Protease-activated receptor-1 (hPar1), a survival factor eliciting tumor progression

Although ample evidence point to the central involvement of protease activated receptor-1 (PAR1) in tumor progression, little is known about the fate of the tumor when hPar1 is being silenced. We observed that hPar1 antisense clones exhibit low PAR1 levels, attenuated cell proliferation and invasion in vitro, and tumor formation in vivo. These clones showed noticeably reduced paxillin phosphorylation compared with the parental A375SM cells, whereas no change in the integrin levels was noticed. Antisense clones injected into the mice resulted in very few and only occasional small tumors, whereas advanced and vascularized tumors were observed in A375SM cells. The antisense-derived tumor sections expressed active caspase-3, increased terminal deoxynucleotidyl transferase-mediated nick-end labeling staining, and a markedly reduced proliferating cell nuclear antigen level compared with A375SM cell-derived tissue sections. Likewise, ablation of the hPar1 gene in a tetracycline-inducible hPar1 system leads to apoptosis in immature blood vessels, whereas mature vessels were unaffected. The activation of PAR1-induced pAkt/protein kinase B abrogated serum-deprived Bim(EL) induction and also markedly inhibited Bax levels. On the other hand, small interfering RNA silencing of the hPar1 gene induced the expression of Bim(EL), a direct substrate of Akt/protein kinase B and also induced expression of active caspase-9 and caspase-3. These results altogether identify PAR1 as a survival factor that protects cells from undergoing apoptosis. We conclude that whereas PAR1 gene expression correlates with tumor progression, its neutralization effectively initiates an apoptotic pathway leading at least in part to significantly reduced tumor formation.     

Nasal Staphylococcus aureus and S. pseudintermedius carriage in healthy dogs and cats: a systematic review of their antibiotic resistance,virulence and genetic lineages of zoonotic relevance

The molecular ecology of Staphylococcus aureus, Staphylococcus pseudintermedius and their methicillin-resistant strains in healthy dogs and cats could serve as good models to understand the concept of bacterial zoonosis due to animal companionship. This study aims to provide insights into pooled prevalence, genetic lineages, virulence and antimicrobial resistance (AMR) among healthy dogs and cats. Original research and brief communication articles published from 2001 to 2021 that reported the nasal detection of S. aureus and S. pseudintermedius in healthy dogs and cats in the community, homes and outside veterinary clinics were examined and analysed. Forty-nine studies were eligible and included in this systematic review. The pooled prevalence of nasal carriage of S. aureus/methicillin-resistant S. aureus (MRSA) in healthy dogs and cats were 10.9% (95% CI: 10.1–11.9)/2.8% (95% CI: 2.4–3.2) and 3.2% (95% CI: 1.9–4.8)/0.5% (95% CI: 0.0–1.1), respectively. Conversely, the pooled prevalence of S. pseudintermedius/methicillin-resistant S. pseudintermedius (MRSP) in healthy dogs and cats were 18.3% (95% CI: 17.1–19.7)/3.1% (95% CI: 2.5–3.7) and 1.3% (95% CI: 0.6–2.4)/1.2% (95% CI: 0.6–2.3), respectively. Although highly diverse genetic lineages of S. aureus were detected in healthy dogs and cats, MSSA-CC1/CC5/CC22/CC45/CC121/CC398 and MRSA-CC5/CC93/CC22/CC30 were mostly reported in dogs; and MSSA-CC5/CC8/CC15/CC48 and MRSA-CC22/CC30/CC80 in cats. Of note, MSSA-CC398 isolates (spa-types t034 and t5883) were detected in dogs. Genetic lineages often associated with MSSP/MRSP were ST20/ST71, highlighting the frequent detection of the epidemic European MRSP-ST71 clone in dogs. S. aureus isolates carrying the luk-S/F-PV, tst, eta, etb and etd genes were seldomly detected in dogs, and luk-S/F-PV was the unique virulence factor reported in isolates of cats. S. pseudintermedius isolates harbouring the luk-S/F-I, seint and expA genes were frequently found, especially in dogs. High and diverse rates of AMR were noted, especially among MRSA/MRSP isolates. There is a need for additional studies on the molecular characterization of isolates from countries with under-studied nasal staphylococci isolates.     

