Semitendinosus and patellar tendons shear modulus evaluation by supersonic shearwave imaging elastography.

PURPOSE: Shear modulus (µ) is directly correlated to the tissue stiffness and can predict the tendon ultimate force to failure. With the knee extended 0° (K0), semitendinosus tendon (ST) is tensioned while patellar tendon (PT) is relaxed. At 80o , knee flexion (K80) tendons present an opposite stress pattern; however, the relation between ST and PT µ in both situations was not studied yet. METHOD: We accessed the µ of the ST and PT at 0o and 80o knee flexion by supersonic shear wave imaging (SSI) elastography from 18 healthy males. Relative µ indexes were calculated for relaxed and tensioned conditions. RESULT: The average µ for ST was µST-K0  = 197·62 ± 31·93 kPa and µST-K80  = 77·76 ± 30·08. For TP, values were µTP-K0  = 23·45 ± 5·89 and µTP-K80  = 113·92 ± 57·23 kPa. Relative µ indexes were calculated for relaxed (IR = µST-K80 /µTP-K0 ) and tensioned conditions (IT = µST-K0 /µPT-K80 ). The relative µ indexes were IR = 3·63 ± 1·50 and IT = 2·00 ± 0·96 (P<0·05). CONCLUSION: Semitendinosus tendon µ was significantly higher than PT µ in both tensioned and relaxed positions. This can predict a higher ultimate force to failure and a less elastic behaviour in ST grafts when compared to PT grafts. This new parameter could aid physicians in graft choice previous to anterior cruciate ligament reconstruction.

Analysis of the correlation between knee extension torque and patellar tendon elastic property.

PURPOSE: Quadriceps strength and patellar tendon (PT) are directly linked and intimately related to daily activities and lower limb function. However, the correlation between knee extension torque (KT) and PT Young's modulus (E) measured directly is still unknown. METHOD: We used supersonic shearwave imaging (SSI) to evaluate the elastic property of PT in healthy young men and analysed its correlation with KT. Twenty-two healthy young males were included and both knees were examined. The E of the PT in the dominant and non-dominant legs was assessed by SSI elastography. KT in maximal voluntary isometric contraction was measured with an isokinetic dynamometer. RESULT: No correlations between KT and PT E were observed in dominant or non-dominant side (P = 0·458 and 0·126, respectively). No significant differences in KT or PT E were observed between both legs (P = 0·096 and 0·722, respectively). Intra-day ICC was rated good (D1 - 0·886, P<0·001 and 0·88, P<0·001) and excellent (D2 - 0·928, P<0·001 and 0·900, P<0·001) for both legs. Inter-day ICC was rated moderate for both legs (0·651, P = 0·016 and 0·630, P = 0·018, respectively). CONCLUSION: No significant correlations were found between KT and PT E, suggesting that quadriceps strength is not an accurate predictor for PT mechanical properties in subjects with no specific training engagement. Habitual loading pattern can play a determinant role in PT mechanical properties, regardless of quadriceps strength. Further investigation on SSI acquisition protocols should be conducted to guarantee higher inter-day ICC values.

Vasodilator effects of Diocleinae lectins from the Canavalia genus.

This study investigated and compared vascular actions of leguminous lectins obtained from the Canavalia genus (Canavalia brasiliensis, Canavalia gladiata, and Canavalia maritima) in the rat models of paw edema and isolated aorta. Paw edema was induced by subcutaneous injection of lectins (0.01-1 mg/kg) in animals pre-treated or not with indomethacin or L-NAME. In isolated aorta, cumulative concentration curves of C. gladiata or C. brasiliensis (1-100 microg/ml) were performed at the contraction plateau induced by phenylephrine or at tissue basal tonus. The mechanism of the lectin relaxant action was investigated by previous addition of L-NAME, indomethacin, or tetraethylammonium. In both models, the lectin domain involvement was evaluated by incubation of lectins with their ligand and non-ligand sugars. The lectins induced paw edema paralleled by protein leakage. The edematogenic activity elicited by C. gladiata and C. brasiliensis involves prostaglandins and nitric oxide (NO), while that of C. maritima occurs without NO interference. C. gladiata and C. brasiliensis elicited aorta relaxation involving NO and prostacyclin, while that of C. gladiata included EDHF. All lectin effects were prevented by their binding sugars. The present study demonstrated important vasodilator effects of different degrees and mechanisms in vivo and in vitro of Canavalia lectins. In vivo, the edematogenic activity was paralleled by plasma exudation, and in vitro, aorta relaxation was strictly dependent on intact endothelium. All effects occurred via interaction with lectin domains and participation of NO and/or prostanoids.

Chemical analysis of lipid-modified membrane proteins in Acholeplasma laidlawii.

