Biomedical subjects

G Baldini

Publications and source records attributed to G Baldini.

At least 19 recordsLinked to original sources

PubMed2003

Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method.

We report on the construction details of a compact autocorrelator set-up for the measurement of the width of infrared laser pulses at the focal plane of a microscope for two-photon excitation fluorescence imaging. One of the novelties of the set-up, which leads to an improved measurement accuracy, is the use of a modulation technique that is achieved by mounting one of the interferometer mirrors on a loudspeaker driven by a sinusoidal bias at low frequency. A non-linear least-square routine selects only that part of the fluorescence signal that is modulated at the same frequency as the loudspeaker bias. To further increase the accuracy, the laser pulse width is obtained from a series of measurements at different values of the modulation bias. The autocorrelator is a compact single bread-board (10 x 20 cm); it is PC-controlled both for the acquisition and the analysis of the data and can be coupled to different ports of the microscope. The increase in the pulse width measured for three different ports of the microscope is well accounted for by the group velocity dispersion and the glass thickness of the optics found along these paths.

Equipment Design↗

PubMed2002

Benefits of immunotherapy with a standardized Dermatophagoides pteronyssinus extract in asthmatic children: a three-year prospective study.

BACKGROUND: Although widely practiced for over 80 years, the role of specific immunotherapy (SIT) in pediatric asthma treatment is still controversial. We assessed the effects of a 3-year period of subcutaneous administration of a standardized preparation of Dermatophagoides pteronyssinus (D pt) on the respiratory health in a group of asthmatic children monosensitized to house dust mite (HDM). METHODS: A randomized clinical trial was performed after 1-year run-in period. Fifteen children receiving SIT for HDM and 14 controls (four drop-outs), matched for age, allergen sensitization, asthma severity, lung function, and non-specific bronchial reactivity (BHR), were studied during the 3-year treatment period. During the whole trial, respiratory symptoms, pharmacological and respiratory function parameters were regularly evaluated. Skin prick tests and methacholine challenge were performed at the beginning and end of the study. RESULTS: In the SIT group significant improvement in asthmatic symptoms and marked reduction in drug intake was observed. The SIT group also showed a significant decrease in non-specific bronchial BHR. No new sensitivity occurred during the study period in the SIT group only. No major local or systemic side-effects were reported during the study. CONCLUSIONS: Our results confirm that SIT is effective in asthmatic children sensitive to mites. It is associated with a decrease in BHR and it may prevent the development of new sensitizations in monosensitized subjects.

Adolescent↗

PubMed2001

Single molecule studies by means of the two-photon fluorescence distribution.

We have characterized a commercial confocal scanning head for the detection of single molecule fluorescence by two-photon excitation. We have verified that the distribution of the fluorescence emitted by dyes and labeled proteins on glass substrates is discrete with quanta proportional to a common reference signal. We describe and test a simple and quantitative tool to discriminate between single molecules and molecular aggregates on single snapshots based on the analysis of the intensity distribution. We have verified the square dependence of the fluorescence intensity vs. the excitation power, suggesting that no appreciable saturation and fast photo-damage of the chromophores takes place at the excitation power employed here.

Fluorescence↗

PubMed2001

Phosphoinositide-specific phospholipase Cbeta1 expression is not linked to nerve growth factor-induced differentiation, cell survival or cell cycle control in PC12 rat pheocromocytoma cells.

Recent reports have highlighted that phosphoinositide-specific phospholipase Cbeta1 expression is linked to neuronal differentiation in different experimental models. We sought to determine whether or not this is also true for nerve growth factor (NGF)-induced neuronal differentiation of rat PC12 cells. However, we did not find differences in the expression of both the forms of phosphoinositide-specific phospholipase Cbeta1 (a and b) during sympathetic differentiation of these cells. Also, PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 were not more susceptible to the differentiating effect of NGF. Furthermore, since it is well established that phosphoinositide-specific phospholipase Cbeta1 affects cell proliferation, we investigated whether or not PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 showed differences in survival to serum deprivation and cell cycle, when compared to wild type cells. Nevertheless, we did not find any differences in these parameters between wild type cells and the overexpressing clones. Interestingly, in PC12 cells the overexpressed phosphoinositide-specific phospholipase Cbeta1 did not localize to the nucleus, but by immunofluorescence analysis, was detected in the cytoplasm. Therefore, our findings may represent another important clue to the fact that only when it is located within the nucleus phosphoinositide-specific phospholipase Cbeta1 is able to influence cell proliferation.

