Biomedical subjects

A Messina

Publications and source records attributed to A Messina.

At least 55 records · Page 3Linked to original sources

PubMed2000

Haematopoietic progenitors from umbilical cord blood.

BACKGROUND/AIM: The aim of this study is to improve the obstetrician-based cord blood collection system and an efficient recovery of CD34+ haematopoietic progenitor stem cells. METHODS: CD34+ cells were purified from total blood using a positive selection enrichment method, called Mini-Macs. RESULTS: The final yield of CD34+ cells we obtained was 10(4) cells/ml, with a CD34+ purity of 99%. CONCLUSION: Our results confirm that, by using this method, it is possible to get a significant stem cell number, thus improving transplanting both peripheral stem cells and umbilical cord ones.

Antigens, CD34↗

PubMed2000

Genomic localization of the human genes TAF1A, TAF1B and TAF1C, encoding TAF(I)48, TAF(I)63 and TAF(I)110 subunits of class I general transcription initiation factor SL1.

Human SL1 is a general transcription initiation factor (GTF) essential for RNA polymerase I to start rRNA synthesis at class I promoters. It is comprised of the TATA box-binding protein (TBP) and three TBP-associated factors (TAF(I)48, TAF(I)63 and TAF(I)110). We have determined that the human genes TAF1A, TAF1B and TAF1C, encoding these three TAF(I) polypeptides, are localized at lq42, 2p25 and 16q24, respectively. All three genes are present as single copies in the human genome and map to different chromosomes, as shown by somatic cell hybrid panel and radiation hybrid panel analysis and FISH. Two of these genes, TAF1C and TAF1B, are transcribed into multiple RNAs, as determined through Northern analysis of mRNA from various human organs and cell lines. If translated into different polypeptides, this could result in production of variant isoforms of SL1 with different activation potentials.

Alleles↗

PubMed1999

Porin is present in the plasma membrane where it is concentrated in caveolae and caveolae-related domains.

Mitochondrial porin, or voltage-dependent anion channel, is a pore-forming protein first discovered in the outer mitochondrial membrane. Later investigations have provided indications for its presence also in other cellular membranes, including the plasma membrane, and in caveolae. This extra-mitochondrial localization is debated and no clear-cut conclusion has been reached up to now. In this work, we used biochemical and electrophysiological techniques to detect and characterize porin within isolated caveolae and caveolae-like domains (low density Triton-insoluble fractions). A new procedure was used to isolate porin from plasma membrane. The outer surface of cultured CEM cells was biotinylated by an impermeable reagent. Low density Triton-insoluble fractions were prepared from the labeled cells and used as starting material to purify a biotinylated protein with the same electrophoretic mobility and immunoreactivity of mitochondrial porin. In planar bilayers, the porin from these sources formed slightly anion-selective pores with properties indistinguishable from those of mitochondrial porin. This work thus provides a strong indication of the presence of porin in the plasma membrane, and specifically in caveolae and caveolae-like domains.

Animals↗

PubMed1999

A method for processing fluorescent labelled tissue into methacrylate: a qualitative comparison of four tracers.

A technique for preserving fluorescence in retrogradely labelled neurons embedded in resin was developed. Four retrograde tracers were tested, Fast Blue (FB); Diamidino Yellow (DY); tetramethylrhodamine dextran (fluoro-ruby) (TMRD) and fluorescein dextran (fluoro-emerald) (FD). These tracers were applied to the cut end of the sciatic nerves in rats either by: (a) direct application of tracer crystals, or (b) dipping the nerve into an aqueous solution containing the tracer. Each lumbar spinal cord was removed and dehydrated by one of two methods: (a) conventional alcohol dehydration, or (b) dehydration through a graded series of aqueous methacrylate infiltration solutions (inert dehydration). Specimens were embedded in methacrylate and horizontal sections cut. The location of labelled motoneurons was mapped using a fluorescence microscope. Direct application of tracer crystals labelled more motoneurons than dipping. Fast Blue labelled considerably more motoneurons than tetramethylrhodamine. Labelling by all tracers was retained following methacrylate embedding. Fast Blue and Diamidino Yellow required inert dehydration, while tetramethylrhodamine dextran and fluorescein dextran were preserved using conventional dehydration. These results indicate that tissue labelled with commonly used fluorescent tracers can be processed and embedded in methacrylate, thereby permitting quantitative analysis by modern stereological methods.

