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Biomedical subjects

F Paoletti

Publications and source records attributed to F Paoletti.

At least 37 records · Page 2Linked to original sources
PubMed2001

A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts.

Cysteine-proteinases (CP) of the papain family can be affinity-adsorbed by egg white cystatin C coupled to Sepharose 4B, thus allowing their selective isolation from either tissue or cultured cell extracts as well as biolological fluids and culture media. CP complexed by immobilized cystatin are further analyzed by means of SDS-PAGE and Western blot followed by serial or parallel immunological detection. The single-step affinity adsorption of papain-like enzymes has the advantage, over immunoprecipitation techniques, of yielding the simultaneous and comprehensive picture of most CP, as both precursor and mature forms, in a given sample. Moreover, cell extraction in the presence of immobilized cystatin ensures a fast complexation of CP, avoiding artifacts, due to conversion, degradation, and, eventually, subtraction of constitutive enzymes from the sample because of their interactions with endogenous inhibitors. This will provide a pattern that might reflect more closely the real CP levels in intact cells. The method may be useful in the field of biochemistry, cell biology, and, possibly, clinical chemistry to perform rapid analyses of papain-like enzymes and to monitor changes in both cellular and extracellular CP profiles along with different physiopathological conditions.

Adsorption↗
PubMed2001

Reduced growth and skeletal changes in zinc-deficient growing rats are due to impaired growth plate activity and inanition.

We investigated the effects of dietary zinc deficiency on skeletal metabolism in an animal model. Thirty 21-d-old male Sprague-Dawley rats were fed for 28 d either a zinc-deficient (ZD) diet (1 mg zinc/kg) or a normal diet ad libitum (AL, 50 mg zinc/kg) or in the same quantity as the ZD (pair-fed, PF). Only in the ZD group were general physical signs of zinc deficiency observed. Compared with the AL and PF rats, ZD rats showed significantly lower mean values in ponderal growth rate, femur weight and length, circulating levels of insulin-like growth factor-I, bone mechanical properties and concentration of zinc and, on histomorphometry, a decrease in the thicknesses of the overall growth plate and hypertrophic cartilage. In contrast, although bone volume was significantly lower in the ZD and PF rats than in the AL rats, no difference was observed between the ZD and PF rats. Osteoclast surface/bone surface and osteoclast number/bone surface ratios were significantly greater in PF rats than in the other two groups and not different in ZD and AL rats. Collectively, these data indicate that zinc deficiency has profound effects on the skeletal system of growing rats. In particular, the effects of zinc deficiency on bone growth and mass are the result of the reduced activity of the growth plate, likely mediated by impairment in the insulin-like growth factor-I system. We did not demonstrate an effect on bone mass via increased bone resorption.

Animals↗
PubMed2000

Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice.

We have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ)-induced hemolytic anemia. TER-119(+)/4A5(+) cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119(+)/4A5(+) cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia. (Blood. 2000;95:2559-2568)

Animals↗
PubMed2000

Combined effect of sourdough lactic acid bacteria and additives on bread firmness and staling.

The effect of various sourdoughs and additives on bread firmness and staling was studied. Compared to the bread produced with Saccharomyces cerevisiae 141, the chemical acidification of dough fermented by S. cerevisiae 141 or the use of sourdoughs increased the volume of the breads. Only sourdough fermentation was effective in delaying starch retrogradation. The effect depended on the level of acidification and on the lactic acid bacteria strain. The effect of sourdough made of S. cerevisiae 141-Lactobacillus sanfranciscensis 57-Lactobacillus plantarum 13 was improved when fungal alpha-amylase or amylolytic strains such as L. amylovorus CNBL1008 or engineered L. sanfranciscensis CB1 Amy were added. When pentosans or pentosans, endoxylanase enzyme, and L. hilgardii S32 were added to the same sourdough, a greater delay of the bread firmness and staling was found. When pentosans were in part hydrolyzed by the endoxylanase enzyme, the bread also had the highest titratable acidity, due to the fermentation of pentoses by L. hilgardii S32. The addition of the bacterial protease to the sourdough increased the bread firmness and staling.

