Convergent evolution of pain-inducing defensive venom components in spitting cobras.

Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

Isolation and characterization of renin-like aspartic-proteases from Echis ocellatus venom.

Three aspartic proteases (SVAPs) have been isolated from venom of the saw-scaled viper, Echis ocellatus. In confirmation of prior transcriptomic predictions, all three forms match to sequences of either of the two SVAP transcripts (EOC00051 and EOC00123), have a molecular weight of 42 kDa and possess a single N-glycan. The SVAPs act in a renin-like manner, specifically cleaving human and porcine angiotensinogen into angiotensin-1 and possess no general protease activity. Their activity is completely inhibited by the aspartyl protease inhibitor Pepstatin A.

Expression of Schwann cell-specific proteins and low-molecular-weight neurofilament protein during regeneration of sciatic nerve treated with neurotrophin-4.

Neurotrophin-4 acts as a potent survival factor for subpopulations of motoneurons. To investigate its effect on Schwann cell sheath and axonal proteins during peripheral nerve regeneration, sciatic nerves in adult rats were transected and repaired, and fibrin glue containing neurotrophin-4 injected around the repair site. At 5, 15, 30 and 60 days after repair, 5-mm nerve segments distal to the repair were collected, and western blotting was used to measure myelin-associated glycoprotein, myelin basic protein and low-molecular-weight neurofilament protein. In control groups these dramatically declined at 5 and 15 days then increased from 30 and 60 days. However, in the neurotrophin-4 group there was a significant increase (to several times basal values) in myelin-associated glycoprotein and myelin basic protein at 5-15 days. The relatively small increases (<7%) in Schwann cell numbers suggest that this is mainly due to increased synthesis per cell. The neurotrophin-4 group also showed a small but significant increase at 15 days in low-molecular-weight neurofilament protein, which however remained much lower than basal. We conclude that neurotrophin-4 regulates the expression of myelin-associated glycoprotein, myelin basic protein, and to a lesser extent low-molecular-weight neurofilament protein, during peripheral nerve regeneration.

Neurotrophin-4 delivered by fibrin glue promotes peripheral nerve regeneration.

Neurotrophin-4 (NT-4) is a recently identified neurotrophic factor with potential trophic effects on subpopulations of neurons. Little is known about its role in peripheral nerve regeneration following nerve injury. To investigate this, 48 Sprague-Dawley rats underwent left sciatic nerve transection and immediate repair. Fibrin glue mixed with either NT-4 or vehicle (control) was injected around the nerve repair site. Nerve regeneration was assessed both functionally and histomorphometrically. The results showed that the NT-4-treated group had a significant increase compared with the control in the regeneration distance at 5 days. The sciatic function index was significantly greater in the NT-4 group from 40 to 60 days after nerve repair. Morphometric analysis revealed that nerves treated with NT-4 had significant improvement in the number of regenerated axons, axonal diameter, and myelin thickness. These results suggest that NT-4 is a potent factor improving rat sciatic nerve regeneration.

Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone.

The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca(2+)](i) elevation in these cells, indicating no G(q) coupling, however, activation of G(q)-coupled P2Y(1) receptors resulted in characteristic [Ca(2+)](i) release. PTH potentiated this nucleotide-induced Ca(2+) release, independently of Ca(2+) influx. PTH-(1-31), which activates only G(s), mimicked the actions of PTH-(1-34), whereas PTH-(3-34), which only activates G(q), was unable to potentiate nucleotide-induced [Ca(2+)](i) release. Despite this coupling of the PTHR to G(s), cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca(2+)](i) release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y(1) agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone.

A CD36-binding peptide from thrombospondin-1 can stimulate resorption by osteoclasts in vitro.

Thrombospondin-1 (TSP-1), purified from platelets, stimulates resorption by avian osteoclasts in an in vitro resorption assay. TSP-1 binds to a number of different cellular receptors via different domains of the molecule and several short receptor-binding sequences have been identified within the TSP-1 molecule. In this study, we have used synthetic peptides representing these various sequences in order to identify the cellular receptor and TSP domain responsible for stimulation of resorption. We show that one peptide CSVTCG, which represents the CD36-binding region of TSP-1, stimulates resorption in a fashion similar to the intact molecule, while the peptides RGDS, RFYVVMWK, and RFYVVM, representing other cell-binding domains of TSP, have no effect on resorption. Using RT-PCR and immunoblotting, we further demonstrate expression of CD36 in human osteoclastoma (giant cell tumour), primary human bone derived cells, and clonal osteoblastic cells. These studies suggest that CD36 is involved in regulation of resorption by osteoclasts and is the receptor responsible for the resorption-promoting effects of TSP-1.

Extracellular ATP activates multiple signalling pathways and potentiates growth factor-induced c-fos gene expression in MCF-7 breast cancer cells.

In the human breast cancer cell line MCF-7, the nucleotides ATP gamma S and UTP, acting extracellularly through the purinergic receptor P2Y(2), lead to elevated intracellular calcium levels and increased proliferation. ATP gamma S and UTP treatment of MCF-7 cells activated transcription of the immediate early gene c-fos, an important component in the response to proliferative stimulation. c-fos induction was enhanced by co-treatment with ATP gamma S and a variety of proliferative agents including growth factors, tumour promoters and stress. Stimulation with ATP gamma S or epidermal growth factor (EGF) led to extracellular signal-regulated kinase (ERK) activation and phosphorylation of the transcription factors CREB and Elk-1. Co-stimulation synergistically activated fos expression and notably led to increased levels of ERK, CREB and EGF receptor phosphorylation, as well as hyperphosphorylation of ternary complex factor. Nevertheless, the ERK pathway does not fully account for this synergy, since fos induction was differentially sensitive to the MEK inhibitor U0126, indicating that these two agonists signal differently to this immediate early gene. Thus, extracellular nucleotides co-operate with growth factors to activate genes linked to the proliferative response in MCF-7 cells through activation of specific purinergic receptors, which thereby represent important potential targets for arresting the neoplastic progression of breast cancer cells.

Expression of members of the thrombospondin family by human skeletal tissues and cultured cells.

We have previously shown that the multifunctional platelet glycoprotein thrombospondin-1 (TSP-1) promotes resorption in an in vitro resorption assay. However, TSP-1 is one of a family of multifunctional TSP molecules, and the current study was undertaken to investigate whether it is TSP-1 or another TSP family member which may be involved in regulation of resorption in vivo. RT-PCR was performed on cultured human bone cells, cultured human chondrocytes, and three separate samples of human osteoclastoma tissue using primers specific for each TSP family member. mRNA for TSP-2 was detected in almost all samples, and significantly in all osteoclastomas in the above tissues, while TSP-1 was detected less frequently and was only seen in one of three osteoclastomas. TSP-3, -4, and COMP were detected only in a minority of cases. These results indicate that TSP-2 is the most common TSP family member found in skeletal tissues and that TSP-2, rather than TSP-1, may be the molecule responsible for promoting resorption in vivo.