Cloning and characterization of Aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in Aplysia californica

Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.     

Monitoring of infectious diseases among wild boars

Sixty-seven serum samples and 43 pathological material samples from wild boars, taken in 5 regions of Russia and in the Kharkov Region of the Ukraine in 2002 to 2005 were studied. Wild boars in some regions of Russia were shown to be carriers of Aujeszky's disease virus, porcine parvovirus, porcine circovirus type 2, lymphotropic herpesvirus-1, porcine cytomegalovirus, Mycoplasma hyopneumoniae and Pasteurella multocida. The classical swine fever (CSF) virus genome was detected by polymerase chain reaction in the samples from 10 wild boars from 2 Russian regions (the Tver and Moscow Regions). Sequencing of the E2 gene 5'-terminal region of detected CSF virus isolates showed that they were closely related to two field virus isolates early found in domestic pigs in the Moscow and Vladimir Regions, which suggests that there is an epizootic relation between the SCF outbreaks among wild boars and domestic pigs in these regions. Tests for porcine reproductive and respiratory syndrome, transmissible porcine gastroenteritis, porcine influenza, enteroviruses, and actinobacillus-induced pleuropneumonia were negative in all the regions under study.     

Methods underpinning national clinical guidelines for hypertension: describing the evidence shortfall

Background  

To be useful, clinical practice guidelines need to be evidence based; otherwise they will not achieve the validity, reliability and credibility required for implementation.