The lipid modification of membrane proteins was investigated in Acholeplasma laidlawii by metabolic labeling and by chemical analysis. A S-glycerylcysteine residue was identified from membrane proteins and we reported the strong preference for saturated acyl chains into the lipid modification. Differential release of fatty acids revealed a ratio [(O-ester- + amide-bound acyl chains)/O-ester-linked chains] close to 1.1 which suggests the involvement of only two O-ester linked fatty acids in the acylation process. Present data indicate that acyl proteins in A. laidlawii are true lipoproteins (mainly diacylated) probably processed by a mechanism analogous to that described for eubacteria and other mycoplasmas.

Biochemical and antigenic characterisation of Mycoplasma gallisepticum membrane proteins P52 and P67 (pMGA).

Two membrane proteins from the avian pathogen Mycoplasma gallisepticum have been previously purified using a simple, efficient and non-denaturing method: a lipoprotein P67 (pMGA) and P52. In the current study, the lipid part of P67 was chemically analysed. The molecular structure of the lipoprotein-lipid component was determined to be S-glyceryl cysteine with two O-ester-linked acyl chains. Fatty acid analysis of the purified P67 indicated a heterogeneous composition: palmitic acid (16:0)>stearic acid (18:0)>oleic acid (18:1c)>myristic acid (14:0), with 16:0 as the major component. These findings, along with previously published results, support the conclusion that P67 is pMGA1.2, a true membrane-associated lipoprotein although not N-acylated. In contrast to P67, P52 is not a lipoprotein. Topological experiments using in situ treatment with proteases and growth inhibition in the presence of anti-P52 serum provided evidence of the surface exposition of the polypeptide. The N-terminal sequence of P52 was found to be similar to the dihydrolipoamide acetyltransferase from several mollicutes; this enzyme is a membrane-associated component of the pyruvate dehydrogenase complex. Immunoblotting techniques revealed that the surface antigens P52 and P67 were specific to the species M. gallisepticum and the closely related species M. imitans. No antigenic difference was revealed within these species with the anti-P52 serum, while anti-P67 serum confirmed the antigenic variability of P67. The potential of P52 and P67 as antigens in serological diagnosis tests or as candidates for anti-mycoplasma subunit vaccines is discussed.

Chemical analysis of processing of spiralin, the major lipoprotein of Spiroplasma melliferum.

The plasma membrane of Spiroplasma melliferum contains a major membrane-associated lipoprotein called spiralin. In this study, the processing pathway of spiralin was investigated by chemical analysis of the purified protein and by using [35S]cysteine, [35S]methionine, [14C]myristic acid (14C-14:0), [14C]palmitic acid (14C-16:0), and globomycin. SDS-PAGE analysis of membrane proteins showed the leader peptide cleavage of prospiralin and provided evidence for an apparent selectivity in the acylation: the unprocessed protein was labelled with 14C-16:0 only (O-ester-linked acyl chains), and the mature form with both 14C-labelled fatty acids (O-ester-linked + amide-linked chains). Chemical analysis of the purified protein revealed that spiralin contains S-glycerylcysteine and is covalently modified with two O-ester-linked acyl chains and one amide-linked fatty acid chain. However, a specific selectivity in the O- and the N-acylations was not confirmed; palmitate and stearate were the major components. The amounts of O-ester- and amide-linked acyl chains, the resistance to Edman degradation and the presence of S-glycerylcysteine together indicate that spiralin is a "classical" lipoprotein (i.e. is triacylated) and is probably processed by a mechanism similar to that described for gram-negative eubacteria. On the basis of these findings, a biogenesis pathway for spiralin is proposed.

Selective acylation of plasma membrane proteins of Mycoplasma agalactiae: the causal agent of agalactia.

Revealed by in vivo labeling with (14)C-palmitic acid, about 15 acylated proteins were identified in the plasma membrane of Mycoplasma agalactiae (type strain PG2), including the major component p40. Triton X-114 phase partitioning and Western blotting demonstrated the amphiphilic properties of the acyl proteins and showed that they were also antigenic components. Chemical analyses of fatty acids bound to proteins revealed the following selectivity order within acylation: stearic acid (18:0) > linoleic acid (18:2c) approximately palmitic acid (16:0) > oleic acid (18:1c) > myristic acid (14:0), with 16:0 and 18:1c preferred for the O-acylation and 18:0 for the N-acylation. The ratio [O-ester- + amide-bound acyl chains]/O-ester-linked chains being close to 1.4 as well as the presence of S-glycerylcysteine suggest that acyl proteins in M. agalactiae are true lipoproteins containing N-acyl diacyl glycerylcysteine, probably processed by a mechanism analogous to that described for Gram-negative eubacteria.

Inhibition of spiralin processing by the lipopeptide antibiotic globomycin.

