Application of Fluorescence Spectroscopy to Quantify Shear-Induced Protein Conformation Change

Rapid and robust methods are required to quantify the effect of hydrodynamic shear on protein conformation change. We evaluated such strategies in this work and found that the binding of the fluorescent probe 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) to hydrophobic pockets in the blood protein von Willebrand factor (VWF) is enhanced upon the application of fluid shear to the isolated protein. Significant structural changes were observed when the protein was sheared at shear rates ≥ 6000/s for ∼3.5 min. The binding of bis-ANS to multimeric VWF, but not dimeric VWF or control protein bovine serum albumin, was enhanced upon fluid shear application. Thus, high-molecular-weight VWF is more susceptible to conformation change upon tensile loading. Although bis-ANS itself did not alter the conformation of VWF, it stabilized protein conformation once it bound the sheared molecule. Bis-ANS binding to VWF was reduced when the sheared protein was allowed to relax before dye addition. Taken together with functional data in the literature, our results suggest that shear-induced conformation changes in VWF reported by bis-ANS correlate well with the normal function of the protein under physiological/pathological fluid flow conditions. Further, this study introduces the fluorescent dye bis-ANS as a tool that may be useful in studies of shear-induced protein conformation change.     

Spectrophotometric assays for measuring redox biomarkers in blood

The assessment of redox status is most frequently performed by measuring redox biomarkers. The spectrophotometer is the most commonly used analytical instrument in biochemistry. There is a huge number of spectrophotometric redox biomarkers and assays, thus distinguishing the most appropriate biomarkers and protocols is overwhelming. The aim of the present review is to propose valid and reliable spectrophotometric assays for measuring redox biomarkers in blood. It is hoped that this work will help researchers to select the most suitable redox biomarkers and assays.     

The mitogenic effect of testosterone and 17β-estradiol on airway smooth muscle cells

Airway disease distribution and/or severity exhibit sex differences suggesting that sex hormones are involved in the respiratory system physiology and pathophysiology. The implication of airway smooth muscle cells (ASMCs) in the physiology of the airways and the pathogenetic mechanism of airway remodeling is of great interest. Therefore, we studied the effect of testosterone and 17β-estradiol on ASMC proliferation and the mechanisms involved.Cell proliferation was estimated using the methyl-[³H]thymidine incorporation and Cell Titer 96^® AQueous One Solution Assay methods. ASMC isolated from adult male or female rabbit trachea were incubated with testosterone (1 pM-1 μM) or 17β-estradiol (1 pM-1 μM), in the presence or absence of the androgen receptor antagonist flutamide (10 nM) or estrogen receptor antagonist ICI182780 (10 nM), as well as of the PI3K inhibitors LY294002 (20 μM) or wortmannin (1 μM), or the MAPK inhibitors PD98059 (100 μM) or U0126 (1 μM).After 24 h of incubation, testosterone and 17β-estradiol increased methyl-[³H]thymidine incorporation and cell number, in ASMC isolated from male or female animals. The induction of ASMC proliferation by testosterone or 17β-estradiol was inhibited by flutamide or ICI182780 respectively, as well as by LY294002, wortmannin, PD98059 or U0126.In conclusion, testosterone and 17β-estradiol have a mitogenic effect on ASMC, which is receptor-mediated and involves the MAPK and PI3K signaling pathways. Moreover, their effect is the same for ASMC from male and female animals. It is possible that gender-related differences in ASMC remodeling, may be influenced by the different patterns of sex steroid hormone secretion in males and females.     

Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae)

Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.     

p75NTR and TROY: Uncharted Roles of Nogo Receptor Complex in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), have been on the forefront of drug discovery for most of the myelin inhibitory molecules implicated in axonal regenerative process. Nogo-A along with its putative receptor NgR and co-receptor LINGO-1 has paved the way for the production of pharmaceutical agents such as monoclonal antibodies, which are already put into handful of clinical trials. On the other side, little progress has been made towards clarifying the role of neurotrophin receptor p75 (p75NTR) and TROY in disease progression, other key players of the Nogo receptor complex. Previous work of our lab has shown that their exact location and type of expression is harmonized in a phase-dependent manner. Here, in this review, we outline their façade in normal and diseased central nervous system (CNS) and suggest a role for p75NTR in chronic axonal regeneration whereas TROY in acute inflammation of EAE intercourse.     

Decorin modulates matrix mineralization in vitro

Decorin (DCN), a member of small leucine-rich proteoglycans, is known to modulate collagen fibrillogenesis. In order to investigate the potential roles of DCN in collagen matrix mineralization, several stable osteoblastic cell clones expressing higher (sense-DCN, S-DCN) and lower (antisense-DCN, As-DCN) levels of DCN were generated and the mineralized nodules formed by these clones were characterized. In comparison with control cells, the onset of mineralization by S-DCN clones was significantly delayed; whereas it was markedly accelerated and the number of mineralized nodules was significantly increased in As-DCN clones. The timing of mineralization was inversely correlated with the level of DCN synthesis. In these clones, the patterns of cell proliferation and differentiation appeared unaffected. These results suggest that DCN may act as an inhibitor of collagen matrix mineralization, thus modulating the timing of matrix mineralization.