Evidence of charge transfer from Na adatoms to the Cs substrate: A collisionally excited autoionization electron-emission study.
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Journal Article↗
Biomedical subjects
A Oliva
Publications and source records attributed to A Oliva.
Biochemical characterization of p16INK4- and p18-containing complexes in human cell lines.
The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G1 cell cycle phase. One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs. We have evaluated the amount of two such inhibitors, namely p16(INK4) and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins. The results obtained indicated that (i) p18 gene expression, unlike p16(INK4) gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16(INK4) and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase. Moreover, employing the human native partially purified p16(INK4)or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16(INK4) bimolecular complex. Our findings suggest that during the cell division cycle the members of the p16(INK4) protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G1 --> S transition.
Carrier Proteins↗
Purification and characterization of recombinant human 5'-methylthioadenosine phosphorylase: definite identification of coding cDNA.
5'-Methylthioadenosine phosphorylase gene maps on the 9p21 chromosome, strictly linked to the important tumor suppressor gene p16INK4A. Chromosomal deletions encompassing both the phosphorylase and p16INK4A genes cause the complete absence of the enzymatic activity in a large number of tumors, thus resulting in well-defined metabolic differences between malignant and normal cells. Recently, the cloning of the phosphorylase gene has been reported on the basis of indirect evidence. In order to demonstrate definitely the identification of 5'-methylthioadenosine phosphorylase gene, we have cloned the putative enzyme coding sequence in a prokaryotic expression vector and expressed the protein in bacteria. The recombinant phosphorylase has been purified to homogeneity and its physicochemical, immunological and kinetic features have been characterized. The results obtained allowed the conclusive demonstration of 5'-methylthioadenosine phosphorylase gene cloning and the use of recombinant protein for further characterization.
Chromatography, Gel↗
Antineutrophil cytoplasmic antibodies are present in long standing type 1 diabetics but do not correlate with selective proteinuria.
Antineutrophil cytoplasmic antibodies (ANCA) are present in systemic vasculitis with or without renal involvement and in inflammatory bowel diseases, conditions which share damage in proteoglycan content of basal membrane. In diabetes, there is a reduction in proteoglycans in the kidney basal membrane, responsible for the decrease in fixed anionic charges and, consequently, for the prevalent anionic proteinuria (albumin, IgG4) even in the early preclinical stage of nephropathy. The aims of this study were to search for the presence of ANCA in long-standing type 1 diabetic patients and to evaluate possible correlations with size- and/or charge-selective proteinuria. Twenty-two type 1 diabetic patients (duration of diabetes 24 years, range 9-30) selected and grouped according to albumin excretion rate values, were studied together with 21 age and sex comparable normal subjects. ANCA, albumin excretion rate, and the clearances of albumin, of prevalently cationic total IgG (IgG) and of anionic IgG4 were evaluated. ANCA were measured using ELISA and indirect immunofluorescence methods; albumin, IgG and IgG4 were tested with RIA or ELISA methods developed in our laboratory. ANCA were found in five patients, three of whom showed proteinuria. 33.3% and 18.2% of patients with normal IgG and albumin clearances respectively had elevated IgG4 clearance. This study shows for the first time the presence of ANCA in long-standing type 1 diabetic patients and confirms a prevalent anionic protein excretion in these patients, but does not show a correlation between the presence of ANCA and proteinuria, even if the presence of ANCA in diseases sharing alterations in proteoglycan content of vascular basal membrane is noteworthy.
Adult↗
Hepatitis C virus infection in Italian patients with hypogammaglobulinemia.
