The effect of cations on the amidase activity of human tissue kallikrein: 1-linear competitive inhibition by sodium, potassium, calcium and magnesium. 2-linear mixed inhibition by aluminium

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide by human tissue kallikrein (hK1) was studied in the absence and in the presence of increasing concentrations of the following chloride salts: sodium, potassium, calcium, magnesium and aluminium. The data indicate that the inhibition of hK1 by sodium, potassium, calcium and magnesium is linear competitive and that divalent cations are more potent inhibitors of hK1 than univalent cations. However the inhibition of hK1 by aluminium cation is linear mixed, with the cation being able to bind to both the free enzyme and the ES complex. This cation was the best hK1 inhibitor. Aluminium is not a physiological cation, but is a known neurotoxicant for animals and humans. The neurotoxic actions of aluminium may relate to neuro-degenerative diseases.     

Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46

Human herpesvirus 6 (HHV-6) employs the complement regulator CD46 (membrane cofactor protein) as a receptor for fusion and entry into target cells. Like other known herpesviruses, HHV-6 encodes multiple glycoproteins, several of which have been implicated in the entry process. In this report, we present evidence that glycoprotein H (gH) is the viral component responsible for binding to CD46. Antibodies to CD46 co-immunoprecipitated an approximately 110-kDa protein band specifically associated with HHV-6-infected cells. This protein was identified as gH by selective depletion with an anti-gH monoclonal antibody, as well as by immunoblot analysis with a rabbit hyperimmune serum directed against a gH synthetic peptide. In reciprocal experiments, a monoclonal antibody against HHV-6 gH was found to co-immunoprecipitate CD46. Studies using monoclonal antibodies directed against specific CD46 domains, as well as engineered constructs lacking defined CD46 regions, demonstrated a close correspondence between the CD46 domains involved in the interaction with gH and those previously shown to be critical for HHV-6 fusion (i.e. short consensus repeats 2 and 3).     

Comparison of three fungal laccases from Rigidoporus lignosus and Pleurotus ostreatus: correlation between conformation changes and catalytic activity

The conformation changes in solution of three fungal laccases in different environmental conditions were examined by circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy. CD measurements indicate that the secondary structure of proteins depends slightly on the pH or ionic strength, though the presence of salt could interfere in the molecular recognition process between substrates and enzymes. The enzymes, however, are highly destabilized by prolonged exposure to low pH or high temperature. The observed unfolding of the proteins coincides with their inactivation and, in some cases, with precipitation. On the other hand, these conditions do not determine the disruption of the geometric arrangement of their metal centres, and this fact suggests that these centres represent the more stable core of the proteins.     

Diagnostic criteria for diabetes revisited: making use of combined criteria

Background  

Existing cut-offs for fasting plasma glucose (FPG) and post-load glucose (2hPG) criteria are not equivalent in the diagnosis of diabetes and glucose intolerance. Adjusting cut-offs of single measurements have not helped so we undertook this project to see if they could be complementary.     

The neuron-specific Rai (ShcC) adaptor protein inhibits apoptosis by coupling Ret to the phosphatidylinositol 3-kinase/Akt signaling pathway

Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals.     

Generation and characterization of novel monoclonal antibodies to the Ret receptor tyrosine kinase

Ret is a tyrosine kinase receptor involved in several human diseases germ-line mutations are responsible for multiple endocrine neoplasia type 2 syndromes while somatic mutations of Ret are found in sporadic medullary thyroid carcinomas. In the present work, we describe the generation and characterization of a panel of novel monoclonal antibodies to Ret obtained by immunizing mice with a Ret-FC fusion protein. Fifty-five independent monoclonal antibodies recognize Ret-FC by enzyme linked immunosorbent assay but not a non-related FC fusion protein. Twenty antibodies further characterized recognize Ret expressing cells by flow cytometry. Finally, immunoprecipitation analysis showed that these antibodies recognize Ret mature glycosylated and immature forms. Thus, these monoclonal antibodies could be used as diagnostic tools to detect Ret expression, as well as therapeutic tools to downmodulate Ret or to deliver cytotoxic drugs to malignancies that overexpress Ret as neuroblastomas, medullary and papillary thyroid carcinomas, seminomas, and leukemia.     

The RET receptor: function in development and dysfunction in congenital malformation

Germline mutations in the RET proto-oncogene are responsible for two unrelated neural crest disorders: Hirschsprung disease, a congenital absence of the enteric nervous system in the hindgut, and multiple endocrine neoplasia type 2, a dominantly inherited cancer syndrome. Moreover, somatic rearrangements of RET are causally involved in the genesis of papillary thyroid carcinoma. The receptor tyrosine kinase encoded by the RET gene acts as the subunit of a multimolecular complex that binds four distinct ligands and activates a signalling network crucial for neural and kidney development. Over the past few years, a clearer picture of the mode of RET activation and of its multifaceted role during development has started to emerge. These findings, which provide new clues to the molecular mechanisms underlying RET signalling dysfunction in Hirschsprung disease, are summarized in this review.     

Preface

Preface

Preface     

Human serine racemase: moleular cloning, genomic organization and functional analysis

High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.