cDNA sequences of three sheep myeloid cathelicidins

AbstractSeveral myeloid antimicrobial peptide precursors have been shown to consist of a N-terminal proregion similar to a protein named cathelin and a structurally varied C-terminal antimicrobial domain. Proteins with these features have been named cathelicidins. In this paper we report the cDNA sequences of three ovine cathelicidins of 155, 160 and 190 residues, respectively, with cationic C-terminal sequences corresponding to putative antimicrobial domains. These are structurally varied and include a Cys-rich sequence of 12 residues, which is similar to the bovine antimicrobial cyclic dodecapeptide, a novel 29 residue sequence named SMAP-29 with a possible α-helical conformation, and a 60 residue sequence named Bac7.5, which appears to be a new member of the Pro- and Arg-rich group of mammalian antimicrobial peptides

Voluntary Exercise Prevents Lead-Induced Elevation of Oxidative Stress and Inflammation Markers in Male Rat Blood

Regular mild exercise enhances antioxidant and anti-inflammatory systems of the body. The present study investigates voluntary exercise effects on lead toxicity as a known oxidative stressor. Male Sprague-Dawley rats were randomly divided into 2 groups. Sedentary control: the animals were housed 7 weeks in the regular cages. Exercise group: the animals were housed 7 weeks in the running wheel equipped cages, that is, the animal model of voluntary exercise. During the 7th week, all animals were administered lead acetate. Blood samples were collected at the end of the 6th week and 7th week (before and after lead administrations). Glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and tumor necrosis factor (TNF-) were measured in the samples. Our results showed that lead administration reduced blood SOD, GPx and CAT and increased TNF-; in the controls, but in the exercise group, changes were not statistically significant. MDA in both groups increased after lead injections but it was significantly lower in exercise group compared to the sedentary animals. We concluded that voluntary exercise may be considered as a preventive tool against lead-induced oxidative stress and inflammation

Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in <i>Arabidopsis</i>

Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation

ENZO: A Web Tool for Derivation and Evaluation of Kinetic Models of Enzyme Catalyzed Reactions

We describe a web tool ENZO (Enzyme Kinetics), a graphical interface for building kinetic models of enzyme catalyzed reactions. ENZO automatically generates the corresponding differential equations from a stipulated enzyme reaction scheme. These differential equations are processed by a numerical solver and a regression algorithm which fits the coefficients of differential equations to experimentally observed time course curves. ENZO allows rapid evaluation of rival reaction schemes and can be used for routine tests in enzyme kinetics. It is freely available as a web tool, at http://enzo.cmm.ki.si

Effect of Fluorosis on Liver Cells of VC Deficient and Wild Type Mice

For decades, mouse and other rodents have been used for the study of oxidative or related studies such as the effect of fluoride. It is known that rodents normally synthesize their own vitamin C (VC) due to the presence of a key enzyme in ascorbic acid synthesis, l-gulono-lactone--oxidase (Gulo), while humans do not have the capacity of VC synthesis due to the deletion of most parts of the GULO gene. The spontaneous fracture (sfx) mouse recently emerged as a model for study of VC deficiency. We investigated the effect of fluoride on liver cells from wild type Balb/c and sfx mice. We found that activities of SOD, GPx, and CAT were reduced in both wild type and sfx mice; however, the amount of reduction in the sfx cells is more than that in Balb/c cells. In addition, while both cells increased MDA, the increase in the sfx cells is greater than that in Balb/c cells. Gene networks of Sod, Gpx, and Cat in the liver of humans and mice are also different. Our study suggests that reaction to fluoride in vitamin C deficient mice might be different from that of wild type mice

Structural Basis for Specificity of Propeptide-Enzyme Interaction in Barley C1A Cysteine Peptidases

C1A cysteine peptidases are synthesized as inactive proenzymes. Activation takes place by proteolysis cleaving off the inhibitory propeptide. The inhibitory capacity of propeptides from barley cathepsin L and B-like peptidases towards commercial and barley cathepsins has been characterized. Differences in selectivity have been found for propeptides from L-cathepsins against their cognate and non cognate enzymes. Besides, the propeptide from barley cathepsin B was not able to inhibit bovine cathepsin B. Modelling of their three-dimensional structures suggests that most propeptide inhibitory properties can be explained from the interaction between the propeptide and the mature cathepsin structures. Their potential use as biotechnological tools is discussed

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H2O2), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes

A programmed cell death pathway in the malaria parasite Plasmodium falciparum has general features of mammalian apoptosis but is mediated by clan CA cysteine proteases

Several recent discoveries of the hallmark features of programmed cell death (PCD) in Plasmodium falciparum have presented the possibility of revealing novel targets for antimalarial therapy. Using a combination of cell-based assays, flow cytometry and fluorescence microscopy, we detected features including mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in parasites induced with chloroquine (CQ) and staurosporine (ST). The use of the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (zVAD), and the mitochondria outer membrane permeabilization (MOMP) inhibitor, 4-hydroxy-tamoxifen, enabled the characterization of a novel CQ-induced pathway linking cysteine protease activation to downstream mitochondrial dysregulation, amplified protease activity and DNA fragmentation. The PCD features were observed only at high (μM) concentrations of CQ. The use of a new synthetic coumarin-labeled chloroquine (CM-CQ) showed that these features may be associated with concentration-dependent differences in drug localization. By further using cysteine protease inhibitors z-Asp-Glu-Val-Asp-fmk (zDEVD), z-Phe-Ala-fmk (zFA), z-Phe-Phe-fmk (zFF), z-Leu-Leu-Leu-fmk (zLLL), E64d and CA-074, we were able to implicate clan CA cysteine proteases in CQ-mediated PCD. Finally, CQ induction of two CQ-resistant parasite strains, 7G8 and K1, reveals the existence of PCD features in these parasites, the extent of which was less than 3D7. The use of the chemoreversal agent verapamil implicates the parasite digestive vacuole in mediating CQ-induced PCD

Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris–HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells

Optogenetic acidification of synaptic vesicles and lysosomes

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes