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I Colombo

Publications and source records attributed to I Colombo.

33 records · Page 2Linked to original sources
PubMed1991

cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase.

We have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase (CPTase; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21), an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH2-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH2-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hamster somatic cell hybrids.

Amino Acid Sequence↗
PubMed1990

Purification, characterization and partial amino acid sequences of carnitine palmitoyl-transferase from human liver.

Carnitine palmitoyl-transferase has been extracted with 0.5% Tween-20 from human liver homogenate and purified to homogeneity. The purified enzyme has a native Mr of 274 kDa. The subunit Mr is of 66 kDa, as shown by SDS-PAGE and immunoblots obtained with antibodies raised against human CPT. Purified CPT shows high affinity for palmitoyl-CoA and palmitoyl-carnitine and is not inhibited by malonyl-CoA. Seven tryptic peptides and the N-terminal of purified human CPT have been sequenced, and found homologous to rat CPT sequence. Both antibodies and peptide sequences are important tools for the investigation of the molecular basis of CPT deficiency in man.

Acyl Coenzyme A↗
PubMed1990

Purification and properties of carnitine acetyltransferase from human liver.

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit.mg-1 with acetyl-CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl-CoA and a pH optimum of 8.7. Apparent Km values for acetyl-CoA are three times lower than for decanoyl-CoA. Km values for L-carnitine in the presence of acetyl-CoA are six times lower than in the presence of decanoyl-CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine. The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5-kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver. Immunoprecipitation and SDS/PAGE analysis of 35S-labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa. We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet-extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.

Acetyltransferases↗
PubMed1981

The effect of compression on the capillary microstructure of tablets.

The effect of compaction pressure on the capillary characteristics of compacts of a crystalline drug and of a pure polymer was studied by means of penetration and porosimetry measurements. Not only the constant but also the exponent m of the general penetration volume-time relationship V = Kmtm proved to be strongly dependent on the pressure: as the compaction pressure is increased, the value of m tends to decrease. The capillary penetration data also showed the development of a marked heterogeneity in the structure of the polymer compacts at the lower compaction pressures: at these pressures the surface layers showed a much lower penetrability than the inner core of the compacts, whereas at the higher pressures the structure of the compacts was homogeneous. Furthermore, on the basis of a generally used equation describing the powder consolidation process, it was possible to relate the most relevant variation of the kinetics of capillary penetration into the drug compacts to the deformation of the particles. It is concluded that the capillary penetration measurements can give useful information in the understanding of the behaviour of pharmaceutical powders under compression.

Permeability↗
PubMed1976

[Controlled clinical trial of parsalmide in patients with subacute and chronic arthro-neuro-mypathies].

Controlled experiments were carried out to compare the anti-inflammatory, analgesic, muscle-relaxing and anxiolytic-tranquillizing activities of a new compound, parsalmide, and indomethacin. The study was conducted on 40 patients of both sexes suffering from syndromes involving neuromotor deficiency of various origin and the double blind between patient, technique was used. Overall evaluation of the activities of the two drugs did not reveal statistically significant differences although parsalmide was more effective with "pain", "tumefaction" and "anxiety" symptoms. This latter finding is particularly interesting owing to the frequent association of neuroanxiety disturbances in such patients. General and local tolerance was good with both drugs.

Adolescent↗
PubMedDate not supplied

Modelling of drug-release from poly-disperse microencapsulated spherical particles.

The topic of this paper is the experimental and theoretical study of drug-release from a system of polydisperse microencapsulated particles that, for the sake of simplicity, are assumed to be spherical. The theoretical analysis performed yields of a mathematical model to describe the physical phenomena involved in drug-release from such a system. In particular, the model is based on the hypothesis of a progressive dissolution of the internal solid drug core (due to solvent penetration through the coating) that gives a liquid solution in the region between the coating and the dissolving solid core. The existence of a concentration gradient between the inner solution and the outer release environment determines drug diffusion through the coating. The coacervation technique was adopted to microencapsulate the solid drug cores (theophylline, a bronchodilator for the treatment of chronic asthma and chronic obstructive lung disease) by an insoluble polymeric layer of ethylcellulose. The amount of drug released from these microencapsulated particles to the external receiver phase is monitored by means of a UV spectrophotometer. As the proposed model fits the experimental results well, it was concluded that it can be a good tool to design and to study this kind of drug-release system.