We investigated the presence of hepatitis C virus (HCV) infection in 58 patients with humoral immunodeficiencies. Forty-three of these patients had common variable immunodeficiency (CVI), 2 had sporadic hyperimmunoglobulin M (HIM) syndrome, 2 had immunoglobulin G subclass deficiency, 4 had ataxia-telangiectasia (AT), and 7 had X-linked agammaglobulinemia (XLA). Patients with late-onset hypogammaglobulinemia (those with CVI, HIM, or immunoglobulin G subclass deficiency) had a 38.2% prevalence of HCV infection. In patients with XLA or AT, HCV infection was not detectable. Most of the HCV-infected patients had persistent viremia, with histologic findings of chronic hepatitis. Although patients positive for HCV ribonucleic acid (RNA) had received several lots of immunoglobulin, we were unable to detect any correlation between the time that alanine aminotransferase levels increased and the time that intravenous immunoglobulin therapy was given, except in one patient with CVI. Moreover, we found no differences in the number of blood transfusions, surgical procedures, or administrations of intravenous or intramuscular immunoglobulin between HCV RNA-positive and HCV RNA-negative groups. We concluded that: (1) the incidence of HCV infection in patients with hypogammaglobulinemia is much higher than that reported in the Italian general population; (2) although patients with hypogammaglobulinemia have persistent viremia, they do not show an aggressive course of HCV disease, nor does hepatocarcinoma develop; and (3) intravenous immunoglobulins are only one of several possible causes of HCV transmission in patients with humoral immunodeficiencies.
Adult↗
Biocompatibility studies on glass ionomer cements by primary cultures of human osteoblasts.
Glass ionomer cements (GICs) are materials largely employed in the dental field that have been considered recently as cements in orthopaedic surgery for their proven osteogenic features. The aim of this study was to compare the response of cultured human osteoblastic cells to a number of commercial glass ionomer cements in order to provide indications useful for the further development of formulations that have potential for use as cements or implants in repair and replacement of bone tissue. The GICs tested were: Ketac-Fil Aplicap, lonocem lonocap 1,0, GC Fuji II, GC Fuji II LC and Vitremer 3M. Several features such as plating efficiency, adhesion and morphology of the cells were studied, as well as the only specific biochemical parameter of osteoblastic phenotype, namely osteocalcin production. In addition, the colonisation of materials by osteoblastic cells was verified by means of scanning electron microscopy. Altogether, the results obtained indicate that four of the five glass ionomer cements tested are biocompatible, showing vital cells adhering to the materials, proliferating and expressing the biochemical markers of osteoblastic phenotype, whereas Vitremer 3M, although currently employed in the dental field, exhibits a great cytotoxicity toward the cells. The adverse reaction of this GIC can be attributed to the leaching of at least two components of the polyacidic phase evidenced by protonic magnetic resonance analysis (PMR), namely 2-hydroxyethylmethacrylate (HEMA), and an unidentified acidic species. The addition of pure HEMA at the same concentrations found by means of PMR to cultures of osteoblastic cells resulted in a complete cell death. Our results also show that in vitro methods employing primary cultures of human cells specific to the implant sites of prostheses are appropriate and suitable tools for evaluating biocompatibility of materials. Furthermore, this kind of approach can provide indications useful in the design of novel materials as well as in improving the characteristics of the formulations already available.
Biocompatible Materials↗
Apoptosis-related mortality in vitro of mononuclear cells from patients with HIV infection correlates with disease severity and progression.
Peripheral blood mononuclear cells (PBMC) from 103 HIV-infected patients were tested for their mortality rate (MR) when incubated in vitro for 3 days in a culture medium. MR was related to apoptosis as shown by DNA analysis and morphological evaluation of ethidium bromide-stained PBMC by flow cytometry. MR was significantly higher in patients in CDC stage IV as compared to patients in stage II or III (p = 0.017). MR was also higher in patients with low CD4 cells/mm3 (p = 0.014 for patients with < 400 cells; p = 0.001 for patients with < 200 CD4 cells/mm3) and with low percentage of CD4 cells (p = 0.001 for patients with < 10% of CD4 cells). A significant negative correlation was observed between MR and both absolute numbers or percentages of CD4 cells (p < 0.001). The addition of interleukin-2 (IL-2) and fibro-blast-conditioned medium (FCM) to the cultures significantly reduced MR. However, the ability of both IL-2 and FCM to preserve viability was significantly associated with p24 negativity. Clinical and immunological follow-up was available for 60 patients for a mean period of 26 months. MR at the beginning of the study was significantly higher in the group of patients who clinically progressed (according to the CDC classification) or died during the follow-up (p < 0.0001). Our data suggest that MR correlates with both disease severity and progression and that MR is directly related to the depletion of CD4 cells in cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
5'-Deoxy-5'-methylthioadenosine phosphorylase and p16INK4 deficiency in multiple tumor cell lines.
