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Biomedical subjects

A Orlacchio

Publications and source records attributed to A Orlacchio.

At least 55 records · Page 3Linked to original sources
PubMed1997

Activity levels of a beta1,6 N-acetylglucosaminyltransferase in lymphomonocytes from multiple sclerosis patients.

The activity of the Golgi glycosyltransferase beta1,6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T), which plays a role in T-cell activation and cell-cell adhesion, appears to be modulated in resting lymphomonocytes during different phases of multiple sclerosis (MS). In particular, a significant decrease (25-30%) of the enzyme activity was observed, with respect to healthy subjects, in MS patients who were in relapse or in the very early stages of remission. A similar trend was found to be associated with patients affected by active lesions. A statistically significant decrease in the enzyme activity was also observed in patients with the progressive form. By contrast, core 2 GlcNAc-T activity did not appear correlated with duration of the disease. Interestingly, MS individuals under treatment with IFN-beta1a, an immunosuppressive agent, showed levels of activity which were comparable with those observed in healthy subjects. Together, these observations suggest that down-regulation of core 2 GlcNAc-T activity is linked to the occurrence of acute phases in the relapsing-remitting form and to the progressive form of the disease, probably caused by altered expression of glycoproteins which are involved in lymphomonocyte activation and/or interaction with the endothelium. Additionally, it appears that the enzyme assay may provide a useful marker of the disease activity and the effects of therapeutical approaches.

Adult↗
PubMed1997

Constitutive expression of beta-N-acetylhexosaminidase in a microglial cell line: transcriptional modulation by lipopolysaccharide and serum factors.

We investigated the expression of the alpha- and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase in the BV-2 microglial cell line under different culture conditions. Beta-N-acetylhexosaminidase from BV-2 microglia cells was separated into its constituent isoenzymes on diethylaminoethyl (DEAE) cellulose, and its activity was monitored with 4-methylumbelliferyl-beta-N-acetylglucosamine and 4-methylumbelliferyl-beta-N-acetylglucosamine-6-sulphate substrates. Forms corresponding to the mouse isoenzymes A and B were present in the cells incubated in serum-supplemented medium as well as in serum-free medium. Lipopolysaccharide, a well-known activator of microglia in vitro, added to the BV-2 cells in serum-supplemented medium induced a decrease in the specific enzymatic activity determined with the 4-methylumbelliferyl-beta-N-acetylglucosamine substrate. Lipopolysaccharide had no effect on hexosaminidase isoenzyme pattern of BV-2 cells in serum-supplemented medium. The level of alpha-subunit mRNA was increased and the level of beta-subunit mRNA was decreased in BV-2 cells incubated in serum-supplemented medium plus lipopolysaccharide. In the cells incubated in a serum-free medium no significant changes in the hexosaminidase-specific activities towards the above substrates were observed. Interestingly, increased expression of alpha- and beta-subunit mRNA was evident in comparison with cultures in serum-supplemented medium. The present results suggest that the BV-2 cell line may be a useful tool to study the possible role of microglia in the metabolism of brain glycolipids.

Animals↗
PubMed1997

Lysosomal alpha-mannosidases of mouse tissues: characteristics of the isoenzymes, and cloning and expression of a full-length cDNA.

Lysosomal alpha-d-mannosidase from mouse tissues was separated into its constituent isoenzymes by DEAE-cellulose chromatography. Forms corresponding to the human isoenzymes B and A were present in testis, brain, spleen and kidney, whereas in epididymis and liver only the B form was present. Murine alpha-mannosidases A and B are glycoproteins and have pH optima, thermal stabilities and molecular masses similar to those of the human isoenzymes. A full-length cDNA (3.1 kb) containing the complete coding sequence for alpha-mannosidase was isolated from a mouse macrophage cDNA library. Comparison of the deduced amino acid sequences of human and mouse alpha-mannosidases showed that they had 75% identity and 83% similarity. Expression of this cDNA in COS cells showed that both the A and the B isoenzymes can arise from a single transcript. Northern blotting analysis showed a 10-fold range in the abundance of alpha-mannosidase mRNA in mouse tissues, with the highest levels found in epididymis, and the lowest in liver.

