A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome

The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.     

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.     

Gelatinase B is involved in the in vitro wound repair of human respiratory epithelium

Following epithelial injury, extracellular matrix undergoes imposing remodelings. We examined the contribution of matrix metalloproteinases, gelatinases A and B, in an in vitro wound repair model of human respiratory epithelium. Confluent human surface respiratory epithelial (HSRE) cells cultured from dissociated surface cells of human nasal polyps were chemically injured. Over the next 3 to 5 days, cells migrated onto the injured area to repair the circular wound. Repair kinetics of these wounds was monitored until wound closure occurred. Gelatinolytic activities were analysed in culture supernates and in cell protein extracts derived from repairing migratory and non repairing stationary cells. Small amounts of gelatinase A were expressed by HSRE cells, and variations of this gelatinase remained very weak for the time of the wound repair. In contrast, gelatinase B was upregulated during the wound repair process, with a maximum peak observed at wound closure. A marked gelatinase B activation occurred only in cells involved in the repair process. Gelatinase B was localized in some migratory basal cells, recognized by an anti-cytokeratin 14 antibody and located around the wound. We could not detect any gelatinase A in repairing or in stationary HSRE cells. Addition of the 6-6B monoclonal antibody, known to inhibit gelatinase B activation, to the culture medium during the repair process resulted in a dose-dependent decrease of the wound repair speed. These results suggest that gelatinase B, produced by epithelial cells, actively contributes to the wound repair process of the respiratory epithelium. © 1996 Wiley-Liss, Inc.     

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.     

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.     

Balance control during an arm raising movement in bipedal stance: which biomechanical factor is controlled?

In order to obtain new insight into the control of balance during arm raising movements in bipedal stance, we performed a biomechanical analysis of kinematics and dynamical aspects of arm raising movements by combining experimental work, large-scale models of the body, and techniques simulating human behavior. A comparison between experimental and simulated joint kinematics showed that the minimum torque change model yielded realistic trajectories. We then performed an analysis based on computer simulations. Since keeping the center of pressure (CoP) and the projection of the center of mass (CoM) inside the support area is essential for equilibrium, we modeled an arm raising movement where displacement of one or the other variable is limited. For this optimization model, the effects of adding equilibrium constraints on movement trajectories were investigated. The results show that: (a) the choice of the regulated variable influences the strategy adopted by the system and (b) the system was not able to regulate the CoM for very fast movements without compromising its balance. Consequently, we suggest that the system is able to maintain balance while raising the arm by only controlling the CoP. This may be done mainly by using hip mechanisms and controlling net ankle torque.