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Biomedical subjects

M Panaccio

Publications and source records attributed to M Panaccio.

18 recordsLinked to original sources
PubMed1995

Rapid identification of Acremonium lolii and Acremonium coenophialum endophytes through arbitrarily primed PCR.

Using a random decamer 5'-CCGAGGTGAC-3' in an arbitrarily primed PCR, similar band patterns were observed between Acremonium lolii and A. coenophialum DNA, which were somewhat different from those formed by other fungal DNA. Despite sharing bands of around 0.7, 0.9 and 2.1 kb, A. lolii can be distinguished from A. coenophialum by the presence of an additional band at around 0.5 kb in the arbitrarily primed PCR.

Acremonium↗
PubMed1995

Biochemical analysis of recombinant glutathione S-transferase of Fasciola hepatica.

Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins purified for biochemical analyses. The rGST proteins are 95% pure as indicated by Coomassie staining of proteins separated by SDS-PAGE. Molecular sieving by HPLC infers that, like the native protein, the rGST proteins form homodimers under non-denaturing conditions. The rGST proteins are recognised by antisera raised to the native GST of F. hepatica. All four rGST proteins from F. hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene. The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs (trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation (trans-2-nonenal, trans,trans-2,4-decadienal) and for epoxy-3-(p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST. No rGST were active with EPNP. rGST47 and 51 showed activity with the other four substrates. rGST7 was active with three substrates whereas rGST1 showed relatively low activity with all substrates except trans,trans-2,4-decadienal. The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the rGSTs with rGST1 showing a 800-fold difference in sensitivity between the inhibitors. These results show that F. hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profiles.

Amino Acid Sequence↗
PubMed1995

Characterisation of a novel Kunitz-type molecule from the trematode Fasciola hepatica.

A low molecular mass monomeric protein termed Fh-KTM (Fasciola hepatica Kunitz-type molecule) was isolated from the trematode Fasciola hepatica. Fh-KTM is a single polypeptide of 58 amino acids and a Mr of 6751. The complete amino acid sequence of Fh-KTM was determined and revealed significant similarity to the Kunitz-type (BPTI) family of proteinase inhibitors. Several polymorphisms were observed suggesting that more than one Fh-KTM molecule may be expressed by this parasite. Modified proline residues were shown to occur at all four positions in this protein as 3-hydroxy derivatives. This is the first report of 3-hydroxyproline residues in a Kunitz-type molecule. Indirect immunofluorescence and immunogold labelling revealed that Fh-KTM is an abundant molecule within the parasite localised to the gut, the parenchymal tissue and the tegument of adult F. hepatica. Serine protease inhibition assays revealed that Fh-KTM exhibited little or no inhibition against chymotrypsin, kallikrein, urokinase or key serine proteases of the blood coagulation pathways. However, Fh-KTM was able to inhibit trypsin even though the P1 reactive amino acid of Fh-KTM was a leucine residue.

Amino Acid Sequence↗
PubMed1994

The secreted cathepsin L-like proteinases of the trematode, Fasciola hepatica, contain 3-hydroxyproline residues.

The cysteine proteinases synthesized by the adult stage of the trematode Fasciola hepatica were found to be a very heterogeneous group of proteins as demonstrated by one- and two-dimensional gel analyses. N-terminal amino acid sequencing indicated the presence of at least two distinct gene products among the secreted cysteine proteinases. Enzymic studies and peptide sequence analysis of the excreted/secreted cysteine proteinases suggested a close relationship to the plant thiol cathepsins and the mammalian cathepsin L subfamily. The cloning of a representative cDNA for a putative Fasciola cathepsin confirmed similarities to the cathepsin L subfamily but revealed low identity with the cathepsin-like proteinases of the related trematode, Schistosoma, nematode cathepsins and the mammalian cathepsin B subfamily. Furthermore, peptide and protein sequencing revealed the modification of certain highly conserved prolines to unusual 3-hydroxyproline derivatives. This is the first report of modified prolines in any proteinase. This finding, as well as the high activities of these cathepsins at neutral to alkaline pH values, raises a number of questions as to the physiological function of these thiol cathepsins and their interaction with host tissues.

Amino Acid Sequence↗
PubMed1993

FoLT PCR: a simple PCR protocol for amplifying DNA directly from whole blood.

FoLT (formamide low temperature) PCR is a protocol for amplifying DNA directly from whole blood without any preparative steps. Up to 10% (vol/vol) whole blood can be added directly into the tube containing the PCR mixture. There is no need for transfers, centrifugations, pre-boiling or any preparative step. It involves the use of formamide (18% vol/vol) as well as reduced incubation temperatures (cycles of 85 degrees, 40 degrees, 60 degrees C). The type of anticoagulant used was critical: sodium heparin or EDTA being superior to lithium or fluoride heparin. Our studies indicate that FoLT PCR probably works by reducing the amount of protein coagulation and allowing more DNA template to be accessible for amplification. The sensitivity of FoLT PCR is such that a single copy gene from 5.5 nucleated cells in 1 microliter of whole blood can be detected.