5'-Deoxy-5'methylthioadenosine phosphorylase (MTA-Pase) gene is localized at the 9p21 region linked to the recently identified putative tumor suppressor gene, p16INK4, which appears implicated in the control of cell division cycle. The phosphorylase is a housekeeping enzyme involved in the purine and amino acid metabolism whose activity is evidentiable in all the normal tissues. Chromosomal deletions encompassing both MTAPase and p16INK4 genes cause the total absence of the enzymatic activity only in malignant cells, thus resulting in defined metabolic differences between malignant and normal cells. MTAPase deficiency was investigated by direct radiochemical assay method and by immunochemical techniques in 35 different human malignant cell lines established from several tumor types. The enzyme-deficient cells derived from breast, lung, ovary and liver cancer, malignant melanomas, malignant gliomas and liposarcomas. Two of the MTAPase-deficient cell preparations (from a liver carcinoma and from a melanoma) are primary cultures thus directly representing the original cancer genotypes. Several of the MTAPase-negative cells were studied for p16INK4 gene deletions and for p16INK4 protein deficiency. In all the examined samples a full correlation exists between the lack of MTAPase and that of p16INK4. A similar result was obtained analysing extracts of Vero cell line, which is a fibroblast MTAPase-negative cell line established from the kidney of a normal adult monkey. Conversely, Cos cells, which also are fibroblasts derived from monkey kidney, show both MTAPase and p16INK4 protein. These results: (i) demonstrate that the phosphorylase deficiency is distributed among almost all the most important human cancers; (ii) confirm and extend the tumor types were p16INK4 gene inactivation is observable and (iii) suggest that deletions at 9p21 (in humans) or at syntenic chromosomes (in other species) might represent a general mechanism of p16INK4 gene loss of function and possibly, in turn, of cancer development and/or progression.
Animals↗
Randomized double-blind placebo-controlled trial of early octreotide in patients with postoperative enterocutaneous fistula.
Somatostatin and octreotide both enhance closure of gastrointestinal fistulas. The present trial was undertaken to test whether early combined treatment with parenteral nutrition and octreotide 100 micrograms every 8 h by subcutaneous injection had a beneficial effect compared with parenteral nutrition plus placebo. Thirty-one patients with post-operative gastrointestinal or pancreatic fistula were randomly assigned to receive parenteral nutrition plus octreotide (14 patients) or placebo (17) within 8 days of fistula onset. The percentage reduction in output and rate of spontaneous closure within 20 days were analysed. Mean(s.d.) reduction in output was similar after octreotide and placebo at 24 h (66(43) versus 68(47) per cent, P = 0.9), 48 h (60(46) versus 57(43) per cent, P = 0.8) and 72 h (62(50) versus 66(49) per cent, P = 0.9) after starting the combined treatment. Closure within 20 days was observed in eight of 14 fistulas in patients given octreotide and in six of 17 in those receiving placebo (P = 0.4). Administration of octreotide, within 8 days of fistula onset, associated with parenteral nutrition does not significantly increase the spontaneous fistula closure rate compared with parenteral nutrition plus placebo.
Aged↗
A new model of intraabdominal abscess: usefulness for hydrosaline metabolism studies in parenteral nutrition associated with sepsis.
The present study was set up to develop a new model of intraabdominal abscess (IAA) useful for hydrosaline metabolism studies based on the ligation of the appendix (AL) and wrapping of the appendix tip with omentum. Two experiments were designed: (1) to characterize the model and (2) to investigate extracellular volume (ECV) changes during parenteral nutrition (PN). Four groups of rabbits were studied at 3 (3DA) and 7 days (7DA) after AL or sham operation. PN was given for 6 days to two groups of septic rabbits: high volume HV) and low volume (LV) groups received 100 and 70 ml/kg.day of water with 7 and 0 meq/day of ClNa, respectively. Serum albumin (SA), ECV, and weight, water and sodium balances were determined. In 3DA, weight loss, reduced spontaneous intake, negative water balance, and reduction in SA were noted. Low SA, higher weight loss, and reduced intake were still observed in 7DA. SA correlated with ECV (r2 = 0.61, P = 0.003) in 7DA. Positive nitrogen balance was achieved during PN. The HV group had higher water and sodium balances than LV. In the HV group only, SA negatively correlated with sodium balance and with ECV at the end of PN (r2 = 0.87, P = 0.0007 and r2 = 0.9, P = 0.0001). The impact on hydrosaline metabolism of IAA in this model resembles that of moderate sepsis in humans. SA decrease appears to have two major components: escape around the inflammatory area and dilution. ECV expansion after PN is influenced by the initial SA concentration.
Abdominal Abscess↗
p16INK4 gene deletions in childhood acute lymphoblastic leukemias.
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Base Sequence↗
Immunodeficiency with hyperimmunoglobulinemia M in two female patients is not associated with abnormalities of CD40 or CD40 ligand expression.
The immunologic defect in X-linked immunodeficiency and hyperimmunoglobulinemia M (HIM) are related to defective expression of the CD40 ligand (CD40L). We have studied two female patients with HIM to evaluate the role of CD40/CD40L in the pathogenesis of impaired immunoglobulin switching. In addition to recurrent infections characteristic of humoral immunodeficiencies, the two patients had chronic hepatitis caused by type C virus. Phenotypic characterization of peripheral blood mononuclear cells showed a similar picture in both patients, with a reduction in the absolute numbers of CD4 cells and increased numbers of CD8 and CD3/DR cells. B cells (CD19+) were reduced in one patient, but CD40 was expressed on all CD19+ cells in both patients. The expression of CD40L was normal on peripheral blood mononuclear cells from the two patients with HIM on both resting and stimulated cells. The combination of anti-CD40 and cytokines (interleukin-2, interleukin-4, and interleukin-10) was able to restore proliferative capacity to anti-IgM. Peripheral blood mononuclear cells from the two patients with HIM showed a high spontaneous production of IgM in vitro and no production of IgG or IgE. Our data suggest that the defect of isotype switching in female patients with HIM is not related to defective expression of the CD40/CD40L receptor system. A possible role for chronic hepatitis C virus infection in the pathogenesis of the disease is suggested by the detection of specific production of anti-hepatitis C virus IgM.
Adolescent↗
Calcific myonecrosis following compartment syndrome: report of two cases, review of the literature, and recommendations for treatment.
Calcific myonecrosis of lower extremity muscles has been identified as an uncommon late sequela of posttraumatic compartment syndromes or ischemic myonecrosis. Previous reports have detailed a small number of cases with similar clinical and radiographic features. Two cases of calcific myonecrosis diagnosed more than 30 years following trauma to an extremity are presented. Treatment consisted of serial debridement of all infected and necrotic tissue followed by wound closure using a rectus abdominis muscle microvascular transplant in one patient and delayed primary closure in the other. Both patients have resumed active lifestyles with no recurrence of drainage or infection. Aggressive debridement and definitive soft tissue coverage are the mainstay of effective treatment for this entity.
Anti-Bacterial Agents↗
Tyrosine phosphorylation pathway is involved in interferon-gamma (IFN-gamma) production; effect of sodium ortho vanadate.
The molecular mechanisms regulating IFN-gamma production have yet to be well characterized. We describe here how treatment of activated cultures of peripheral blood mononuclear cells (PBMC) with the phosphotyrosine phosphatases (PTP) inhibitor sodium ortho vanadate results in greatly enhanced IFN-gamma production. Conversely, cellular proliferation of the same cultures is profoundly inhibited by treatment with vanadate, while the expression of IL-2R and DR molecules on activated lymphocytes remains substantially unmodified. Increased IFN-gamma production, but not inhibition of cellular proliferation, was also observed in mitogen-activated vanadate-treated Jurkat cells. On the other hand, IFN-gamma production induced in cultures of PBMC treated or not with vanadate, was strongly inhibited by incubation with the protein tyrosine kinase (PTK) inhibitor herbimycin A. As a result of the inhibited phosphatase activity, substrates for PTK become hyperphosphorylated on tyrosine residues, as shown by Western blot analysis of cell lysates from cultures of PBMC treated with vanadate. We suggest that the tyrosine phosphorylation pathway plays a role in regulating IFN-gamma production.
