Biomedical subjects

A Pugnaloni

Publications and source records attributed to A Pugnaloni.

At least 19 recordsLinked to original sources

PubMed1999

Expression of vascular endothelial growth factor related to 72-kilodalton metalloproteinase immunostaining in patients with serous ovarian tumors.

BACKGROUND: The aim of the current study was to investigate the expression of vascular endothelial growth factor (VEGF) by neoplastic cells in patients with serous ovarian tumors. The correlation between neoangiogenesis and 72-kilodalton metalloproteinase (MMP2) immunostaining also was evaluated. METHODS: Fifty-eight patients with serous ovarian tumors who were treated at the Institute of Gynecology and Obstetrics of Ancona University (Ancona, Italy) were used as the study population; 10 women had serous cystoadenoma, 16 women had a serous borderline tumor, and 32 women had invasive cystoadenocarcinoma. Expression of VEGF and MMP2 was evaluated immunohistochemically by polyclonal antibody anti-VEGF (dilution, 1:100) and affinity purified, rabbit anti-MMP2, formalin fixed, paraffin embedded tissue. Positive staining was expressed as a percentage of positive cells per 10(3) counted neoplastic cells. RESULTS: Compared with cystoadenomas and borderline tumors, the tessutal VEGF immunostaining was significantly higher in cystoadenocarcinomas, with the highest values detected in architectural International Federation of Gynecology and Obstetrics Grade 3 neoplasms (P2 < 0.001). A direct relation was observed between VEGF and MMP2 immunostaining (correlation coefficient, 0.44; P2 = 0.013). A relation was found between VEGF expression and disease free survival as evaluated by Cox hazards analysis (P2 = 0.03). CONCLUSIONS: Neoangiogenesis detected by VEGF immunostaining appears to be a promising indicator of aggressiveness in serous ovarian tumors. In cystoadenocarcinomas, VEGF expression appears to be related to MMP2 index.

Adult↗

PubMed1999

Vascular endothelial growth factor expression as a prognostic index in serous ovarian cystoadenocarcinomas: relationship with MIB1 immunostaining.

OBJECTIVE: The aim of our study was to investigate the expression of vascular endothelial growth factor (VEGF) by neoplastic cells in serous ovarian cystoadenocarcinomas; the correlation between this marker of angiogenesis, histopathologic parameters, disease-free survival, and MIB1 immunostaining was also evaluated. MATERIALS AND METHODS: Thirty-two patients with serous ovarian cystoadenocarcinoma, treated at the Institute of Gynecology and Obstetrics, Ancona University (Italy), were used as study population; 10 women with serous cystoadenoma were also analyzed. The expression of VEGF was immunohistochemically evaluated by polyclonal antibody anti-VEGF (Santa Cruz, CA, dilution 1:100) on formalin-fixed paraffin-embedded tissue. RESULTS: Compared to cystoadenomas, the tissutal VEGF immunostaining was significantly higher in cystoadenocarcinomas, with the highest values in architectural grade 3 neoplasms (P < 0. 001). A direct relationship was observed between VEGF immunostaining and MIB1 index (r = 0.44, P = 0.013). A relationship was defined between VEGF expression and disease-free survival, evaluated by Cox hazards analysis (P < 0.001). CONCLUSIONS: Angiogenesis, evaluated by VEGF immunostaining, seems to be an interesting prognostic indicator in serous ovarian cystoadenocarcinoma, involved in neoplastic proliferation.

Adult↗

PubMed1999

Tyrosine phosphorylation in type-1 diabetes by immunogold detection: an in vitro human aortic endothelial cell (HAEC) study in the presence of diabetic low density lipoproteins (LDL).

An immunomorphometric study of tyrosine phosphorylation was performed by the immunogold technique on cultured human aortic endothelial cells (HAEC) with a view to demonstrating their impaired signal transduction status, induced in vitro by incubation with low-density lipoproteins from the plasma of Type-1 diabetic patients. The results seem to sustain the hypothesis that extranuclear bioenergetic derangement induced by low-density lipoproteins from Type-1 diabetic patients may be associated with an up-regulation of the nuclear energetic machinery aimed at maintaining intracellular metabolic equilibrium. Our data demonstrate that phosphorylated tyrosine is a useful marker to monitor this metabolic condition.

