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Biomedical subjects

A Orlacchio

Publications and source records attributed to A Orlacchio.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Beta-N-acetylhexosaminidase in the urine, kidney and serum of bromobenzene-intoxicated mice.

beta-N-Acetylhexosaminidase isoenzymes were separated from the kidney, serum and urine of normal mice and mice intoxicated with bromobenzene, using DEAE-cellulose chromatography. Both mouse serum and urine showed hexosaminidase profiles similar to the human counterparts with the presence of B (basic), I (intermediate) and A (acidic) isoenzymes. A notable feature was the presence of a high proportion of an intermediate form in mouse urine which is not always present in human urine. Hexosaminidase activity increased significantly in urine of mice intoxicated with bromobenzene. Its increase was time-dependent and due to kidney damage with a release in the urine of hexosaminidase A, I and, in higher proportion, B. No significant differences were observed in mouse kidney and serum profiles following intoxication with bromobenzene. The total activity of hexosaminidase, using 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as substrate, did not increase in the serum of mice intoxicated with bromobenzene. Both hexosaminidase activity and the isoenzyme pattern in urine can be used as indicators of kidney damage by bromobenzene intoxication.

Animals↗
PubMed1992

Pregnancy modulates the expression of beta-N-acetylhexosaminidase in rat serum and tissues.

1. beta-N-Acetylhexosaminidases in maternal rat serum were separated by DEAE-cellulose chromatography and compared with those of adult rat serum. 2. In pregnant serum there is an increase of the isoenzymes which are entirely composed of beta-subunits (B and intermediate forms). 3. These alterations could be compared to those already described in human pregnancy. 4. The levels of beta-N-acetylhexosaminidase and the relative expression of alpha- and beta-subunits in normal and pregnant serum correlate with the above isoenzyme expression. 5. The increase of B and intermediate forms as well as the increase of specific activity during pregnancy was not peculiar to maternal serum but was also demonstrated in several foetal tissues and in maternal tissues, in which cases the beta-N-acetylhexosaminidase isoenzyme patterns closely resemble the foetal ones rather than those of the adult rat tissues. 6. These analogies strongly suggest that the expression of beta-subunit of beta-N-acetylhexosaminidase is regulated by hormones or other factors related to pregnancy.

Animals↗
PubMed1992

Calcium ionophore A-23187 inhibits the secretion of beta-hexosaminidase from the GG2EE mouse macrophage cell line.

Secretion of the lysosomal enzyme beta-N-acetylhexosaminidase is inhibited by calcium ionophore A-23187 in the GG2EE macrophage cell line. Such inhibition is time and dose dependent. Calcium ionophore A-23187 treatment causes a change in the pattern of hexosaminidase isoenzymes detectable in the cell extract, as assessed by DEAE-cellulose chromatography. In particular, control cells show two hexosaminidase isoenzymes corresponding to hexosaminidase A and B, whereas cells treated with calcium ionophore A-23187 express a third isoenzyme form with properties similar to hexosaminidase S.

Animals↗
PubMed1992

beta-N-acetylhexosaminidases from Serratia marcescens.

Two forms of beta-N-acetylhexosaminidase from Serratia marcescens with an optimum pH of 5.0 and 6.5, respectively, to 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside were separated by DEAE-cellulose chromatography and Sephacryl S-200 chromatography. On the basis of their molecular weights, thermal stability, substrate specificity and isoelectric points, the form with an acidic pH optimum resembled hexosaminidase B, whereas the form with a neutral pH optimum resembled hexosaminidase C. Lectin binding studies showed that the acidic form does not bind to concanavalin-A-Sepharose, Tetragonolobus purpurea-agarose, wheat germ-agglutinin-Sepharose or Ricinus communis-agglutinin-agarose, whereas the neutral form binds to the last two lectin columns.

Hexosaminidases↗
PubMed1992

[Vascular radiology and physics of fluids].

