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P Pizzo

Publications and source records attributed to P Pizzo.

At least 19 recordsLinked to original sources
PubMed2001

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes.

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.

Animals↗
PubMed1999

Development and evaluation of an improved method for screening of amphetamines.

We developed a homogeneous immunoassay method to eliminate false-positive amphetamine results caused by cross-reactive substances, including over-the-counter allergy and cold medications. This method uses a neutralizing antibody that binds to amphetamines but does not bind to the labeled amphetamine conjugate used in the assay. The amount of neutralizing antibody is sufficient to reduce the assay signal resulting from authentic amphetamine and methamphetamine, but not the signal resulting from cross-reactants. This concept was implemented using the CEDIA DAU Amphetamines assay on Hitachi 747 and 717 clinical chemistry analyzers. Urine samples were tested using the standard, unmodified reagents in one channel and reagents containing the neutralizing antibody in a second channel. The difference in rate between the two tests was calculated by the analyzer; true-positive samples showed a significantly greater decrease in assay signal in response to neutralizing antibody as compared with false-positive samples. The neutralization method was evaluated in two studies using 448 samples that tested positive in the initial CEDIA DAU Amphetamines screening test. The samples were separated into categories of 154 true-positive samples and 294 false-positive samples based upon a secondary screen with the Abbott FPIA Amphetamines assay followed by gas chromatography-mass spectrometry (GC-MS) testing using the HHS (SAMHSA) cutoff criteria. The CEDIA neutralization test successfully identified all 154 of the GC-MS confirmed positive samples. The test successfully identified as false positive 251 out of the 294 (85.4%) samples that failed to confirm by GC-MS.

Amphetamines↗
PubMed1998

Delayed activation of the store-operated calcium current induced by calreticulin overexpression in RBL-1 cells.

Calreticulin (CRT) is a high-capacity, low-affinity Ca2+-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca2+ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca2+ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca2+ current (ICRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca2+-binding domain, markedly retards the ICRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca2+-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of ICRAC activation.

Animals↗
PubMed1997

Dynamic properties of an inositol 1,4,5-trisphosphate- and thapsigargin-insensitive calcium pool in mammalian cell lines.

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate- and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate-sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+-ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+-ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed.

Animals↗
PubMed1997

1997 guidelines for the use of antimicrobial agents in neutropenic patients with unexplained fever. Infectious Diseases Society of America.

This is the first in a series of practice guidelines commissioned by the Infectious Diseases Society of America through its Practice Guidelines Committee. The purpose of these guidelines is to provide assistance to clinicians when making decisions on treating the conditions specified in each guideline. The targeted providers are internists, pediatricians, and family practitioners. The targeted patients and setting for the fever and neutropenia guideline are hospitalized individuals with neutropenia secondary to cancer chemotherapy. Panel members represented experts in adult and pediatric infectious diseases and oncology. The guidelines are evidence-based. A standard ranking system was used for the strength of the recommendations and the quality of the evidence cited in the literature reviewed. The document has been subjected to external review by peer reviewers as well as by the Practice Guidelines Committee and was approved by the IDSA Council. An executive summary, algorithms, and tables highlight the major recommendations. The guideline will be listed on the IDSA home page at http://www.idsociety.org.

Adult↗
PubMed1997

Use of fluoroquinolones for empirical management of febrile neutropenia in pediatric cancer patients.

BACKGROUND: Empiric antibiotic therapy has become a standard of care for the febrile neutropenic patient. Many clinical trials over the previous three decades have demonstrated that a variety of antibiotic combinations and more recently potent antibiotic monotherapies may preserve the patient through the critical time of fever and neutropenia. Recently attempts have been made to identify "low risk" patients who may not require traditional, intensive, hospitalized intravenous antimicrobial therapy. Therefore the need for new treatment alternatives for the febrile neutropenic pediatric cancer patient in particular revolves around the desire for less complex regimens, agents with minimal toxicity and expense and the option of an oral formulation for outpatient management. OBJECTIVE: Fluoroquinolones, especially ciprofloxacin and ofloxacin, are examined in this paper as potential oral alternatives for managing the low risk neutropenic pediatric cancer patient population. Attention must be paid to their antibacterial spectra, however, and in some cases fluoroquinolones should be combined with a second agent for additional Gram-positive coverage. RESULTS: Several studies, including one ongoing trial at the National Cancer Institute, have shown the potential benefits of oral fluoroquinolone therapy among low risk febrile neutropenic patients. Joint complaints in children after ciprofloxacin therapy in the National Cancer Institute trial thus far have been minimal, reversible and felt to be unrelated to ciprofloxacin treatment. CONCLUSION: The use of outpatient therapy, such as the fluoroquinolones, to manage febrile neutropenic episodes must be approached with caution and should be undertaken only in selected low risk patients.

