PubMed Health⌕ Search

Biomedical subjects

G Chirico

Publications and source records attributed to G Chirico.

At least 19 recordsLinked to original sources
PubMed2006

Unfolding time distribution of GFP by single molecule fluorescence spectroscopy.

We have studied the unfolding of single molecules of GFP-mut2 mutant trapped in wet silica gels in a wide range of GuHCl concentration. After the addition of denaturant, the number of fluorescent molecules decreases with unfolding rates (of the order of 0.01 min(-1)) that are in very good agreement with bulk fluorescence and circular dichroism data. Unexpectedly, single molecule experiments show rare fluctuations in the number of fluorescent proteins at equilibrium. On the other hand, although a first approximate description of the number decays can be reasonably performed by single exponential functions, the distributions of the single molecule unfolding times show a maximum at times congruent with 50-100 min up to the denaturation midpoint concentration of [GuHCl] congruent with 2.5 M. A theoretical analysis of the distributions indicates that this feature is a fingerprint of the competition between unfolding and refolding processes when the protein is very far from the midpoint denaturant concentration.

Gels↗
PubMed2005

A randomised, double blind, placebo controlled trial of the effect of theophylline in prevention of vasomotor nephropathy in very preterm neonates with respiratory distress syndrome.

BACKGROUND: Vasomotor nephropathy is a common renal dysfunction in very preterm neonates. OBJECTIVE: To determine whether theophylline could prevent vasomotor nephropathy in very preterm infants with respiratory distress syndrome. METHODS: A randomised, double blind, placebo controlled trial of 50 preterm infants of gestational age < or = 32 weeks needing assisted ventilation. Infants received an intravenous dose of theophylline (1 mg/kg) or placebo for three days. The 24 hour urine volume was measured daily. On days 2, 5, and 11, blood samples and 12 hour urine collections were analysed for electrolytes, creatinine, and urea. RESULTS: On day 1, urine output was significantly higher in the theophylline (2.4 (0.9) ml/kg/h) than the placebo (1.6 (1.0) ml/kg/h; p = 0.023) group (values are mean (SD)). The incidence of oligoanuria was significantly lower in the theophylline treated (5%) than the placebo (33%) group. Twenty four hours after the first administration of theophylline/placebo, serum creatinine concentration was significantly lower in the theophylline (0.76 (0.23) mg/dl) than the placebo (1.0 (0.41) mg/dl; p = 0.025) group. On day 5 an increase in serum creatinine was observed in both groups. On day 11 a significant reduction in serum creatinine was observed, compared with day 5, with no difference between the two groups. CONCLUSION: The results suggest that, in very preterm infants with respiratory distress syndrome, early theophylline administration improves renal function during the first two days of life.

Creatinine↗
PubMed2005

Selective fluorescence recovery after bleaching of single E2GFP proteins induced by two-photon excitation.

We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.

Animals↗
PubMed2005

The increase of oxygen requirement as index to identify the infants at high risk of pneumothorax during nasal continuous positive airway pressure.

AIM: The aim of this study was to evaluate the inspiration fraction of oxygen (FiO2) trend as an indicator of timing to suspend nasal continuous positive airway pressure (N-CPAP) and shift the babies to mechanical ventilation, in order to reduce the incidence of pneumothorax, comparing a similar population admitted in our division during the previous year. METHODS: Seventy-five newborns (mean gestational age 33.5 weeks, mean birth weight 2,072 g) admitted during 2003 in our Neonatal Intensive Care Unit, treated with Infant Flow System Nasal-CPAP, were included. Patients with more than 40% increase of the starting FiO2 in the first 24 h of treatment, were intubated and shift on mechanical ventilation. Seventy-seven infants, admitted during the previous year, with similar characteristics (mean gestational age 33.7 weeks, mean birth weight 2,047 g) were considered as control. RESULTS: Fifty-six neonates improved, 19 worsened and required mechanical ventilation. One of these developed pneumothorax (1.3%). Of the 77 infants admitted during the previous year, 26 worsened and were mechanically ventilated, and 8 developed pneumothorax (10.3%). The difference of incidence of pneumothorax was significant (P =0.0337). CONCLUSIONS: An increase of FiO2 more than 40% of the initial value during the first 24 h of N-CPAP may be considered a useful marker to identify infants at high risk of pneumothorax.

Age Factors↗
PubMed2004

Multiphoton switching dynamics of single green fluorescent proteins.

Multi-photon driven photo-switching between dark and bright (fluorescent) states of a green fluorescent protein (GFP) mutant is demonstrated. A single-protein investigation shows the existence of two distinct bright states that display sharp two-photon cross-section bands peaked at 780 nm and at 870 nm. Fluorescence of these two species can be independently switched on and off. These results highlight a new photoconversion pathway for photochromic GFPs and can have significant applications in multi-photon confocal microscopy and in optical data-storage architectures.