Animals↗

PubMed1999

Mapping of the human Voltage-Dependent Anion Channel isoforms 1 and 2 reconsidered.

Eukaryotic porins or VDACs (Voltage-Dependent Anion-selective Channels) are integral membrane proteins forming large hydrophilic pores. Three functioning genes for VDAC isoforms have been detected in mouse and the corresponding cDNAs are known also in humans. Tissue-specific VDAC isoform 1 (HVDAC1) deficiency in human skeletal muscle is responsible of a rare mitochondrial encephalomyopathy, fatal in childhood. Since coding sequences are not affected in the patient, we focused our interest in the gene structure. HVDAC1 and 2 have been previously mapped at chromosomes Xq13-21 and 21, respectively. Screening of an human chromosome X cosmid library resulted only in the isolation of processed pseudogenes, finely mapped at Xq22 and Xp11.2. Here, we report the mapping of HVDAC1 to chromosome 5q31 and HVDAC2 to chromosome 10q22 by FISH. Exon/intron probes, designed on the basis of the mouse gene structures, were obtained by long extension PCR amplification using the whole genomic DNA as a template. The sequence of the probe extremities clearly pointed to a genuine VDAC genomic sequence. Human and mouse regions where VDAC 1 and 2 genes were mapped are known to be synthetic, thus reinforcing the mapping of the human homologues.

Chromosome Mapping↗

PubMed1999

Anterograde transport of leukemia inhibitory factor within transected sciatic nerves.

Disappointing functional recovery following peripheral nerve repair can be improved by neurotrophic growth factors. Leukemia inhibitory factor (LIF) is unique in that it has independent neurotrophic and myotrophic actions. The aim of this study was to explain this finding by establishing the existence of anterograde axonal transport of LIF from the site of nerve division to denervated muscles. Using 125I LIF, administered topically via an entubulation repair of divided rat sciatic nerve, we monitored its subsequent distribution by measuring the radioactivity associated with nerve segments and denervated muscles. We established that LIF preferentially accumulated in denervated muscles, a process we could reduce by 70% after tightly ligating the intervening nerve, confirming the presence of anterograde axonal transport. This was most likely an active mode of transport that ceased approximately 24 h after nerve division, establishing a narrow clinical time frame within which the myotrophic action of LIF could be optimized following nerve repair.

Animals↗

PubMed1999

Buffer effect on the kinetics of ornithine carbamyl transferase by HPLC.

A kinetic study of ornithine carbamyl transferase (OCT) from Streptococcus faecalis has been carried out, employing a new HPLC method based on the direct determination of citrulline. A different kinetic pattern was observed in tris(hydroxymethyl)-aminomethane (TRIS) and triethanolamine (TEA) buffers: a ping-pong or an ordered sequential mechanism are suggested, respectively. The inhibition of Tris buffer on OCT reaction has also been demonstrated. The inhibition of Tris buffer on OCT reaction has also been demonstrated.

Buffers↗

PubMed1999

Direct resolution of optically active isomers on chiral packings containing ergoline skeleton. 6. Enantioseparation of profens.

The enantioselective behaviour of some underivatized 2-arylpropionic acids (profens) and flobufen by HPLC using a terguride-based chiral stationary phase was tested. X-ray analysis of crystals of the chiral selector and its complexes with naproxen allowed a deeper insight into the enantiodiscriminative process. The column stability and reproducibility, and the potential of the packing for semipreparative scale separations were also determined. A method for determining flobufen enantiomers and metabolites in plasma samples is described.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

Role of interferon regulatory factor 1 in monocyte/macrophage differentiation.