Bread↗
PubMed2000

Prevention of post-herpetic neuralgia: acyclovir and prednisolone versus epidural local anesthetic and methylprednisolone.

BACKGROUND: Treatment of herpes zoster (HZ) includes the use of acyclovir with or without steroids. An alternative therapy is the epidural administration of local anesthetics with or without steroids. This trial compared the efficacy of these two treatment regimens in the prevention of post-herpetic neuralgia (PHN). METHODS: Six hundred adults over 55 years of age with a rash of less than 7 days duration, and severe pain due to HZ, were enrolled and randomized to receive either intravenous acyclovir (10 mg/kg three times daily) for 9 days+prednisolone (60 mg per day with progressive reduction) for 21 days, or 6-12 ml bupivacaine (0.25%) every 6-8 or 12 h+methylprednisolone 40 mg every 3-4 days by epidural catheter during a period ranging from 7 to 21 days. Efficacy was evaluated at 1, 3, 6 and 12 months. PHN was assessed as pain and/or allodynia, and "abnormal sensations" (hypoesthesia, burning, itching, etc.). Statistical analysis was performed based on the intent-to-treat population. RESULTS: In the 485 patients who completed the study, the incidence of pain after 1 year was 22.2% (51 patients of 230) after acyclovir+steroids, and 1.6% (4 patients of 255) after epidural analgesia+steroids. The incidence of abnormal sensations was 12.2% (28 patients) after acyclovir+steroids, and 4.3% (11 patients) in group B. CONCLUSIONS: Epidural administration of local anesthetic and methylprednisolone is significantly more effective in preventing PHN at 12 months compared to intravenous acyclovir and prednisolone.

Acyclovir↗
PubMed1999

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells.

Binding experiments with the specific muscarinic ligand [3H]N-methylscopolamine (3H-NMS) have shown the presence of constitutive muscarinic acetylcholine receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1-M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K(D)) for 3H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B+) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B+ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells.

Acetylcholinesterase↗
PubMed1999

Shoulder involvement in rheumatic diseases. Sonographic findings.

OBJECTIVE: To distinguish using shoulder sonography the different changes present in rheumatoid arthritis (RA), polymyalgia rheumatica (PMR), and periarticular disorders (PD) of soft tissue of the shoulder. METHODS: Ninety shoulders of patients with RA, 32 with PMR, 122 with PD, and 108 controls were studied sonographically, using a 7.5 MHz linear probe. The following structures were evaluated: long head of biceps tendon, supraspinatus, infraspinatus and subscapularis tendons, subacromial and subscapularis bursae, rotator cuff (thickness), calcifications, and glenohumeral and acromioclavicular joints. Statistical analysis was by Student's t test and chi-squared test. RESULTS: Involvement of long head of biceps tendon (peritendinous fluid collection, changes of thickness, and/or echotexture) was significantly different between RA and PMR and between PD and PMR. Alterations in thickness and/or fibrillar pattern were evaluated in rotator cuff tendons: supraspinatus tendon was involved with significant differences between PD and both RA and PMR; the changes of subscapularis tendon were present, with significant differences between PD and both the other groups; the alterations of infraspinatus tendon were not statistically different between the 3 groups. Effusion within bursae was present, with significant differences only between RA and PD. The mean thickness of rotator cuff was significantly different between controls (6.2 mm) and both PD (5.3 mm) and RA (5.8 mm), and between PMR (6 mm) and PD. Evaluation of effusion within the glenohumeral joint (capsule-bone distance) showed significant differences between controls (2.4 mm) and both RA (4.2 mm) and PMR (4 mm), between RA and PD (2.6 mm), and between PMR and PD. Calcifications were present only in PD (21.3%) and RA (6.7%), with significant differences. Effusion within the acromioclavicular joint was present in RA (35.5%) and PD (20.5%), with significant differences. CONCLUSION: Shoulder sonography showed involvement of all structures in RA, the prevalence of effusion in PMR, and involvement mainly of tendons in PD.

