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.     

The haemodynamic, renal excretory and hormonal changes induced by resting in the left lateral position in normal pregnant women during late gestation

Objective  To characterise the haemodynamic, renal-electrolyte and hormonal parameters in normal near-term pregnancy.
Design  Observational prospective case-series study.
Setting and population  Eleven women with normal pregnancies at 35–39 weeks gestation.
Methods  Following baseline laboratory assessments and placement of a right-atrial catheter, serial measurements were obtained for 2 hours in the supine position (SP) followed by a change to the (LLP) and subsequent observations for 2 hours.
Main outcome measures  Blood pressure (BP), central venous pressure (CVP), atrial natriuretic peptide (ANP), plasma renin activity (PRA), plasma aldosterone (ALDO), diuresis, creatinine clearance, sodium and potassium excretion.
Results  In the SP, the subjects' BP remained stable while their CVP decreased. In the LLP, the subjects' systolic and diastolic BP consistently decreased by about 15 mmHg and their CVP increased within the first 60 minutes. ANP levels doubled in the subjects while they rested in the LLP, whereas the subjects' PRA and ALDO levels decreased by half compared with when they rested in the SP. In the LLP, the subjects' creatinine clearance significantly increased by 12% and their sodium excretion and diuresis increased by 38% and 59% respectively.
Conclusion  Rest in the LLP induces systemic and intra-renal haemodynamic and hormonal changes that may play a central physiological role in the renal excretory response to restore excessive sodium/water retention in late pregnancy.     

Brainstem glioma progression in juvenile and adult rats

OBJECT： Brainstem gliomas are common in children and have the worst prognosis of any brain tumor in this age group. On the other hand, brainstem gliomas are rare in adults, and the authors of some clinical studies have suggested that this lesion behaves dif ferently in adults than in children. In the present study, the authors test an orthotopic C6 brainstem glioma model in juvenile and adult rats, and investigate the biological behavior of this lesion in the 2 age groups. METHODS： The C6 glioma cells were stereotacti eally implanted into the pons of juvenile or adult male rats. Neurological presentation and survival time were recorded.     

Expression of granulocyte colony-stimulating factor and granulocyte colony-stimulating factor receptor genes in partially overlapping monoclonal B-cell populations from chronic lymphocytic leukemia patients

B lymphocytes were purified from the peripheral blood of 30 B-cell chronic lymphocytic leukemia (B-CLL) patients and tested for the ability to produce granulocyte colony-stimulating factor (G-CSF) in vitro. Fifteen Staphylococcus aureus Cowan I (SAC)-stimulated, but not unstimulated, B-cell suspensions produced G-CSF in short-term cultures. Accordingly, G-CSF mRNA was detected only in SAC-stimulated B cells. Five CLL B-cell fractions that released G-CSF following exposure to SAC were also incubated with CD40 or anti-mu antibodies in the presence or absence of recombinant (r) interleukin-2 (IL-2) or IL-4. The 5 cell suspensions produced G-CSF only on culture with CD40 monoclonal antibody in combination with rIL-2 or rIL-4. CD5+ B lymphocytes, which represent the normal counterparts of most B-CLL proliferations, did not produce G-CSF under any of the above culture conditions. G-CSF produced by leukemic B lymphocytes was biologically active, because conditioned media of SAC-stimulated cells supported the in vitro growth of myeloid colonies from normal bone marrow progenitors. The colony stimulating activity of CLL B-cell supernatants was ascribed to both G-CSF and granulocyte-macrophage colony stimulating factor. G-CSF receptors (G- CSFRs) were detected on freshly isolated B lymphocytes from 7 of 11 B- CLL patients; 5 of these cell suspensions produced G-CSF in culture, whereas 2 did not. rG-CSF rescued 3 of the 7 G-CSFR+ cell fractions from spontaneous apoptosis but had no effect on their in vitro proliferation.