Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae).

A thrombin (E.C. 3.4.21.5) inhibitor, savignin, was isolated from the salivary glands of Ornithodoros savignyi by a combination of size exclusion, anion-exchange, and reversed-phase chromatography. The inhibitor has a molecular mass of 12,430.4 Da as determined by electrospray mass spectrometry. The behavior of savignin during anion-exchange chromatography indicated that it has an acidic pI. The available N-terminal sequence (residues 1-11) differed from that of ornithodorin with only one residue. Savignin inhibits thrombin-induced platelet aggregation, but has no effect on ADP- or collagen-induced aggregation. Kinetic studies indicated that savignin is a competitive, slow-, tight-binding inhibitor of alpha-thrombin (K(i) = 4.89 +/- 1.39 pM). Tight-binding kinetics showed that the inhibitor has a lower affinity for gamma-thrombin (K(i) = 22.3 +/- 5.9 nM). Plasmin, factor Xa, and trypsin are not inhibited by savignin.     

Characterization of a centromere-linked recombination hot spot in Saccharomyces cerevisiae.

A 1.5-kilobase-pair SalI-HindIII (SH) restriction fragment from the region of Saccharomyces cerevisiae chromosome XIV immediately adjacent to the centromere appears to contain sequences that act as a hot spot for mitotic recombination. The presence of SH DNA on an autonomously replicating plasmid stimulates homologous genetic exchange between yeast genomic sequences and those present on the plasmid. In all recombinants characterized, exchange occurs in plasmid yeast sequences adjacent to rather than within the SH DNA. Hybridization analyses reveal that SH-containing plasmids are present in linear as well as circular form in S. cerevisiae and that linear forms are generated by cleavage at specific sites. Presumably, it is the linear form of the plasmid that is responsible for the stimulation of genetic exchange. Based on these observations, it is proposed that this DNA fragment contains a centromere-linked recombination hot spot and that SH-stimulated recombination occurs via a mechanism similar to double-strand-gap repair (J. W. Szostak, T. Orr-Weaver, J. Rothstein, and F. Stahl, Cell 33:25-35 1983).     

Spectral mechanisms in the tree squirrel retina

Summary The retina of the gray squirrel (Sciurus carolinensis) contains rods and cones in a ratio of about 23. The spectral mechanisms in this retina were examined in behavioral and electrophysiological experiments. Tests of color vision revealed that this animal has a spectral neutral point at about 500 nm and, thus, dichromatic color vision. Recordings made from single optic nerve fibers and results obtained from an analysis of the flicker photometric electroretinogram (ERG) indicated that vision in the gray squirrel is based on three spectral mechanisms. One of these, presumably rod-based, has peak sensitivity at about 502 nm. The other two mechanisms reflect the presence of two classes of cone having average peak sensitivity of about 444 nm and 543 nm.     

Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae.

A functional centromere located on a small DNA restriction fragment from Saccharomyces cerevisiae was identified as CEN14 by integrating centromere-adjacent DNA plus the URA3 gene by homologous recombination into the yeast genome and then by localizing the URA3 gene to chromosome XIV by standard tetrad analysis. DNA sequence analysis revealed that CEN14 possesses sequences (elements I, II, and III) that are characteristic of other yeast centromeres. Mitotic and meiotic analyses indicated that the CEN14 function resides on a 259-base-pair (bp) RsaI-EcoRV restriction fragment, containing sequences that extend only 27 bp to the right of the element I to III region. In conjunction with previous findings on CEN3 and CEN11, these results indicate that the specific DNA sequences required in cis for yeast centromere function are contained within a region about 150 bp in length.     