Green Fluorescent Proteins↗
PubMed2004

Neonatal outcome in patients with rheumatic disease.

Rheumatic autoimmune diseases have a higher prevalence in women, particularly during their childbearing age. Due to improved management, an increasing number of patients plan and carry out one or more pregnancies. Therefore, a growing interest is being paid to the possible consequences of maternal disease and associated treatment on the fetus and newborn infant. If maternal disease is characterized by the presence of IgG isotype autoantibodies, these can cross the placenta with possible antibody-mediated damage to the fetus. This is typically the case of the so called neonatal lupus erythematosus (NLE); a similar mechanism has been shown in infants of patients with immune thrombocytopenic purpura (ITP) and, less frequently, in those from mothers with antiphospholipid syndrome (APS). Indeed, this last condition is often responsible for placental, rather than neonatal, pathology. In addition, immunosuppressive and other drugs administered to the mothers during pregnancy and lactation might affect the fetal and neonatal immune system development. Finally, mothers disease and/or treatment could be related to neuropsychological alteration reported in some of their children.

Antiphospholipid Syndrome↗
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↗
PubMed2003

Early use of Nasal-BiPAP in two infants with Congenital Central Hypoventilation syndrome.

AIM: To reduce the problems caused by prolonged artificial ventilation in babies with Congenital Central Hypoventilation syndrome (CCHS). METHODS: Two term infants with CCHS, weighing 4030 g and 3100 g, respectively, at the beginning of treatment and aged 53 and 31 d, respectively, were successfully ventilated with a Nasal Bilevel Positive Airway Pressure (N-BiPAP) device. RESULTS: In the first patient the tcPO2 recordings (mean +/- SD) during sleep were 46 +/- 12 mmHg before using N-BiPAP and 58 +/- 13 mmHg after using the device, while those for tcPCO2 were 75 +/- 9 mmHg and 49 +/- 11 mmHg, respectively. In the second patient tcPO2 during sleep was 42 +/- 3 mmHg before, and 55 +/- 5 after N-BiPAP, and for tcPCO2 the recordings were 119 +/- 24 mmHg and 55 +/- 6 mmHg, respectively, showing a significant improvement. One infant had persistent gastro-oesophageal reflux, and frontal skin abrasion caused by the face mask. Nevertheless, these complications did not necessitate the discontinuation of N-BiPAP ventilation, thus precluding prolonged use of intubation and tracheotomy. CONCLUSION: In infants with CCHS, early use of non-invasive, positive-pressure ventilation with N-BiPAP, in association with careful monitoring, can decrease problems caused by prolonged intubation and tracheotomy.

Apnea↗
PubMed2003

Autoimmune haemolytic anaemia in a newborn infant.

The case is reported of an infant with autoimmune haemolytic anaemia of perinatal onset. Combined treatment with steroids and cyclosporin was necessary to improve haemolysis and reduce the high transfusion requirements. Treatment was discontinued at 13 months of age. The child was healthy at the follow up at 24 and 36 months of age.

Anemia, Hemolytic, Autoimmune↗
PubMed2002

Effect of general and epidural anaesthesia on thyroid hormones and immunity in neonates.

BACKGROUND: The aim of this study was to verify if variations of thyroid hormones related to circumstances of delivery and mode of maternal anaesthesia can contribute to neonatal neutrophil respiratory burst and natural killer cell activity. METHODS: We evaluated 10 infants born by vaginal delivery (group A), 10 infants born by caesarean section after epidural anaesthesia with lidocaine (group B) and 10 infants born by caesarean section after general anaesthesia with sevoflurane (group C). RESULTS: A significant reduction of neutrophil respiratory burst test was found in groups A and C compared with group B. Natural killer cell (NK) activity with an effector : target ratio of 30 : 1 (NK30) and 10 : 1 (NK10) was significantly higher in group A compared with the B and C groups. In addition, thyroid stimulating hormone (TSH) concentration was significantly reduced in group A compared with the B and C groups. A significant negative correlation was found between TSH and NK30 or NK10. CONCLUSIONS: Our results suggest that the mode of delivery and anaesthesia can significantly modify the endocrine-immune system in the newborn. Caesarean section delivery with regional anaesthesia seems to produce fewer modifications of neonatal immune function compared with general anaesthesia.

Anesthesia, Epidural↗
PubMed2002

Late-onset neutropenia in very low birthweight infants.

