Glycosilated nucleolin as marker for human gliomas.

Nucleolin is a multifunctional DNA and RNA binding protein involved in regulation of gene transcription, chromatin remodeling, RNA metabolism, and ribosomal RNA synthesis. Nucleolin seems to be over-expressed in highly proliferative cells and is involved in many aspect of gene expression: DNA recombination and replication, RNA transcription by RNA polymerase I and II, rRNA processing, mRNA stabilization, cytokinesis, and apoptosis. Although nucleolin is localized predominantly in the nucleolus, it has also been shown to be localized in a phosphorylated/glycolsilated form on the cell surface of different cells. Numerous articles dealing with surface nucleolin targeting for tumor therapy have been recently published. However, at present, no extensive informations are so far available for the presence of nucleolin in human gliomas. In the present work we investigated on the presence and localization of nucleolin in glioma on glioma specimens at different grade of malignancy and on primary glioma cell cultures derived by surgical resection, trying to correlate the presence of glycosilated membrane nucleolin with the malignancy grade. To this purpose an antibody produced by us against gp273 protein, demonstrated to recognized the glycosilated surface nucleolin, has been used. The results obtained demonstrate that surface nucleolin increase with the malignancy grade thus suggesting that it may constitute a histopathological marker for glioma grading and a possible tool for targeted therapy.

Protein of insect sperm mitochondrial crystals. Crystallomitin.

Mitochondrial derivatives of insect sperm usually contain a crystalline protein that shows a 45-nm main period, made up of 20-nm subperiods, determined by the coiling of filament bundles. Filaments are 2 nm thick and have a globular appearance. The crystals contain two main polypeptides, 52,000 and 55,000 daltons. These polypeptides are closely related, contain a high percentage of proline, and are insoluble in sodium dodecyl sulfate due to disulfide cross links. We suggest for this class of protein the name crystallomitin.

Sperm-egg interaction at fertilization: glycans as recognition signals.

This article first examines the events occurring in male and female genital tracts, which prepare human sperm to encounter the egg. Central is a glycoprotein, gp20, homologous to the leukocyte antigen CD52. This protein is secreted in the epididymal cells, inserted in the sperm plasma membrane and exposed in the equatorial region of the head at the end of the capacitation process. The mechanisms and molecules of the first interaction event between gametes in the mollusk bivalve Unio elongatulus and the current state of our knowledge of the same interaction in other species is then considered. The egg of Unio is very peculiar because it is highly polarized. Similar to other well-known egg models, the ligand for recognition is located on the egg coat which is a sort of fibrous network made up of very few glycoproteins, while the receptor is on the sperm surface. The difference is that in this egg, the ligand molecules are not uniformly distributed but are restricted to an area of the egg coat at the vegetal pole, the crater area. The role of carbohydrates in ligand function and of a specific type of oligosaccharide chain in particular, is discussed in the wider context of glycans acting as recognition signals.

Density and size distributions of the intramembranous particles in the cytoplasmic membrane of human peripheral blood lymphocytes. II. Ultrastructural differences between T and B cells.

Separated T and B lymphocytes from human peripheral blood were studied using the freeze-fracture technique. Quantitative analysis performed on density and size of intramembranous particles (IMPs) present on both fracture faces of the plasma membrane has revealed remarkable differences between cells belonging to the two main lymphocyte populations. In particular: (a) both fracture faces of the cytoplasmic membrane of B lymphocytes exhibit larger particles than T lymphocytes; (b) the mean densities, on both protoplasmic (PF) and external (EF) fracture faces, in B lymphocytes are lower than in T lymphocytes; (c) in B cells the partition ratio of particles between PF and EF is reversed with respect to T cells; (d) on both fracture faces of B lymphocytes, the IMP densities present a normal distribution while on T cells, density values show bimodal distributions indicating the existence of two cell subsets differing in particle density.

Density and size distributions of the intramembranous particles in the cytoplasmic membrane of human peripheral blood lymphocytes.

A freeze-fracture study has been carried out on human peripheral blood lymphocytes (hPBL) from healthy donors. Lymphocytes were frozen either from 37 degrees C or 4 degrees C. Quantitative analysis performed on density and size of intramembranous particles (IMPs) present on both fracture faces of the plasma membrane has revealed: a) a difference in size between IMPs on the external face (EF) and those on the protoplasmic face (PF); b) a remarkable influence of temperature either on size or density of the IMPs; c) the existence at 4 degrees C of two lymphocyte populations differing in IMP density.

GSH system in relation to redox state in dystrophic skin fibroblasts.

Glutathione and GSH-related enzymes were determined in human Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) skin fibroblasts in order to relate muscular dystrophy to the redox state of the cell. The analysis of GSH, GSSG and total GSH levels in normal and dystrophic-cultured fibroblasts shows no differences in normal growth condition. However, the specific activity of two GSH-related enzymes, glutathione S-transferases (GST) and gamma-glutamylcysteine synthetase (gamma-GCS), shows significant variations between normal and both types of dystrophic skin fibroblasts. These results suggest that even in normal growth condition some components of GSH metabolism may be altered. A condition of sublethal oxidation obtained by H(2)O(2) treatment was able to show a difference in the cellular response of GSH system components between normal and dystrophic cells. While in DMD cells there is a decrease of roughly 55% in GSH and of 30% in total GSH concentration, no changes are measured in normal and BMD cells. The remarkable increase in glutathione peroxidase (GPx) activity and decrease in GSH-reductase (GR) activity measured in DMD cells can in part explain these changes. These results indicate a different capacity of DMD cells to support oxidative stress with respect to BMD and normal cells, and suggest a possible role of the GSH-antioxidant system in dystrophic pathology.

Acylphosphatase expression during macrophage differentiation and activation of U-937 cell line.

The two acylphosphatase isoenzymes (muscle type and common type) are differently involved in cell differentiation processes. In this paper we investigate the expression of the two isoenzymes during macrophage differentiation and activation. The U-937 human promonocytic cell line is a model for cell differentiation induced by the tumor promoter phorbol myristic acetate (PMA). Here we show that only the expression of the muscle type acylphosphatase increases during U-937 differentiation and macrophage activation, confirming that the two isoenzymes are differently regulated. Moreover, we determined, in the same conditions, the level of specific mRNA. Results show that after an initial two-fold decrease during PMA stimulation, the muscle type acylphosphatase mRNA levels remain constant also after the treatment with lipopolysaccharide and gamma-interferon, treatments that lead to macrophage activation. It is possible that post-transcription regulation is responsible for the regulation of muscle type acylphosphatase in the cell during differentiation and macrophage activation.

Human peripheral eosinophils with receptors for IgM: demonstration and ultrastructural morphology.

Receptors for IgM were detected on peripheral blood human eosinophils by a rosette technique with ox red blood cells coated with the IgM fraction of the specific immunserum. Between 14% and 43% (mean 27%) FcmuR positive cells were found after an overnight incubation period at 37 degrees C by using this technique. The specificity of the receptors for IgM was assessed by studying the inhibitory capacity of purified human IgM in the rosette assay. From an ultrastructural point of view, the EAM rosette-forming cells are mature eosinophilic granulocytes characterized by a nucleus with a variable number of lobes and a certain number of "first type" granules partially or totally devoid of their content.

Sialyglycoconjugates release during in vitro capacitation of human spermatozoa.

Modifications of the human sperm surface during incubation in capacitating conditions were studied by radiolabeling terminal sialic acid residues of cell surface glycoconjugates using the sodium metaperiodate/sodium tritiated borohydride method. During in vitro capacitation, sialyglycoconjugates were released from the human sperm surface according to well reproducible kinetics. This release could be inhibited by the presence of seminal plasma in the capacitation buffer. Two principal size classes of sialyglycoconjugates were detected in the capacitation medium and analyzed by gel filtration chromatography and SDS-PAGE. The smaller class was characterized by glycopeptides less than 5,000 Da, whereas the larger class was characterized by two sialyglycoproteins of approximately 15,000-16,000 and 22,000-23,000 Mr. The role of human albumin, a key component of the capacitation buffer, in the removal of these molecules from the sperm surface was studied in light of its constant association with large amounts of released material.

Gas chromatography/mass spectrometry of catechol estrogens.

Catecholestrogens (CCEs), namely 2- or 4-hydroxyestradiol and hydroxyestrone, are highly polar, reactive, and extremely labile estrogen metabolites in many experimental conditions. For these reasons, indirect assay methods mainly have been used. Some experimental evidence suggests that CCEs are synthesized and biologically active mostly in target cells. At this level, unfortunately, the indirect assays cannot be used. We present a method of gas chromatographic/mass spectral (GC/MS) analysis for the identification of individual CCEs; the major fragmentation ions of authentic estrogen standards as trimethylsilylether derivatives, and the MS patterns of the major CCEs, namely, 2-hydroxyestradiol and hydroxyestrone, are included. Few examples of CCEs detected in human breast cancer tissues and in breast cyst fluids are reported. Sample extracts were submitted to reversed-phase, high-performance liquid chromatography (RP-HPLC) and were quantified by "on line" electrochemical (EC) detection; thereafter, either crude extracts or single eluted peaks were submitted to GC/MS, by which detection limits of less than 5 pmol were attained. As expected, the molecular ion was the most relevant molecule in all but one case. On the contrary, the other relative intensities of major fragmentation ions M -15, M -30, M -90, and M -15 + (-90) were unevenly distributed, although represented in the majority of cases. In all cases, the GC/MS of peak fractions, purified by RP-HPLC and UV detection, confirmed the results of liquid chromatographic analysis combined with EC detection. In contrast, GC/MS of crude extracts was not equally satisfactory. Comparison of a liquid chromatography system with EC detection and the GC/MS approach revealed some inconsistency in quantitation of individual CCEs.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid screening method for determination of Ecstasy and amphetamines in urine samples using gas chromatography-chemical ionisation mass spectrometry.