Interferon regulatory factor-1 (IRF-1) has been recognized as an important tumor suppressor and growth regulatory transcription factor, which is also involved in cell differentiation. In this study we investigated the role of IRF-1 in phorbol 12-myristate 13-acetate (PMA)-induced monocyte/macrophage differentiation of human monoblastic U937 cells. For this purpose U937 cells were stably transfected with a vector overexpressing IRF-1 antisense mRNA (U937 IRF-1A cells) and with the SV-40 empty vector (U937-SV40 e.v. cells). We report here that U937 and U937-SV40 e.v. cells differentiated into macrophage-like cells upon PMA stimulation and showed IRF-1 up-regulation. On the contrary, U937 IRF-1A cells stimulated with PMA kept an undifferentiated phenotype and proliferated actively. A direct correlation between induction of IRF-1 and up-regulation of IRF-1 gene targets such as ornithine decarboxylase (ODC) and WAF-1/CIP-1 was also observed in U937 cells. On the other hand U937 IRF-1A cells down-regulated ODC and did not express WAF-1. Results show that IRF-1 plays a pivotal role in PMA-induced monocyte/macrophage differentiation.

Cell Differentiation↗

PubMed1999

Ornithine decarboxylase gene expression in Castleman's disease.

Castleman's disease (CD) is a rare atypical lymphoproliferative disorder that is clinically and histologically heterogeneous and is associated with the risk of developing malignant lymphoma. Based on pathological findings CD is divided into two types: a localized form and a multicentric form. The clinical course differs in these two forms. We examined the molecular mechanisms that lie between benign and malignant disease, evaluating a possible implication of oncogenes in the pathogenesis. Since deregulated expression of the gene for ornithine decarboxylase (ODC) has been observed in a variety of human malignancies, we compared ODC expression between the localized and multicentric forms. Using northern blot analysis we found that ODC gene expression clearly differs between the localized and multicentric forms. The findings in this report indicate that the variable pattern of ODC gene expression in the different types of CD could be useful for examining the evolution of this disease.

Adult↗

PubMed1999

Muscle cells become necrotic rather than apoptotic during reperfusion of ischaemic skeletal muscle.

While necrosis is known as a major mechanism for the loss of viability of skeletal muscle following ischaemia and reperfusion, much less is known of the role of apoptosis. In this study rat hind limbs were subjected to 2 h of tourniquet ischaemia, then reperfused for either 0, 0.25, 0.5, 1, 3, 8, 16 or 24 h (n = 6 per group). Mean viability of muscle, assessed by tetrazolium dye reduction, after 2 h ischaemia and 24 h reperfusion was 17%. Histological examination revealed disrupted, necrotic muscle fibres from 30 min to 24 h reperfusion. Apoptotic nuclei were identified by haematoxylin staining and TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labelling. No TUNEL-positive cells were observed at the end of the ischaemic period, but a small number of TUNEL-positive endothelial and smooth muscle cells were found at 30 min reperfusion, with a progressive increase in their number up to 24 h reperfusion. Apoptotic neutrophils were detected after 8-24 h reperfusion. At no stage was apoptosis seen in the nuclei of skeletal muscle fibres. It appears that apoptosis plays no role in the death of muscle fibres after ischaemia-reperfusion injury to skeletal muscle.

Animals↗

PubMed1998

Sequence and expression pattern of the Drosophila melanogaster mitochondrial porin gene: evidence of a conserved protein domain between fly and mouse.