Cell Line↗

PubMed1998

Histomorphometric studies in rat cerebral cortex: normal aging and cell loss.

One characteristic feature of the aged central nervous system (CNS) is neuron loss. Programmed cell death (PCD) has been implicated in neuronal death during development and may be involved in a number of age-related neurodegenerative diseases of the CNS. Cell death in the aging cerebral cortex was investigated in the present morphometric and immunohistochemical study of rat frontal cortex by detection of bcl-2 as the factor preventing PCD. The results were interpreted in the light of the bioenergetic features of aged motoneuron cells. Our results showed that 1) bcl-2 does not influence neuronal survival, and ii) the presence in aging frontal cortex of minor cellular morphometric and bioenergetic modifications, confirming the difference between normal aging and neurodegenerative disease.

Aging↗

PubMed1997

Regulation of cellular tyrosine phosphorylation by stimulatory and inhibitory muscarinic acetylcholine receptors.

Tyrosine phosphorylation is a key signaling event in transmembrane and cytoplasmic signal transduction. The m5 muscarinic receptor (m5AChR) responds to ligand stimulation with calcium influx and protein phosphorylation. In contrast, neither of these responses has been associated with m4AChR signaling. We hypothesized that activation of the m5AChR would alter tyrosine phosphorylation patterns spatially within the cell and in a calcium influx-sensitive manner. CHO cells stably transfected with m4- or m5AChRs were assessed for spatial localization and quantity of phosphotyrosylated proteins in response to receptor activation. Results were confirmed by immunoblot of whole cell lysates and cytosol and membrane fractions. m5AChR activation increased tyrosine phosphorylation in all subcellular compartments; coincubation with CAI, a calcium influx inhibitor, reduced phosphorylation below basal levels. Western blot confirmed the change of phosphotyrosylated proteins of M(r) 70, 85, 120, and 180 kDa in whole and fractionated cells. PLC-gamma, used as a marker of m5AChR activity, was increased in quantity and degree of phosphorylation in CHOm5 cell membranes and microvilli in response to receptor activation. Both the quantitative increase and tyrosine phosphorylation of PLC-gamma in membrane fractions was inhibited by CAI. In contrast, CC treatment of CHOm4 cells reduced tyrosine phosphorylation throughout the cell. CC-stimulation of m5AChR cells caused a calcium influx-sensitive increase in phosphotyrosylated proteins throughout the cell, though predominantly in the membrane and microvilli. Activation of the m5AChR induces tyrosine phosphorylation, whereas activation of the m4AChR inhibited tyrosine phosphorylation below baseline, further demonstrating the dichotomy between signaling of these two AChRs.

Animals↗

PubMed1997

Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats.

We investigated NADH oxidation in non-synaptic and synaptic mitochondria from brain cortex of 4- and 24-month-old rats. The NADH oxidase activity was significantly lower in non-synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone-binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.

Aging↗

PubMed1996

In vitro study of gingival fibroblasts from normal and inflamed tissue: age-related responsiveness.

The aim of this study was to characterize some phenotypic expressions of fibroblasts from the human oral mucosa. Gingival and lower forearm fibroblasts from young (20-30 years) and elderly (> 60 years) subjects were analyzed. Gingival fibroblasts were taken from donors with (P) and without (NP) periodontal disease, while skin biopsies were taken from healthy subjects. Cell proliferation was assessed by evaluating the cell multiplication coefficient (C.M.C.). The proliferation potential of gingival fibroblasts from elderly individuals with and without periodontopathy did not differ from that of young subjects in the same condition but differed significantly in the skin samples. Enzyme neutral endopeptidase (EC 3.4.24.11) (NEP) activity, studied as a possible marker of cell ageing, showed an age-related increase in human skin fibroblasts but not consistently in gingival fibroblasts from individuals with or without periodontal disease. Cell area and substrate adhesion were evaluated by morphometric analysis. There were no significant differences between elderly P and NP subjects, while significant differences were observed between young and elderly P subjects. In conclusion, proliferative capacity and NEP activity in gingival fibroblasts did not appear to be age-related, probably because their microenvironment is continually moistened by saliva, which continues to contain growth factors, notably EGF, even into senescence. Tissue reaction and repair are important clinical and therapeutic implications.

