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A Minelli

Publications and source records attributed to A Minelli.

At least 37 records · Page 2Linked to original sources
PubMed2000

Molecular and binding characteristics of IP3 receptors in bovine spermatozoa.

We have shown the presence of inositol 1,4,5-triphosphate (IP3) receptors in bovine spermatozoa. These receptors are mainly localized and functionally associated with the acrosome region. Molecular characterization of these bovine IP3 receptors has shown that the functional size of the IP3 binding domain is a protein of 66+/-2 kDa, in agreement with the size of both bovine adrenal cortex and bovine adrenal medullar chromaffin cells IP3 receptors. In contrast, bovine cerebellum IP3 receptor displays molecular weight of 220+/-5 kDa, a value in agreement with data in the literature. Bovine IP3 receptors have a one-affinity state characterized by a low affinity (Kd 750 nM) and a relatively high density (7.5 pmol/mg protein). They are functional and release internal calcium upon the binding of the second messenger. Moreover, the finding that the specific A1 adenosine receptor agonist R-PIA elicits almost the same effect as IP3 might be of some help in understanding the physiological role of these inhibitory adenosine receptors in mammalian spermatozoa.

Acrosome Reaction↗
PubMed2000

Ammonium triggers calcium elevation in cultured mouse microglial cells by initiating Ca(2+) release from thapsigargin-sensitive intracellular stores.

Microglial cells are thought to serve as sensors for pathologic events in the brain. In the present study we demonstrate that these cells respond with an increase in intracellular calcium concentration ([Ca2+]i) to intracellular alkaline shifts induced by either application of NH3/NH4+ or by an extracellular alkaline shift. The cytoplasmic pH (pHi) and [Ca2+]i in cultured mouse microglial cells were studied employing the fluorescent probes BCECF and fura-2, respectively. Application of NH3/NH4+ caused an initial rapid alkalinization followed by a slow recovery towards the resting level, while application of alkaline (pH 8.2) solution triggered a slower rise in pHi. The [Ca2+]i elevation triggered by NH3/NH4+ and extracellular alkaline shift were caused by different mechanisms: extracellular alkalinization induced a transmembrane Ca2+ entry, whereas NH3/NH4+ triggered Ca2+ release from thapsigargin- and ATP-sensitive intracellular pools. The mobilization of intracellular Ca2+ caused by NH3/NH4+ was blocked by a specific inhibitor of phospholipase C, U-73122, but was not affected by an inhibitor of G-protein, pertussis toxin. This implies that NH3/NH4 interacts with phospholipase C and leads to an increase in the intracellular level of inositol 1,4,5-trisphosphate (InsP3). In contrast to a previous study using a microglial cell line, application of NH3/NH4+ did not result in a release of tumor necrosis factor alpha (TNF-alpha), a marker of microglial activation, in the primary microglial cells. This implies that ammonium does not lead to activation of microglia in the culture model.

Adenosine Triphosphate↗
PubMed2000

Isochromosome (7)(q10) in Shwachman syndrome without MDS/AML and role of chromosome 7 anomalies in myeloproliferative disorders.

Shwachman syndrome (SS) is an autosomal recessive disorder in which bone marrow dysfunction is observed, with development of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) in up to one third of the cases. Inconclusive data are available as to increased chromosome breakage in SS, while chromosome 7 anomalies, and often an isochromosome (7)(q10), are frequent in cases with MDS/AML. We report on the consistent presence of an i(7)(q10) in the bone marrow and blood lymphocytes in one of two sisters affected with SS without any clinical or cytological signs of MDS/AML. Thus, this patient was either a case of constitutional mosaicism for the i(7)(q10), or this had to be acquired in a nondysplastic and non-neoplastic marrow clone. DNA polymorphism analysis demonstrated the paternal origin of the i(7q). We postulate that the SS mutation acts as a mutator gene, and causes karyotype instability; abnormal clones would thus arise in the marrow, and chromosome 7 anomalies, i(7q) in particular, will in turn lead to MDS/AML. If this interpretation is correct, it would be also an indication to consider chromosome 7 anomalies in general, out of SS, as primary changes in MDS/AML pathogenesis.