AIM: To evaluate the incidence and duration of late-onset neutropenia (defined as an absolute neutrophil count (ANC) <1500 mm(-3) at a postnatal age of >3 wk) in a population of infants with birthweight <2000 g, and to determine whether copper deficiency, a possible cause of both anemia and neutropenia, may be associated with this complication. METHODS: Complete blood cell count and differential were assessed in 247 low (LBW) and very low birthweight (VLBW) infants who were discharged after 3 wk of life. In neutropenic infants plasma copper and ceruloplasmin levels were also measured. RESULTS: Late-onset neutropenia was detected in 11 out of 147 VLBW infants (7.5%) and in 7 out of 127 LBW infants (5.5%). A neutrophil count of <1000 mm(-3) was observed in 14 infants (5.1%). A significantly lower gestational age was found in neutropenic infants compared with non-neutropenic infants. In neutropenic infants ANCs were significantly correlated with hemoglobin and hematocrit. In addition, a significant negative correlation was found between neutrophil and reticulocyte counts. Plasma copper concentration was significantly correlated with birthweight. Oral copper sulfate was administered to infants with plasma copper concentration <50 microg dl(-1), and did not seem to affect ANC, hemoglobin, hematocrit or reticulocyte counts. CONCLUSION: Late-onset neutropenia appears to be a benign condition that is not associated with any particular complication and does not require specific treatment. Reference ranges after the early neonatal period and during the first few months of life in LBW and VLBW infants should probably be set at lower values.

Blood Cell Count↗
PubMed2002

The immune development in a child born to a cyclosporin A-treated woman with systemic lupus erythematosus/polymyositis.

The case of a woman affected by an overlap syndrome systemic lupus erythematosus/polymyositis (PM), who presented with active myositis at the start of the pregnancy, is reported. Therapy with cyclosporin, corticosteroids, hydroxychloroquine and high-dose intravenous immunoglobulin induced a progressive remission of clinical and laboratory signs of myositis. At 33 weeks of gestation, after a premature pre-term rupture of membrane, a male child was delivered. Although premature, and small for gestational age, he had a normal growth, and did not show any clinical sign suggestive of immune deficiency. Lymphocyte phenotypical and functional studies, as well as response to vaccination, were also normal.

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

Rotational dynamics of curved DNA fragments studied by fluorescence polarization anisotropy.

The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data.

Algorithms↗
PubMed2001

Brownian dynamics simulations of fluorescence fluctuation spectroscopy.

We have developed a program for the simulation of the fluorescence fluctuations as detected from highly diluted samples of (bio)molecules. The model is applied to translational diffusion and takes into account the hydrodynamic interactions. The solution concentration is kept constant by assuming periodic boundary conditions and spans here the range 0.5< C < 10 nM. We show that the fluorescence correlation functions can be accurately computed on systems of limited size (a few molecules per simulation box) by simulating for a total time approximately 100-300 times the diffusion relaxation time of the fluorescence autocorrelation function. The model is applied also to the simulation of the scanning fluorescence correlation spectroscopy (FCS) and of the photon counting histograms for the confocal collection configuration. Scanning FCS simulations of highly diluted samples (C approximately equals 0.5 nM) show anticorrelation effects in the autocorrelation functions of the fluorescence signal that are less evident for higher concentrations. We suggest here that this effect may be due to the non-uniform occupancy of the scanning area by the fluorophores.

Algorithms↗
PubMed2001

Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy.

O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation. We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case. Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity. At least two components were detected, with lifetimes of approximately 2.5 and 0.5 ns. The lifetimes are very close to the values measured in bulk solutions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited state.

Biochemistry↗
PubMed2001

Regulation of p21waf1/cip1 expression by intracellular redox conditions.

Reactive oxygen species (ROS) have been considered for a long time only as molecules for inducing oxidative damage to proteins, lipids, and nucleic acids. However, in the last few years some physiological effects of ROS have been hypothesized, consisting of the redox regulation of several biological processes, including the transduction of mitogenic signals. This means that intracellular generation of ROS could be necessary to maintain homeostasis, as well as that their formation/scavenging should be controlled processes. We developed an experimental procedure that causes redox perturbations in intact cells, based on the exposure of living cells to diethylmaleate (DEM), a GSH-depleting agent. By this procedure we demonstrated that ROS generated following DEM treatment induces a G1 arrest, that is accompanied by several redox-dependent changes in cell cycle-related proteins. One of these is the p53-independent accumulation of p21waf1/cip1, which requires the integrity of the ras-MAPK pathway. Accordingly, DEM treatment strongly activates ERK2. On the other hand, redox perturbations provoked by DEM induce several early phenomena, including p21waf1/cip1 and Rb dephosphorylation.

Animals↗