In the cardiovascular system, morphology and functionality are closely related. The observation that atherosclerotic lesions appear with different incidence in the various sites and prefer bifurcations, suggested that vascular architecture might be an important pathogenic factor. Vascular geometry deals with the evaluation of the "form" element from a theoretical point of a view, pointing out the angles and diameters which can make the arterial system physiologically more efficient. The key element of the system is the bifurcation: depending on whether bifurcations are considered as a single entity or as a whole, either "local" or "global" geometry is employed. The morphology of the bifurcation directly affects blood flow patterns, influencing both blood flow velocity and wall shear stress. Experimental studies evidenced that high blood flow velocities and low wall shear stress have, respectively, a preventive or favorable role in atheroma development. By observing these altered flow sites, the areas which are most likely to develop atherosclerotic damage have been detected in the various bifurcations; the areas correspond to angiographic and autoptic findings. Based on their experience, the authors discuss the contribution that the different imaging techniques can give to the study of vascular geometry.

Angiography↗
PubMed1991

beta-N-acetylhexosaminidases in human cerebrospinal fluid and serum of patients with multiple sclerosis.

Beta-N-Acetylhexosaminidase activity and isoenzyme have been investigated in normal human cerebrospinal fluid and that of patients with multiple sclerosis. beta-N-acetylhexosaminidase activity in normal cerebrospinal fluids has been resolved into five components. The major component was in a form that eluted from DEAE cellulose at the same salt concentration as hexosaminidase As, the isoenzyme previously identified in human serum. Cerebrospinal fluid from patients exhibited a different isoenzyme profile, showing a remarkable increase in a form having a pI which was more acidic than that of As. These changes have a potential use in the diagnosis and further biochemical characterization of multiple sclerosis.

Chromatography, Ion Exchange↗
PubMed1991

[Role of computerized tomography in endosseous implantology].

The Authors are proposing the use of computerized tomography for the evaluation of osseous structures of the maxilla and mandible before installation of titanium fixtures. The results indicate that the CT scan can give information about the structure and bone density. The height and the width of the alveolar osseous crests and the relationship with the incisive canal and the mandibular canal are clearly demonstrated. The CT scan can facilitate the measurement of the space available in order to install the titanium fixtures.

Alveolar Process↗
PubMed1991

Analysis of the patterns of expression of mRNAs for the alpha- and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase in mouse epididymis and testis.

mRNA for the alpha- and beta-subunits of mouse beta-N-acetylhexosaminidase were detected in tissue sections of epididymis and testis using 3H-labelled antisense RNA probes by hybridization in situ. There were clear differences in the intensity of the signals given by the two tissues with alpha-subunit mRNA being more abundant in testis than epididymis, while the opposite was so with beta-subunit mRNA. Differences in the isoenzyme patterns of the two tissues are likely to be due, at least in part, to differences in transcription by the Hexa and Hexb genes.

Animals↗
PubMed1991

beta-Hexosaminidase expression in chick embryo fibroblasts in vitro.

1. Two forms of beta-hexosaminidase, similar to hexosaminidase A and hexosaminidase C, were separated by DEAE-cellulose chromatography in chick embryo skin fibroblasts in vitro. 2. beta-Hexosaminidase specific activity increases during development in cultured chick embryo skin fibroblasts in vitro. 3. Concanavalin-A treatment determines the increase of the neutral form, hexosaminidase C, during development. 4. Concanavalin-A reduces the specific activity of beta-hexosaminidase during development.

Animals↗
PubMed1991

Increase of intermediate forms of beta-N-acetylhexosaminidase during rat liver development and regeneration.

1. Rat liver beta-N-acetylhexosaminidase was separated into several different molecular forms by DEAE-cellulose chromatography. 2. The subunit composition of the isoenzymes, as well as the similarities to human hexosaminidases, were determined by using the specific active alpha subunit substrate 4-methylumbelliferyl-beta-N-acetylglucosamine-6-sulphate. 3. As in human tissues, the intermediate form lacked the active alpha subunit and resembled hexosaminidase B rather than A. 4. The intermediate form was markedly increased in foetal liver and in regenerating liver after partial hepatectomy. 5. The variations in isoenzyme expression were accompanied by variations in specific activity of hexosaminidase. 6. Sulphated substrate analysis and thermal stability experiments indicated that the rapid cell proliferation had a greater effect on the formation of beta-subunit of hexosaminidase than on that of alpha-subunit.