Bone Marrow Diseases↗
PubMed2000

Immunolocalization of A1 adenosine receptors in mammalian spermatozoa.

The presence of A1 adenosine receptors (A1AR) in mammalian spermatozoa was previously demonstrated by radiochemical and immunochemical detection. This study was performed to investigate the cellular location of the A1AR to determine whether these receptors were somehow connected with ecto-adenosine deaminase and to evaluate their function in calcium uptake. By immunofluorescence staining we showed that in mammalian spermatozoa A1AR were constantly localized in the acrosomal region. This finding was confirmed by immunogold detection. Confocal analyses with anti-A1 and anti-ADA antibodies showed a high degree of co-localization. Calcium loading assay showed that this association was functional and affected calcium accumulation in mammalian spermatozoa. Therefore, we concluded that the acrosomal localization of A1AR was a constant feature in mammalian sperm. Moreover, these A1 receptors were functionally coupled to ecto-ADA and were able to modulate calcium uptake into an IP3-gated store.(J Histochem Cytochem 48:1163-1171, 2000)

Adenosine Deaminase↗
PubMed1999

Neuronal, glial, and epithelial localization of gamma-aminobutyric acid transporter 2, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in the cerebral cortex and neighboring structures.

Neuronal and glial high-affinity Na+/Cl(-)-dependent plasma membrane gamma-aminobutyric acid (GABA) transporters (GATs) contribute to regulating neuronal function. We investigated in the cerebral cortex and neighboring regions of adult rats the distribution and cellular localization of the GABA transporter GAT-2 by immunocytochemistry with affinity-purified polyclonal antibodies that react monospecifically with a protein of 82 kDa. Conventional and confocal laser-scanning light microscopic studies revealed intense GAT-2 immunoreactivity (ir) in the leptomeninges, choroid plexus, and ependyma. Weak GAT-2 immunoreactivity also was observed in the cortical parenchyma, where it was localized to puncta of different sizes scattered throughout the radial extension of the neocortex and to few cell bodies. In sections double-labeled with GAT-2 and glial fibrillary acidic protein (GFAP) antibodies, some GAT-2-positive profiles also were GFAP positive. Ultrastructural studies showed GAT-2 immunoreactivity mostly in patches of varying sizes scattered in the cytoplasm of neuronal and nonneuronal elements: GAT-2-positive neuronal elements included perikarya, dendrites, and axon terminals forming both symmetric and asymmetric synapses; nonneuronal elements expressing GAT-2 were cells forming the pia and arachnoid mater; astrocytic processes, including glia limitans and perivascular end feet; ependymal cells; and epithelial cells of the choroid plexuses. The widespread cellular expression of GAT-2 suggests that it may have several functional roles in the overall regulation of GABA levels in the brain.

Animals↗
PubMed1999

Activity of IMP- and AMP-preferring isoforms of 5'-nucleotidase from human seminal plasma with AMP analogues.

AMP analogues modified at various positions of the molecule were checked as substrates for the two soluble isoforms of 5'-nucleotidase from human seminal plasma. These isoforms were isolated to near homogeneity by affinity chromatographies. AMP derivatives were differently dephosphorylated by both the isoforms depending on the site of modification in the natural compound. Changes in the phosphate moiety reduced significantly hydrolysis by the IMP-preferring form, whereas the AMP-preferring form was less affected. The AMP-preferring form was characterized by a relatively broad specificity toward substrate analogues indicating that the binding domains for the phosphate moiety of these isoforms are not identical. Substitutions at the C-8 adenine base reduced the hydrolysis rate of both the enzymes and variations of the syn-anti conformational equilibrium resulted in different effects on catalysis by both forms. Therefore, the orientation of the heterocyclic base around the glycosidic bond may not be the crucial factor affecting binding and catalytic activity. Hydrogen bonding potential of base N-7 was essential for the binding and catalysis of the IMP- but not of the AMP-preferring form. This was the most striking difference between the studied isoforms. Modifications and substitutions of 6-amino function, better accepted by the IMP-preferring form than by the AMP-preferring form, indicated that no essential hydrogen bonding is required for catalytic activity. The binding was however significantly slowed in 6-SH-PuMP. Hydrogen bonding potential of N-1 was significant for the hydrolysis rate of the IMP- but not of the AMP-preferring form. We suggest that these human seminal plasma isoforms of soluble 5'-nucleotidase, characterized by unique features, may represent the tissue-specific expression of the polymorphic gene.