A shared epitope found in the major paralysis inducing tick species of Africa

Cross-reactivity between all the paralysis inducing tick species of veterinary relevance in Africa was demonstrated, by using a monoclonal antibody directed against the paralysis inducing toxin of Rhipicephalus evertsi evertsi. Western blot results, together with amino acid composition studies indicated that this monoclonal antibody recognizes protein bands of similar molecular mass and amino acid composition in R. evertsi evertsi and Ixodes rubicundus. This suggests that the Karoo paralysis toxin of I. rubicundus is possibly also a trimer with a high degree of homology to the spring lamb paralysis toxin of R. evertsi evertsi. The conclusive identity of these protein bands of 1. rubicundus could not be shown. Bio-assay studies performed on 1-day-old chickens suggested that the anti-spring lamb paralysis toxin monoclonal antibody also recognizes the paralysis toxin present in Argas (Persicargas) walkerae, by rendering some degree of protection against the effect of this toxin.     

Identification of anticoagulant activities in the salivary glands of the soft tick,Ornithodoros savignyi

Salivary gland extracts of the sand tampan, Ornithodoros savignyi, prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) significantly in a concentration-dependent manner. There was also a pronounced inhibition of human activated factor Xa (fXa) by salivary gland extracts. The salivary gland extracts inhibited chromogenic assays specific for both fXa and thrombin. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the salivary gland proteins followed by elution of specific areas or bands from a polyvinylidene difluoride (PVDF)-membrane, showed that various anticoagulant factors are present when screened by means of the APTT assay. The most active component was associated with a band of M _r of 14 kDa. Partial purification of this component was achieved using isoelectric focusing (IEF) and size-exclusion highperformance liquid chromatography (HPLC).     

Structural and functional characterization of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant, and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxy‐terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os–C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94–15 µg/ml) to both Gram‐positive and Gram‐negative bacteria, whereas the parent peptide only exhibited Gram‐positive antibacterial activity. The Os peptide was found to be two‐fold more active than Os–C against three of the four tested bacteria but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os–C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane‐mimicking environment, the cysteine‐containing peptide has a higher α‐helical content. Both peptides were found to be non‐toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The mechanism of alphaIIbbeta3 antagonism by savignygrin and its implications for the evolution of anti-hemostatic strategies in soft ticks

Savignygrin, a alphaIIbbeta3 antagonist presents the RGD sequence on the substrate-binding loop of the (BPTI-fold). This study investigated whether this is the only integrin-targeting motif associated with its mechanism. It forms a tight-binding complex with alphaIIbbeta3 that is resistant to SDS dissociation under reducing and non-reducing conditions, but not to temperature or EDTA. The same complex is formed on resting and activated platelets, as well as aggregated platelets that have been disaggregated with savignygrin. Binding of FITC labeled savignygrin to platelets show that the binding kinetics and affinity of savignygrin is similar for resting and activated platelets (Kd approximately 50-70 nM). Binding to resting or activated platelets was significantly inhibited by two savignygrin peptide fragments, S2 (GSRGDEDATFG) and S3 (FDREDGGSRQG) that correspond with two specific loop-like areas in the structure of savignygrin that together form a continuous binding interface. The inability of S3 to inhibit platelet aggregation indicates that it targets a novel ligand-binding site. A model of alphaIIbbeta3 based on the recent crystal structure of alphavbeta3 into which the RGD sequence of savignygrin was docked shows that savignygrin lies along the interface formed by the two subunits. A novel mode of integrin antagonism is indicated that includes the targeting of distinct sites on the alphaIIbbeta3 subunits. The S2 and S3 loops are not involved in the mechanisms of the related soft tick blood coagulation inhibitors and suggest that this allowed their evolution as integrin targeting motifs.     

Apyrase Activity and Platelet Aggregation Inhibitors in the Tick Ornithodoros Savignyi (Acari: Argasidae)

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activities supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.     

Highly selective c-Jun N-terminal kinase (JNK) 3 inhibitors with in vitro CNS-like pharmacokinetic properties II. Central core replacement

In this Letter, we describe the evolution of selective JNK3 inhibitors from 1, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs. Strong SAR was found for substitution of the naphthalene ring, as well as for inhibitors adopting different central scaffolds. Significant potency gains were appreciated by inverting the polarity of the thione of the parent triazolothione 1, resulting in potent compounds with attractive pharmacokinetic profiles.