Cell Division↗
HCV infection in patients with primary defects of immunoglobulin production.
We tested for infection with hepatitis C virus (HCV) in 58 patients affected by humoral immunodeficiencies: 43 common variable immunodeficiency (CVI), two hyper IgM syndrome (HIM), two IgG subclass deficiency, four ataxia-telangiectasia (AT), and seven X-linked agammaglobulinaemia (XLA). While the assessment of serum specific HCV antibodies in some of these patients was not informative because of the impairment in specific antibody production, the reverse transcriptase polymerase chain reaction (RT-PCR) assay used to detect serum HCV RNA was a useful method for diagnosing infection. We found that 38% of late onset hypogammaglobulinaemic patients (CVI, HIM or IgG subclass deficiency) had evidence of HCV infection. HCV infection was not detectable in patients with XLA or AT. The majority of our patients had persistent viraemia, and those who underwent liver biopsy showed histological findings of chronic hepatitis. Moreover, we could demonstrate in vitro that eight of 18 HCV-infected patients were actively producing anti-HCV antibodies, despite their impaired antibody production. The high rate of HCV infection in hypogammaglobulinaemic patients could be related to several nosocomial routes of transmission, including intravenous immune globulin administration. Despite the persistent viremia only two patients had cirrhosis and none had hepatocarcinoma.
Adolescent↗
Oral cephalosporins: clinical results in lower respiratory tract infections.
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Administration, Oral↗
Evaluation of the invasive techniques for diagnosing bacterial respiratory infections.
Bacterial community-acquired respiratory infections are usually sustained by strains highly responsive to antibiotic therapy. Thus, the clinical approach is based on an empirical treatment and does not require the isolation of the causative pathogen and the determination of the bacterial susceptibility to antibiotics. On the other hand, Gram-negative bacteria, most commonly multidrug resistant, frequently affect immunocompromised and nosocomial patients and their identification in cultures is absolutely necessary for proper antibacterial treatment. To this aim, two conventional methods are used, i.e. the blood culture, which is positive only in 20% of pneumonia cases, and the sputum culture, which is not invasive but easily contaminated by oropharyngeal flora. Consequently, invasive techniques for sampling the pathologic specimen, such as the BAL and the PSB, performed with the help of fiberoptic bronchoscope, are needed. The diagnostic power and the limits of both these techniques are analyzed. Moreover, the opportunity to obtain quantitative cultures, which may discriminate between contamination and infection is considered.
Biopsy, Needle↗
Topoisomerase II DNA cleavage stimulation, DNA binding activity, cytotoxicity, and physico-chemical properties of 2-aza- and 2-aza-oxide-anthracenedione derivatives.
The cytotoxic activity of mitoxantrone and related anthracenediones has been ascribed to the ability of these compounds to interfere with DNA topoisomerase II function, resulting in DNA cleavage stimulation. The molecular details of enzyme inhibition by these intercalating agents remain to be defined. In an attempt to identify the structural determinants for optimal activity, the molecular and cellular effects of a series of heteroanalogues bearing different side-chains were examined in relation to the physico-chemical and DNA binding properties of these compounds. The results indicated that substitution of a pyridine ring for the dihydroxyphenylene ring in the planar chromophore caused a marked reduction of cytotoxic activity and of the ability to stimulate topoisomerase II-mediated DNA damage in intact cells and with simian virus 40 DNA in vitro. Although all tested derivatives were shown to intercalate into DNA, their DNA binding affinities were appreciably lower than that of mitoxantrone. The behavior of 2-aza derivatives more closely resembled that of ametantrone, suggesting that the potency of agents of this class is influenced more by the presence of hydroxyl groups than by the phenylene ring. The observation that a dramatic reduction (or loss) of the ability of aza derivatives to stimulate DNA cleavage is associated with a marked reduction of cytotoxic potency supports a primary role of topoisomerase II-mediated effects in the mechanism of action of the effective agents of this class. Because appreciable cytotoxic activity and significant in vivo antitumor efficacy are retained by compounds inactive (or poorly active) in inhibition of topoisomerase II, these results are consistent with multiple effects of anthracenediones at the cellular level.
Antineoplastic Agents↗