Angela Ingianni

Distribution of GABAB receptor mRNAs in the rat brain and peripheral organs

GABA, the predominant inhibitory neurotransmitter present in the mammalian CNS, is also found in the periphery. GABA actions are mediated by the ionotropic GABA(A)/GABA(C) receptors, as well as the metabotropic GABA(B) receptor. The rat GABA(B) receptor has recently been cloned and two cDNA clones have been isolated encoding two isoforms of the receptor, GABA(B)R1a and R1b. Northern blot analysis revealed the presence of both transcripts in the rat brain using specific cDNA probes for GABA(B)R1a and R1b, respectively. However, Northern blot analysis, hybridized with a probe containing a sequence common to both isoforms, revealed specific RNAs in the rat brain and in testis, but not in other peripheral tissues. In the present study, by using the more sensitive reverse transcriptase-polymerase chain reaction with a specific set of primers for each isoform and Southern blot analysis, we found that both isoforms of the GABA(B) receptor are expressed not only throughout the brain but also in all peripheral organs examined, including heart, spleen, lung, liver, small intestine, large intestine, kidney, stomach, adrenal, testis, ovary and urinary bladder. The peripheral distribution of GABA(B)R1 mRNAs supports the notion of a physiological role for GABA in the control of a wide range of peripheral organs.

Enterococcus flavescens sp. nov., a New Species of Enterococci of Clinical Origin

Four yellow-pigmented group D enterococci of uncertain taxonomic position were isolated from several humans with severe infections. The results of DNA composition, DNA-DNA hybridization, fatty acid content, and biochemical property studies demonstrated that these organisms were slightly related to other previously described yellow-pigmented enterococcal species and constitute a new species, for which we propose the name Enterococcus flavescens. The type strain of E. flavescens is strain CCM 4239 [corrected].

Expression of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Receptors and PACAP in Human Fetal Retina

Abstract: Specific receptors for pituitary adenylate cyclase‐activating polypeptide (PACAP), a novel peptide with neuroregulatory and neurotrophic functions, have recently been identified in the retinas of different mammalian species. In the present study, expression of PACAP receptors and PACAP was investigated in the retinas of 12–18‐week human embryos. Radioligand binding studies showed that the two forms of PACAP with 38 and 27 amino acids (PACAP 38 and PACAP 27, respectively) displaced the binding of 125I‐PACAP 27 with IC50 values in the picomolar range, whereas functional receptor assays demonstrated that the two peptides were potent and effective stimulators of adenylyl cyclase activity. In contrast, vasoactive intestinal peptide (VIP) and human peptide histidine‐isoleucine, which are homologous to PACAP, displayed lower affinities for the 125I‐PACAP 27 binding site and were much less potent stimulators of cyclic AMP formation. Glucagon and secretin were inactive in both receptor assays. The expression of specific PACAP receptors was further investigated by reverse transcription‐polymerase chain reaction technique, which showed the presence of mRNAs coding for PACAP type I and for nonselective PACAP type II (both VIP1 and VIP2) receptors. By the same technique, expression of PACAP mRNA was also detected. These data indicate that the developing human retina synthesizes PACAP and that the peptide may act on retinal cells by predominantly stimulating PACAP type I receptors coupled to cyclic AMP formation.

[Phe1phi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 acts as a partial agonist at ORL1 receptor endogenously expressed in mouse N1E-115 neuroblastoma cells.

The nociceptin derivative [Phe1phi(CH2-NH)Gly2]-nociceptin-(1-13)-NH2 (Phe(phi)noc) has been reported to act either as a simple antagonist or as a full agonist at the opioid receptor-like (ORL1) receptor. In the present study, we identified the expression of the ORL1 receptor in murine N1E-115 neuroblastoma cells and used this neuronal system to investigate the pharmacological activity of Phe(phi)noc. Like nociceptin, Phe(phi)noc stimulated the binding of [35S]GTPgammaS (EC50 = 120 nM) and inhibited forskolin-stimulated [3H]cAMP formation (EC50 = 3.3 nM). However, Phe(phi)noc elicited maximal effects lower than those induced by nociceptin, and when combined with nociceptin potently antagonized the responses to the natural agonist (Ki = 0.9 nM). These data indicate that Phe(phi)noc acts as a partial agonist at the ORL1 receptor endogenously expressed in N1E-115 cells.

