubMed1993

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes.

[1-14C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP3) and calcium, is concerned, substantial differences were found: EGF increased IP3 production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP3 and calcium transients, suggesting that this transduction pathway is not activated during fetal life.

Amino Acids↗
PubMed1993

A novel X-linked member of the human zinc finger protein gene family: isolation, mapping, and expression.

We report the partial characterization of a novel putative zinc finger gene of the Krüppel-type (ZNF81), isolated from an X Chromosome (Chr) specific library. The pattern of segregation in human-hamster somatic cell hybrids of sequences homologous to the ZNF81 finger domain has established that it resides within the Xp22.1-Xp11 region. ZNF81 represents yet another example, together with ZFX, ZNF41, and ZNF21, of members of the zinc finger gene family residing within the short arm of the human X Chr. Sequence analysis showed that ZNF81 may encode a polypeptide(s) containing tandem arrays of 12 canonical C2H2 zinc fingers of the Krüppel-type at the C-terminus. Northern analysis indicated that probes from the ZNF81 finger domain hybridize to polyadenylated transcripts present in several cell lines, a result that supports the hypothesis that it is an expressed, functional member of this multigene family.

Amino Acid Sequence↗
PubMed1993

Intracellular signalling of epinephrine in rat hepatocytes during fetal development and hepatic regeneration.

The changes in intracellular calcium concentration and IP3 production after the addition of epinephrine were analysed in adult, fetal (20th-22nd day of intrauterine life), and regenerating rat hepatocytes (4 h-24 h after partial hepatectomy) to determine whether the signal transduction is the same in quiescent proliferating and differentiating cells. The epinephrine treatment causes a significative cytosolic calcium transient in hepatocytes isolated in the last day of fetal life (22-day old) and in the early stage of regeneration (4 h). This effect is not significant in the previous stage of fetal life (20-day old) and at the onset of M phase of cell cycle after partial hepatectomy (24 h). [3H]myo inositol incorporation into IP3 and IP4 is higher in 20 day fetal and regenerating hepatocytes with respect to the control. In these cells the epinephrine does not affect basal level of IP3 and IP4, while it causes a substantial increase of these inositol phosphates in adult hepatocytes. [3H]myo inositol incorporation into PIP2 is very low at the 20th day of fetal life. Epinephrine has no effect on this parameter in fetal and regenerating hepatocytes. Our results show that the epinephrine signal is mediated differently in proliferating and in quiescent hepatocytes.

Animals↗