Animals↗

PubMed2001

Eosinophil cationic protein in infants with respiratory syncytial virus bronchiolitis: predictive value for subsequent development of persistent wheezing.

Infants with acute bronchiolitis during the first months of life are at increased risk of developing persistent wheezing and bronchial asthma later in life. The study of eosinophil cationic protein (ECP) suggests that eosinophil-related inflammatory mechanisms may play a role in respiratory syncytial virus (RSV) bronchiolitis. The aim of our study was to verify whether serum ECP (s-ECP) measurements are useful in predicting the development of persistent wheezing in children affected by RSV bronchiolitis during a 5 years follow-up period. Forty-eight infants were enrolled prospectively (mean age: 153.5 days). All had a clinical and radiological diagnosis of acute bronchiolitis and confirmed RSV infection. Peripheral eosinophil counts, levels of s-ECP, and serum IgE concentrations were measured during bronchiolitis. Five years later the children were re-evaluated in regard to their respiratory symptoms (standardized questionnaires) and atopic status (specific IgE levels). We observed significantly higher s-ECP levels (P < 0.001) at enrollment in subjects who developed persistent wheezing compared to subjects who did not show late wheezing. Initial s-ECP values allowed significant and correct prediction of persistent wheezing (P < 0.001). The risk to develop respiratory symptoms was 9.73 higher for infants with s-ECP levels > or = 8 microg/L than for those with s-ECP levels <8 microg/L (P < 0.0001). In conclusion, our study suggests that s-ECP levels in infants with bronchiolitis are useful in predicting the risk to develop wheezing in the subsequent 5 years.

Asthma↗

PubMed2001

Primary ciliary dyskinesia: diagnosis in children with inconclusive ultrastructural evaluation.

The purpose of this study was to distinguish between acquired and genetically determined ciliary abnormalities in children with severe chronic respiratory diseases. Samples of nasal ciliated epithelium from 50 subjects (25 male, 25 female; age-range 2-19 years) with severe chronic respiratory diseases were examined using transmission electron microscopy (TEM). Based on TEM findings, patients were divided into two groups: A and B. Group A comprised 39 children with ciliary alterations compatible with a condition probably occurring secondary to chronic inflammation (alterations of peripheral pairs, swollen cilia, and compound cilia). The other 11 patients, Group B, exhibited a greater number of alterations of the central pair and dynein arms (p< 0.001), which were qualitatively similar to, but less numerous than, those observed in primary ciliary dyskinesia (PCD). In both groups, analysis of ciliary beat frequency and waveform was performed by phase contrast microscopy (PCM). All the children with a ciliary beat frequency of < 7 Hz were treated with daily physiotherapy and with antibiotics, as recommended for PCD, for a 6-month period. After this treatment, the children were reexamined by PCM. Almost 50% of the children from Group B (i.e. those with a small proportion of specific ultrastructural defects) showed permanence of low ciliary beat frequency. This was also observed in two children of Group A. These children were considered to be affected by PCD. Our study describes a method for the diagnosis of PCD in the absence of specific ultrastructural defects or when these defects are present in only a small proportion of the cilia.

Adolescent↗

PubMed2001

Localization of the small monomeric GTPases Rab3D and Rab3A in the AtT-20 rat pituitary cell line.