5'-Nucleotidase↗
PubMed1999

Early onset of gastric carcinoma and constitutional deletion of 18p.

We report on the association of a gastric carcinoma and a constitutional deletion of the short arm of chromosome 18 in a 14-year-old patient. The phenotype of the patient, including microcephaly, ptosis, micrognathia, tetralogy of Fallot, and mental retardation, fits well with previously reported cases of del(18p); she also showed a positive serology against Helicobacter pylori. The comparison of the alleles of polymorphic loci located on the short arm of chromosome 18 between the patient and her parents showed a maternal origin of the abnormal chromosome. Loss of heterozygosity (LOH) for loci located in the long arm of chromosome 18 is a frequent event in gastric carcinomas; it was observed in the tumoral mass of our patient and again, the alleles lost were of maternal origin. We postulate that the constitutional chromosomal abnormality may have favored the loss of the abnormal chromosome in some cells and that the loss of the deleted chromosome 18 (demonstrated by LOH for this chromosome in the tumoral mass) has been an early step in the pathogenesis of the gastric carcinoma of our patient with Helicobacter pylori infection acting as a cofactor.

Abnormalities, Multiple↗
PubMed1999

CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa.

Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, which showed the localization of CD26 at the postacrosomal region and at the midpiece of the spermatozoa after incubation with vesicles. Moreover, a similar localization of CD26 and ecto-ADA on the spermatozoa was evidenced after fusion. Our results suggest that the interaction CD26/ecto-ADA might be responsible for fusion. Since A(1)ARs are said to be second receptors for ecto-ADA to form ecto-ADA/A(1)AR complexes, and since horse spermatozoa have A(1)ARs at their surface, the interaction CD26/ecto-ADA/A(1)AR during the fusion process cannot be ruled out.

Adenosine Deaminase↗
PubMed1998

Neuronal and glial localization of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in human cerebral cortex: with a note on its distribution in monkey cortex.

High-affinity gamma-aminobutyric (GABA) plasma membrane transporters (GATs) influence the action of GABA, the main inhibitory neurotransmitter in the human cerebral cortex. In this study, the cellular expression of GAT-1, the main cortical GABA transporter, was investigated in the human cerebral cortex by using immunocytochemistry with affinity-purified polyclonal antibodies directed to the C-terminus of rat GAT-1. In temporal and prefrontal association cortex (Brodmann's areas 21 and 46) and in cingulofrontal transition cortex (area 32), specific GAT-1 immunoreactivity (ir) was localized to numerous puncta and fibers in all cortical layers. GAT-1+ puncta were distributed homogeneously in all cortical layers, although they were slightly more numerous in layers II-IV, and appeared to have a preferential relationship to the somata and proximal dendrites of unlabeled pyramidal cells, even though, in many cases, they were also observed around nonpyramidal cells. Electron microscopic observations showed that GAT-1+ puncta were axon terminals that formed exclusively symmetric synapses. In addition, some distal astrocytic processes also contained immunoreaction product. Analysis of the patterns of GAT-1 labeling in temporal and prefrontal association areas (21 and 46), in cingulofrontal transition areas (32), and in somatic sensory and motor areas (1 and 4) of the monkey cortex revealed that its distribution varies according to the type of cortex examined and indicated that the distribution of GAT-1 is similar in anatomically corresponding areas of different species. The present study demonstrates that, in the human homotypical cortex, GAT-1 is expressed by both inhibitory axon terminals and astrocytic processes. This localization of GAT-1 is compatible with a major role for this transporter in GABA uptake at GABAergic synapses and suggests that GAT-1 may contribute to determining GABA levels in the extracellular space.

