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Biomedical subjects

A Orlacchio

Publications and source records attributed to A Orlacchio.

At least 73 records · Page 4Linked to original sources
PubMed1995

A fluorescent assay for the determination of UDP-GlcNAc: Gal beta 1,3GalNAc-R (GlcNAc to GalNAc) beta 1,6-N-acetylglucosaminyltransferase activity.

A fluorescent assay for UDP-GlcNAc: Gal beta 1,3Gal-NAc-R beta 1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) activity has been developed involving dansylation of the enzyme reaction product. Core 2 GlcNAc-T detection was performed using unlabeled UDP-GlcNAc as the donor and Gal beta 1,3GalNAc alpha-pAp as the acceptor. The product, Gal beta 1,3(GlcNAc beta 1,6)-GalNAc alpha-pAp, was quantitatively derivatized with dansyl chloride at the NH2 moiety of the pAp group and the resultant fluorescent trisaccharide was separated on a Spherisorb ODS2 HPLC column. This method, rapid and economical, was found to be sensitive enough for the detection of 1 pmol of reaction product and therefore represents a reliable alternative to assays which use radiolabeled substrates. Additionally, the approach described here can be adapted for the assay of other glycosyltransferases, where the acceptor substrate has a pAp group as a hydrophobic aglycon linker.

Acetylgalactosamine↗
PubMed1995

Partial sequence of the purified protein confirms the identity of cDNA coding for human lysosomal alpha-mannosidase B.

Human lysosomal alpha-mannosidase has been purified by a simple and rapid method in sufficient quantities for the analysis of its subunit composition and partial protein sequencing. Analysis of the N-terminal residues of the 30 kDa polypeptide has enabled us to confirm the identity of the recently cloned cDNA that was tentatively identified as that of lysosomal alpha-mannosidase [Nebes and Schmidt (1994) Biochem. Biophys. Res. Commun. 200, 239-245] and to locate the position of this polypeptide within the total deduced amino acid sequence. This finding will therefore provide a firm foundation for the characterization of alpha-mannosidosis mutations.

Amino Acid Sequence↗
PubMed1995

Platelet glycohydrolase activities: characterization and release.

Granules containing acid hydrolases have been detected in human platelets but have not been thoroughly characterized. We have studied the activity and characteristics of glycohydrolases present in normal human platelets, evaluated their release upon stimulation with thrombin, and assessed the contribution of platelet - released lysosomal contents to the glycohydrolase activity present in normal serum. Platelets contained a remarkable glycohydrolase activity with a prevalence of beta - N-acetylhexosaminidase. All glycohydrolases were released to some extent upon stimulation with thrombin and contributed to the glycohydrolase activity found in human serum. alpha-Mannosidase and alpha-galactosidase were partially inactivated after release by a mechanism as yet undefined. In addition, thrombin stimulation affects the intraplatelet isoenzyme pattern of beta-N-acetylhexosaminidase by producing the appearance of a new form.

Blood Platelets↗
PubMed1995

No evidence of the hook effect in peritoneal fluid CA 125 measurement using immunoenzymatic second generation assays: comparison with immunoradiometric assays.

OBJECTIVE: To monitor the occurrence of the hook effect in measurements of peritoneal fluid CA 125 levels using two different immunoenzymatic second generation assays (ETI-II and EIA-II), and to compare these results with those obtained using the respective immunoradiometric versions of the assays (IRMA-II and ELSA-II). STUDY DESIGN: CA 125 levels were determined in peritoneal fluid and serum samples obtained from 45 women with gynecological diseases. The assays were carried out using IRMA-II and ETI-II (Sorin Biomedica) and ELSA-II and EIA-II (CIS Bio International) assays. Occurrence of the hook effect and linearity of the assays were evaluated. Statistical analyses were performed by Wilcoxon's test and linear regression analysis. RESULTS: Undiluted peritoneal fluids, assayed for their CA 125 content, showed falsely underestimated values of the antigen when IRMA-II and ELSA-II assays were performed. The phenomenon disappeared only at high dilutions of the sample (> 50). Conversely, immunoenzymatic ETI-II and EIA-II assays performed on undiluted peritoneal fluids did not show underestimated CA 125 values. CA 125 values obtained by immunoenzymatic assay were lower than those obtained using their respective immunoradiometric versions at a dilution of 1:100 (P < 0.001). A good correlation was observed between ELSA-II and EIA-II (r = 0.929) CA 125 values. CONCLUSION: The EIA-II immunoenzymatic assay appears to be more suitable for CA 125 measurement in peritoneal fluid in that it is not subject to the hook effect and its results correlated well with those obtained via its immunoradiometric version.

Ascitic Fluid↗
PubMed1995

[Computerized tomography assessment of bone damage following injury of the anterior cruciate ligament].