Adult↗

PubMed1996

Activation of endothelial cells during insulin-dependent diabetes mellitus: a biochemical and morphological study.

Plasma membrane lipid dynamics and cellular morphology were evaluated in endothelial cells obtained from umbilical cords of five women affected by insulin-dependent diabetes mellitus (IDDM) and six healthy pregnant women of similar age and gestational age. Endothelial cells were prepared by an adaptation of the method of Jaffe et al. Membrane fluidity was studied by means of the steady-state fluorescence anisotropy (r) of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), a fluorescent probe specifically anchoring at the membrane surface. Fluid phase endocytosis was evaluated by the measurement of the changes in fluorescence intensity of TMA-DPH at various times, owing to the internalization of the fluorescent marker in endocytic vesicles. The morphological and morphometric studies were performed by means of transmission electron microscopy (TEM). Endothelial cells obtained from IDDM women showed: (a) increased fluidity of the superficial region of the plasma membrane; (b) a more active fluid phase endocytosis compared with cells from healthy women; (c) increase in mitochondrial area, Weibel-Palade bodies and rough reticulum with wide cisternae. No statistically significant correlation was found between metabolic control and membrane fluidity and endocytosis. All the observed modifications suggest the presence of endothelial cell activation with membrane reshaping during IDDM. These alterations might play a central role in the pathophysiology of atherosclerosis and microangiopathy associated with diabetes mellitus.

Adult↗

PubMed1996

An electron microscopic study of clinical and laboratory-derived strains of teicoplanin-resistant Staphylococcus haemolyticus.

Staphylococcal resistance to glycopeptides (which involves more teicoplanin than vancomycin) is uncommon and largely confined to Staphylococcus haemolyticus, an emerging nosocomial pathogen with a tendency to develop antibiotic resistance. In this study, six S. haemolyticus strains, including two isogenic pairs of teicoplanin-susceptible/-resistant strains and two resistant clinical isolates, were used in a morphologic and morphometric electron microscope investigation. Cells from both clinical and laboratory-derived teicoplanin-resistant strains exhibited abnormally roughened, irregular outlines when observed by transmission electron microscopy. However, no significant differences in cell wall thickness resulted from morphometric analysis when the susceptible/resistant cells of the two isogenic pairs were compared. By scanning electron microscopy, an abnormally roughened, blistered surface was associated with teicoplanin-resistant cocci. A certain variability was noted between strains, not clearly related to the resistance level. In freeze-fracture investigations, a higher number per square micrometer of intramembrane particles, more significant in the E than in the P membrane fracture face, was observed in the laboratory-derived resistant clones as compared to susceptible parent strains. Further studies are needed to understand the cause-effect relation between these ultrastructural alterations and staphylococcal resistance to teicoplanin (but not to vancomycin).

Anti-Bacterial Agents↗

PubMed1995

Morpho-functional modifications of human syncytiotrophoblast plasma membrane during pregnancy induced hypertension.

A decrease of Na+/K(+)-ATPase activity has been reported in syncytiotrophoblast plasma membrane (SPM) obtained from pregnancy induced hypertensive (PIH) women. The aim of the present work was to verify if the reported modifications in activity are due to a decreased number of enzymatic molecules or to a conformational change of the enzyme itself. Morphological studies were performed in order to better understand the relations between the enzymatic protein and the lipid bilayer. Kinetic studies were also performed. SPM obtained from PIH showed: i) an increased affinity of Na+/K(+)-ATPase for ouabain binding, ii) a significant change in the maximum velocity of the enzyme, iii) a higher distribution factor (DF) of intramembrane particles (IMPs) in the exoplasmic face of the membrane, iv) a decreased mean diameter of IMPs both in the protoplasmic and exoplasmic faces, v) a decreased number of IMPs in the exoplasmic face. In conclusion, a conformational modification seems to be at the basis of the decreased Na+/K(+)-ATPase activity during PIH as suggested by binding, ultrastructural and kinetic data herein reported.

Adult↗

PubMed1995

Biochemical-morphological modifications of platelet membranes during pregnancy-induced hypertension.

A platelet (PLT) function modification has been reported in normal pregnancies compared with the nonpregnant condition and it has been hypothesized to play a central role in the pathogenesis of pregnancy-induced hypertension (PIH). The aims of the present study were (i) to evaluate the lipid composition, fluidity at different depths, transport functions, and ultrastructural features of the PLT membrane in PIH and (ii) to ascertain whether similar modifications may be determined by the state of pregnancy in comparison with the nonpregnant condition. The platelets of healthy pregnant women (HPW) showed: (i) an increase in Ca2+ ATPase activity, (ii) a decreased fluidity of the deeper site of the membrane, (iii) a reduced cholesterol concentration, with an increased ratio between unsaturated and saturated fatty acids; (iv) a decreased intramembranous particles (IMP) distribution factor (DF) of the plasma membrane E face in comparison with healthy nonpregnant women. When comparing women affected by PIH with HPW, we observed (i) reduced Na+/K+ ATPase activity and enhanced Ca2+ ATPase activity and intraplatelet calcium concentrations, (ii) an increased membrane fluidity, (iii) an increased cholesterol concentration and ratio between unsaturated and saturated fatty acids, (iv) a reduction of the IMP number and the DF. Pregnancy produces a deep modification of the platelet plasma membrane, and PIH produces a more pronounced alteration of the maternal platelets, which can be responsible for the observed modifications in placental blood flow and in the fetomaternal exchange.

Blood Platelets↗

PubMed1995

The human umbilical vein in normal, hypertensive and diabetic pregnancies: immunomorphological and ultrastructural evidence.

Mother-fetus exchanges at the placental level are found to be altered in women affected by hypertensive or diabetic pregnancies following the onset of microenvironmental, circulatory, trophic or tissue disorders. Our aim was therefore to assess the alterations occurring within the umbilical cord, particularly its venous endothelial component and underlying smooth muscle layer, using transmission (TEM) and scanning electron microscopy (SEM) and immunohistochemical analyses. Immunohistochemical data appear to support the ultrastructural evidence for an activated state of these vascular structures, in both conditions (hypertension and diabetes). Furthermore, mainly during diabetic pregnancies, extracellular matrix molecules such as tenascin and fibronectin also quantitatively increase at the vein wall level. The umbilical cord seems to be a structure capable of responding actively to abnormal microenvironmental conditions which seriously threaten the health of the fetus and also the mother herself.

Cell Adhesion Molecules↗

PubMed1995

Umbilical veins in dichorionic twins. A morpho-functional assessment.

Investigations on singleton and twin pregnancies show different functional behaviour on maternal-fetal relationship. In some ways twin pregnancies may be considered at risk and they may develop associated pathologies such as hypertension. The aim of this work was to evaluate the morpho-functional behaviours of umbilical cord veins in twin and singleton gestations to better understand the role of these extra-embryonic tissues in the regulation of pregnancies. The umbilical cords were studied from singleton pregnancies and from dichorionic twin pregnancies. Biochemical and morphological investigations were carried out. A significant decrease in the anisotropy values was observed in endothelial cells from twins compared with singletons. Our ultrastructural data show immaturity features at the vein vessel wall level in twins. Furthermore, immunohistochemical investigations showed a lower degree of expressivity concerning adhesion molecules such as ICAM-1 and ELAM. Morphogenetic extracellular glycoproteins like fibronectin and tenascin seem over-expressed in twin pregnancies. Our morpho-functional data well testify the lower maturation degree of umbilical cord veins in twins with respect to singletons.

Anisotropy↗

PubMed1995

Membrane fluidization by animycotic bifonazole.

Calorimetry, nuclear magnetic resonance and X-ray diffraction techniques have been used to obtain thermodynamic and structural information on dipalmitoyl phosphatidylcholine (DPPC) liposomes doped by the antimycotic drug bifonazole in the range 0 < R < 1, where R = moles of bifonazole/moles of DPPC. The technique of spin labeling electron spin resonance (ESR) has also been used to study permeability and fluidity properties. The decrease of the cooperativity at the gel to liquid crystalline phase transition, as shown by ESR an DSC measurements, indicates that bifonazole imparts higher fluidity to the lipid matrix. Increase in permeability of ascorbate ions, after incorporation of bifonazole in the membrane, has been detected by ESR experiments using spin label 5-SASL. 13C NMR spectra indicate that the drug molecule is highly immobilized. X-ray diffraction and freeze fracture TEM results show that the equilibrated phase at room temperature is lamellar and unidimensional together with the presence of small particles and pits of uniform size. A marked hysteresis is evident in the formation of this phase.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

Human skeletal muscle mitochondria in aging: lack of detectable morphological and enzymic defects.