Amino Acid Sequence↗
PubMed1997

Distribution of active alpha- and beta-subunits of beta-N-acetylhexosaminidase as a function of leukaemic cell types.

beta-N-Acetylhexosaminidase isoenzymes, and the distribution of the alpha- and beta-subunits forming the enzyme in a representative series of fresh leukaemic cells and in established leukaemic cell lines, were obtained by using a combination of DEAE-cellulose chromatography and assay with the fluorogenic substrates 4-methylumbelliferyl-beta-N-acetylglucosaminide hydrolyzed by both alpha and beta subunits, and 4-methylumbelliferyl-beta-N-acetylglucosaminide-6-SO4 hydrolyzed only by hexosaminidase isoenzymes containing alpha-subunits. The presence of hexosaminidase S (the alpha alpha dimer), was found in all the leukaemic cell populations we surveyed, but not in normal human cells. The presence of this isoenzyme can therefore be considered as an additional marker of leukaemic cells. A prevalence of hexosaminidase A and A-like intermediate forms (alpha beta structure), characterize leukaemic cells of myeloid origin, whereas greater amounts of hexosaminidase B and B-like intermediate forms (beta beta structure), were consistent attributes of leukaemic cells of lymphoid origin. An over-expression of beta-subunits in blasts might be related to their undifferentiated status. These changes in the isoenzymes of hexosaminidase may prove informative about a variety of changes in the biology of leukaemic cells that could range from chromosomal alterations to changes in the proteolytic processing and glycosylation.

Hexosaminidase A↗
PubMed1997

Cytokine secretion and nitric oxide production by mononuclear cells of patients with multiple sclerosis.

Several experimental findings suggest a potential role of excessive nitric oxide (NO) production by macrophages, microglia and astrocytes in the pathogenesis of demyelinating lesions in MS. We assessed the production of nitrites by peripheral blood mononuclear cells (PBMCs) of 15 MS patients (10 F and 5 M) with the R-R form (EDSS: 1-3.0) and in 15 age-matched control subjects. 9 out of the 15 MS patients showed active lesions in MRI at the time of examination. 7 patients were also monitored at the onset, during and following a clinical relapse. Secretion of cytokines by PBMCs was assessed at the basal time and after 24 h of incubation with lipopolysaccharide (LPS). The production of nitrites in the supernatants of PBMCs stimulated and not stimulated with lipopolysaccharide was evaluated. The secretion of IL1 beta, IFN-gamma, TNF-alpha, IL-6 IL-10 and TGF-beta by PBMCs was detected using ELISA methods. The production of NO, both basal and stimulated, was significantly higher in the patients with active lesions than in those without active lesions (p < 0.01). No significant difference was evident between the basal and LPS-stimulated production of NO between control subjects and MS patients without active lesions. During relapses there was a significant increase in NO production by PBMCs compared to the clinical stable stage of the disease (p < 0.0001). This increase was significantly greater in the early stage of relapse than in the late stage (p < 0.04). A decline of NO levels was observed during recovery. Steroid treatment induced a significant decrease in the PBMC NO production of MS patients during exacerbations (p < 0.01). The levels of IL-1 beta, IFN-gamma and TNF-alpha are significantly higher in the supernatants of the PBMCs which produced greater amounts of NO (p < 0.02, p < 0.03, p < 0.01, respectively). On the other hand, NO levels were negatively related to IL-10 and TGF-beta production (R = -75, p < 0.0001 and R = -0.79, p < 0.0001, respectively). The increase production of NO by peripheral blood mononuclear cells demonstrated in our study to be associated with increased production of proinflammatory cytokines could therefore be considered to be a marker of mononuclear cell activation in the peripheral blood of MS patients and, indirectly, of disease activity. Its increased secretion during T cell and monocyte homing in the CNF could contribute to the damage to the blood-brain barrier and the subsequent cytokine-mediated cytotoxic effect to myelin and oligodendrocytes in the white matter of MS patients.

Adult↗
PubMed1997

Structural organization and expression of the gene for the mouse GM2 activator protein.

