Rachel Zamostiano

Complete Sequence of a Novel Protein Containing a Femtomolar‐Activity‐Dependent Neuroprotective Peptide

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, pl 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 neuron—astrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the β‐amyloid peptide (the Alzheimers 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.

Cloning and Characterization of the Human Activity-dependent Neuroprotective Protein*

We have recently cloned the mouse activity-dependent neuroprotective protein (ADNP). Here, we disclose the cloning of human ADNP (hADNP) from a fetal brain cDNA library. Comparative sequence analysis of these two ADNP orthologs indicated 90% identity at the mRNA level. Several single nucleotide polymorphic sites were noticed. The deduced protein structure contained nine zinc fingers, a proline-rich region, a nuclear bipartite localization signal, and a homeobox domain profile, suggesting a transcription factor function. Further comparative analysis identified an ADNP paralog (33% identity and 46% similarity), indicating that these genes belong to a novel protein family with a nine-zinc finger motif followed by a homeobox domain. The hADNP gene structure spans ∼40 kilobases and includes five exons and four introns with alternative splicing of an untranslated second exon. The hADNP gene was mapped to chromosome 20q12–13.2, a region associated with aggressive tumor growth, frequently amplified in many neoplasias, including breast, bladder, ovarian, pancreatic, and colon cancers. hADNP mRNA is abundantly expressed in distinct normal tissues, and high expression levels were encountered in malignant cells. Down-regulation of ADNP by antisense oligodeoxynucleotides up-regulated the tumor suppressor p53 and reduced the viability of intestinal cancer cells by 90%. Thus, ADNP is implicated in maintaining cell survival, perhaps through modulation of p53.

The identification of secreted heat shock 60 -like protein from rat glial cells and a human neuroblastoma cell line

The intracellular stress-induced proteins provide protection against toxic insults. Here, a 60,000-Da heat shock 60 (hsp60)-like protein was detected, with five different antibodies, in conditioned media derived from rat cortical astrocytes and a human neuroblastoma cell line. Extracellular neuroblastoma hsp60-like immunoreactivity was increased 3-fold in the presence of the neuropeptide vasoactive intestinal peptide (VIP) and was augmented 2-fold after temperature elevation. Intracellular hsp60 immunoreactivity was reduced 2-3-fold in the presence of VIP; this reduction was attenuated in the presence of brefeldin A, an inhibitor of protein secretion. In contrast, the activity of lactate dehydrogenase (LDH), an intracellular marker, did not change in the presence of VIP. Essentially no extracellular LDH activity was detected, indicating no cellular damage. A novel aspect for stress proteins having extracellular protective roles is suggested.

Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia

ABSTRACT Tilapia are an important global food source due to their omnivorous diet, tolerance for high-density aquaculture, and relative disease resistance. Since 2009, tilapia aquaculture has been threatened by mass die-offs in farmed fish in Israel and Ecuador. Here we report evidence implicating a novel orthomyxo-like virus in these outbreaks. The tilapia lake virus (TiLV) has a 10-segment, negative-sense RNA genome. The largest segment, segment 1, contains an open reading frame with weak sequence homology to the influenza C virus PB1 subunit. The other nine segments showed no homology to other viruses but have conserved, complementary sequences at their 5′ and 3′ termini, consistent with the genome organization found in other orthomyxoviruses. In situ hybridization indicates TiLV replication and transcription at sites of pathology in the liver and central nervous system of tilapia with disease. IMPORTANCE The economic impact of worldwide trade in tilapia is estimated at

A femtomolar-acting neuroprotective peptide induces increased levels of heat shock protein 60 in rat cortical neurons: a potential neuroprotective mechanism

7.5 billion U.S. dollars (USD) annually. The infectious agent implicated in mass tilapia die-offs in two continents poses a threat to the global tilapia industry, which not only provides inexpensive dietary protein but also is a major employer in the developing world. Here we report characterization of the causative agent as a novel orthomyxo-like virus, tilapia lake virus (TiLV). We also describe complete genomic and protein sequences that will facilitate TiLV detection and containment and enable vaccine development. The economic impact of worldwide trade in tilapia is estimated at

A Novel Signaling Molecule for Neuropeptide Action: Activity‐dependent Neuroprotective Protein

7.5 billion U.S. dollars (USD) annually. The infectious agent implicated in mass tilapia die-offs in two continents poses a threat to the global tilapia industry, which not only provides inexpensive dietary protein but also is a major employer in the developing world. Here we report characterization of the causative agent as a novel orthomyxo-like virus, tilapia lake virus (TiLV). We also describe complete genomic and protein sequences that will facilitate TiLV detection and containment and enable vaccine development.

Identification of a Novel RNA Virus Lethal to Tilapia

Activity-dependent neurotrophic factor (ADNF) was recently isolated from conditioned media of astrocytes stimulated with vasoactive intestinal peptide (VIP). ADNF provided neuroprotection at femtomolar concentration against a wide variety of toxic insults. A nine amino acid peptide (ADNF-9) captured with even greater potency the neuroprotective activity exhibited by the parent protein. Utilizing Northern and Western blot analyses, it was now shown that ADNF-9 increased the expression of heat shock protein 60 (hsp60) in rat cerebral cortical cultures. In contrast, treatment with the Alzheimers toxin, the beta-amyloid peptide, reduced the amount of intracellular hsp60. Treatment with ADNF-9 prevented the reduction in hsp60 produced by the beta-amyloid peptide. The protection against the beta-amyloid peptide-associated cell death provided by ADNF-9 may be mediated in part by intracellular increases in hsp60.

A novel VIP responsive gene. Activity dependent neuroprotective protein.

Abstract: 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.

Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR

ABSTRACT Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with >80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide.

Synonymous site conservation in the HIV-1 genome

Abstract: Activity dependent neuroprotective protein (ADNP, 828 amino acids, pI 5.99) is a glial‐derived protein that contains a femtomolar active neuroprotective peptide, NAPVSIPQ (NAP). VIP induces a two‐ to threefold increase in ADNP mRNA in astrocytes, suggesting that ADNP is a VIP‐responsive gene. ADNP is widely distributed in the mouse hippocampus, cerebellum, and cerebral cortex. VIP has been shown to possess neuroprotective activity that may be exerted through the activation of glial proteins. We suggest that ADNP may be part of the VIP protection pathway through the femtomolar‐acting NAP and through putative interaction with other macromolecules.