We have investigated structural and functional properties of skeletal muscle mitochondria obtained from biopsies from young and old individuals. The morphometric analysis of muscle sections revealed a tendency to an increase of total area, numerical density and volume density of mitochondria in the aged. The enzymatic activities of NADH-Coenzyme Q reductase, succinate cytochrome c reductase, ubiquinol-cytochrome c reductase exhibited a high variability of specific activities without any correlation with age. Expression of the values as enzyme turnovers reduced the variability but was unable to reveal any age-dependent modification.

Adolescent↗

PubMed1994

A biochemical-morphological study on microvillus plasma membrane development.

The microvillus plasma membrane of the human placental syncytiotrophoblast at term has been extensively studied, while little is known about the characteristics of its development. The aim of the present work was to compare functional and structural properties of this membrane at early and term gestational age. Ten normal term placentas (40 weeks) and ten placentas at 10 weeks of gestational age were studied. The Na+/K+-ATPase activity is significantly decreased in the syncytiotrophoblast plasma membrane obtained from term placentas as compared to the early ones, with significant variation of maximum velocity (Vmax). The microviscosity, evaluated by the P parameter of DPH and Sn parameters of 5- and 16-NS, is increased in the term placentas compared to the early placentas. This alteration is accompanied by an increased cholesterol to phospholipids ratio in term placentas, while there is a decreased unsaturated to saturated fatty acid ratio. As follows from morphological studies, an increased mean diameter in the E face was observed in the term placenta with respect to the early placenta. The distribution factor DF, which indicates the particle aggregation state, decreased in the E face in the term placenta as compared to the early one. The present biochemical morphological study shows that a deep modification of the membrane is at the basis of the syncytiotrophoblast plasma membrane development.

Female↗

PubMed1994

The use of acellular autologous skeletal muscle grafts in peripheral nerve repair: a morphometrical-morphological study.

Alternative materials for autologous nerve grafts in peripheral nerve surgery have long been investigated. This report describes the use of acellular (freeze-thawed) autologous skeletal muscle grafts (ASMG) to bridge a 2 cm gap in the sciatic nerve of rats. Neurophysiologic and morphometric studies performed 6 weeks after surgical procedure showed for the nine treated Sprague-Dawley rats (Group A) a good axon regeneration rate, although this was lower than that for the control group (Group B), a lower Nerve Conduction Velocity (NCV) and a thinner myelin sheath than in group B (n = 9 Sprague-Dawley rats). In the latter case, complete resection of 2 cm of the sciatic nerve was followed by an immediate coaptation of the proximal and distal ends.

Animals↗

PubMed1994

Characterization of large unilamellar vesicles as models for studies of lipid peroxidation initiated by azocompounds.

The aim of this work was to characterize large unilamellar vesicles (LUVETs) prepared by a hand-driven extrusion device in order to use them for studies of lipid peroxidation and antioxidant activity. Vesicle structure and size were examined by electron microscopy. Lipid and antioxidant content was determined before and after the extrusion procedure. Then LUVETs were subjected to autoxidation initiated by both the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) and the water-soluble 2,2'-azobis(2-amidinopropane hydrochloride) azocompounds. The results demonstrated that: i) LUVETs prepared with lipid concentrations ranging between 25 and 150 mM were essentially unilamellar and reasonably homogeneous, with an average diameter of 90 nm; ii) the phospholipid, cholesterol and antioxidant amounts retained by filters were about 10-15%; iii) LUVETs were suitable for autoxidation studies initiated by the water-soluble azocompound both in the absence and presence of antioxidants. The lipid-soluble azocompound could be used only at low concentrations and its vesicle content had to be determined since part of the initiator was not incorporated into the lipid bilayer. These data suggest that LUVETs seem to be recommended for studies of lipid peroxidation and antioxidant activity.

Antioxidants↗