Sweet as honey, bitter as bile: Mitochondriotoxic peptides and other therapeutic proteins isolated from animal tissues, for dealing with mitochondrial apoptosis.

Mitochondria are subcellular organelles involved in cell metabolism and cell life-cycle. Their role in apoptosis regulation makes them an interesting target of new drugs for dealing with cancer or rare diseases. Several peptides and proteins isolated from animal and plant sources are known for their therapeutic properties and have been tested on cancer cell-lines and xenograft murine models, highlighting their ability in inducing cell-death by triggering mitochondrial apoptosis. Some of those molecules have been even approved as drugs. Conversely, many other bioactive compounds are still under investigation for their proapoptotic properties. In this review we report about a group of peptides, isolated from animal venoms, with potential therapeutic properties related to their ability in triggering mitochondrial apoptosis. This class of compounds is known with different names, such as mitochondriotoxins or mitocans.

Identifying the toxins in hornet (Vespa basalis) venom that induce rat pain responses.

The black-bellied hornet Vespa basalis is responsible for the large quantity of accidents and severe wasp envenomation in China. This study aims to identify the rat pain responses induced by experimental V. basalis sting and related-components in the venom. It was observed that unilateral intraplantar injection of crude V. basalis venom could induce several kinds of pain related behaviors in a dose-dependent manner including spontaneous pain, unilateral thermal and unilateral mechanical hypersensitivity at different time courses. Fourteen main fractions were separated from the crude venom of V. basalis using high performance liquid chromatography, among them, five components (1, 3, 4, 9 and 12) could absolutely mimic the crude venom-induced pain behaviors. According to the molecular mass and N-terminal sequence, the component 3 and 4 were identified as Mastoparan B and HP-1 respectively, the component 9 was speculated as a novel variant of HP-1/2. In addition, the other two sub-components (1-1 and 1-2) purified from component 1 cannot be determined. The results offered the key information about six active polypeptides from V. basalis contributing to pain responses, which might provide a basis for exploring mechanisms of wasp sting injury.

Structure and Function of the Bacterial Protein Toxin Phenomycin.

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime toward mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell-free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phenomycin, however, have remained unanswered. In this paper, we have used morphological profiling to show that direct inhibition of translation underlies the toxicity of phenomycin in cells. We have performed studies of the cellular uptake mechanism of phenomycin, showing that endosomal escape is the toxicity-limiting step, and we have solved a solution phase high-resolution structure of the protein using NMR spectroscopy. Through bioinformatic as well as functional comparisons between phenomycin and two homologs, we have identified a peptide segment, which constitutes one of two loops in the structure that is critical for the toxicity of phenomycin.

Study of mastoparan analog peptides against Candida albicans and safety in zebrafish embryos (Danio rerio).

Aim: In this work, mastoparan analog peptides from wasp venom were tested against Candida albicans and safety assays were performed using cell culture and model zebrafish. Materials & methods: Minimal inhibitory concentration was determined and toxicity was performed using human skin keratinocyte and embryo zebrafish. Also, permeation of peptides through embryo chorion was performed. Results: The peptides demonstrated anti-C. albicans activity, with low cytotoxicity and nonteratogenicity in Danio rerio. The compounds had different permeation through chorion, suggesting that this occurs due to modifications in their amino acid sequence. Conclusion: The results showed that the studied peptides can be used as structural study models for novel potential antifungal agents.

Effects of apelin on reproductive functions: relationship with feeding behavior and energy metabolism.

Apelin is an adipose tissue derived peptidergic hormone. In this study, 40 male Sprague-Dawley rats were used (four groups; n = 10). Apelin-13 at three different dosages (1, 5 and 50 µg/kg) was given intraperitoneally while the control group received vehicle the same route for a period of 14 days. In results, apelin-13 caused significant decreases in serum testosterone, luteinizing hormone and follicle-stimulating hormone levels (p < 0.05). Administration of apelin-13 significantly increased body weights, food intake, serum low-density lipoprotein and total cholesterol levels (p < 0.05), but caused significant decreases in high-density lipoprotein levels (p < 0.05). Serum glucose and triglyceride levels were not significantly altered by apelin-13 administration. Significant decreases in both uncoupling protein (UCP)-1 levels in the white and brown adipose tissues and UCP-3 levels in the biceps muscle (p < 0.05) were noted. The findings of the study suggest that apelin-13 may not only lead to obesity by increasing body weight but also cause infertility by suppressing reproductive hormones.

Skin fibrosis: Models and mechanisms.