The cyclic lipopeptide globomycin, a specific inhibitor of signal-peptidase II (Lsp A), proved toxic for the mollicute Spiroplasma melliferum with a minimal inhibitory concentration (MIC) in the range 6.25-12.5 microM, about one order of magnitude higher (that is, less efficient) than bee-venom mellitin. SDS-PAGE analysis of cell proteins followed by immunolabeling ("Western blotting") and by crossed immunoelectrophoresis demonstrated that the cleavage of the prespiralin leader peptide was prevented by globomycin. Cell fractionation experiments showed that prespiralin was membrane bound and did not accumulate in the cytoplasm or in the culture medium. Furthermore, the use of the potential-sensitive fluorescent dye 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide (diS-C3-[5]) revealed that, in contrast to valinomycin and mellitin, globomycin up to 30 microM has no effect on the electrical transmembrane potential of S. melliferum. This indicates that the toxicity of globomycin for spiroplasma cells is mainly if not exclusively owing to the inhibition of spiralin processing. Added to previously published data, these results suggest that spiralin and probably other lipoproteins of mollicutes are acylated and membrane targeted by a mechanism involving notably the processing of the prelipoprotein precursor by a type II, globomycin-sensitive signal peptidase.

Selective acylation of plasma membrane proteins of Mycoplasma mycoides subsp. mycoides SC, the contagious bovine pleuropneumonia agent.

The plasma membrane of Mycoplasma mycoides subsp. mycoides SC (strain KH3J) contains over 160 polypeptides with apparent molecular masses ranging from 14 to 125 kDa and isoelectric point values (pIs) from 5 to 9. In vivo labeling with [14C]-fatty acids revealed about 35 acylated polypeptides including the two major components p42 and p65 and displaying pIs between 5.5 and 9.0, with a majority between 6.5 and 8. The amphiphilic nature of most of these acyl proteins was confirmed by Triton X-114 phase partitioning. Gas-liquid chromatography analyses showed that the order of preference for protein acylation was 16:0 > 18:2c > 18:1c > 18:0 > 14:0, with 16:0 being the major O-ester-bound fatty acyl chain and 18:2c the major N-linked chain. The presence of S-glycerylcysteine and a ratio of [O-ester-bound acyl chains + N-linked chains]/O-ester bound chains of approximately 1.2 in M. mycoides subsp. mycoides SC membrane proteins are consistent with a lipid modification similar to that occurring in lipoproteins of Gram-negative eubacteria that contain an N-terminal acyl S-glycerylcysteine.

Acylation and immunological properties of Mycoplasma gallisepticum membrane proteins.

The acylation of Mycoplasma gallisepticum membrane proteins was studied by electrophoresis after in vivo labelling with different 14C-fatty acids and by chemical analysis. The immunological properties of these proteins were investigated by Western blotting and crossed immunoelectrophoresis. Among the ca. 200 membrane polypeptides resolved by two-dimensional electrophoresis, 35 components (including the major protein p67) were covalently modified with acyl chains. These acylated proteins displayed lower pls than average (5.0-7.4 vs. 5.0-9.0) and proved to be the major membrane protein antigens and immunogens of M. gallisepticum. The apparent selectivity of fatty acid incorporation into proteins was, as suggested by in vivo labelling: palmitic acid (16:0) > myristic acid (14:0) > oleic acid (18:1c) > stearic acid (18:0) > linoleic acid (18:2c). However, the true order of selectivity, as revealed by chemical analysis, proved to be 18:2c > 16:0 > 18:1c > 18:0 > 14:0. More specifically, palmitic acid was the major O-ester-bound fatty acid and linoleic acid the major amide-linked fatty acid. The observed average ratio [O-ester-bound + amide-linked acyl chains]/O-ester-bound chains approximately 1.4 and the presence of S-glycerylcysteine suggest that, in M. gallisepticum, membrane proteins are lipid-modified according to a mechanism identical to that depicted for lipoproteins of Gram-negative eubacteria.

[Does spiralin has a cleavable N-terminal signal sequence?]. / La spiraline possède-t-elle une séquence signal N-terminale clivable?

The amino acid sequence of spiralin deduced from the nucleotide sequence of its gene was fictitiously shortened by 1 to 50 residues from each terminus and the compositions of both series of theoretical polypeptides were calculated. The two series of compositions thus obtained were compared to that of the purified protein, with the use of the Marchalonis and Weltman index (S delta Q). The results of this analysis, which permits the difficulty resulting from the blocking of the N-terminal amino acid to be overcome, show that spiralin is probably synthesized as a 241-residue precursor containing an N-terminal signal sequence cleaved close to cysteine-24. Since spiralin is acylated and since the sequence Val-Val-Ala-Cys24 shares some similarity with the consensus sequence of bacterial lipoprotein modification/processing site, the hypothesis of a cleavage just before cysteine-24 seems plausible.

Antigenic relatedness between the spiralins of Spiroplasma citri and Spiroplasma melliferum.

Four spiralins were compared by rocket immunoelectrophoresis, quantitative immunoblotting techniques, and the spiroplasma deformation test with the use of antispiralin (polyclonal) monospecific antibodies. This investigation revealed that the spiralins of Spiroplasma citri and S. melliferum are antigenically related and that probably no more than two epitopes simultaneously saturable with antibodies are shared by the two proteins. One at least of these epitopes is accessible to antibodies on the spiroplasma cell surface.