We investigated the cellular localization of the small GTPases Rab3D and Rab3A in AtT-20 cells treated with the drug Brefeldin A. Brefeldin A induces the redistribution of the Golgi complex into the endoplasmic reticulum and tubulation of endosomes. However, in Brefeldin A-treated wild-type AtT-20 cells, both Rab3D and Rab3A retained their distribution, indicating that they belong to a nonendosomal, post-Golgi compartment. Immunoelectron microscopy experiments indicated that both Rab3D and Rab3A localized to the ACTH-containing, large dense core granules. In contrast, in cell clones overexpressing a mutated form of Rab3D (Rab3D N135I), Rab3A did not localize to the dense core granules. Moreover, since our previous results showed that overexpression of Rab3D N135I severely impaired regulated ACTH secretion in AtT-20 cells, we sought to determine whether the impairment could depend on a redistribution of two key components of the regulated exocytosis machinery, synaptotagmin and SNAP-25. As far as synaptotagmin was concerned, in cell clones overexpressing Rab3D N135I, the protein did not localize close to the plasma membrane, in agreement with the previously reported defective docking of dense core granules to the plasma membrane. Immunofluorescence experiments showed that SNAP-25 did not change its localization in these cell clones. All in all, our findings strengthen the notion that both Rab3D and Rab3A are associated with the dense core granule compartment of AtT-20 cells, and that the impairment in the ACTH secretion caused by overexpression of a mutated Rab3D form is likely to be due to a lacking of granule docking to the plasma membrane, possibly because Rab3A fails to associate with the granules.

Adrenocorticotropic Hormone↗

PubMed2000

Enhanced nuclear diacylglycerol kinase activity in response to a mitogenic stimulation of quiescent Swiss 3T3 cells with insulin-like growth factor I.

Results from several laboratories have established the existence in the nucleus of an autonomous polyphosphoinositide cycle, which is involved in both cell proliferation and differentiation. A key step of intranuclear polyphosphoinositide metabolism is the phospholipase C-mediated generation of diacylglycerol (DAG). In insulin-like growth factor (IGF)-I-stimulated Swiss 3T3 cells, a transient elevation of intranuclear DAG levels is essential for attracting the alpha isoform of protein kinase C (PKC) to the nucleus. Previous evidence has shown that the nucleus also contains DAG kinase, i.e., the enzyme that yields phosphatidic acid from DAG, thus terminating PKC-mediated signaling events. Here we show that IGF-I treatment of quiescent Swiss 3T3 cells results in the stimulation of nuclear DAG kinase activity. Time course analysis showed an inverse relationship between nuclear DAG mass and DAG kinase activity levels. After IGF-I treatment, maximal enhancement of DAG kinase activity was measured in the internal matrix domain of the nucleus. PKC-alpha remained within the nuclear compartment, even when nuclear DAG mass returned to basal levels. This was conceivably due to interactions with specific nuclear PKC-binding proteins, some of which were identified as lamins A, B, and C and protein C23/nucleolin. Treatment of cells with two DAG kinase inhibitors, R59022 and R59949, blocked the IGF-I-dependent rise in nuclear DAG kinase activity and maintained elevated intranuclear levels of DAG. The two inhibitors also markedly potentiated the mitogenic effect of IGF-I. These results suggest that nuclear DAG kinase plays a key role in regulating the levels of DAG present in the nucleus and that DAG is a key molecule for the mitogenic effect that IGF-I exerts on Swiss 3T3 cells.

3T3 Cells↗

PubMed2000

New insight on beta-lactoglobulin binding sites by 1-anilinonaphthalene-8-sulfonate fluorescence decay.

The fluorescence time decay parameters of the beta-lactoglobulin-1-anilinonaphthalene-8-sulfonate complex have been investigated under physical and chemical perturbations (2 < pH < 8 and added electrolyte 0 < NaCl < 0.5 M) to obtain new insight on the nature of the protein binding interactions. A double exponential decay of the bound probe lifetime has been confirmed by the presence of a longer component, 11 to 14.5 ns, and a shorter component, 2.5 to 3.5 ns. The two lifetimes are ascribed to different binding modes associated also with different exposure to the solvent; in particular, the longer component is attributed to binding inside the hydrophobic beta barrel, while a "surface" site is suggested for the shorter component. A detailed analysis of the lifetime fractional intensities correlates the binding constants with ionic strength and supports the presence of electrostatic effects at both sites. A Debye-Hückel approach, applied to extrapolate the electrostatic free energy contribution vs. pH at vanishing ionic strength, gives interesting clues on the effective charge felt by the ANS ligands in the proximity of each site. In particular, binding is found to parallel the aspartate and glutamate titrations between pH 3 and pH 4.5; the "surface" site mainly responds to the presence of these local titrating charges while the "internal" site more closely follows the overall protein net charge.

Anilino Naphthalenesulfonates↗

PubMed2000

Rab3A and Rab3D control the total granule number and the fraction of granules docked at the plasma membrane in PC12 cells.

Rab proteins are Ras-like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immunoelectron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild-type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.

Animals↗

PubMed1999

The pro-apoptotic drug camptothecin stimulates phospholipase D activity and diacylglycerol production in the nucleus of HL-60 human promyelocytic leukemia cells.

It has recently been reported (T. Shimizu et al., J. Biol. Chem., 273: 8669-8674, 1998) that the pro-apoptotic drug, camptothecin, an inhibitor of topoisomerase I, induces a protein kinase C-alpha-mediated phosphorylation of lamin B in HL-60 cells, which precedes both degradation of lamin B and fragmentation of DNA. In this paper, we report that, in HL-60 cells exposed to camptothecin, there is a rapid and sustained increase of nuclear protein kinase C-alpha activity that is due to an increase in the amount of protein kinase C-alpha present in the nucleus. The enhancement of nuclear kinase C activity is preceded by an increase in the mass of nuclear diacylglycerol. As demonstrated by its sensitivity to propranolol, the nuclear diacylglycerol mass increase is due to the activation of a phospholipase D. Indeed, inhibitors of neither phosphatidylcholine-specific phospholipase C nor phosphoinositide-specific phospholipase C blocked the rise in nuclear diacylglycerol. In vitro assays also demonstrated the activation of a nuclear phospholipase D, but not of a phosphoinositide-specific phospholipase C, after treatment with camptothecin. Propranolol was also able to block the rise in nuclear protein kinase C-alpha activity, thus suggesting that the increase in diacylglycerol mass is important for the activation of the kinase at the nuclear level. Moreover, propranolol was capable of drastically reducing the number of HL-60 cells that underwent apoptosis after treatment with camptothecin. Our results show the activation during apoptosis of a phospholipase D-mediated signaling pathway operating at the nuclear level. This pathway may represent an attractive therapeutic target for the modulation of apoptotic events in human disease.

Antineoplastic Agents, Phytogenic↗

PubMed1999

Evidence of heterogeneous 1-anilinonaphthalene-8-sulfonate binding to beta-lactoglobulin from fluorescence spectroscopy.

Steady-state and dynamic fluorescence titrations show that: (a) the complex between beta-lactoglobulin (BLG) and 1-anilinonaphthalene-8-sulfonate (ANS) displays a heterogeneous equilibrium with large changes in the binding strength vs. pH and ion concentration; and (b) the fluorescence response of bound ANS reveals two separate lifetimes that suggest two different sites (or binding modes). While steady-state fluorescence titrations yield effective values of the binding constant and of the bound ANS quantum efficiency, it is shown that, by combining steady-state fluorescence and lifetime decay of ANS, it is possible to give quantitative estimates of the association constants for each site. When heading from the acid (pH approximately 2) to the native state (pH approximately 6) the main result is a very large reduction of the effective binding constant. This and the results of titrations vs. ionic strength suggest that electrostatic interactions are a major contribution to ANS binding to BLG.

Anilino Naphthalenesulfonates↗

PubMed1999

Role of the cysteine-rich domain of the t-SNARE component, SYNDET, in membrane binding and subcellular localization.

Wild-type syndet is efficiently recruited at the plasma membrane in transfected AtT-20 cells. A deletion at the cysteine-rich domain abolishes palmitoylation, membrane binding, and plasma membrane distribution of syndet. Syndet, SNAP-25A, and SNAP-25B share four cysteine residues, of which three, Cys2, Cys4, and Cys5, are absolutely conserved in all three homologs. Mutations at any pair of cysteines within cysteines 2, 4, and 5 shift syndet from the cell surface into the cytoplasm. Thus, at least two cysteines within the conserved triplet are necessary for plasma membrane localization. Syndet C1S/C3S, with substitutions at the pair Cys1 and Cys3, distributes to the plasma membrane, a Golgi-like compartment, and the cytosol. We conclude that Cys1 and Cys3 are not absolutely necessary for membrane binding or plasma membrane localization. Our results show that the cysteine-rich domain of syndet plays a major role in its subcellular distribution.

Animals↗

PubMed1999

Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells.

