Cloning, sequence analysis, and processing of the rat and human atrial natriuretic peptide precursors

Complementary DNA sequences and structural genes encoding the atrial natriuretic peptide precursor (prepro-ANP) have been cloned. Analysis of DNA sequences, complementary to rat atrial prepro-ANP mRNA, has revealed that the various natriuretic peptides isolated from rat atrium reside at the carboxy terminus of a 152-amino-acid precursor protein. The human gene, comprised of three exons and two intervening sequences, encodes a protein of 151 amino acids highly homologous to the rat precursor. Although putative proteolytic processing sites can be identified throughout the prepro-ANP amino acid sequence, the natural form of the mature ANP has not been identified. Therefore, the sites and mechanisms of prepro-ANP processing to mature peptides forms are unknown. However, the successful cloning of the prepro-ANP gene and corresponding cDNAs provide the necessary molecular tools to address these fundamental questions relating to the regulation of ANP synthesis and processing in atrial and extraatrial tissues.     

Recombinant expression of a secreted form of the atrial natriuretic peptide clearance receptor

A general structure for the atrial natriuretic peptide clearance receptor (ANP C-receptor) has been proposed based on hydropathicity analysis of the deduced amino acid sequence of this membrane protein (Fuller, F., Porter, J.G., Arfsten, A., Miller, J., Schilling, J., Scarborough, R.M., Lewicki, J.A., and Schenk, D.B. (1988) J. Biol. Chem. 263, 9395-9401). The ANP C-receptor is believed to possess a large amino-terminal extracellular domain (436 amino acids), a single hydrophobic transmembrane anchor (23 amino acids), and a short cytoplasmic tail (37 amino acids). As a means of testing the structure and proposed cellular orientation of this protein, we have employed the technique of in vitro mutagenesis to prepare a receptor mutant (anc-) lacking the transmembrane and cytoplasmic domains. Expression of this mutant in mammalian cells using a vaccinia virus vector results in secretion of a truncated soluble form of the ANP C-receptor which binds native ANP and synthetic ANP analogs with a specificity similar to that of the native ANP C-receptor. In contrast to the native ANP C-receptor that exists predominantly as a homodimer on the cell surface, the secreted receptor exists as a monomeric species. The results are consistent with the proposed structure of this receptor with the amino-terminal domain containing the ANP-binding site oriented extracellular to the plasma membrane. In addition, these data demonstrate that the receptor does not require association with the plasma membrane or its native dimeric configuration in order to bind ANP ligands with high affinity and specificity.     

Concanavalin A distorts the beta-GlcNAc-(1-->2)-Man linkage of beta- GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man- (1-- >6)]-Man upon binding

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1-->2)-alpha- Man-(1-->3)-[beta- GlcNAc-(1-->2)-alpha-Man-(1-->6)]-Man) of N-linked oligosaccharides with a Ka of 1.41 x 10(6 )M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta- GlcNAc-(1-->2)-alpha-Man- (1-->6)]-Man to 2.7A. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1-->6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1-->3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1-->2)-Man glycosidic linkage PSI torsion angle on the 1-->6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1-->6 arm GlcNAc site.      

Effects of small C-ANF receptor ligands on plasma levels of atrial natriuretic factor, blood pressure, and renal function in the rat.

In this article, after a very brief review on ANF receptors, we report our study on the effects of small C-ANF receptor ligands in the rat. Two small ligands were synthesized: 2-naphthoxyacetyl-isonipecotyl-rANF11-15-NH2 (5 aa), containing 5 amino acids; and Ala7-rANF8-17-NH2 (10 aa), containing 10 amino acids from the ring structure of ANF1-28. After control periods, 5 aa or 10 aa were infused i.v. at a dose of 10 micrograms.min-1.kg-1 body weight for 70 min in anesthetized rats, followed by a 60-min recovery period. The 5 aa and 10 aa peptides significantly and reversibly increased plasma levels of endogenous immunoreactive ANF by 106 +/- 29 and 52 +/- 24 pg/mL, respectively. Infusion of the 5 aa peptide significantly decreased mean arterial blood pressure from 113 +/- 1 to 100 +/- 3 mmHg (1 mmHg = 133.32 Pa) and increased glomerular filtration rate from 1.6 +/- 0.2 to 2.3 +/- 0.2 mL/min, sodium excretion from 0.6 +/- 0.3 to 3.4 +/- 0.4 mumol/min, and potassium excretion from 0.5 +/- 0.2 to 1.2 +/- 0.2 mumol/min. Similar results were obtained with the 10 aa peptide. The effects of both peptides on blood pressure and sodium excretion persisted throughout the recovery period. The results confirm and extend previous observations showing that C-ANF receptors mediate the removal of ANF from the circulation. The shortening of the minimal peptide length necessary to bind to C-ANF receptors markedly enhances the possibility of developing orally active C-ANF receptor ligands for the treatment of cardiovascular and renal diseases.     

Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct viral epitopes restricted by a single major histocompatibility complex molecule.

Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.     

A Simple Model of Optimal Population Coding for Sensory Systems

A fundamental task of a sensory system is to infer information about the environment. It has long been suggested that an important goal of the first stage of this process is to encode the raw sensory signal efficiently by reducing its redundancy in the neural representation. Some redundancy, however, would be expected because it can provide robustness to noise inherent in the system. Encoding the raw sensory signal itself is also problematic, because it contains distortion and noise. The optimal solution would be constrained further by limited biological resources. Here, we analyze a simple theoretical model that incorporates these key aspects of sensory coding, and apply it to conditions in the retina. The model specifies the optimal way to incorporate redundancy in a population of noisy neurons, while also optimally compensating for sensory distortion and noise. Importantly, it allows an arbitrary input-to-output cell ratio between sensory units (photoreceptors) and encoding units (retinal ganglion cells), providing predictions of retinal codes at different eccentricities. Compared to earlier models based on redundancy reduction, the proposed model conveys more information about the original signal. Interestingly, redundancy reduction can be near-optimal when the number of encoding units is limited, such as in the peripheral retina. We show that there exist multiple, equally-optimal solutions whose receptive field structure and organization vary significantly. Among these, the one which maximizes the spatial locality of the computation, but not the sparsity of either synaptic weights or neural responses, is consistent with known basic properties of retinal receptive fields. The model further predicts that receptive field structure changes less with light adaptation at higher input-to-output cell ratios, such as in the periphery.     

Expanded potential for recombinant trisegmented lymphocytic choriomeningitis viruses: protein production, antibody production, and in vivo assessment of biological function of genes of interest

The recombinant engineering of trisegmented lymphocytic choriomeningitis virus (LCMV) to express two genes of interest was recently reported. We used this technology to efficiently express green fluorescent protein (GFP) and the immunoregulatory gene product interleukin-10 (IL-10) in vitro, assess IL-10 function in vivo during viral meningitis, and generate specific, robust monoclonal antibody responses to IL-10. Tripartite viruses were attenuated in wild-type and TLR7(-/-) mice. However, IFNAR1(-/-) mice sustained systemic viral replication when 2 nucleotide substitutions from a persistent LCMV variant were present. These findings demonstrate the utility of tripartite LCMV in vitro and in vivo to study genes in the context of a well-defined model system.     

Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring

Background  

The rapid and accurate identification of species is a critical component of large-scale biodiversity monitoring programs. DNA arrays (micro and macro) and DNA barcodes are two molecular approaches that have recently garnered much attention. Here, we compare these two platforms for identification of an important group, the mammals.     

Measles virus infection in a transgenic model: virus-induced immunosuppression and central nervous system disease.

Measles virus (MV) infects 40 million persons and kills one million per year primarily by suppressing the immune system and afflicting the central nervous system (CNS). The lack of a suitable small animal model has impeded progress of understanding how MV causes disease and the development of novel therapies and improved vaccines. We tested a transgenic mouse line in which expression of the MV receptor CD46 closely mimicked the location and amount of CD46 found in humans. Virus replicated in and was recovered from these animals' immune systems and was associated with suppression of humoral and cellular immune responses. Infectious virus was recovered from the CNS, replicated primarily in neurons, and spread to distal sites presumably by fast axonal transport. Thus, a small animal model is available for analysis of MV pathogenesis.