Aged↗
PubMed1998

Premature induction of aging in sublethally H2O2-treated young MRC5 fibroblasts correlates with increased glutathione peroxidase levels and resistance to DNA breakage.

Human MRC5 fibroblasts, at different passages in cultures, were used as an in vitro model to assess variations and/or induction of aging parameters under basal conditions or following sublethal oxidative stress by H2O2. DNA sensitivities to oxidatively-induced breakage, rather than basal levels of damaged DNA, were significantly different between cultures at low and high population doubling level (PDL): old cells maintained most of their DNA integrity even at high concentrations of H2O2, while young cells showed more extensive DNA damage which developed in a dose-dependent fashion. However, young cells pretreated with low doses of H2O2 exhibited increased resistance against further oxidative damage to DNA thus reproducing a senescent-like profile of sensitivity. In turn, DNA from old cultures incubated in a NAD precursor-free medium was more prone to H2O2-induced strand breaks mimicking DNA sensitivity of young cells. The extent of oxidatively-induced DNA damage in MRC5 populations correlated inversely with the levels of glutathione peroxidase (GPx) activity that almost doubled when cells passed from the young to the senescent stage. In addition, H2O2-pretreatment of young cells induced an increase in GPx expression approaching old cell values and promoted also the premature appearance of neutral beta-galactosidase activity and decreased c-fos expression upon serum stimulation, both of which were assumed to be characteristic traits of the senescent phenotype.

Cell Line↗
PubMed1998

An in vivo, ex vivo and in vitro comparative study of activity of copper oligopeptide complexes vs Cu(II) ions.

The tetrapeptide-Cu(II) complex H-(l-His-Gly)2-OH/Cu(II), indicated as L-Cu(II), has been investigated, as compared to the Cu(II) inorganic salt CuSO4, for its antioxidative and anti-inflammatory properties under a panel of experimental conditions. Both inorganic and organic Cu(II) compounds showed comparable activities in vitro and ex vivo by: (i) protecting, in a dose-dependent manner, rat brain homogenates from Fe(III)/ascorbate- or haemoglobin-induced lipid peroxidation; (ii) inhibiting the superoxide-mediated ferricytochrome c reduction by activated macrophages. CuSO4 and L-Cu(II) also exhibited similar anti-inflammatory effects in vivo by reducing significantly the extent of carrageenan-induced edema in the rat paw. The activities of the two compounds diverged strikingly only in the xanthine/xanthine oxidase system at low phosphate buffer concentration. L-Cu(II) decreased the rate of NBT reduction by superoxide in a true SOD-like fashion without affecting urate production. Instead, Cu(II) ions caused the rapid xanthine oxidase inactivation thus inhibiting both urate and superoxide production; this effect might be ascribed to the superoxide-mediated generation of the strong oxidant Cu(III) and its interaction with the enzyme. The administration of Cu(II), whether complexed with linear oligopeptides or as an inorganic salt, to animals or tissue extracts, conferred protection against oxidation and ought, conceivably, to interact with endogenous biological molecules and form highly bioavailable complexes which serve, subsequently, as the real scavengers. Moreover, the claimed prominent scavenger activities of Cu(II)-oligopeptide complexes over inorganic copper ions could be realised only in very simple in vitro systems through mechanisms which, although of biochemical interest, are unlikely to be of physiopathological significance.

Animals↗
PubMed1998

Coexpression of erythroid and megakaryocytic genes in acute erythroblastic (FAB M6) and megakaryoblastic (FAB M7) leukaemias.

There is evidence to suggest a close relationship between the erythroid and megakaryocytic lineages. Using RT-PCR, we evaluated the coexpression of erythroid and megakaryocytic genes in blasts from 25 acute myeloid leukaemia (AML) cases (FAB M1-M7) and three unclassifiable leukaemias with trilineage dysplasia (trilineal AML). All FAB M6 and M7 and trilineal leukaemias expressed mRNAs for alpha-globin, glycoprotein IIb (GpIIb), erythropoietin receptor (Epo-R) and thrombopoietin receptor (c-mpl), but not for myeloperoxidase (MPO) which in contrast was expressed in the other FAB-subtype leukaemias. These data support the hypothesis that blasts from M7 and M6 leukaemias may derive from (or represent) a common progenitor cell with resident bipotentiality towards the megakaryocytic and erythrocytic lineages.

