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.

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.

Different glycoforms of the human GPI-anchored antigen CD52 associate differently with lipid microdomains in leukocytes and sperm membranes.

CD52 is a human GPI-anchored antigen, expressed exclusively in the immune system and part of the reproductive system (epididymal cells). Sperm cells acquire the antigen from the epididymal secretions when transiting in the epididymal corpus and cauda. The peptide backbone of CD52, consisting of only 12 aminoacids, is generally considered no more than a scaffold for post-translational modifications, such as GPI-anchor and especially N-glycosylation which occur at the third asparagine. The latter modification is highly heterogeneous, especially in the reproductive system, giving rise to many different glycoforms, some of which are tissue specific. A peculiar O-glycan-containing glycoform is also found in reproductive and immune systems. We determined to locate CD52 in microdomains of leukocytes and sperm membranes using two antibodies: (1) CAMPATH-1G, the epitope of which includes the last three aminoacids and part of the GPI-anchor of glycoforms present in leukocytes and sperm cells; (2) anti-gp20, the epitope of which belongs to the unique O-glycan-bearing glycoform also present in both cell types. Using a Brij 98 solubilization protocol and sucrose gradient partition we demonstrated that the CD52 glycoforms recognized by both antibodies are markers of typical raft microdomains in leukocytes, whereas in capacitated sperm the O-glycoform is included in GM3-rich microdomains different from the cholesterol and GM1-rich lipid rafts with which CAMPATH antigen is stably associated. The importance of the association between GM3 and O-glycans for formation of specialized microdomains was confirmed by heterologous CD52 insertion experiments. When prostasomes from human seminal fluid were incubated with rat sperm from different epididymal regions, the CD52 glycoform recognized by anti-gp20 decorated rat epididymal corpus and cauda sperm, associated with the same low-cholesterol GM3-rich sperm membrane fractions as in human sperm. The glycoforms recognized by CAMPATH-1G were not found in rat sperm. The relationship between this differential insertion and differences in glycosylation of rat and human CD52 is discussed.

Epitope analysis of immunoglobulins against gp20, a GPI-anchored protein of the human sperm surface homologous to leukocyte antigen CD52.

Gp20 is a sialylglycoprotein of the human sperm surface related to maturation and capacitation and is homologous to CD52, a glycosyl- phosphatidyl-inositol (GPI)-anchored protein highly expressed in lymphocytes, monocytes, eosinophils, and epididymal cells, described by the monoclonal antibody family CAMPATH. The CAMPATH antigen is characterized by a very short peptide (12 amino acids) and an N-linked oligosaccharide chain bound to the asparagine located in the third position and a GPI anchor bound to the C-terminal serine. The CAMPATH epitope includes three amino acids at the C-terminus and part of the GPI anchor. It has been suggested that anti-gp20 interacts with the same peptide recognized by CAMPATH antibodies but with a different epitope, since it describes the corresponding antigen in a different way. For example, it localizes the corresponding antigen in the equatorial region of the sperm head when sperm are capacitated, whereas CAMPATH antibodies bind all over the sperm surface. Our results indicate that the anti-gp20 epitope does not include the peptide backbone, the GPI anchor, or the N-glycans but consists of O-linked oligosaccharide chains bound to a unique CD52 glycoform present both in sperm and leukocytes. This is suggested by results obtained using many different approaches, such as immunoblot analysis of gp20 after removal of N- and O-glycans and after jacalin (Artocarpus integrifolia agglutinin)-affinity chromatography.

Expression of gp20, a human sperm antigen of epididymal origin, is reduced in spermatozoa from subfertile men.

gp20, a sialylglycoprotein of human sperm homologous to CD52, is present everywhere on the surface of the freshly ejaculated sperm but is prevalently localized in the equatorial region of the head of capacitated sperm. In the present study, we confirmed this feature on large scale and correlated equatorial exposure of the antigen to the presence of serum albumin (SA) in the capacitation medium. Furthermore, we analyzed the relationship between the presence of the antigen and its equatorial exposure after capacitation and fertility, by comparing immunostaining for gp20 in the motile fraction of spermatozoa from fertile and subfertile men. A significantly higher percentage of nonimmunostained spermatozoa before capacitation (38.5% +/- 23 vs. 12% +/- 7, P < 0.0001) and a lower increase in the percentage of sperm with equatorial localization after capacitation (19.3% +/- 25 vs. 34.6% +/- 22, P = 0.039) were observed in subfertile men (n = 60) compared to fertile men (n = 15). In the whole study group, a positive correlation was also found between the percentage of spermatozoa exhibiting equatorial localization in capacitated samples and normal head forms (R = 0.50; P < 0.0001).

Transvaginal sonographic appearance of peritoneal pseudocysts.