Administration, Oral↗
PubMed1996

Mitochondrial alterations induced by aspirin in rat hepatocytes expressing mitochondrially targeted green fluorescent protein (mtGFP).

Mitochondria in primary living hepatocytes were visualized in cells transfected with a chimeric plasmid encoding for the green fluorescent protein (GFP) of Aequorea victoria engineered to be specifically targeted to mitochondria, as described recently (Rizutto et al. (1995) Curr. Biol. 5, 635-642). The identification of the fluorescent organelles as authentic mitochondria was confirmed by double labeling with rhodamine 123. Acetylsalicylate treatment of hepatocytes induced in mitochondria typical morphological alterations closely analogous to the swelling promoted by acetylsalicylate in isolated mitochondria. Cyclosporin A, which in isolated mitochondria prevents the changes induced by acetylsalicylate, had no protective action but induced per se specific alterations in the morphology of mitochondria. Moreover, exposure of hepatocytes to cyclosporin A followed by acetylsalicylate caused the same mitochondrial changes induced by each of the two compounds separately. The structural alterations caused by acetylsalicylate were constantly associated with a decrease in mitochondrial urea synthesis and cell viability.

Animals↗
PubMed1996

Targeting aequorin and green fluorescent protein to intracellular organelles.

Two proteins of Aequorea victoria were molecularly engineered and produced in mammalian cells, in order to serve as specific reporters of subcellular microenvironments. Aequorin (AEQ), a Ca(2+)-sensitive photoprotein, was successfully targeted to three intracellular locations: cytosol, nucleus and mitochondria. The recombinant apoprotein, reconstituted into active AEQ by the addition of the prosthetic group to the culture medium, allows the direct measurement of [Ca2+] within those compartments, thus directly addressing questions of large biological interest. The same approach was utilized for the green fluorescent protein (GFP) for specific labelling, in vivo, of the various subcellular structures. GFP was targeted to mitochondria: the recombinant protein, strongly fluorescent in a highly reducing environment, provides a powerful tool for visualizing these organelles in living cells, and may represent the prototype of a new family of intracellularly targeted fluorescent probes.

Aequorin↗
PubMed1996

Reduced levels of dystrophin associated proteins in the brains of mice deficient for Dp71.

Duchenne muscular dystrophy (DMD) is a progressive degenerative lethal muscle disease. A significant proportion of DMD affected children suffer also from mental retardation. The rod shaped protein, dystrophin, which is absent from or defective in the muscle of DMD patients, binds to a number of membrane associated proteins (known collectively as dystrophin associated proteins [DAPs]). The levels of DAPs is greatly reduced in the muscle of DMD patients and mdx mice, which lack dystrophin. In addition to dystrophin isoforms, the DMD gene codes also for several smaller proteins. One of the small proteins, Dp71, is expressed in most or all non-muscle tissues and is the major DMD gene product in the brain. The function of the small DMD gene products is unknown. Here we show that mutant mice which do not express the smaller non-muscle products of the DMD gene have a reduced level of DAPs in their brain. This suggests that Dp71 is important for the formation and/or stabilization of a DAPs complex in brain.

Animals↗
PubMed1996

Resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC) in HIV-1 isolated from paediatric patients.

We conducted detailed virological evaluations of 16 HIV-1-infected paediatric patients treated with 3TC (lamivudine) monotherapy. High-level phenotypic resistance against this compound (up to 2,500-fold) was seen in virtually all cases, usually within 8-12 weeks of initiation of therapy. This was concomitant with the appearance of the M184V mutation in viral reverse transcriptase, previously shown to be responsiblefor such resistance. Viral burden fell in virtually all cases after commencement of therapy, and remained below baseline in each instance studied, despite a rebound effect and the appearance of drug resistance. Viral isolates from some patients underwent a switch from a non-syncytium-inducing (NSI) to a syncytium-inducing (SI) phenotype during the course of the study, although no relationship was apparent between dose of drug employed, time to development of drug resistance or time of appearance of SI phenotype.

Anti-HIV Agents↗
PubMed1995

Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells.

BACKGROUND: It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic (Escherichia coli) and eukaryotic (Caenorhabditis elegans and Drosophila melanogaster) living cells; it can therefore be used as a powerful marker of gene expression in vivo. The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles. RESULTS: In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol. Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide). When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria. We also describe two of the many possible applications of this recombinant GFP in physiological studies. The targeted chimera allows the visualization of mitochondrial movement in living cells. Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential. Moreover, when GFP is cotransfected with a membrane receptor, such as the alpha 1-adrenergic receptor, the fluorescence of the GFP in intact cells can be used in recognizing the transfected cells. Thus, specific changes in intracellular Ca2+ concentration that occur in cells expressing the recombinant receptor can be identified using a classical fluorescent Ca2+ indicator. CONCLUSION: GFP is an invaluable new tool for studies of molecular biology and cell physiology. As a marker of transfection in vivo, it provides a simple means of identifying genetically modified cells to be used in physiological studies. More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters.