The GM2 activator protein is an essential component for the degradation of GM2 ganglioside by hexosaminidase A in vivo. Mutations in the human gene coding for the GM2 activator protein cause the AB variant of GM2-gangliosidosis, a condition that is clinically indistinguishable from Tay-Sachs disease. To understand better factors affecting the expression of the GM2 activator protein gene (Gm2a) in mouse tissues, we have determined its exon-intron organization and analyzed its promoter region. Gm2a is about 14 kb, has four exons, and the 5' flanking region contains a CAAT box, Sp1 binding sites, AP-1, AP-2 sites, and a pair of IRE sites. A 1.2-kb fragment upstream from the initiation codon was shown to have promoter activity in NIH 3T3 cells. Similarities between the elements present in Gm2a and Hexa promoters might in part explain their similar expression patterns in mouse tissues. The different levels of GM2 activator protein mRNA in liver, kidney, brain, and testis are not owing to the use of different transcription start sites, because a single start site was found 50 bp upstream from the initiation codon in each these tissues. Northern blot analysis demonstrated variation in the GM2 activator protein mRNA expression during mouse development. Gm2a was mapped to Chromosome (Chr) 11, where it co-segregated with Csfgm.

3T3 Cells↗
PubMed1997

Hexosaminidase in Trichinella spiralis is a single protein with alpha- and beta-subunits catalytic activities.

Beta-N-acetylhexosaminidase is expressed as a single protein in Trichinella spiralis and has catalytic properties similar to the alpha- and beta-subunits of human and mouse isoenzymes A and B. It can hydrolyze the artificial substrates, 4-methylumbelliferyl-beta-D-glucosamine and 4-methylumbelliferyl-beta-D-glucosamine-6-sulphate which are respectively hydrolyzed by the beta- and alpha-subunits. The enzyme is thermostable, has a basic isoelectric point, and thus is similar to the B isoenzyme. Northern blotting experiments indicate that the enzyme is encoded by a single gene. Hexosaminidase from Trichinella spiralis shows that the substrate specificities of alpha- and beta-subunits precede the duplication of their genes.

Animals↗
PubMed1996

Lysosomal hydrolases in serum from human immunodeficiency virus-infected patients.

beta-Hexosaminidase isoenzymes were separated by DEAE-cellulose chromatography in the serum of 23 patients infected with human immunodeficiency virus at different stage of the disease. Forms corresponding to hexosaminidase B, I and A were present in pathological sera. There is an increase in the percentage of hexosaminidase I in pathological sera, that could be used as an additional marker to monitor the clinical stage of the disease. Furthermore, total activities of some lysosomal enzymes were determined in these sera. Activities of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, alpha-mannosidase and beta-mannosidase were significantly higher in the serum of patients at the C3 stage of disease than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate substrate, beta-glucuronidase and beta-galactosidase.

Adult↗
PubMed1996

Purification and properties of human urinary beta-D-mannosidase.

Two forms of the lysosomal enzyme beta-mannosidase were identified and purified from human urine. The purification strategy employed allowed sufficient quantities of both forms to be obtained for subunit analysis and for further characterizations. The two beta-mannosidases were identified as beta-mannosidase B and A, in order of their elution from an ion-exchange column. In all samples examined, the A form was predominant, and the B/A ratio was consistently 0.14. The two forms displayed the same optimum pH (i.e., 4.3) and both were retained by a Concanavalin-A Sepharose column, but showed different isoelectric points, molecular masses and subunit compositions. Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of pure beta-mannosidases B and A suggest that active protein B (160 kDa) consists of three subunits, one 75 kDa and two 49 kDa subunits. Protein A is smaller and appears to be composed of three subunits of 75 kDa, 49 kDa and 37 kDa. Two forms of beta-mannosidase, exhibiting a chromatographic behaviour comparable to the urinary forms, were also detected in human kidney. Nevertheless, in this tissue their relative distribution was different, the B/A ratio being 19.

Chromatography, Agarose↗
PubMed1996

An approach for fluorometric determination of glycosyltransferase activities.

A new strategy for the fluorometric determination of glycosyltransferase activities is reported. The method involves dansyl chloride derivatization of the reduced form (pNH2phenyl) of a hydrophobic, aglycon moiety covalently linked to a number of acceptor substrates (pNO2phenyl). Focusing on the Golgi enzyme core 2 N-acetyl-glucosaminyltransferase, we found that synthesis and fractionation of the dansylated substrate derivative were rapid, easy and inexpensive. Additionally, the corresponding enzyme assay proved reproducible and very sensitive, as 0.4 pmol of reaction product were readily detected. This fluorometric approach appears therefore to be a valid tool for investigating the monitoring differential expression of glycosyltransferases exhibiting low levels of enzyme activity.

Animals↗
PubMed1996

[Biomechanical changes in and lesions of the lumbosacral spine in horse riding. Magnetic resonance assessment].