Animals↗
PubMed1998

Polycyclic aromatic hydrocarbons enhance the production of phorbol 12-myristate 13-acetate-induced superoxide ions in human monocytes.

Monocytes, separated from peripheral blood, preincubated with a mixture of polycyclic aromatic hydrocarbons (PAHs) show an enhanced production of superoxide ions (O2-.) when the cells are stimulated with phorbol 12-myristate 13-acetate (PMA, direct activator of protein kinase C). When opsonized-zymosan is used as a stimulus (receptor-dependent stimulus), no enhanced production of O2-. is observed. Superoxide production increases dose dependently up to a PAH concentration of 5 microg/ml. Although the effect was rather small (125-145% of the control value), it was significant and reproducible. Similar enhancing activity was also observed in the production of hydrogen peroxide (H2O2) excluding an inhibitory effect of PAHs on the enzyme superoxide dismutase (SOD). Since the effect is related to the concentration of PMA and in the absence of stimulus, the O2-. is undetectable in both the control and in the PAHs-treated cells, it is concluded that the over production of O2-. is due to an increased activity of the NADPH oxidase.

Carcinogens, Environmental↗
PubMed1998

Regulation of agonist-receptor binding by G proteins and divalent cations in spermatozoa solubilized A1 adenosine receptors.

Solubilized A1 adenosine receptor (A1AR) was used to investigate the effect of several cations on agonist-binding characteristics and GTP hydrolysis. It was shown by Western blot with G beta-M14 that this preparation contains both G proteins and receptor. The role of the receptor molecule is to facilitate the activation of G proteins as alpha-GTP complex, and GTP hydrolysis has important consequences for the basic deactivation mechanism. Divalent cations, such as Mn2+, Ca2+, and Mg2+, potentiated the agonist-specific binding: Mn2+ had the highest apparent affinity with half-maximal effect at 50 microM. Binding assays, performed in the presence of 100 microM Mn2+, showed an increase in the apparent affinity of the binding sites, whereas, in the presence of 1 mM Mg2+, significant alteration of the apparent affinity, but not of the number of sites, was detected. Concentrations of 1 mM Mg2+ and 100 microM Mn2+ enhanced GTPase activity, whereas 5 mM Ca2+ resulted in the increase of Vmax values without significant alterations of K(m). In the presence of A1-specific agonists, Mn2+ and Mg2+ caused a decrease of Vmax values and an increase of GTP affinity. Other cations, such as Co2+, Cd2+, Cu2+, and Zn2+, inhibited the binding capacity but caused almost no changes in GTP hydrolysis kinetics.

Animals↗
PubMed1998

Molecules, developmental modules, and phenotypes: a combinatorial approach to homology.

Most traditional views of homology rely on two unwarranted premises: the pervasively hierarchical nature of biology, inclusive of the levels of genes, development, and morphology and the linear mapping of genes onto developmental schedules and of developmental schedules onto phenotypes. These premises are only occasionally verified. Hierarchical behavior is negated by gene duplication and exon shuffling at the level of genes, by the coexistence of autonomous vs nonautonomous gene expression at the level of development, by ontogenetic repatterning at the level of morphology. The linearity of mapping of genes onto development is disturbed by genetic piracy, uncoupling of positional vs spatial control, and pleiotropy. The independence of developmental modules affects the mapping of development onto morphology and, finally, the peculiar topology of the epigenetic code affects the linearity of the gene to phenotype mapping. To cope with this complex behavior, a combinatorial approach to homology is recommended.