OBJECTIVE: To describe the sonographic features of peritoneal pseudocysts (PPCs) in order to determine the particular aspects that distinguish them from true ovarian cysts. METHODS: Thirty-one women with PPCs were investigated using transvaginal sonography immediately before surgery. The diameters of the cysts were measured, and the shape, margins, content, location, presence of septa and echogenic portions were analyzed. RESULTS: The PPCs were monolateral in 20 (65%) and bilateral in 11 (35%) women. A well-defined cystic structure was found in only six (19%) women, while in the other 25 (81%) women the PPCs showed blurred, undefined margins and a bizarre morphology, giving them a star-like tubular or lumpy shape. The ipsilateral ovary was identified in 26 (84%) cases either external to the cyst or entrapped within it like a 'spider in a web'. Septa were present in 25 (81%) cases and were often mobile, resembling a 'flapping sail' when touched by the endovaginal probe. CONCLUSION: Despite having a gross morphology resembling that of a true ovarian cyst, PPCs present some characteristic sonographic findings that allow a correct differential diagnosis in the vast majority of cases.

Gp273, the ligand molecule for sperm-egg interaction in the bivalve mollusk, Unio elongatulus, binds to and induces acrosome reaction in human spermatozoa through a protein kinase C-dependent pathway.

In a previous article, we suggested that gp273, the ligand molecule for sperm-egg interaction in the bivalve mollusk Unio elongatulus has functional carbohydrate epitopes in common with a human zona pellucida glycoprotein, probably ZP3. We demonstrated that: 1) anti-gp273-purified immunoglobulin G (IgG), which recognizes a carbohydrate gp273 epitope including a Lewisa-like structure, interacts with a zona pellucida protein; 2) human sperm specifically bind to gp273; and 3) binding is reversed by anti-gp273 IgG. In the present study, we confirm this suggestion by demonstrating that heat-solubilized zonae pellucidae reverse gp273-human sperm binding, that gp273-binding sites are restricted to the acrosomal region, and that gp273 induces the acrosome reaction in human sperm. We also demonstrated that gp273-binding sites on human sperm function as signaling receptors because exposure of spermatozoa to this glycoprotein results in significant stimulation of protein kinase C (PKC) activity. Because the PKC inhibitor, bisindolylmaleimide I, reverses both PKC activation and the acrosome reaction, this kinase is a key component of the signal transduction pathway activated by gp273 and leading to the exocytotic event.

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.

Surface of human sperm bears three differently charged CD52 forms, two of which remain stably bound to sperm after capacitation.

gp20 is a sialoglycoprotein of the human sperm surface with a core peptide homologous to the leukocyte antigen CD52, a GPI-anchored glycosylated protein which is described by the monoclonal antibody CAMPATH-1. Comparative analyses, by means of CAMPATH and anti-gp20, indicated that they describe it in morphologically and functionally different ways, suggesting that the respective epitopes are different but also casting doubt on the immunological identity of the antigen. In the present study, we used immunodepletion to demonstrate that CAMPATH and anti-gp20 interact with the same antigen, but that anti-gp20 has a much higher avidity for the antigen than CAMPATH. Anion exchange fractionation analysis of the antigen revealed three differently charged gp20-CD52 forms, the least charged of which, was largely without a GPI-anchor. All three forms were associated with freshly ejaculated sperm, whereas capacitated sperm only contained the two GPI-anchored, more charged forms, which were also the ones found in the prostasome fraction of seminal plasma and in leukocytes. The two charged, GPI-anchored forms were described as homogeneous by anti-gp20, since they ran as a singlet; the third form ran as a doublet. When tested for insertion into Jurkat T cells, the medium charged form inserted the most readily and the less charged one could not be inserted at all.

Carbohydrate-mediated sperm-egg interaction and species specificity: a clue from the Unio elongatulus model.

As a first step fertilization requires that sperm bind to the extracellular coat of the egg. It is generally accepted that binding is mediated by complex carbohydrates on the egg coat, recognized by carbohydrate-binding proteins on the sperm surface. In the mollusc bivalve Unio elongatulus, the main functional epitope of the carbohydrate ligand has been determined, and it shares several characteristics with other sugar structures involved in the fertilization process in different animal models. A polyclonal antibody against the Unio epitope reacts with the human ZP3 glycoprotein and the Unio ligand binds human spermatozoa in an in vitro assay. These findings are discussed in the light of the species specificity of sperm-egg binding at fertilization.

Structural characterization of the N-glycans of gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus.