Amino Acid Sequence↗
PubMed1995

Correlation between computed tomographic brain scan abnormalities and neuropsychological function in children with symptomatic human immunodeficiency virus disease.

OBJECTIVE: To evaluate the clinical significance of computed tomographic brain scan abnormalities observed in children with symptomatic human immunodeficiency virus disease. PATIENTS: Eighty-seven previously untreated children with symptomatic human immunodeficiency virus type 1 disease. METHODS: General levels of cognitive functioning, obtained from age-appropriate intelligence tests, and social-emotional behavior were correlated with computed tomographic brain scan abnormality ratings. RESULTS: A significant relation between computed tomographic brain scan abnormalities and cognitive dysfunction as well as aberrant behavior was found, which appeared stronger in (younger) vertically infected children compared with transfusion-infected patients. Calcifications, independent from the degree of brain atrophy, were associated with significantly greater delays in neurocognitive development. CONCLUSION: Computed tomographic brain scan abnormalities, even when mild, were of clinical significance, suggesting that human immunodeficiency virus-associated central nervous system compromise is a continuous process and that scans may be helpful at baseline in defining patients at risk and for monitoring them during therapy.

Brain↗
PubMed1994

Interrelations among patterns of change in neurocognitive, CT brain imaging and CD4 measures associated with anti-retroviral therapy in children with symptomatic HIV infection.

The interrelationships of patterns of change and variability between baseline and after 6 months of anti-retroviral therapy in neurocognitive, brain imaging, and immune measures were studied in 77 children with symptomatic HIV disease. Overall improvement in CNS structure/function after 6 months of anti-retroviral therapy was limited; new intracerebral calcifications tended to occur and old ones tended to progress in young children with vertically acquired HIV infection, despite treatment. Substantial inter-individual differences in change were however observed. Factors which explained part of the variance in the magnitude and direction of change were baseline structural and functional abnormalities, rating of degree of CNS penetration of the drug protocol, and concurrent changes on other variables. These preliminary data suggest that CNS specific effects of therapies as well as pretreatment status of CNS function/structure need to be taken into consideration when evaluating future trials of anti-retroviral therapy for children with symptomatic HIV infection.

AIDS Dementia Complex↗
PubMed1994

Psychologic adjustment of human immunodeficiency virus-infected school-age children.

We investigated the psychosocial adjustment of school-aged, human immunodeficiency virus-positive children and factors associated with level of adjustment. Participants were primarily transfusion-infected children living in middle-class families. We administered measures of depression, anxiety, and self-concept to children, and measures of behavior problems, social functioning, personality characteristics, and life events to parents. An index of disease stage was also collected. Children reported experiencing low levels of depressive and anxious affect and generally felt positively about themselves. By contrast, parents saw their children as more anxious and less socially active than respective standardization samples. A greater than expected proportion of these children, as reported by their parents, scored in the maladaptive range on measures of social functioning, anxiety, and conduct problems. Experience of adversive life events and progression of the disease were associated with more behavioral and social problems. Findings are discussed in terms of their generalizability and implications for future research.

Adaptation, Psychological↗
PubMed1993

Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor.

The effects of oxidized ATP (oATP) on responses triggered by extracellular adenosine 5'-triphosphate (ATPe) were investigated in the mouse macrophage-like cell line J774. ATPe induced in this cell line two kinds of responses mediated by two different P2 purinergic receptors: 1) an early permeabilization of the plasma membrane to extracellular hydrophilic markers of M(r) up to 900 mediated by P2Z receptors; and 2) a fast mobilization of Ca2+ from intracellular stores mediated by P2Y receptors. Low oATP concentrations (100 microM) completely blocked the first response without affecting the second. ATPe-dependent cell swelling, vacuolization, and lysis were also inhibited. Antagonism developed slowly, as an incubation at 37 degrees C for at least 2 h in the presence of oATP was needed and was irreversible, thus suggesting that the inhibitory action was due to covalent modification of the receptor. The rate of hydrolysis of exogenous ATP was slightly decreased by oATP, indicating a minor blocking effect of this compound on plasma membrane ecto-ATPases in the concentration range tested. These observations suggest that oATP may be a potentially very useful tool for isolation and characterization of the P2Z purinergic receptor.

Adenosine Triphosphate↗