Base Sequence↗
PubMed1998

Multicenter trial comparing tramadol and morphine for pain after abdominal surgery.

Seventy patients (40 male, 30 female), mean (SD) age 60.8 +/- 13.7 years were treated with parenteral morphine (10 mg/1 ml ampul) or tramadol (100 mg/2 ml ampul) to verify their analgesic effects in pain following abdominal surgery. The multicenter trial followed an open, controlled experimental design between patients, randomized within the centers. The drugs were given by intramuscular injection, as requested by patients, starting in the postoperative period when pain was more than 70 mm, assessed on a visual analog scale. Patients were allowed up to six ampuls of tramadol or morphine in the 24-h trial but in the first 4 h, if they asked for supplementary analgesic, only diclofenac (75 mg in a 3-ml ampul) was allowed. Both test drugs gave rapid and constant pain relief. After the first dose, pain intensity was reduced 36.2% with tramadol, and 51% with morphine; the pain-free interval was similar for both treatments. The quality of sleep and the number of hours of sleep the night after surgery were similar for both groups. Tramadol was tolerated better, giving rise to no untoward reactions; with morphine there was one case of mild respiratory depression. In abdominal surgery, therefore, tramadol given by intramuscular injection has postoperative analgesic activity similar to morphine, but is better tolerated.

Abdominal Pain↗
PubMed1997

Cysteine proteinases are responsible for characteristic transketolase alterations in Alzheimer fibroblasts.

Cultured fibroblasts from patients affected by Alzheimer's disease (AD) exhibited peculiar alterations of the enzyme transketolase (TK). Abnormalities (dubbed alkaline bands, ab) consisted of enzyme forms having unusually high pl and were proposed as a marker of the disease in living patients. The mechanisms of TK-ab expression were investigated with the use of cysteine proteinase inhibitors and purified preparations of either rat liver or human cysteine proteinases. The cysteine proteinase inhibitors N-acetyl-leu-leu-norleucinal (ALLN), L-trans-Epoxy-succinyl-leucylamido(4-guanidino)butane (E-64), and egg white cystatin added to AD cells just prior to extraction abolished TK abnormalities. Moreover, 1 day incubation of AD cultures with either ALLN (10 micrograms/ml), NH4Cl (10 mM), or KCl (30 mM) prevented TK-ab generation, due, presumably, to an impairment of lysosomal functions. Isolated rat liver cysteine proteinases were able to degrade TK in normal extracts and reproduce the characteristic TK-ab of AD fibroblasts. Moreover, pure human cathepsin H was also shown to partially induce an Alzheimer-like TK pattern and cleave normal TK to a 35 kDa fragment as spontaneously occurring in AD fibroblasts. The explanation of mechanisms of TK-ab formation provided evidence for an underlying imbalance of proteolysis in AD fibroblasts due to a relative increase/derangement of the cysteine proteinases cathepsins which might be also involved in the reported abnormal processing of multiple cellular components.

Alzheimer Disease↗
PubMed1997

Cerebrospinal fluid cytokines in patients with tuberculous meningitis.

The level of tumor necrosis factor (TNF)-alpha, soluble TNF receptors p75 (sTNFR-75) and sTNFR-55, interferon (IFN)-gamma, and interleukin (IL)-10 and IL-12 were measured in 59 cerebrospinal fluid (CSF) samples from 15 patients with tuberculous meningitis (TBM). TBM was associated with elevated concentrations of TNF-alpha, sTNFR-75, sTNFR-55, IFN-gamma, and IL-10, while CSF IL-12 was undetectable in all TBM patients. A significant correlation between cytokines and CSF adenosine deaminase activity was also found. The levels of TNF-alpha did not decrease over time, being still detectable in the CSF 16 months after starting antibiotic therapy, whereas IFN-gamma along with anti-inflammatory mediators sTNFR-75, sTNFR-55, and IL-10 remained elevated in the CSF for 4-8 months. The chronic release of cytokines in the CSF compartment was related neither to the TBM stage nor to the clinical outcome of the disease, thus suggesting the presence of a continuous activity of the inflammatory process at the site of infection.