TT virus infection in Italy: prevalence and genotypes in healthy subjects, viral liver diseases and asymptomatic infections by parenterally transmitted viruses

This study was aimed to evaluate TT virus prevalence in subjects with hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections in patients affected by hepatitis of unknown origin (non‐A–non‐E hepatitis) and in healthy subjects who had not been exposed to HBV, HCV and HIV. A total of 317 subjects were tested; 40 were HBsAg asymptomatic carriers, 57 subjects were anti‐HCV positive (45 without chronic hepatitis and 12 with HCV‐related chronic hepatitis), and 27 had chronic non‐A–non‐E hepatitis. Fifty‐seven subjects were intravenous drug users (IVDUs) (52 with HCV or/and HIV infections), seven patients underwent a liver transplant for fulminant hepatitis and 137 were healthy subjects from the general population. Overall, TTV‐DNA was detected in 62 subjects (19.6%): in 17.9% of the HBsAg carriers, in 14% of the anti‐HCV‐positive patients (in 8.3% and in 15.5% of patients with and without chronic hepatitis, respectively), in 22.2% of non‐A–non‐E hepatitis patients, in 22.8% of IVDUs, in 57.1% of fulminant hepatitis patients. TTV‐DNA was also found in 20.4% healthy subjects. The prevalence in the different subgroups was not statistically different. The genotypes were identified in 40 of the 62 (64.5%) TTV‐DNA positive samples: genotype 1a in 17.5%, 1b in 27.5%, genotype 2 in 27.5%, genotype 3 in 15.0%, genotype 4 in 5.0% and genotype 5 in 7.5%; the genotype distribution in the subsets of patients was not significantly different. In conclusion, this study showed that TTV infection is common in Italy; it is widespread throughout the entire population and five genotypes are present in Sardinia. Our results further dismiss the role of TTV as cofactor in influencing the clinical course of infections with other hepatitis viruses as well as the role of HIV in enhancing TTV transmission and replication.

Production of lysozyme‐enriched biomass from cheese industry by‐products

Cheese whey and cottage cheese whey are by‐products of the milk and cheese industry, resulting from the production of cheese and cottage cheese (ricotta) from milk. They are still rich in organic substances and cannot be discarded into the environment without proper treatment. Whey and cottage cheese whey were used as culture media for some strains of the yeast Kluyveromyces lactis, transformed with the human lysozyme gene. It was found that the yeast strains grew well in both media and produced a considerable amount of recombinant protein. Production kinetics showed that the human lysozyme was produced in a greater amount within 36 h of fermentation (125 μg ml−1vs 25 μg ml−1 in the control) than in the synthetic commercial media used for strain preparation and characterization. The recombinant protein produced was actually shown to be the human lysozyme, using renaturing SDS‐PAGE and Western blot techniques. While producing recombinant protein, the Kluyveromyces strain cleared the cottage cheese whey of most organic substances and produced a considerable amount (almost 3%) of lysozyme‐enriched useful biomass.

Inhibition of herpes simplex virus-induced cytopathic effect by modified hen egg-white lysozymes

Hen egg-white lysozyme and three of its basic derivatives obtained by chemical modification were tested for their activity in vitro against a wild strain of herpes simplex virus type 1. Marked inhibition of the cytopathic effect was exhibited by the three chemical derivatives and the heat-inactivated lysozyme, whereas the native enzyme displayed only modest anticytopathic activity. Enzymatic activity did not appear to be necessary for the antiherpes activity of the lysozyme compounds. Instead, other properties such as their basic nature seemed to be relevant to their antiherpes effectiveness in vitro. At the concentrations used, all compounds but one had no significant effect on cell viability and growth. Some of the compounds tested caused formation of deposits on the surface of the cells. Some correlation between deposit formation and antiherpes cytopathic activity was found. The antiherpes efficacy in vitro and toxicity of the modified lysozymes were compared with those of known antiviral agents. The lysozymes were less toxic than the reference antiviral agents, and some of them were also more active.

Role of G Protein βγ Subunits in Muscarinic Receptor-Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and Gs‐stimulated adenylyl cyclase activities and inhibiting the Ca2+/calmodulin‐ and forskolin‐stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγt) and blocked by putative βγ scavengers. Membrane incubation with βγt caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγt potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin‐releasing hormone. RT‐PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated Gs. In addition, βγt inhibited the Ca2+/calmodulin‐ and forskolin‐stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP‐bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.

Type I interferons impair BDNF-induced cell signaling and neurotrophic activity in differentiated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons

J. Neurochem. (2012) 122, 58–71.

Production and Characterization of a Human Recombinant Monoclonal Fab Fragment Specific for Influenza A Viruses

ABSTRACT A human recombinant monoclonal Fab fragment that specifically recognizes all the influenza A virus strains tested was produced in transformed Escherichia coli using the phage display technique. No strain of influenza B virus reacted with it. It was purified after four cycles of panning and by a single passage through an immunoaffinity column. About 1 mg of pure monoclonal antibody was obtained from 1 liter of culture medium in 3 working days. The Fab fragment reacted with a viral 27-kDa protein, which could reasonably be a matrix protein. Indirect immunofluorescence tests performed on virus-infected MDCK cells showed that this Fab fragment was at least equally efficient as other commercial monoclonal antibody-based systems in detecting influenza A viral infections. The potential advantages of human recombinant Fabs on murine monoclonal antibodies are discussed.