Acetylglucosamine↗
PubMed1991

Amphotericin B stimulates secretion of beta-hexosaminidase from mouse adherent spleen cells.

Secretion of the lysosomal enzyme hexosaminidase is induced by amphotericin B in mouse spleen adherent cells that show a significant increase in their candidacidal activity. The stimulation of beta-hexosaminidase is both time and dose dependent. Amphotericin B treatment did not change hexosaminidase expression that is represented mainly by "A-type" hexosaminidase in macrophages, on the basis of its biochemical properties.

Acetylglucosamine↗
PubMed1990

Treatment of HL-60 cells with dimethyl sulphoxide inhibits the formation of beta-N-acetylhexosaminidase S.

beta-N-Acetylhexosaminidase of HL-60 cells was separated into two main forms, A and S, by chromatography on DEAE-cellulose. Analysis of developmental changes in the isoenzyme pattern was complicated by the fact that the specific activity of beta-N-acetylhexosaminidase underwent a 6-fold change during the normal growth cycle. Two other lysosomal enzymes, beta-galactosidase and alpha-mannosidase, behaved similarly. Induction of differentiation of HL-60 cells with dimethyl sulphoxide at a low cell density (3 x 10(5) cells/ml) had a greater effect on the abundance of alpha-subunits of beta-N-acetylhexosaminidase, measured with 4-methylumbelliferyl-beta-N-acetylglucosaminide 6-sulphate, than of beta-subunits, measured with 4-methylumbelliferyl-beta-N-acetylglucosamine, and resulted in an isoenzyme profile in which A and B were the major forms, with the levels of form S greatly decreased.

Cell Division↗
PubMed1990

Identification of an intermediate form of beta-N-acetylhexosaminidase in chorionic villi.

A minor form of beta-N-acetylhexosaminidase has been found in chorionic villi, in addition to the major forms A and B. This form does not hydrolyze the 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside-6-sulpha te substrate, is thermostable, has a higher mol mass (120,000) than A and B (100,000) and on analytical isoelectric focusing, it shows a microheterogeneity with values ranging between 6.3 and 7.0. For these characteristics, it resembles beta-N-Acetylhexosaminidase P from pregnancy serum, from which is chromatographically indistinguishable.

Anions↗
PubMed1990

An enzyme with properties similar to those of beta-N-acetylhexosaminidase S is expressed in the promyelocytic cell line HL-60.

Extracts of the human promyelocytic cell line HL-60 contain a form of beta-N-acetylhexosaminidase that is not retained on columns of benzeneboronate-agarose ('phenylboronate-agarose') and has a pI value lower than that of beta-N-acetylhexosaminidase A. It is clearly distinct from beta-N-acetylhexosaminidase A in its behaviour on DEAE-cellulose columns, and it requires a higher concentration of salt for its elution. This 'extra' form has a higher ratio of activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate and 4-methylumbelliferyl beta-N-acetylglucosaminide than has beta-N-acetylhexosaminidase A and is less stable when heated at 50 degrees C. It has a pH optimum of 4.5 and is therefore not beta-N-acetylglucosaminidase C. Anti-(human beta-N-acetylhexosaminidase alpha-subunit) serum precipitated both beta-N-acetylhexosaminidase A and the 'extra' form, whereas an anti-(beta-subunit) serum precipitated beta-N-acetylhexosaminidase A but not the 'extra' form. Western blotting and immunodetection of polypeptides derived from the 'extra' form revealed a band corresponding in size to mature alpha-subunits. On the basis of this and of its behaviour on isoelectric focusing, chromatofocusing and its kinetic properties, we conclude that the 'extra' form is composed of alpha-subunits and resembles beta-N-acetylhexosaminidase S, the residual form in Sandhoff's disease.

Blotting, Western↗