The need for analytical screening tests more reliable and valid to detect amphetamine and related "designer drugs" in biological samples is becoming critical, due to the increasing diffusion of these drugs on the European illegal market. The most common screening procedures based on immunoassays suffer a number of limitations, including low sensitivity, lack of specificity and limited number of detectable substances. This paper describes a screening method based on gas-chromatography-mass-spectrometry (GC/MS) using positive chemical ionisation (PCI) detection. Methanol was used as reactant gas in the ionisation chamber. Molecular ions of different compounds were monitored, allowing a sensitivity of 5-10 ng/ml with high selectivity. The sensitivity of the method gives positive results in samples taken 48-72 h after intake of one dose of 50-100 mg. The method is simple and rapid. Sample preparation was limited to one liquid-liquid extraction, without any hydrolysis and derivatisation. Hydrolysis is critical to identify metabolites excreted as conjugates. Blank urine samples spiked with known amounts of amphetamine (AM), methylamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and methylenedioxyphenyl-N-methyl-2-butanamine (MBDB) were analysed. The method was successfully tested on real samples of urine from people, whose use of amphetamine was suspected, and results were compared with results obtained with immunoassays.

Identification of 3,4-methylenedioxyamphetamine analogs encountered in clandestine tablets.

Recently, 3,4-methylenedioxyamphetamine derivatives have been encountered in the Italian illicit market, mainly in form of tablets. Among this class of substances small modifications of the molecule may result in a wide range of derivatives and analogs some of which are not yet listed as controlled substances in the Italian schedules. Due to the structural similarity some of these molecules have a gas chromatographic behavior and mass spectra that only slightly differ. In the present work, an analytical strategy is proposed to achieve the identification of analogs within this class of molecules. In seized material sent by the Court of Law of Rome to our laboratories a number of tablets engraved with different symbols (e.g., 'Dollar', 'Fido Dido' and 'Bomb') were submitted to analysis in order to establish whether they contained drugs of abuse. The analytical techniques employed for this purpose were UV spectrophotometry and thin-layer chromatography which provided information suggesting that the tablets contained a methylenedioxyamphetamine. Gas chromatography with flame ionization detection indicated that the main ingredient differed from the molecules of the same class already known. Finally, capillary gas chromatographic-mass spectrometric analysis of the native molecules and their pentafluoropropionic acid derivatives, performed with both, electron impact and chemical ionization, allowed the identification, in each tablet, of three molecules: the N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MDP-2-MB, MBDB), the 1-(3,4-methylenedioxyphenyl)-2-butanamine (MDP-2-B) and the N,N-dimethyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MDP-2-MMB).

[Determination of 4-bromo-2,5-dimethoxyamphetamine (DOB) found in illicit tablets seized in Italy]. / Identificazione della 4-bromo-2,5-dimetossiamfetamina (DOB) in compresse clandestine sequestrate in Italia.

Some of the molecules belonging to the amphetamines group (4-bromo-2,5-dimethoxyamphetamine, DOB) or to the phenethylamines (4-bromo-2,5-dimethoxy-phenethylamine, 2C-B or Nexus) have closely related structures that make their identification quite difficult. The unambiguous identification is crucial in forensic responses. This paper describes the analytical approach used to achieve the identification of the main ingredient contained in tablets seized in the illicit market of Rome (Italy) and submitted to our laboratory by the Court of Law of Rome. The procedure entails the basic extraction of the main ingredient from the tablets with tert-butyl methyl ether followed by qualitative gas chromatographic mass-spectrometric (GC-MS) analysis using both electron impact detection (EI) and chemical ionization (CI). The examination of the mass spectra obtained from the native molecule and from its pentafluoropropionyl-derivative allows the structural identification of the side chain and the substitutions on the aromatic ring. This analytical approach can thus be useful to distinguish between amphetamine-like and phenetylamine-like compounds using instruments and techniques commonly available in the forensic toxicology laboratories.

Jointly determined saving and fertility behaviour: theory, and estimates for Germany, Italy, UK and USA.

