Abstract:The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pI 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor; (2) a glutaredoxin active site; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuronastrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the ␤-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E-deficient mice accelerated the acquisition of developmental reflexes and prevented short-term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E-deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function. Key Words: Vasoactive intestinal peptide-Apolipoprotein E-Learning and memory-Neuronal survival-Molecular cloning-mRNA.

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The most prevalent lung cancer, non-small cell lung cancer (NSCLC) has receptors for vasoactive intestinal peptide (VIP). Here the effects of a VIP antagonist (VIPhyb) on NSCLC growth were investigated. In vivo, when VIPhyb (10 ,ug, s.c.) was daily injected into nude mice, xenograft formation was significantly inhibited by -40%. In vitro, VIP (100 nM) stimulated colony formation -2-fold, whereas 1 ,uM VIPhyb inhibited colony formation by -50% when adenocarcinoma cel line NCI-H838 was used. The attenuation of tumor proliferation is receptor mediated, as VIPhyb inhibited specific 12sI-labeled VIP binding to cell lines NCI-H157 and NCI-H838 with an IC5s of 0.7 ,M. VIP (10 nM) increased the cAMP levels 5-fold when ceUl line NCI-H838 was used, and 10 ,uM VIPhyb inhibited the increase in cAMP caused by VIP. Northern blot analysis and radioimmunoassays have shown VIP mRNA and VIP-like immunoreactivity in NSCLC cells. These data suggest that VIP may be a regulatory peptide in NSCLC and that VIPhyb is a VIP receptor antagonist that inhibits proliferation.Non-small cell lung cancer (NSCLC), which includes adenocarcinoma, large cell carcinoma, and squamous cell carcinoma, is the most prevalent type of lung cancer, killing ==110,000 people in the United States annually (1). Even with current surgical and chemotherapeutic interventions, NSCLC patients have a median survival time of 5 years. Although the regulation of growth for NSCLC is poorly understood, previous investigations have shown that NSCLC cells possess receptor sites for vasoactive intestinal peptide (VIP; refs. 2 and 3), a substance with recognized effects on cellular proliferation and development (4-6).VIP, a 28-amino acid polypeptide, is derived from a 170-amino acid precursor protein (7,8). PreproVIP may be metabolized to VIP by trypsin-like enzymes, carboxypeptidase B-like enzymes, and peptidyl-a-monooxygenase (9-11). VIP, which is amidated at the C terminus, is localized to neurons and endocrine cells in the central nervous system (CNS) and periphery of normal animals. Upon CNS administration, VIP modulates neuronal activity, stimulates adenylate cyclase activity, and regulates cerebral blood flow (12). Also, VIP maintains survival of spinal cord neurons in primary culture (6). In the periphery, endogenous VIP is present in nerves supplying airway smooth muscle as well as glands and pulmonary vessels within the normal adult lung (13). VIP functions as a bronchodilator and relaxes pulmonary vascular smooth muscles (14-17). These actions may be mediated by the VIP receptors that have been detected in binding assays with plasma membranes derived from the rat, mouse, guinea pig, and human lung (18)(19)(20). By in vitro autoradiographic techniques and lung slices, these VIP receptors have been localized to the alveoli and epithelium of the rat lung and pulmonary artery smooth muscle and alveolar walls of the human lung (21, 22); VIP is deficient in patients with bronchial asthma (23).In the malignant lung, high-affinity 125I-labeled VIP binding sites wer...

Stearyl‐Nle‐17‐VIP (SNV) is a novel agonist of vasoactive intestinal peptide (VIP) exhibiting a 100‐fold greater potency than the parent molecule and specificity for a receptor associated with neuronal survival. Here, mice deficient in apolipoprotein E (ApoE), a molecule associated with the etiology of Alzheimer's disease, served as a model to investigate the developmental and protective effects of SNV. In comparison to control animals, the deficient mice exhibited (a) reduced amounts of VIP messenger RNA; (b) decreased cholinergic activity (c) significant retardation in the acquisition of developmental milestones: forelimb placing behavior and cliff avoidance behavior; and (d) learning and memory impairments. Daily injections of SNV to ApoE‐deficient newborn pups resulted in increased cholinergic activity and marked improvements in the time of acquisition of behavioral milestones, with peptide‐treated animals developing as fast as control animals and exhibiting improved cognitive functions after cessation of peptide treatment. Specificity was demonstrated in that treatment with a related peptide (PACAP), pituitary adenylate cyclase‐activating peptide, produced only limited amelioration. As certain genotypes of ApoE increase the probability of Alzheimer's disease, early counseling and preventive treatments may now offer an important route for therapeutics design. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 329–342, 1997

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The complete coding sequence of a novel protein (828 amino acids, pI 5.99), a potential new mediator of vasoactive intestinal peptide (VIP) activity was recently revealed. The expression of this molecule, activity-dependent neuroprotective protein (ADNP), was augmented in the presence of VIP, in cerebral cortical astrocytes. The mRNA transcripts encoding ADNP were enriched in the mouse hippocampus and cerebellum. The protein deduced sequence contained the following: (1) a unique peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor (ADNF) and exhibiting neuroprotection in vitro and in vivo; (2) a glutaredoxin active site; and (3) a classical zinc binding domain. Comparative studies suggested that the peptide, NAPVSIPQ (NAP), was more efficacious than peptides derived from ADNF. ADNP, a potential mediator of VIP-associated neuronal survival, and the new peptide, a potential lead compound for drug design, are discussed below.

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a b s t r a c tWe study regular expressions that use variables, or parameters, which are interpreted as alphabet letters. We consider two classes of languages denoted by such expressions: under the possibility semantics, a word belongs to the language if it is denoted by some regular expression obtained by replacing variables with letters; under the certainty semantics, the word must be denoted by every such expression. Such languages are regular, and we show that they naturally arise in several applications such as querying graph databases and program analysis. As the main contribution of the paper, we provide a complete characterization of the complexity of the main computational problems related to such languages: nonemptiness, universality, containment, membership, as well as the problem of constructing NFAs capturing such languages. We also look at the extension when domains of variables could be arbitrary regular languages, and show that under the certainty semantics, languages remain regular and the complexity of the main computational problems does not change.