Spinal injuries account for 5-15% of sport-related traumas. Equestrian sports are considered, together with rugby, motor sport and diving, the riskiest sport for severe spinal injuries. We investigated the biomechanical changes and repeated microtraumas in equitation. We examined with MR the lumbar spines of 12 professional horsewomen and horsemen, 18-51 years old (mean: 33.4 years), belonging to F.I.S.E. (the Italian Federation for Equestrian Sports) and classified as Olympic riders, Senior and Young European riders. An 0.3-T resistive and an 0.2-T permanent magnets with dedicated surface coils were used. We also examined a control group of non-professional healthy volunteers homogeneous by age. MR findings were classified as follows: 1) changes in normal bending and angles of the lumbar spine; 2) injuries and changes in lumbar disks; 3) changes in spinal ligaments; 4) vertebral body injuries. MR was very sensitive in the assessment of all lumbosacral components in all the athletes; besides yielding useful findings to integrate with clinical results, MR also has a predictive value relative to both the continuation of sports activity and the possible damage at the end of it.

Adolescent↗
PubMed1996

[Iron accumulation in the liver of patients with thalassemia major assessed with low field strength magnetic resonance: correlation with clinico-instrumental parameters].

Eight transfusion dependent patients (3 women and 5 men) with thalassemia major undergoing long-term treatment with Desferoxamine were submitted to MRI, with T2* GE sequences and low field strength. The ratio between liver mean signal intensity and skeletal muscle (L/M) and the ratio between the former and subcutaneous fat (L/F) were calculated in all patients. The results were compared with those of a control group of 7 healthy volunteers (7 men). L/M and L/F ratios were separately correlated with the following parameters: patient's age, transfusion history, serum ferritin, ferritin peak and its onset, transaminases (AST and ALT) and chelation index. The latter is a complex parameter allowing the actual assessment of iron content and of the real efficacy of chelation therapy. In all patients, both the L/M and the L/F ratios decreased significantly (L/M ratio: 0.67 +/- 0.45 vs. 1.2 +/- 0.21, p < 0.02; L/F ratio: 0.39 +/- 0.15 vs. 0.84 +/- 0.11, p < 0.001) relative to the control group. No significant correlation was found between the ratios and any hematochemical parameter, except for r = 0.77 (p < 0.04) between L/F ratio and the chelation index. Our study demonstrates that MRI may play a major role in the examination of thalassemic patients even at low field strength and with GE sequences, which yield good quality images with a relatively short acquisition time. Thus, MRI can be suggested for routine liver studies thanks to its high quality depiction of the liver and to its qualitative and semiquantitative yield. The good correlation between L/F ratio and the chelation index permits MR evaluation of the efficacy of different chelation treatments.

Adolescent↗
PubMed1996

[Role of computerized tomography in bladder carcinoma treated with simultaneous combined radiotherapy and chemotherapy].

We report on the pneumocystography-CT follow-up of locally aggressive bladder carcinoma patients treated with combined and simultaneous irradiation and chemotherapy. Particular attention was paid to assess residual thickening of the wall involved by the tumor, which is the most critical parameter to discriminate a persistent from a resolved condition. We examined 16 patients (mean age: 66.5 years) with histologically locally advanced urinary bladder cancer staged T2-T3. All patients underwent the first CT exam of the bladder with the pneumocystography technique before therapy, for disease staging, another exam after chemotherapy and a third one after simultaneous irradiation and chemotherapy. The endoluminal mass completely regressed in all cases, with normal endoscopic findings. In 9 of 16 cases, the bladder wall was thickened in the tumor site, with normal mucosal surface. The histology of bioptic specimens obtained during cystoscopy showed a high frequency of phlogistic reactions from foreign bodies, corresponding to the bladder wall thickening seen on CT images. We conclude that, in extensive neoplastic bladder wall involvement, therapy-induced histologic regressive phenomena can cause a granulomatous reaction in the bladder wall, thus thickening it, which is the most frequent sign in these cases but that, in our experience, was never associated with tumor invasion. This should be always considered in the CT studies performed after this kind of combined treatment.

Aged↗
PubMed1996

[Study of prostatic carcinoma with ultrasonography and magnetic resonance with "dedicated" surface coil].