PIP: This study uses new theories of capital accumulation and fertility in a comparative framework to test predictions with time-series data for Germany, Italy, the UK, and the US. The exogenous-fertility model is based on models of Barro and Becker. The endogenous-fertility models are based on models of Veall and Nishimura and Zhang. It is assumed that life cycle periods are youth, middle age, and old age. Several theoretical frameworks are tested with endogenous and exogenous fertility and altruism and nonaltruism. Data are obtained during 1950-90. Dependent variables are the total lifetime fertility rate and real per capita household savings. Explanatory variables include social security, the real social security deficit per capita, the real rate of interest, the real per capita disposable income, the average male real wage rate, the average female real wage rate, and the real child benefit rate. The explanatory variables are individually graphed to show differences by country over time. Findings suggest that fertility is endogenous in a nonaltruistic model. The only model not rejected by the data was the model in which fertility and intergenerational transfers were explained by nonaltruistic concerns. Fertility was positively affected by the male wage rate in all countries. Fertility was negatively affected by the female wage rate in all countries. Disposable income was insignificant in the UK and Germany and positive and significant in Italy and the US. The interest rate was significant in only 1 model. Child benefits had a positive and significant effect on fertility in the UK. In savings models, disposable income was significant and positive, and child benefits and wage rates were insignificant. Social security coverage had a negative effect on fertility and a positive effect on savings, except in Germany. Findings indicate that saving and fertility are jointly determined.^ieng

Behaviour of ovarian tumors of low malignant potential treated with conservative surgery.

OBJECTIVE: Fertility-sparing surgery has been proposed for the treatment of borderline ovarian tumors. The aim of this study was to evaluate the outcome of patients submitted to cystectomy (CYS) compared with patients treated by unilateral salpingo-oophorectomy (USO) or bilateral salpingo-oophorectomy with/without total hysterectomy (radical surgery, RS). METHODS: We reviewed retrospectively the data of patients treated in 3 institutions for borderline ovarian tumors. One hundred and sixty-eight patients underwent laparoscopic or laparotomic surgical treatment from 1985 to 2006. Tumor recurrence rate, disease-free survival and site of recurrences were evaluated. Specific prognostic factors, such as stage, histology, micropapillary subtype, exophytic tumor growth, intraoperative spillage, endosalpingiosis, staging procedures, and route of surgery were analysed. RESULTS: Thirty-five patients underwent cystectomy, 50 unilateral salpingo-oopohorectomy, and 83 radical surgery. Twelve patients in the CYS group (34.3%), 10 in the USO group (20.0%), and 5 (6.0%) in RS group relapsed. Five-year progression-free survival (PFS) was 59.6%, 78.4%, and 93.5% in CYS, USO and RS groups, respectively. None of the relapsed patients died of disease. CONCLUSIONS: Cystectomy is an effective surgical strategy for patients with borderline ovarian tumor. The higher risk of local relapses is not associated with a reduction in the overall survival. The procedure should be offered to young patients with bilateral tumors and to very young ones, considering the higher risk of local relapse.

Seladin-1/DHCR24 protects neuroblastoma cells against Abeta toxicity by increasing membrane cholesterol content.

The role of brain cholesterol in Alzheimer's disease (AD) is currently a matter of debate. Experimental evidence suggests that reducing circulating and brain cholesterol protects against AD, however recent data indicate that low membrane cholesterol results in neurode-generation and that the cholesterol synthesis catalyst seladin-1 is down-regulated in AD-affected brain regions. We previously reported a significant correlation between resistance to amyloid toxicity and content of membrane cholesterol in differing cultured cell types. Here we provide evidence that Abeta42 pre-fibrillar aggregates accumulate more slowly and in reduced amount at the plasma membrane of human SH-SY5Y neuroblastoma cells overexpressing seladin-1 or treated with PEG-cholesterol than at the membrane of control cells. The accumulation was significantly increased in cholesterol-depleted cells following treatment with the specific seladin-1 inhibitor 5,22E-cholestadien-3-ol or with methyl-beta-cyclodextrin. The resistance to amyloid toxicity and the early cytosolic Ca2+ rise following exposure to Abeta42 aggregates were increased and prevented, respectively, by increasing membrane cholesterol whereas the opposite effects were found in cholesterol-depleted cells. These results suggest that seladin-1-dependent cholesterol synthesis reduces membrane-aggregate interaction and cell damage associated to amyloid-induced imbalance of cytosolic Ca2+. Our findings extend recently reported data indicating that seladin-1 overexpression directly enhances the resistance to Abeta toxicity featuring seladin-1/DHCR 24 as a possible new susceptibility gene for sporadic AD.