Anticoagulants↗
PubMed1992

Primary sequence heterogeneity and tissue expression of glutathione S-transferases of Fasciola hepatica.

Glutathione S-transferases (GSTs) from Fasciola hepatica have been purified by glutathione affinity chromatography. Two closely migrating species of Mr 26,000 and 26,500 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several species resolved by two-dimensional gel analysis, indicating substantial heterogeneity among the GSTs. N-terminal amino acid sequencing revealed one core sequence containing three polymorphisms, whereas the sequence of GST peptides implied a minimum of three different GSTs. The amino acid sequence data assigned the F. hepatica GSTs to the mu class of GSTs with high similarities to these proteins in other helminths and mammals. The native GSTs of F. hepatica appeared to behave as dimers as determined by molecular sieving chromatography. The observation that the GSTs of F. hepatica are heterogeneous in sequence and behave as dimers in the native state suggest that these isoenzymes may exhibit considerable functional heterogeneity which may be of importance to the parasite. Immunocytochemical studies suggest that the main source of GST in F. hepatica are the parenchymal cells and peripheral tissues of the parasite. Some extracellular GST is associated with the lamellae of the intestinal epithelium. The identification of an intestinal GST is unique among trematodes studied to date.

Amino Acid Sequence↗
PubMed1990

Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep.

The potential of GST as a vaccine candidate against liver fluke infection in ruminants was studied by vaccinating sheep (n = 9) with GST purified from adult worms of Fasciola hepatica and challenging with 500 F. hepatica metacercariae. The immunization induced a high antibody response to GST in contrast to the poor or undetectable response to this Ag observed in naturally infected sheep. Throughout the trial, the progress of the fluke infection was monitored by measuring RBC hemoglobin levels, the extent of liver damage and the fecal egg output in the sheep. This analysis indicated that a subpopulation (n = 4) of the GST vaccinated animals exhibited no anemia, reduced liver damage and a lower mean fecal egg count relative to the infected control group suggesting a lower fluke burden in these animals. Worm burdens in the livers of the GST vaccine group (107 +/- 22) were 57% lower than in the infected control group (250 +/- 25). The subpopulation of the GST vaccine group demonstrated a 78% reduction in mean worm burdens relative to the control group. These results show that GST of adult F. hepatica is a novel Ag that can significantly protect sheep against liver fluke infection. The results suggest that the immune response to GST is directed to the juvenile worm reducing the number of worms that can establish in the liver of the vaccinated animals.

Animals↗
PubMed1987

Heterogeneity of the human transferrin receptor and use of anti-transferrin receptor antibodies to detect tumours in vivo.

The human transferrin receptor (TFR) which is present on dividing cells, many tumours and erythroid precursors is readily identified using specific monoclonal antibodies. A new anti-human TFR monoclonal antibody, HuLy-m9, is described and its distribution on cell lines, normal and tumour tissue was examined and compared with two other anti-TFR monoclonal antibodies, namely, OKT9 and 5E9. The three antibodies were shown to recognise different epitopes on the surface of the TFR and have different reactivities with in vitro cell lines. Peptide map analyses of the TFR recognised by each monoclonal antibody from the same cell line were identical; however, differences were observed between cell lines. 131I-radiolabelled HuLy-m9 was used to successfully localise a nasopharyngeal carcinoma in vivo.

Antibodies, Monoclonal↗
PubMed1987

Molecular characterization of the murine cytotoxic T-cell membrane glycoprotein Ly-3 (CD8).

The murine Ly-2/3 glycoprotein is a surface marker of T cells restricted by class I major histocompatibility complex antigens. It is a disulfide-bonded heterodimer in which either the alpha or alpha' polypeptide chain encoded by Ly-2 is covalently linked to the beta polypeptide chain encoded by Ly-3. The nucleotide and predicted amino acid sequence of the murine Ly-3 cDNA, isolated by using the rat Ly-3 cDNA clone pX9.15, together with the amino acid sequence of Ly-3.1 peptides and the N terminus, are presented here. The alignment of peptide data from the Ly-3.1 antigen with that of the predicted amino acid sequence of the Ly-3.2 antigen confirmed that the putative Ly-3 cDNA clones do in fact encode the Ly-3 protein. The Ly-3.2 cDNA clones encode a protein of 213 amino acids, which includes a 21-residue leader sequence and structural features in common with immunoglobulin variable, joining, and hinge regions. Searches of protein data bases revealed that Ly-3 is a member of the immunoglobulin superfamily with significant homology to Ly-2, immunoglobulin variable region kappa and lambda light chains, and the beta chain of the T-cell receptor. A single N-linked glycosylation site was found at asparagine-13. The relative expression of two mRNA species (approximately 1.3 and 2.3 kilobases) varied according to the source of mRNA. A murine B1 repeat was located in the 3' untranslated region of Ly-3 cDNA clones.

Amino Acid Sequence↗