Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Translational research of novel hormones: lessons from animal models and rare human diseases for common human diseases.

Since the 1980s, a number of bioactive molecules, now known as cardiovascular hormones, have been isolated from the heart and blood vessels, particularly from the subset of vascular endothelial cells. The natriuretic peptide family is the prototype of the cardiovascular hormones. Over the following decade, a variety of hormones and cytokines, now known as adipokines or adipocytokines, have also been isolated from adipose tissue. Leptin is the only adipokine demonstrated to cause an obese phenotype in both animals and humans upon deletion. Thus, the past two decades have seen the identification of two important classes of bioactive molecules secreted by newly recognized endocrine cells, both of which differentiate from mesenchymal stem cells. To assess the physiological and clinical implications of these novel hormones, we have investigated their functions using animal models. We have also developed and analyzed mice overexpressing transgenic forms of these proteins and knockout mice deficient in these and related genes. Here, we demonstrate the current state of the translational research of these novel hormones, the natriuretic peptide family and leptin, and discuss how lessons learned from excellent animal models and rare human diseases can provide a better understanding of common human diseases.

Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium.

The mammalian main olfactory epithelium (MOE) recognizes and transduces olfactory cues through a G protein-coupled, cAMP-dependent signaling cascade. Additional chemosensory transduction mechanisms have been suggested but remain controversial. We show that a subset of MOE neurons expressing the orphan receptor guanylyl cyclase GC-D and the cyclic nucleotide-gated channel subunit CNGA3 employ an excitatory cGMP-dependent transduction mechanism for chemodetection. By combining gene targeting of Gucy2d, which encodes GC-D, with patch clamp recording and confocal Ca2+ imaging from single dendritic knobs in situ, we find that GC-D cells recognize the peptide hormones uroguanylin and guanylin as well as natural urine stimuli. These molecules stimulate an excitatory, cGMP-dependent signaling cascade that increases intracellular Ca2+ and action potential firing. Responses are eliminated in both Gucy2d- and Cnga3-null mice, demonstrating the essential role of GC-D and CNGA3 in the transduction of these molecules. The sensitive and selective detection of two important natriuretic peptides by the GC-D neurons suggests the possibility that these cells contribute to the maintenance of salt and water homeostasis or the detection of cues related to hunger, satiety, or thirst.

Some aspects of the venom proteome of the Colubridae snake Philodryas olfersii revealed from a Duvernoy's (venom) gland transcriptome.

We investigated the putative toxins of Philodryas olfersii (Colubridae), a representative of a family of snakes neglected in venom studies despite their growing medical importance. Transcriptomic data of the venom gland complemented by proteomic analysis of the gland secretion revealed the presence of major toxin classes from the Viperidae family, including serine proteases, metalloproteases, C-type lectins, Crisps, and a C-type natriuretic peptide (CNP). Interestingly, the phylogenetic analysis of the CNP precursor showed it as a linker between two related precursors found in Viperidae and Elapidae snakes. We suggest that these precursors constitute a monophyletic group derived from the vertebrate CNPs.

Natriuretic peptides, but not nitric oxide donors, elevate levels of cytosolic guanosine 3',5'-cyclic monophosphate in ependymal cells ex vivo.

Atrial natriuretic peptide-(1-28) (ANP), brain natriuretic peptide-(1-32) (BNP) and C-Type natriuretic polypeptide (CNP) occur in the brain, are concentrated in the anteroventral area of the third cerebral ventricle and participate in the regulation of body fluid homeostasis. The ventricles of the mammalian brain are lined by a continuous monolayered epithelium of polyciliated ependymal cells. In the adult rat, the ependymocytes continue to express the intermediate filament vimentin, but do not contain glial fibrillary acidic protein. Ependymal functions are poorly understood, but may extend to osmoregulation and volume sensing. Ependymal cells possess receptors for the natriuretic peptides, and in cell culture respond to them with an increase in their cyclic GMP content. In this study, a cyclic GMP-specific antibody was employed together with an ex vivo brain slice system to assess the ependymal response to ANP, BNP and CNP under close to life-like conditions. While ANP in concentrations of 0.1 nM and 1 nM had no effect, at concentrations of 10nM and 100 nM it increased ependymal cyclic GMP levels in a concentration-dependent manner. The other natriuretic peptides BNP, and CNP, also increased the cyclic GMP content of ependymocytes, while nitric oxide (NO) donors had no effect. However, in contrast to the natriuretic peptides, the NO donors elevated the level of cyclic GMP in the brain parenchyma below the ependymal layer.