Bronchodilator Agents↗
PubMedDate not supplied

Increased tumorigenicity and invasiveness of C6 rat glioma cells transfected with the human alpha-2,8 sialyltransferase cDNA.

Gangliosides are thought to be involved in tumor cell proliferation, migration and invasiveness as so far demonstrated by the addition of exogenous gangliosides to the culture medium. To better understand the direct influence that alterations in ganglioside synthesis can exert on these functional aspects of cell biology, in the present study, we investigated the behaviour of C6 rat glioma cells after stable transfection with the human CMP-NeuAc:NeuAcalpha2-3Galbeta1-4GlcCer alpha2,8-sialyltransferase (SAT-II, EC 2.4.99.8) gene. The enzyme synthesizes ganglioside GD(3) by adding a sialic acid residue to ganglioside GM(3). Stable transfection of the constructs into C6 cells and expression of the human SAT-II gene were evaluated using PCR and RT-PCR amplification, respectively. Qualitative and quantitative analysis of the ganglioside profile was performed by conventional HP-TLC and identity of de novo synthesized species was assessed by TLC immunostaining. Results show that whereas C6 parental cells and C6 cells transfected with the empty expression vector synthesize, almost exclusively, ganglioside GM(3), de novo synthesis of GD(3) is clearly observed in clones expressing the alpha2,8-sialyltransferase. Subcutaneous grafting in athymic nude mice of cells expressing high levels of GD(3) induces tumors growing faster and more aggressively than controls. In in vitro assays, the same cells demonstrate increased proliferation rate, motility and invasiveness. Chemotaxis and chemoinvasion were assayed using the modified Boyden chamber. Data obtained suggest that endogenously neosynthesized GD(3) is able to modify proliferation rate, motility and invasion of C6 rat glioma cells, enhancing the features of malignancy of this tumor cell line.

Animals↗
PubMedDate not supplied

Glycosphingolipid expression in solid tumours and transformed cell lines.

Glycosphingolipids are assumed to play a crucial role in cell-cell and cell-substrate interactions, including cell adhesion, proliferation, differentiation and apoptosis. Furthermore, cell surface glycolipid profile changes in the so called "social disorders", such as malignant transformation. To better investigate these modifications, the ganglioside composition in different solid tumours and in two transformed cell lines was analyzed. In some of these models we also tried to correlate the pattern of gangliosides to the key enzymes involved in their metabolism. The results we obtained can be summarized as follows:(1), meningiomas with or without chromosome 22 deletion: predominance of ganglioside GD3 in the former and of ganglioside GM3 in the latter. Correlation between GM3/GD3 ratio and SAT-2 activity; (2), mammary carcinomas developed in MMTV/c-neu transgenic mice: accumulation of GM3-derived species. The different ganglioside distribution seems to correlate with the tumour size; (3), Sarcoma Galliera-strain cells SGS/3A and normal syngenic murine fibroblasts FG: transformed cells exhibit a lower activity of sialyltransferases (SAT-1, SAT-2, SAT-4) compared to normal fibroblasts, suggesting a possible correlation with the ganglioside pattern. The neuraminidase activity seems to correlate to the glycoprotein sialic acid content; (4), 3T3 normal murine fibroblasts and SVT2 transformed cells: GM3 is absent in 3T3, while it accounts for the main ganglioside species in SVT2. On the contrary, GM2 present in a large amount in normal fibroblasts, is practically absent in transformed cells. No correlation has been observed between ganglioside profile and glycosyltransferase activities so far examined.

Animals↗