Animals↗
PubMed1998

EAAC1, a high-affinity glutamate tranporter, is localized to astrocytes and gabaergic neurons besides pyramidal cells in the rat cerebral cortex.

High-affinity uptake of glutamate from the synaptic cleft plays a crucial role in regulating neuronal activity in physiological and pathological conditions. We have used affinity-purified specific polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of rabbit and rat EAAC1, a glutamate (Glu) transporter believed to be exclusively neuronal, to investigate its cellular and subcellular localization and whether it is expressed exclusively in glutamatergic cells of infragranular layers, as suggested by previous studies. Light microscopic studies revealed that EAAC1 immunoreactivity (ir) is localized to neurons and punctate elements in the neuropil. EAAC1-positive neurons were more numerous in layers II-III and V-VI, i.e. throughout all projection layers. Most EAAC1-positive neurons were pyramidal, although nonpyramidal cells were also observed. Some EAAC1-positive non-pyramidal neurons stained positively with an antiserum to GAD, thus demonstrating that EAAC1 is not confined to glutamatergic neurons. Non-neuronal EAAC1-positive cells were also observed in the white matter, and some of them stained positively with an antiserum to GFAP. Ultrastructural studies showed that EAAC1-ir was in neuronal cell bodies, dendrites and dendritic spines, but not in axon terminals, i.e. exclusively postsynaptic. Analysis of the type of axon terminals synapsing on EAAC1-ir profiles showed that 97% of them formed asymmetric contacts, thus indicating that EAAC1 is located at the very sites of excitatory amino acid release. Unexpectedly, EAAC1-ir was also found in a few astrocytic processes located in both the gray and the white matter. The localization of EAAC1 may explain the pathological symptoms that follow EAAC knockout (seizures and mild toxicity), as seizures could be due to the loss of EAAC1-mediated fine regulation of neuronal excitability at axodendritic and axospinous synapses, whereas the mild toxicity may be related to the functional inactivation of astrocytic EAAC1.

Amino Acid Transport System X-AG↗
PubMed1998

Occurrence of prostasome-like membrane vesicles in equine seminal plasma.

Equine seminal plasma was shown to contain membrane vesicles that are similar to the well characterized prostasomes in human seminal plasma. Determination of nucleoside and nucleotide concentrations of these particles have shown that ATP, ADP and adenosine are the main components of the nucleotidic pool. 5' nucleotidase, endopeptidase and dipeptidyl peptidase i.v. activities have been found on the surface of the particles. The interaction between these prostasome-like vesicles and spermatozoa was demonstrated by electron micrograph scans which revealed the steps of a fusion-like process leading to mixing of the membranes. In addition, endopeptidase activity, a marker enzyme of these seminal vesicles that is normally absent from equine spermatozoa, was shown to be acquired by these cells after interaction with the vesicles. The addition of these vesicles to equine spermatozoa resulted in the modification of adenylate catabolism. Therefore, a role in stabilizing the energy charge of the spermatozoa thus allowing longer viability is proposed for these organelles.

Adenosine↗
PubMed1998

Concurrent cytogenetic and molecular investigations in uterine and ovarian neoplasms.

We studied a group of 24 uterine and ovarian neoplasms with the purpose to verify if any correlation could be established between chromosomal abnormalities, loss of heterozigosity (LOH) and microsatellite instability (MIN). Tumor specimens obtained from 24 women (12 affected by ovarian and 12 by uterine neoplasms) were split in two parts, one was used for short term cultures for cytogenetic investigation while from the second DNA was extracted for molecular studies. We studied 22 polymorphic loci from 19 chromosomes and compared the alleles observed in the tumor with those observed in the DNA obtained from peripheral blood. Extensive loss of heterozigosity was observed when total or partial chromosomal loss was observed in at least 50% of the examined cells; MIN did not correlate with any particular cytogenetic abnormality nor with LOH.

Chromosome Aberrations↗