We investigated the role of an experimental surface coil for low field (0.2 T) MRI of prostatic gland carcinoma; the study was performed comparing MRI with US findings and with bioptic and pathologic results. To this purpose, 16 patients with prostatic cancer suspected at US and demonstrated with US-guided biopsy, were examined with low intensity field (0.2 T) MRI performed with a dedicated saddle-shaped coil and a body coil. This experimental surface coil with perineal application is used to study the prostatic gland with low field MR equipment where no transrectal probe is available. We analyzed the results of this study and found that, in all patients but one, the neoplastic lesions exhibited low signal intensity on T2-weighted images, while the normal gland was hyperintense. Six patients underwent radical prostatectomy and diagnostic imaging findings were correlated with pathologic findings obtained with whole mount specimens and serial histologic slides. The number of tumor foci, their site and size, and stromal reaction were studied on T2-weighted images; the typical low signal intensity was absent in one of 16 patients, in which case the microscopic exam of pathologic specimens showed extensive tumor spread with infiltration of perineural lymphatic vessels, with neither nodular patterns nor stromal reactions. The correlation between MR findings and the whole mount specimens (n = 6) demonstrated MR underestimation of tumor spread. In our opinion, this technique is useful for the detection and characterization of prostatic lesions, in spite of the presence of isointense lesions and the risk of underestimating the disease, especially to the prostatic capsule. To conclude, the dedicated surface coil can improve the diagnostic accuracy of low field MRI.

Adenocarcinoma↗
PubMed1995

Neuromagnetic fields of the brain evoked by voluntary movement and electrical stimulation of the index finger.

Neuromagnetic fields from the left cerebral hemisphere of five healthy, right-handed subjects were investigated under two different experimental conditions: (1) electrical stimulation of the right index finger (task somatosensory evoked fields, task SEF's), and (2) voluntary movement of the same finger referred to as movement-related fields, (MRFs). The two conditions were, performed in random order every 5-8 s. In addition, the task SEF's were compared to control SEF's recorded at the beginning of the experiment in order to find the optimal dewar position for localizing the central sulcus. The magnetic signals of the sources corresponding to the main components of the somatosensory evoked fields (early ones at 24 ms and at 34 ms, and late ones after 50 ms) and movement-related fields (motor field, MF and movement-evoked field I-MEF I) were mapped and localized by means of a moving dipole model. In four out of five subjects the MEF I dipoles were found to be located deeper than the early task SEF dipoles. In addition, all of the task SEF's components were found to exhibit larger amplitudes than the control SEF's components. The results are discussed in respect to the ability to selectively analyze contributions of mainly proprioceptive (area 3a) and cutaneous (area 3b) areas in the primary somatosensory cortex using magnetoencephalography. An additional finding of the study was that all of the task SEF's components were found to exhibit larger amplitudes than the control SEF's components.

Adult↗
PubMed1995

Beta-N-acetylhexosaminidases A and S have similar sub-cellular distributions in HL-60 cells.

In HL-60 cells the most abundant isoenzymes of beta-N-acetyl-hexosaminidase are A (alpha beta) and S (alpha alpha). Sub-cellular fractionation of HL-60 cells by differential centrifugation showed that both A and S forms were present in the lysosomal and post-lysosomal (soluble) fractions in approximately equal abundance. Ion-exchange chromatography showed that a fraction enriched with plasma membranes had the A form, and a form of beta-N-acetylhexosaminidase less acidic than A, but there was no S. Analysis of the alpha-subunits of beta-N-acetylhexosaminidases A and S using Western blotting and immuno-detection with antisera raised to synthetic peptides showed that mature alpha-subunits were present in both A and S isolated from the lysosomal fraction. This observation establishes that the alpha alpha-dimer of beta-N-acetyl-hexosaminidase (S) can be transported to lysosomes in HL-60 cells whereas there is good evidence that this does not take place in fibroblasts. HL-60 cells were not stimulated to secrete lysosomal enzymes by incubating them with NH4Cl and, unlike fibroblasts, are unlikely to use mannose-6-phosphate mediated transport of beta-N-acetylhexosaminidases to lysosomes. Comparison of the sequence of the beta-N-acetylhexosaminidase alpha-subunit with a 43 amino acid sequence of cathepsin D, though to function in the mannose-6-phosphate independent targeting of this enzyme to lysosomes, showed alignment in a region towards the C-terminus in which 21% of the residues were identical with the interposition of a one amino acid gap.

Amino Acid Sequence↗