We have recently cloned a cDNA encoding mitochondrial porin in Drosophila melanogaster and shown its chromosomal localization (Messina et al., FEBS Lett. (1996) 384, 9-13). Such cDNA was used as a probe for screening a genomic library. We thus cloned and sequenced a 4494-bp genomic region which contained the whole gene for the mitochondrial porin or VDAC. It was found that this D. melanogaster porin gene contains five exons, numbered IA (115 bp), IB (123 bp), II (320 bp), III (228 bp) and IV (752 bp). The exons II, III and IV contain the protein coding sequence and the 3' untranslated sequence (3'-UTR). The first base in exon II precisely corresponds to the first base of the starting ATG codon. Exon IA corresponds to the 5'-UTR sequence reported in the published cDNA sequence. Exon IB corresponds to an alternative 5'-UTR sequence, demonstrated to be transcribed by 5'-RACE experiments. The exon-intron splicing borders and the length of the exon III perfectly match a homologous internal exon detected in the mouse genes. Such exon encodes a protein domain predicted by sequence transmembrane arrangement models to contain major hydrophilic loops and it is thus suspected to have a conserved distinct function. In situ hybridization experiments confirmed the localization of the genomic clone on the chromosome 2L at region 32B3-4. Together with genomic Southern blotting at various stringencies, the same experiment did not confirm the presence of a second genetic locus on D. melanogaster chromosomes. Northern blots demonstrated that the porin gene is a housekeeping one: three messages of approx. 1.2-1.6 kbp are transcribed in every fly developmental stage that was studied. They were shown to derive by an alternative usage of different promoters and polyadenylation sites.

Alternative Splicing↗

PubMed1998

Genomics and transcription analysis of human TFIID.

TFIID, a multisubunit protein comprised of TBP (TATA box-binding protein) and TAF(II)s (TBP-associated factors), has a central role in transcription initiation at class II promoters. TAF(II)s role as mediators of regulatory transcription factors, such as pRb and p53, and their involvement in signal transduction pathways suggest that some may participate in the control of cell proliferation and differentiation: therefore, they could be considered potential protooncogenes or antioncogenes. With the aim of starting to analyse these potential roles, we have determined the genomic position of nine human TAF(II) genes (TAF[II]250, TAF[II]135, TAF[II]100, TAF[II]80, TAF[II]55, TAF[II]43, TAF[II]31, TAF[II]28, TAF[II]20/15) and of two previously unknown sequences related to TAF(II)250 and TAF(II)31, respectively. Except for those encoding TAF(II)250 and TAF(II)31, these genes are present in a single copy and, with the exclusion of those for TAF(II)43 and TAF(II)28 (both at 6p21), are localized in different segments of the genome. Indeed, six of them map to a chromosomal region commonly altered in specific neoplasias, which defines them as candidates for involvement in oncogenesis. Our experiments also demonstrate that TAF(II) transcripts are synthesized ubiquitously, mostly at low levels similar to those of TBP. Interestingly, the amount of the major mRNA species detected by TAF(II)20/15 cDNA is higher, which suggests that the polypeptide it encodes may also perform functions independently of TFIID. TAF(II) isoforms, indicated by additional bands on Northern blots, may play a role in modulation of TFIID function. These data will be useful for analysing variations of TAF(II) mRNA phenotype during cell proliferation, differentiation and development, both normal and pathological.

Animals↗

PubMed1998

Novel aspects of the electrophysiology of mitochondrial porin.

The recent findings that mitochondrial porin, VDAC, participates in supramolecular complexes and is present in the plasmamembrane need to be reconciled with its biophysical properties. We report here that VDAC often displays previously unobserved or unappreciated behaviors. Reconstituted VDAC can: a) exhibit fast gating when in any of many conductance substates; b) close completely, although briefly, on its own; c) close for a long periods, in the presence of König's polyanion; d) take several milliseconds to re-open when an applied transmembrane potential is switched off; e) be desensitized by prolonged exposure to high voltages, so that it will not re-open to the full conductance state upon subsequent return to zero voltage; f) display polarity-dependent voltage-induced closure. These behaviors are especially noticeable when the observations are conducted on a single reincorporated channel, suggesting that interactions between copies of VDAC may play a role in determining its electrophysiological properties. Any model of VDAC's structure, gating and function should take these observations into account.

Animals↗

PubMed1998

Use of a microquantity enzyme immunoassay in a large-scale study of measles, mumps and rubella immunity in Italy.