An ACL tear causes an anterior subluxation of the tibial bone which rotates with its fulcrum on the medial collateral ligament; consequently, the lateral femoral condyle impacts on the external tibial plateau. The presence of a subcortical lesion of the spongiform bone in the posterior external tibial plateau is an indirect sign of an ACL tear. On MR images, traumatic changes are depicted as changes in bone marrow signals. To assess the diagnostic capabilities of CT in demonstrating the bone lesion subsequent to ACL trauma, 23 patients with positive MR exams of the knee were submitted to CT. Fifteen patients had complete ACL tears, 8 had partial tears and 20 exhibited an occult fracture of the posterior portion of the external tibial plateau. This study pointed out a significant correlation between ACL tears and changes in the spongiform structure of the posterolateral tibial plateau. Such changes are depicted not only by MRI but also by CT: the latter method is also a valuable tool to study these conditions long after the traumatic event. No more than 5 slices, 1-1.5 mm thick, acquired at the tibial plateau allowed the trabecular structure and its abnormal changes to be studied without markedly lengthening examination time.

Anterior Cruciate Ligament Injuries↗
PubMed1995

Influence of cell differentiation and protein kinase C activation on sub-cellular distribution of beta-N-acetylhexosaminidases of HL 60 cells.

There have been several accounts regarding the alterations of the lysosomal enzyme beta-N-acetylhexosaminidase in human leukaemic cells. In addition to Hex A (alpha beta) and Hex B (beta beta) forms, leukaemic cells contain a third isoenzyme displaying many characteristics in common with Hex S, the alpha alpha dimer representing the residual activity in patients with Sandhoff's disease. In the human leukaemic cell line HL 60, A (alpha beta) and S (alpha alpha) are the most abundant forms. Sub-cellular fractionation of HL 60 cells showed that both A and S forms were present in the lysosomal and post-lysosomal fractions, however, a proportion of activity was found to be associated with the plasma membrane. The phorbol ester 12-O-tetra-decanoylphorbol-13-acetate (TPA) exerts complex effects on the physiology of HL 60 cells, leading to cell differentiation along the macrophage pathway and including activation of Protein Kinase C (PKC). In order to assess the extent to which cell differentiation and PKC activation plays a role in modulating the expression of hexosaminidase during cell differentiation, we treated HL 60 cells with TPA and in parallel with the more specific activator of PKC, 1-oleoyl-2-acetyl diglycerol (OAG) which does not cause cell differentiation. We observed that 24 h exposure of HL 60 cells to TPA or OAG produced significant modification of the hexosaminidase isoenzyme pattern of HL 60 cells. The most remarkable effect was seen in both cases in the plasma membrane fraction. Taken together, our results suggest a correlation between hexosaminidase expression and kinase(s) activation.

Cell Differentiation↗
PubMed1994

GM2 activator protein expression in mouse tissues.

The GM2 activator protein is an essential cofactor of hexosaminidase A in the degradation of GM2 ganglioside and it is responsible for the variant AB of GM2 gangliosidosis in man. In this study GM2 activator protein and its mRNA were determined in different mouse tissues. It was found that this protein is expressed mostly in the spleen and testis followed by brain and kidney which represent the main source in man. It is also interesting that in mouse testis there is a higher expression of the alpha subunit of hexosaminidase, thus suggesting a relationship between alpha subunit and GM2 activator protein. Furthermore the results indicate that the expression of GM2 activator protein is regulated, at least in part, at the transcriptional level.

Animals↗
PubMed1994

Different expression of beta-N-acetylhexosaminidase in mouse tissues.

The expression of genes encoding for the alpha and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase was investigated in different mouse tissues. It was found, using fluorogenic substrates, that the amounts of alpha and beta subunits were not the same in different tissues: alpha-subunit was more abundant in the brain, beta-subunit in epididymis and brain. The different isoenzyme patterns and specific activities in mouse tissues are due to the differences in the amount of hexosaminidase subunits. The mRNA, evaluated by Northern blotting analyses, revealed a greater expression of alpha-subunit in the testis and of beta-subunit in the brain and epididymis. The results indicate, therefore, that gene expression and the amount of subunits are in good relationship for beta-subunit, whereas there is no correlation for alpha-subunit.

Animals↗
PubMed1994

Advances in interventional radiology in oncology.

Interventional radiology (IR) includes all procedures performed percutaneously under US, fluoroscopy, CT or DSA guidance by a radiologist to obtain diagnostic and therapeutic achievements. According to the pathology under examination, these procedures may be applied alone, as a definite therapeutic tool, or together with other traditional techniques permitting an easier and more successful application. In this report the authors discuss briefly the IR possibilities in many oncological branches.

Chemoembolization, Therapeutic↗
PubMed1994

[Magnetic resonance imaging in denervated muscle. A preliminary study].

Ten patients with various forms of peripheral neuropathy and spinal radiculopathy were examined with MRI using a low-field permanent magnet (0.2 T) and spin echo (SE)/inversion recovery (IR) sequences, with an adequate inversion time to suppress healthy muscle signal. In acute denervation MR sensitivity was low on both sequences; in subacute denervation the damaged muscle was more intense than the healthy muscle only on IR sequences. MRI adequately depicted fatty infiltration in chronic denervation. In conclusion, MRI is a promising tool for mapping and noninvasively monitoring denervated motor units in skeletal muscles, whose role is currently complementary to that of electromyography.