Gp273, a glycoprotein of the egg extracellular coats of the mollusc bivalve Unio elongatulus, is the ligand molecule for sperm-egg interaction during fertilization. In this study we have analyzed the N-glycans from gp273. N-glycans were enzymatically released by PNGase F digestion and their structures were elucidated by normal phase HPLC profiling of the 2-aminobenzamide-labeled N-glycans, MALDI-TOF mass spectrometry and 1H NMR spectroscopy. The combined data revealed that the N-glycans of gp273 consist of Glc1Man9GlcNAc2 and Man9GlcNAc2. In Unio, the presence of noncomplex-type N-glycans parallels the inefficacy of these glycans in the ligand function. Their role in the protection of the polypeptide chain from proteolytic attack is suggested by the electrophoretic patterns obtained after enzymatic digestion of the native and the N-deglycosylated protein. These results are discussed in the light of the evolution of the recognition and adhesion properties of oligosaccharide chains in the fertilization process.

[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.

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.

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.

A 140-kDa glycopeptide from the sperm ligand of the vitelline coat of the freshwater bivalve Unio elongatulus, only contains O-linked oligosaccharide chains and mediates sperm-egg interaction.

In previous studies we found that sperm binding activity in the vitelline coat of the freshwater bivalve Unio elongatulus is located on the O-linked oligosaccharide chains of gp273, one of the two major components of the extracellular coat, and that fucose plays a key role in this interaction. In this paper we report the partial characterization of a large glycopeptide (about 140 kDa) obtained by cyanogen bromide fragmentation of gp273, that maintains sperm binding activity. Lectin blotting revealed that the glycopeptide reacted with lectins from Arachis hypogaea (PNA) and Lotus tetragonolobus (LTA) but not Canavalia ensiformis (ConA). No other PNA-positive fragments could be detected in the electrophoretic pattern of fragmented gp273 but several ConA-positive fragments of lower molecular weight were present indicating that all the O-linked chains are clustered together in this fragment. Two-dimensional gel electrophoresis of the fragment revealed it to be acidic in nature in contrast with the neutral character of the whole gp273 molecule. Competition binding assay showed that this fragment is a strong inhibitor of the interaction, whereas no effect was detected using the ConA-positive peptides. This confirms that the sperm receptor activity of gp273 is related to its O-linked chains. The immunodominance of this fragment is also discussed.

Biochemical and MALDI analysis of the human sperm antigen gp20, homologue of leukocyte CD52.

In previous work we demonstrated that gp20, a sialoglycoprotein of human sperm is homologous to the leukocyte antigen CD52 and that anti-gp20 recognizes an antigen of the same molecular weight as that recognized by CAMPATH-1 (anti CD52) in leukocytes and sperm, but with some differences. In this study we used anti-gp20 to perform immunoblot analysis of many different sperm, seminal plasma and leukocyte samples. The sperm and seminal plasma antigens were similar and appeared to consist of two components, whereas the leukocyte antigen is unique. Evidence of the presence of two components of the sperm antigen, running respectively at about 19 and 21 kDa, was obtained by analyzing the purified antigen stained with Coomassie brilliant blue and by immunoblot analysis of the antigen after two-dimensional electrophoresis. Both components had an isoelectric point (pI) between 3 and 6. MALDI analysis of the purified antigen confirmed the presence of two components and indicated masses (Mr) of 8243 and 10908. The possible relationship between these findings and the presence of two forms of the CD52 gene differing at two aminoacids C-terminal to the GPI-anchor site has been discussed.

A sialoglycoprotein, gp20, of the human capacitated sperm surface is a homologue of the leukocyte CD52 antigen: analysis of the effect of anti-CD52 monoclonal antibody (CAMPATH-1) on capacitated spermatozoa.

In this study we performed N-terminal sequence analysis of gp20, a 20 kDa sialoglycoprotein on the human sperm surface previously identified by radiolabelling of the sialic acid residues of sperm surface. We found 100% identity with the N-terminus of CD52, an antigen expressed on almost all human leukocytes. We also show that, like CD52, gp20 behaves as a glycosylphosphatidylinositol (GPI)-anchored protein and that anti-gp20 antiserum reacts with an antigen on leukocytes of the same molecular weight as CD52. Using CAMPATH-1, the monoclonal antibody against CD52, in fluorescent staining of capacitated spermatozoa, Western blot analysis and the zona-free hamster egg penetration test, we found that the effect of this antibody was different from that of our anti-gp20. Western blot analysis revealed a well-defined 20 kDa band with anti-gp20, whereas a 14-20 kDa band was detected with CAMPATH-1. Anti-gp20 stained the equatorial region of the sperm head, whereas CAMPATH-1 stained the tail in immunofluorescence analysis of capacitated spermatozoa. A dose-dependent inhibitory effect was seen with CAMPATH-1, similar to that previously detected with anti-gp20, in a zona-free hamster egg penetration test. However, with CAMPATH-1 agglutination of motile spermatozoa was detected, and this was not present with anti-gp20. This suggests that the epitopes recognized by the two antibodies are different.