Matrix synthesis, deposition and remodeling are complex biological processes that are critical in development, maintenance of tissue homeostasis and repair of injured tissues. Disturbances in the regulation of these processes can result in severe pathological conditions which are associated with tissue fibrosis as e.g. in Scleroderma, cutaneous Graft-versus-Host-Disease, excessive scarring after trauma or carcinogenesis. Therefore, finding efficient treatments to limit skin fibrosis is of major clinical importance. However the pathogenesis underlying the development of tissue fibrosis is still not entirely resolved. In recent years progress has been made unraveling the complex cellular and molecular mechanisms that determine fibrosis. Here we provide an overview of established and more recently developed mouse models that can be used to investigate the mechanisms of skin fibrosis and to test potential therapeutic approaches.

Gremlin Induces Ocular Hypertension in Mice Through Smad3-Dependent Signaling.

PURPOSE: Transforming growth factor-ß2 induces extracellular matrix (ECM) remodeling, which likely contributes to the defective function of the trabecular meshwork (TM) leading to glaucomatous ocular hypertension. Bone morphogenetic proteins (BMPs) inhibit these profibrotic effects of TGFß2. The BMP antagonist gremlin is elevated in glaucomatous TM cells and increases IOP in an ex vivo perfusion culture model. The purpose of this study was to determine whether gremlin regulates ECM proteins in the TM, signals through the Smad3-dependent pathway, and induces ocular hypertension in mice. METHODS: Ad5.Gremlin or Ad5.TGFß2 was injected intravitreally into one eye of each mouse. Intraocular pressure measurements were taken using a TonoLab tonometer. Gremlin, TGFß2, fibronectin (FN), and collagen-1 (Col-1) expression in the TM was determined by immunofluorescence, Western immunoblot, and quantitative (q)PCR analyses. RESULTS: Ad5.Gremlin or Ad5.TGFß2 each caused significant IOP elevation in mice. Immunofluorescence and Western blot analysis demonstrated that gremlin and TGFß2 reciprocally increased the expression of each other, and both increased FN expression in the TM and surrounding tissues. Ad5.Gremlin elevated IOP and increased Fn and Col-1 gene expression in the TM of Smad3 wild-type (WT) mice, but had no effect in Smad3 HET or Smad3 KO mice. CONCLUSIONS: Our results demonstrate that intravitreal injections of either Ad5.Gremlin or Ad5.TGFß2 elevate IOP and upregulate the ECM protein FN in the TM of mice. These data show that gremlin signals through the Smad3-dependent pathway in the TM to elevate IOP. We determined for the first time gremlin's role in inducing ocular hypertension in an in vivo model system.

Apelin-13 deteriorates hypertension in rats after damage of the vascular endothelium by ADMA.

Asymmetric dimethylarginine (ADMA) is a risk factor for endothelial dysfunction. The polypeptide apelin has biphasic effects on blood vessels in vivo and in vitro. We investigated the effect of apelin-13 on ADMA-damaged vessels. Rats were divided among ADMA-treated and control groups, which were treated with ADMA (10 mg·(kg body mass)(-1)·day(-1)) or saline, respectively, for 4 weeks. Systolic blood pressure (SBP) was measured before and after the injection of apelin-13. The ultrastructure of endothelial cells in caudal arteries was examined using transmission electron microscopy. The reactivities of isolated caudal artery rings were observed after exposure to apelin-13, and myosin light chain (MLC) phosphorylation was assessed by immunohistochemistry in rings treated with or without apelin-13. ADMA induced hypertension and endothelial dysfunction. After injection of apelin-13, SBP declined in the control group but was elevated in the ADMA-treated group. In vitro, apelin-13 caused relaxation in rings in the control group, but it contracted rings in the ADMA-treated group. Apelin-13 promoted MLC phosphorylation in vascular smooth muscle cells (VSMCs) in the ADMA group. These results indicate that apelin-13 might pass through ADMA-damaged endothelium and act on VSMCs to increase MLC phosphorylation, thus contributing to vasoconstriction and exacerbating hypertension.

Tat-collapsin response mediator protein 2 (CRMP2) increases the survival of neurons after NMDA excitotoxity by reducing the cleavage of CRMP2.

Collapsin response mediator protein 2 (CRMP2) is a brain-specific multifunctional adaptor protein involved in neuronal polarity and axonal guidance. Our previous results showed CRMP2 may be involved in the hypoxic preconditioning and ischemic injury, but the mechanism was not clear. This study explored whether CRMP2 was involved in NMDA-induced neural death, and the possible mechanism. Western blot analysis demonstrated that NMDA reduced the phosphorylation of CRMP2 and inspired the cleavage of CRMP2. Also, it was detected that NMDA treatment did not affect the phosphorylation of CRMP2 in early stage (<6 h). Over-expression of CRMP2 aggravated the NMDA-induced injury, suggesting the vital role of CRMP2 in excitotoxicity. Tat-CRMP2 was designed to provide the cleavage site of calpain. Thiazolyl blue tetrazolium bromide assay, Hoechst33342/Propidium Iodide staining and Western blot assay showed that Tat-CRMP2 pretreatment increased cell viability compared with the control group against NMDA exposure by decreasing the cleavage of CRMP2. In conclusion, these studies indicated that cleavage of CRMP2 plays an important role involved in the NMDA-induced injury. The cleavage of CRMP2 may be a promising target for excitatory amino acid-related ischemic and hypoxic injury.