The seroprevalence of antibodies to measles, mumps, and rubella viruses (MMR) was determined in 1498 subjects in Catania, Italy, ranging in age from 1 month to 25 years. The study population was divided into seven age groups and screened by enzyme immunoassay using microquantities (10 microl) of whole blood collected by fingerprick on filter paper discs. The results showed that seroconversion for measles (87.6%) and mumps (73.2%) occurred between 6 and 10 years of age. The seroprevalence of antibodies to rubella virus increased slowly through the age groups, reaching the highest rate (93.3%) between 16 and 20 years of age. Passively transmitted maternal antibodies to mumps and rubella were absent in babies between 5 and 8 months of age, and a few cases positive for measles antibodies were found among babies 6 and 7 months of age. The enzyme immunoassay was demonstrated to be suitable for low-cost large-scale screening for MMR immunity. The rate of vaccine failure was also evaluated and found to be 9.5% for the measles virus, 12.9% for the mumps virus and 0.0% for the rubella virus.

Adolescent↗

PubMed1998

The metabotropic glutamate receptor mGlu5 controls the onset of developmental apoptosis in cultured cerebellar neurons.

Cultured cerebellar granule cells grown in medium containing 10 mM K+ undergo apoptosis after 4-5 days in vitro (DIV), and, at that time, the activity of metabotropic glutamate (mGlu) receptors coupled to polyphosphoinositide (PI) hydrolysis begins to decline. In granule cells at 4 DIV, the mGlu receptor subtype mGlu5 was expressed at high levels. The expression of another PI-coupled mGlu receptor, the mGlu1a, was low at 4 DIV but increased during the following days. In cultures at 4-5 DIV, the few cells that already showed an apoptotic phenotype were devoid of mGlu5 receptors, but they all expressed mGlu1a receptors. The development of apoptosis was accelerated after treating the cultures with: (i) mGlu5 antisense oligonucleotides; (ii) the mixed mGlu receptor antagonist, (+)-alpha-methyl-4-carboxyphenylglycine; or (iii) the glutamate depleting enzyme, alanine aminotransferase. In contrast, an induced overexpression of mGlu5 receptors protected cultured granule cells against apoptotic death. We suggest that the activity of mGlu5 receptors supports cell survival, and a decline in the expression of mGlu5 receptors gives access to programmed cell death in cerebellar granule cells developing in primary cultures.

Animals↗

PubMed1998

Brain tissue autofluorescence: an aid for intraoperative delineation of tumor resection margins.

The intrinsic autofluorescence properties of biological tissues can change depending on alterations induced by pathological processes. Evidence has been reported concerning the application of autofluorescence as a parameter for in situ cancer detection in several organs. In this paper, autofluorescence properties of normal and tumor tissue in the brain are described, suitable for a real-time diagnostic application. Data were obtained both on ex vivo resected samples, by microspectrofluorometric techniques, and in vivo, during surgical operation, by means of fiberoptic probe. Significant differences were found in autofluorescence emission properties between normal and tumor tissues, in terms of both spectral shape and signal amplitude, that confirm the potential of autofluorescence as a parameter to distinguish neoplastic from normal condition. The noninvasiveness of the technique opens up interesting prospects for improving the efficacy of neurosurgical operations, by allowing an intraoperative delineation of tumor resection margins.

Brain↗

PubMed1997

Simultaneous assay for aspartate aminotransferase and guanase in human serum by high-performance liquid chromatography.

A simple high-performance liquid chromatography (HPLC) assay for the simultaneous determination of guanase and aspartate aminotransferase (AST) activities in a single serum sample is described. The method is based on direct detection of enzymatically formed products xanthine and glutamate, respectively. The procedure is sensitive, precise (C.V. below 2% for guanase and 3% for AST), suitable for routine purposes and requires only 100 microliters of sample. Kinetic measurements have shown the guanase activity to have an apparent Michaelis constant of 24.5 microM and the AST activity of 11.1 and 0.18 mM for aspartate and oxoglutarate, respectively, at 37 degrees C in Tris-HCl buffer (pH 7.5).

Aspartate Aminotransferases↗