Insulin-secreting cells express four GTPases of the Rab3 family. After separation of extracts of INS-1 cells on a sucrose density gradient, the bulk of the A, B, and C isoforms was recovered in the fractions enriched in insulin-containing secretory granules. Rab3D was also mainly associated with secretory granules, but a fraction of this isoform was localized on lighter organelles. Analyses by confocal microscopy of immunostained HIT-T15 cells transfected with epitope-tagged constructs confirmed the distribution of the Rab3 isoforms. Transfection of HIT-T15 cells with GTPase-deficient mutants of the Rab3 isoforms decreased nutrient-induced insulin release to different degrees (D>B>A>>C), while overexpression of Rab3 wild types had minor or no effects. Expression of the same Rab3 mutants in PC12 cells provoked an inhibition of K+-stimulated secretion of dense core vesicles, indicating that, in beta-cells and neuroendocrine cells, the four Rab3 isoforms play a similar role in exocytosis. A Rab3A/C chimera in which the carboxyterminal domain of A was replaced with the corresponding region of C inhibited insulin secretion as Rab3A. In contrast, a Rab3C/A chimera containing the amino-terminal domain of C was less potent and reduced exocytosis as Rab3C. This suggests that the degree of inhibition obtained after transfection of the Rab3 isoforms is determined by differences in the variable amino-terminal region.

Animals↗

PubMed1998

Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface.

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by approximately 40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.

3T3 Cells↗

PubMed1998

Expression of Rab3D N135I inhibits regulated secretion of ACTH in AtT-20 cells.

Rab proteins are small molecular weight GTPases that control vesicular traffic in eucaryotic cells. A subset of Rab proteins, the Rab3 proteins are thought to play an important role in regulated exocytosis of vesicles. In transfected AtT-20 cells expressing wild-type Rab3D, we find that a fraction of the protein is associated with dense core granules. In the same cells, expression of a mutated isoform of Rab3D, Rab3D N135I, inhibits positioning of dense core granules near the plasma membrane, blocks regulated secretion of mature ACTH, and impairs association of Rab3A to membranes. Expression of Rab3D N135I does not change the levels of ACTH precursor or the efficiency with which the precursor is processed into ACTH hormone and packaged into dense core granules. We also find that cells expressing mutated Rab3D differentiate to the same extent as untransfected AtT-20 cells. We conclude that expression of Rab3D N135I specifically impairs late membrane trafficking events necessary for ACTH hormone secretion.

Adrenocorticotropic Hormone↗

PubMed1998

Intranuclear distribution of HMGI/Y proteins. An immunocytochemical study.

The intranuclear distribution of HMGI/Y proteins was analyzed by immunofluorescent staining in several cell lines using a polyclonal antibody that stained a fibrogranular network. In actively growing 3T3 fibroblasts, HMGI/Y proteins were mainly localized to heterochromatin masses, whereas in quiescent cells they were more diffusely distributed. Double labeling experiments showed a co-localization of HMGI/Y with DNA topoisomerase IIalpha. These results are in agreement with previously published biochemical data and indicate a possible involvement of HMGI/Y proteins in several nuclear functions, including chromatin organization and gene expression.

3T3 Cells↗

PubMed1997

Botulinum neurotoxin types A and E require the SNARE motif in SNAP-25 for proteolysis.

Botulinum neurotoxins type A and E (BoNT/A and BoNT/E) are metalloproteases with a unique specificity for SNAP-25 (synaptosome-associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It has been suggested that this specificity is directed through the recognition of a nine residue sequence, termed SNARE motif, that is common to the other two SNARE proteins: VAMP (vesicle-associated membrane protein) and syntaxin, the only known substrates of the other six clostridial neurotoxins. Here we analyse the involvement of the four copies of the SNARE motif present in SNAP-25 in its interaction with BoNT/A and BoNT/E by following the kinetics of proteolysis of SNAP-25 mutants deleted of SNARE motifs. We show that a single copy of the motif is sufficient for BoNT/A and BoNT/E to recognise SNAP-25. While the copy of the motif proximal to the cleavage site is clearly involved in recognition, in its absence, other more distant copies of the motif are able to support proteolysis. Also, a non-neuronal isoform of SNAP-25, Syndet, is shown to be sensitive to BoNT/E, but not BoNT/A, whilst the SNAP-25 isoforms from Torpedo marmorata and Drosophila melanogaster were demonstrated not to be substrates of these metalloproteases.

Amino Acid Sequence↗