Acute Disease↗
PubMed1993

Cloning and sequence analysis of a cDNA clone coding for the mouse GM2 activator protein.

A cDNA (1.1 kb) containing the complete coding sequence for the mouse GM2 activator protein was isolated from a mouse macrophage library using a cDNA for the human protein as a probe. There was a single ATG located 12 bp from the 5' end of the cDNA clone followed by an open reading frame of 579 bp. Northern blot analysis of mouse macrophage RNA showed that there was a single band with a mobility corresponding to a size of 2.3 kb. We deduce from this that the mouse mRNA, in common with the mRNA for the human GM2 activator protein, has a long 3' untranslated sequence of approx. 1.7 kb. Alignment of the mouse and human deduced amino acid sequences showed 68% identity overall and 75% identity for the sequence on the C-terminal side of the first 31 residues, which in the human GM2 activator protein contains the signal peptide. Hydropathicity plots showed great similarity between the mouse and human sequences even in regions of low sequence similarity. There is a single N-glycosylation site in the mouse GM2 activator protein sequence (Asn151-Phe-Thr) which differs in its location from the single site reported in the human GM2 activator protein sequence (Asn63-Val-Thr).

Amino Acid Sequence↗
PubMed1993

Blood testosterone levels are correlated with beta-N-acetylhexosaminidase activity in human caput epididymis.

beta-N-acetylhexosaminidase exhibits a relatively high activity in human epididymis. Its isoenzymatic profile and immunological properties led us to conclude that the increased activity was not due to the expression of an isoform unrelated to the HEX A and B present in other human tissues. Measurement of enzyme levels in the three distinct epididymal regions revealed that in any given sample, activity was higher in the caput than in the corpus or cauda. Moreover, we found a striking correlation between beta-HEX activity in the caput region and concentrations of blood testosterone, suggesting a possible involvement of the hormone in modulating enzyme expression. Since the caput epididymis plays a role in the maturation of sperm cells, our data may be an indication that beta-HEX activity in the caput has a physiological relevance in human epididymis functions.

Chromatography, Ion Exchange↗
PubMed1993

[MR staging in renal neoplasms. Comparison with surgical data].

Forty-six patients bearing renal lesions were studied with magnetic resonance (MR) imaging. A superconductive magnet (1.5 T) was used to stage the lesions according to Robson's criteria. A positive correlation between MR and pathologic results was observed in 40 cases. MR imaging overstaged 4 lesions: 2 of them for suspected infiltration of perirenal fat (MR stage II, versus pathologic stage I), one was a false-positive finding for lymph node metastasis (MR stage IIIb, versus pathologic stage II) and another one for suspected bowel loop infiltration (MR stage IVa, versus pathologic stage II). Two lesions were understaged: in one case bowel loop infiltration was missed by MR imaging (MR stage II, versus pathologic stage IVa) and another one for a false-negative lymph node metastasis (MR stage II, versus pathologic stage IIIb). In all the cases with involvement of renal vein and vena cava, MR imaging correctly demonstrated the presence of neoplastic thrombi.

Adult↗
PubMed1992

A coupled assay for UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc).

UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc) (i.e., core 2 GlcNAc-T) is a developmentally regulated enzyme of the O-linked oligosaccharide biosynthesis pathway. We have developed a coupled-enzyme assay for core 2 GlcNAc-T that is approximately 100 times more sensitive than the standard assay using UDP-[3H]GlcNAc as a sugar donor. Core 2 GlcNAc-T reactions were performed using unlabeled UDP-GlcNAc donor and Gal beta 1-3GalNAc alpha-paranitrophenyl (pNp) as acceptor. The product, Gal beta 1-3(GlcNAc beta 1-6)GalNAc alpha-pNp was then further reacted with purified bovine beta 1-4Gal-T and UDP-[3H]Gal to produce Gal beta 1-3([3H]Gal beta 1-4GlcNAc beta 1-6) GalNAc alpha-pNp, which was separated on an Ultrahydrogel HPLC column. Approximately 10% of the available GlcNAc-terminating acceptor was substituted in the Gal-T reaction, allowing 1 pmol of product to be readily detected. The increased sensitivity of the coupled assay should facilitate studies of core 2 GlcNAc-T activity where material is limiting or specific activity is low.

Animals↗
PubMed1992

Cloning and sequence analysis of a cDNA encoding the alpha-subunit of mouse beta-N-acetylhexosaminidase and comparison with the human enzyme.

cDNAs encoding the mouse beta-N-acetylhexosaminidase alpha-subunit were isolated from a mouse testis library. The longest of these (1.7 kb) was sequenced and showed 83% similarity with the human alpha-subunit cDNA sequence. The 5' end of the coding sequence was obtained from a genomic DNA clone. Alignment of the human and mouse sequences showed that all three putative N-glycosylation sites are conserved, but that the mouse alpha-subunit has an additional site towards the C-terminus. All eight cysteines in the human sequence are conserved in the mouse. There are an additional two cysteines in the mouse alpha-subunit signal peptide. All amino acids affected in Tay-Sachs-disease mutations are conserved in the mouse.

Amino Acid Sequence↗