Péptidos natriuréticos en insuficiencia cardiaca / Natriuretic peptides in heart failure

El hecho de que el corazón sea capaz de segregar hormonas, y que éstas se liberen en cantidades importantes ante determinadas situaciones cardiacas, ha abierto un horizonte lleno de esperanzas e incertidumbres. Estas hormonas, denominadas péptidos natriuréticos, poseen propiedades diuréticas, natriuréticas y vasodilatadoras. Las utilizadas en clínica son el ANP, BNP y sus porciones terminales (NT-proANP y NT-proBNP). Actualmente, la mayoría de estudios están realizados con el BNP, pero debido a su menor variabilidad y mayor vida media es esperable que en los próximos años se incrementen en gran medida el número de trabajos con NT-proBNP. De cualquier forma, parece con suficiente evidencia en la actualidad que el nivel plasmático de estas hormonas será de gran ayuda en el diagnóstico, pronóstico, despistaje, control farmacológico y tratamiento de la insuficiencia cardiaca (AU)

The fact that the heart is able to secrete hormones, which are released in significant amounts in advance of certain cardiac conditions, has resulted in a wide range of opportunities and raised a multitude of questions. These hormones, named natriuretic peptides, possess diuretic, natriuretic and vasodilatory properties. The ones used in daily clinical practice are atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and their N-terminal fragments NT-proANP and NT-proBNP, respectively. Although most studies currently involve the use of BNP, the number involving NT-proBNP is expected to increase substantially in coming years because its level is less variable and its half-life longer. Nevertheless, at present there appears to be sufficient evidence to suggest that the plasma levels of these hormones will be extremely useful for the diagnosis, prognosis, screening, pharmacological monitoring, and treatment of patients with heart failure (AU)

Natriuretic peptides and the dialysis patient.

The natriuretic peptide family consists of four structurally similar, but genetically distinct molecules with pronounced cardiovascular and renal actions. They are counterregulatory hormones playing an important role in fluid volume homeostasis. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) cause diuresis, natriuresis, and vasodilatation. C-type natriuretic peptide (CNP) has antimitogenic effects and causes vascular smooth muscle relaxation. Dendroaspis natriuretic peptide (DNP) shares many of the actions of ANP and BNP, but its function in humans is not yet fully understood. Natriuretic peptides have been extensively investigated as biochemical markers of the fluid state. Levels are elevated in disease conditions characterized by fluid overload and are closely related to survival in various cardiac disease states. In the dialysis population, BNP correlates significantly with cardiac function, whereas ANP is sensitive to volume changes during dialysis. However, changes in concentration do not predict achievement of euvolemia, and short half-life, combined with complicated assay techniques, make ANP a less than satisfactory tool for assessing hydration. BNP is a superior prognosticator for risk stratification in dialysis patients, and serial estimations will help in the identification of occult cardiac disease.

Natriuretic peptides: markers or modulators of cardiac hypertrophy?

The natriuretic peptides (NPs), through their diuretic, vasodilatory and anti-mitogenic properties, play an important role in the regulation of cardiovascular, renal and endocrine homeostasis. Recent studies suggest that they have utility in both the diagnosis and management of heart failure. Plasma brain NP (BNP) levels have been used to establish prognosis in patients with heart failure and those at risk for heart failure post-myocardial infarction. They have been used to establish a cardiac etiology for acute shortness of breath, and to guide and assess the efficacy of therapy in patients with established heart failure. BNP is also approved for use in the management of acute decompensated heart failure. Of note, recent studies suggest that cardiac NPs suppress myocyte hypertrophy and interstitial fibrosis in the heart, arguing for an important autocrine-paracrine role of these peptides in controlling the cardiac response during hypertrophy. Therefore, the existing evidence supports a role for BNP as both a marker and a modulator of hypertrophy.