Structure and proteoglycan composition of specialized regions of the elastic tendon of the chicken wing

The elastic tendon of the avian wing has been described by others as a unique structure with elastic properties due to the predominance of elastic fibers in the midsubstance. Further analyses of the tendon have shown it to possess five anatomically distinct regions. Besides the major elastic region, a distally located fibrocartilage and three tendinous regions are present. The tendinous regions connect: (1) the muscle to the elastic region, (2) the elastic region to the fibrocartilage and (3) the latter to the insertion site. The elastic region possesses thick and abundant elastic fibers and very thin, interconnecting collagen fibers. The collagen fibers in the sesamoid fibrocartilage are thick and interwoven, defining spaces occupied by fibrochondrocytes embedded in a non-fibrillar and highly metachromatic matrix. Biochemical analyses have shown that the fibrocartilage has about tenfold the amount of glycosaminoglycans (GAGs) found in the other regions. The main GAG in this region was chondroitin sulfate (CS) (plus keratan sulfate as detected immunocytochemically), while the other regions showed variable amounts of CS, dermatan sulfate (DS) and heparan sulfate. Further analyses have shown that a large CS-bearing proteoglycan is found in the fibrocartilage. The elastic region possesses two main proteoglycans, a large CS-bearing proteoglycan (which reacted with an antibody against keratan sulfate after chondroitinase ABC treatment) and a predominant DS-bearing proteoglycan, which showed immunoreactivity when assayed with an anti-biglycan antibody. The results demonstrate that the elastic tendon is a complex structure with complex regional structural and compositional adaptations, suited to different biomechanical roles.     

Effects of 24 weeks of progressive resistance training on knee extensors peak torque and fat-free mass in older women

This study examined the effects of resistance training (RT) on knee extensor peak torque (KEPT) and fat-free mass (FFM) in older women. Seventy-eight volunteers (67.1 ± 5.9 years old) underwent 24 weeks of progressive RT (RTG) while 76 (67.4 ± 5.9 years old) were studied as controls (CG). Dominant knee extension peak torque was assessed using an isokinetic dynamometer (Biodex System 3) and FFM measurements were performed by dual-energy x-ray absorptiometry. Muscle strength and FFM were evaluated before and after the intervention in all volunteers. Participants in the RTG trained major muscle groups 3 times per week during 24 weeks. Training load was kept at 60% of 1 repetition maximum in the first 4 weeks, 70% in the following 4 weeks, and 80% in the remaining 16 weeks, with repetitions, respectively, decreasing from 12, 10, and 8. A Split-plot analysis of variance was performed to examine between- and within-group differences, and the level of significance was accepted at p ≤ 0.05. It was observed that the RTG showed significant increases in KEPT (from 89.9 ± 21.8 to 102.8 ± 22.6 N·m; p < 0.05) and FFM (from 36.4 ± 4.0 to 37.1 ± 4.2 kg, p < 0.05). Appendicular FFM was also significantly increased after the intervention period in the RTG (13.9 ± 1.8 to 14.2 ± 1.9 kg, p < 0.05). None of these changes were observed for the CG. Consistent with the literature, it is concluded that a progressive RT program promotes not only increases in muscle strength, as evaluated by an isokinetic dynamometer, but also in FFM as evaluated by the DXA, in elderly women.     

Interaction of the Two Structural Domains of Calmodulin with Mature and Immature Rat Brain Microtubules

The inhibitory effect of calmodulin on the assembly of mature and immature rat brain microtubules was compared with that of the two major structural domains of this protein, the COOH-terminal fragment (amino acids 78-148) and the NH2-terminal fragment (amino acids 1-77), to determine the calmodulin structural domain responsible for the inhibitory effect on microtubule assembly. Microtubules prepared during the early stages of brain development, i.e., during intensive neurite outgrowth, are more sensitive to inhibition by the Ca2(+)-calmodulin complex than those obtained from adult brain. Significant inhibition of immature microtubule assembly was observed with both fragments in the absence of Ca2+, but the effects were more important when Ca2+ was present. With adult brain microtubules, the two fragments remained without effect on assembly in the absence of Ca2+, whereas some inhibition was seen in its presence but only with the COOH-terminal polypeptide. Under all these conditions, the COOH-terminal fragment was always more active than the NH2-terminal fragment on microtubule polymerization, albeit to a lesser extent than native calmodulin.     

Coordination of lapachol to bismuth(III) improves its anti-inflammatory and anti-angiogenic activities

Complex [Bi(Lp)₂]Cl was obtained with 4-hydroxy-3-(3-methylbut-2-enyl)naphthalene-1,2-dione, “lapachol” (HLp). Lapachol, [Bi(Lp)₂]Cl and BiCl₃ were evaluated in a murine model of inflammatory angiogenesis induced by subcutaneous implantation of polyether polyurethane sponge discs. Intraperitoneal (i.p.) administration of lapachol or [Bi(Lp)₂]Cl reduced the hemoglobin content in the implants suggesting that reduction of neo-vascularization was caused by lapachol. In the per os treatment only [Bi(Lp)₂]Cl decreased the hemoglobin content in the implants. Likewise, N-acetylglucosaminidase (NAG) activity decreased in the implants of the groups i.p. treated with lapachol and [Bi(Lp)₂]Cl while in the per os treatment inhibition was observed only for [Bi(Lp)₂]Cl. Histological analysis showed that the components of the fibro-vascular tissue (vascularization and inflammatory cell population) were decreased in lapachol- and complex-treated groups. Our results suggest that both lapachol and [Bi(Lp)₂]Cl exhibit anti-angiogenic and anti-inflammatory activities which have been attributed to the presence of the lapachol ligand. However, coordination to bismuth(III) could be an interesting strategy for improvement of lapachol’s therapeutic properties.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin,Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser⁴⁷³ while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser⁴⁷³ and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.     

The antimicrobial activity of lapachol and its thiosemicarbazone and semicarbazone derivatives

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.