Immunochemical cross-reactivity between cyanogen bromide fragments of human serum albumin

The antigenic structure of human albumin was investigated in order to establish whether or not there was any similarity between its antigenic sites. Using immunoadsorbent columns prepared with cyanogen bromide fragments of human serum albumin, antibodies directed against different portions of the albumin molecules were isolated. Measurement of the amount of the antibodies isolated and study of their specificity by inhibition techniques show that these subpopulations of antibodies reacted not only with the fragment used for their isolation (homologous) but also with the other fragments (heterologous). Heterologous fragments were inhibiting only at a very high concentration with regard to the homologous ones. These results show that there is a weak cross-reactivity between different portions of the albumin molecule. This reaction is most probably due to the homology existing in the sequence of the human albumin molecule which has arisen by gene duplication. The same type of behavior can be predicted to extent to other molecules which have evolved by similar mechanisms.     

An imbalance in autophagy contributes to retinal damage in a rat model of oxygen-induced retinopathy

In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP.     

Measuring expansion from macro‐ to nanoscale using NPC as intrinsic reporter

Expansion microscopy is a super‐resolution method that allows expanding uniformly biological samples, by increasing the relative distances among fluorescent molecules labeling specific components. One of the main concerns in this approach regards the isotropic behavior at the nanoscale. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters i.e. scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC), as well‐known nanoscale component endowed of a preserved and symmetrical structure localized on the nuclear envelope. Here, we show that Nup153 is a good reporter to quantitatively address the isotropy of the expansion process. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale to measure subcellular features with an accuracy from 10 to 5 nm. Therefore, it is an excellent method for structural studies of macromolecular complexes.     

Endocrine modulation of Brucella abortus-infected osteocytes function and osteoclastogenesis via modulation of RANKL/OPG

Osteoarticular brucellosis is the most frequent complication of active disease. A large amount of cells in bone are osteocytes. Since bone remodeling process is regulated by hormones we sought to study the effect of cortisol and DHEA in Brucella abortus-infected osteocytes. Cortisol treatment inhibited the expression of IL-6, TNF-α, MMP-2 and RANKL in B. abortus-infected osteocytes. DHEA could reverse the inhibitory effect of cortisol on MMP-2 production. B. abortus infection inhibited connexin 43 (Cx43) expression in osteocytes. This expression was increased when cortisol was incorporated during the infection and DHEA treatment partially reversed the effect of cortisol. Osteocytes-infected with B. abortus induced osteoclast's differentiation. Yet, the presence of cortisol, but not DHEA, during osteocyte infection inhibited osteoclastogenesis. Glucocorticoid receptor (GR) is implicated in the signaling of cortisol. Infection with B. abortus was able to increase GRα/β ratio. Levels of intracellular cortisol are not only dependent on GR expression but also a result of the activity of the isoenzymes 11β-hydroxysteroid dehydrogenase (11β-HSD)-1 (cortisone to cortisol conversion), 11β-HSD2 (cortisol to cortisone conversion). B. abortus infection increased 11β-HSD 1/2 ratio and cortisone mimicked the effect of cortisol. Our results indicated that cortisol and DHEA could modulate osteocyte responses during B. abortus infection.     

Verbascoside down‐regulates some pro‐inflammatory signal transduction pathways by increasing the activity of tyrosine phosphatase SHP‐1 in the U937 cell line

Polyphenols are the major components of many traditional herbal remedies, which exhibit several beneficial effects including anti‐inflammation and antioxidant properties. Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) is a redox sensitive protein tyrosine phosphatase that negatively influences downstream signalling molecules, such as mitogen‐activated protein kinases, thereby inhibiting inflammatory signalling induced by lipopolysaccharide (LPS). Because a role of transforming growth factor β‐activated kinase‐1 (TAK1) in the upstream regulation of JNK molecule has been well demonstrated, we conjectured that SHP‐1 could mediate the anti‐inflammatory effect of verbascoside through the regulation of TAK‐1/JNK/AP‐1 signalling in the U937 cell line. Our results demonstrate that verbascoside increased the phosphorylation of SHP‐1, by attenuating the activation of TAK‐1/JNK/AP‐1 signalling. This leads to a reduction in the expression and activity of both COX and NOS. Moreover, SHP‐1 depletion deletes verbascoside inhibitory effects on pro‐inflammatory molecules induced by LPS. Our data confirm that SHP‐1 plays a critical role in restoring the physiological mechanisms of inducible proteins such as COX2 and iNOS, and that the down‐regulation of TAK‐1/JNK/AP‐1 signalling by targeting SHP‐1 should be considered as a new therapeutic strategy for the treatment of inflammatory diseases.     

High‐ and low‐frequency mechanical properties of living starfish oocytes

We studied the mechanical properties of living starfish oocytes belonging to two species, Astropecten Auranciacus and Asterina pectinifera, over a wide range of timescales. We monitored the Brownian motion of microspheres injected in the cytoplasm using laser particle‐tracking (LPT) and video multiple‐particle‐tracking (MPT) techniques, to explore high‐ and low‐frequency response ranges, respectively. The analysis of the mean‐square‐displacements (MSD) allowed us to characterize the samples on different timescales. The MSD behavior is explained by three power‐law exponents: for short times (τ < 1 ms) it reflects the semiflexible behavior of the actin network; for intermediate timescales (1 ms < τ < 1 s) it is similar to that of a soft‐glass material; finally for long times (τ > 1 s) it behaves mainly like a viscous medium. We computed and compared the viscoelastic moduli using a recently proposed model describing the frequency response of the cell material. The large fluctuations found in the MSD over hundreds of trajectories indicate and confirm the significant cytoplasm heterogeneity. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)