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.     

Substrate specificity characterization of recombinant metallo oligo-peptidases thimet oligopeptidase and neurolysin

We report a systematic and detailed analysis of recombinant neurolysin (EC 3.4.24.16) specificity in parallel with thimet oligopeptidase (TOP, EC 3.4.24.15) using Bk sequence and its C- and N-terminal extensions as in human kininogen as motif for synthesis of internally quenched fluorescent substrates. The influence of the substrate size was investigated, and the longest peptide susceptible to TOP and neurolysin contains 17 amino acids. The specificities of both oligopeptidases to substrate sites P(4) to P(3)' were also characterized in great detail using seven series of peptides based on Abz-GFSPFRQ-EDDnp taken as reference substrate. Most of the peptides were hydrolyzed at the bond corresponding to P(4)-F(5) in the reference substrate and some of them were hydrolyzed at this bond or at F(2)-S(3) bond. No restricted specificity was found for P(1)' as found in thermolysin as well for P(1) substrate position, however the modifications at this position (P(1)) showed to have large influence on the catalytic constant and the best substrates for TOP contained at P(1), Phe, Ala, or Arg and for neurolysin Asn or Arg. Some amino acid residues have large influence on the K(m) constants independently of its position. On the basis of these results, we are hypothesizing that some amino acids of the substrates can bind to different sub-sites of the enzyme fitting P-F or F-S bond, which requires rapid interchange for the different forms of interaction and convenient conformations of the substrate in order to expose and fit the cleavage bonds in correct position for an efficient hydrolysis. Finally, this plasticity of interaction with the substrates can be an essential property for a class of cytosolic oligopeptidases that are candidates to participate in the selection of the peptides to be presented by the MHC class I.     

Structure of the Mesorhizobium huakuii and Rhizobium galegae Nod factors: a cluster of phylogenetically related legumes are nodulated by rhizobia producing Nod factors with alpha,beta-unsaturated N-acyl substitutions

Rhizobia are symbiotic bacteria that synthesize lipochitooligosaccharide Nod factors (NFs), which act as signal molecules in the nodulation of specific legume hosts. Based on the structure of their N-acyl chain, NFs can be classified into two categories: (i) those that are acylated with fatty acids from the general lipid metabolism; and (ii) those (= alphaU-NFs) that are acylated by specific alpha,beta-unsaturated fatty acids (containing carbonyl-conjugated unsaturation(s)). Previous work has described how rhizobia that nodulate legumes of the Trifolieae and Vicieae tribes produce alphaU-NFs. Here, we have studied the structure of NFs from two rhizobial species that nodulate important genera of the Galegeae tribe, related to Trifolieae and Vicieae. Three strains of Mesorhizobium huakuii, symbionts of Astragalus sinicus, produced as major NFs, pentameric lipochitooligosaccharides O-sulphated and partially N-glycolylated at the reducing end and N-acylated, at the non-reducing end, by a C18:4 fatty acid. Two strains of Rhizobium galegae, symbionts of Galega sp., produced as major NFs, tetrameric O-carbamoylated NFs that could be O-acetylated on the glucosamine residue next to the non-reducing terminal glucosamine and were N-acylated by C18 and C20 alpha,beta-unsaturated fatty acids. These results suggest that legumes nodulated by rhizobia synthesizing alphaU-NFs constitute a phylogenetic cluster in the Galegoid phylum.     

Retention and Degradation of N-Glycoproteins in the Rough Endoplasmic Reticulum

Recent studies have shown that newly synthesized proteins and glycoproteins are submitted to a quality control mechanism in the rough endoplasmic reticulum (ER). In this report we present two models: One model will illustrate a transient retention in rough ER leading to a further degradation of glycoproteins in the cytosol, (soluble alkaline phosphatase expressed in Man-P-Dol deficient CHO cells lines). The second model will illustrate a strict retention of glycoproteins in rough ER without degradation nor recycling through the Golgi (E1, E2 glycoproteins of Hepatitis C virus in stably transfected UHCV-11.4 cells and in infected Hep G2 cells).In both cases, oligomannoside structures are markers of these phenomena, either as free soluble released oligomannosides in the case of degradation, or as N-linked oligomannosides for strict retention in rough ER.