[Apelin signalisation and vascular physiopathology]. / Signalisation apeline et physiopathologie vasculaire.

The formation of the vascular system is an early step in organogenesis that involves the participation of various signalling pathways. Integration of the extracellular signals decoded by their cognate membrane receptors orchestrate the cell events, which act at different stages, from the primitive network formed by vasculogenesis to the arborescent network remodeled by angiogenesis. Our laboratory showed the participation of a new signalling pathway in physiological angiogenesis and tumour neovascularisation. This signalling pathway named apelin comprises a G protein-coupled receptor and a peptide ligand. Expression of apelin receptors is observed during the embryonic formation of blood vessels where it is localized in the endothelium. In HUVECs, which endogenously express apelin receptors, apelin promotes the phosphorylation of ERKs, Akt and p70 S6 Kinase. In addition, apelin increases in vitro the proliferation of these endothelial cells. Finally, injection of apelin in the vitreous induces in vivo the sprouting and the proliferation of endothelial cells from the retinal vascular network. Accordingly, all these results led us to study the role of apelin signalling in tumour neovascularisation. In two tumoral cell lines, we showed that hypoxia induces the expression of apelin gene. In addition, the overexpression of apelin gene resulting from stable transfection of these cell lines clearly accelerates in vivo tumour growth, as a consequence of an increased number of vessels irrigating these tumours. The pathological relevance of these data has been validated by the characterization of an overexpression of apelin gene in one third of human tumours. Taken together, apelin signalling is both involved in physiological angiogenesis and pathological neoangiogenesis, and therefore represents an interesting pharmacological target for anti-angiogenic therapies.

Toxicokinetic study of recombinant human heparin-binding epidermal growth factor-like growth factor (rhHB-EGF) in female Sprague Dawley rats.

PURPOSE: To determine the toxicity and pharmacokinetics of recombinant heparin-binding epidermal growth factor-like growth factor in female Sprague Dawley rats following intra-bladder and intravenous administration. MATERIALS AND METHODS: rhHB-EGF was administered once daily for 6 or 27 days at doses of 3, 10, or 30 microg/kg. 125I-rhHB-EGF was administered on day 7 or 28 for pharmacokinetic analysis. Toxicity was assessed by general appearance and behavior, gross necropsy, blood chemistry and microscopic evaluation. RESULTS: Plasma AUCss of [125I] rhHB-EGF equivalents following IB administration for 7 days were 4.28+/-2.29, 7.75+/-2.70, and 7.11+/-1.42 ng ml(-1) h(-1) at doses of 3, 10, and 30 microg/kg, respectively. Following IV administration, the AUCss on day 7 increased from 27.0+/-2.66 to 124+/-5.09 and 385.11+/-7.57 ng ml(-1) h(-1) with increasing the dose from 3 to 10 and 30 microg/kg. Similar AUCss data was obtained after 28 day administration. No toxicity was evident upon gross examination. Histologic examination revealed subacute inflammation and lymphocytic infiltration of the urinary bladder in animals from all groups dosed by the IB route. CONCLUSIONS: Plasma and bladder concentrations of recombinant human [125I] rhHB-EGF equivalents were significantly lower following the IB route than following IV administration. Histologic tissue examination indicated no toxicity attributable to rhHB-EGF.

Direct hepatotoxic effect of KC chemokine in the liver without infiltration of neutrophils.

KC is a mouse homolog of human chemokine gro-alpha (CXCL1), expression of which is increased in liver diseases. We show that activated, but not quiescent, hepatic stellate cells (HSCs) express KC. Hepatic stellate cells constitutively express the KC receptor, CXCR2. Addition of recombinant KC to HSCs undergoing activation in culture increases secretion and processing of Type I collagen. Overexpression of endogenous KC in the mouse liver could be achieved by an intraperitoneal injection of CCl(4), followed after 24 hrs by an injection of recombinant KC into circulation. This protocol resulted in about a 14-fold increase in concentration of KC protein in the liver. Overexpression of KC was associated with upregulation of the mRNA for CXCR2 and MIP-2 and with necrosis and increased synthesis of Type I collagen. This suggests that KC has a direct hepatotoxic effect, which led to a massive liver necrosis after 48 hrs. No accumulation of neutrophils was seen in the livers as judged by histology and reverse transcriptase-polymerase chain reaction analysis of myeloperoxidase mRNA. Autostimulation of KC and CXCR2 expression by recombinant KC protein in the mice with preexisting liver injury indicates a positive feedback regulation. Such regulation and direct hepatotoxicity of KC with increased collagen synthesis represent novel findings about the role of KC/ gro-alpha in liver pathology.