Adenosine Deaminase↗
PubMed1997

Constitutive and inducible expression of megakaryocyte-specific genes in Friend erythroleukaemia cells.

Friend murine erythroleukaemia cells (MELC) were analysed by semiquantitative RT-PCR for the constitutive and inducible expression of megakaryocyte-specific genes. Uninduced MELC expressed detectable levels of mRNAs for acethylcholinesterase (AChE), platelet factor-4 (PF4), glycoprotein IIb (GPIIb) and von Willebrand factor (VWF), whereas the erythroid alpha- and beta-globin genes were not transcribed appreciably. However, MELC exposed to 5 mM hexamethylene bisacetamide (HMBA) or 1.5% dimethyl sulphoxide (DMSO) seemed to be channelled towards a mixed erythroid/megakaryocytic phenotype characterized by unaltered levels of VWF mRNA, increased levels of AChE, GPIIb and PF4 mRNA. and simultaneous induction of the globin genes. Megakaryocyte-related genes were expressed. in the absence of globin gene transcription, by MELC treated with either phorbol-12-myristate acetate (PMA; 100 ng/ml) or colcemid (40 nM), an antimicrotubule agent capable of promoting polyploidization in this model. Moreover, PMA and colcemid induced also de novo expression of the thrombopoietin receptor c-mpl. PMA and colcemid did not affect high basal c-myb mRNA levels which, in turn, were down-regulated upon HMBA or DMSO induction. Additionally, both uninduced and induced MELC exhibited significant levels of Epo-R and IL-3R mRNAs, whereas no expression of granulocyte/macrophage-related genes was detected. Megakaryocyte gene expression of MELC was also compared to that of other haemopoietic cell populations from normal mice and mice infected with the anaemic strain of the Friend virus. According to our results, MELC should be seen as an unique erythro-megakaryocytic model of differentiation, potentially useful for studying molecular events governing lineage commitment as well as some steps of megakaryocytopoiesis.

Animals↗
PubMed1996

Regional reductions of transketolase in thiamine-deficient rat brain.

Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0-25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.

Animals↗
PubMed1996

Megakaryocyte-like increase in ploidy of Friend's erythroleukemia cells induced to endoreplication by colcemid.

Previous work from this laboratory has shown that Friend's murine erythroleukemia cells (MELCs) express some bio-chemical traits of the megakaryocytic lineage. The supposed mixed erythroid/megakaryocytic nature of these cells has been investigated further by challenging MELCs with the antimicrotubule agent colcemid. This compound, at the concentration of 40 nM, was found to induce a striking arrest of cell growth without significant effects on viability. At the same time, the bulk of treated MELCs underwent a large increase in size to contain, after 3 days, as much as 4 times more proteins and 5 times more DNA than controls. As shown by high rates of 3H-thymidine incorporation, increase in DNA content was the result of active synthesis without completion of intervening mitosis according to a process that closely resembled endoreplication. Eventually, colcemidinduced MELCs presented multilobed nuclei and were arranged into discrete ploidy groups containing up to 16 N levels of DNA. Moreover, upon colcemid addition, MELCs initiated a polyploid response that was shown to continue, even in the absence of the inducer, to yield cells that became strongly positive for acetylcholinesterase (AChE) in the late stages of culture. These effects were compatible with a colcemid-induced commitment of MELCs to megakaryocyte differentiation, for which these cells seemed to be definitely programmed. The expression of megakaryocyte features in MELCs provided further evidence for the bipotentiality (erythroid/megakaryocytic) of this model.

Animals↗