Mutations in Desmin's Carboxy-Terminal “Tail” Domain Severely Modify Filament and Network Mechanics

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-α-helical carboxy-terminal domain (“tail”), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin “tail” mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l_p. Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the “tail” mutants (l_p = 1.0-2.0 μm) and the wild-type desmin (l_p = 1.1 ± 0.5 μm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered.In order to explore the importance of the “tail” domain as such for filament network properties, we employed a “tail”-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the “tail” domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.     

4-1BB signaling synergizes with programmed death ligand 1 blockade to augment CD8 T cell responses during chronic viral infection

Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection. Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and timing of 4-1BB costimulation are carefully controlled. When PD-L1-blocking Abs are given together with a single low dose of anti-4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs to PD-L1 are given with a repetitive high dose of anti-4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7 posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However, whereas the high dose of anti-4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of anti-4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic infection.     

Hyperphosphorylation and aggregation of tau in experimental autoimmune encephalomyelitis

Axonal damage is a major morphological correlate and cause of permanent neurological deficits in patients with multiple sclerosis (MS), a multifocal, inflammatory and demyelinating disease of the central nervous system. Hyperphosphorylation and pathological aggregation of microtubule-associated protein tau is a common feature of many neurodegenerative diseases with axonal degeneration including Alzheimer's disease. We have therefore analyzed tau phosphorylation, solubility and distribution in the brainstem of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Tau was hyperphosphorylated at several sites also phosphorylated in Alzheimer's disease and became partially detergent-insoluble in EAE brains. Morphological examination demonstrated accumulation of amorphous deposits of abnormally phosphorylated tau in the cell body and axons of neurons within demyelinating plaques. Hyperphosphorylation of tau was accompanied by up-regulation of p25, an activator of cyclin-dependent kinase 5. Phosphorylation of tau, activation of cdk5, and axonal pathology were significantly reduced when diseased rats were treated with prednisolone, a standard therapy of acute relapses in MS. Hyperphosphorylation of tau was not observed in a genetic or nutritional model of axonal degeneration or demyelination, suggesting that inflammation as detected in the brains of rats with EAE is the specific trigger of tau pathology. In summary, our data provide evidence that axonal damage in EAE and possibly MS is linked to tau pathology.     

Role of the mitochondrial DnaJ homolog Mdj1p as a chaperone for mitochondrially synthesized and imported proteins.

Mdj1p, a DnaJ homolog in the mitochondria of Saccharomyces cerevisiae, is involved in the folding of proteins in the mitochondrial matrix. In this capacity, Mdj1p cooperates with mitochondrial Hsp70 (mt-Hsp70). Here, we analyzed the role of Mdj1p as a chaperone for newly synthesized proteins encoded by mitochondrial DNA and for nucleus-encoded proteins as they enter the mitochondrial matrix. A series of conditional mutants of mdj1 was constructed. Mutations in the various functional domains led to a partial loss of Mdj1p function. The mutant Mdj1 proteins were defective in protecting the tester protein firefly luciferase against heat-induced aggregation in isolated mitochondria. The mitochondrially encoded var1 protein showed enhanced aggregation after synthesis in mdj1 mutant mitochondria. Mdj1p and mt-Hsp70 were found in a complex with nascent polypeptide chains on mitochondrial ribosomes. Mdj1p was not found to interact with translocation intermediates of imported proteins spanning the two membranes and exposing short segments into the matrix, in accordance with the lack of requirement of Mdj1p in the mt-Hsp70-mediated protein import into mitochondria. On the other hand, precursor proteins in transit which had further entered the matrix were found in a complex with Mdj1p. Our results suggest that Mdj1p together with mt-Hsp70 plays an important role as a chaperone for mitochondrially synthesized polypeptide chains emerging from the ribosome and for translocating proteins at a late import step.     

Ghrelin ovarian cell expression in patients with polycystic ovary syndrome: an immunohistochemical evaluation.

INTRODUCTION: The influence of ghrelin on different organs has been studied recently, e.g. in the regulation of pituitary hormone release, regulation of energy homeostasis, glucose metabolism and insulin secretion, cell proliferation, and reproductive function. However, the etiology of polycystic ovary syndrome has not been fully explained. The aim of our study was to estimate the presence of ghrelin in polycystic ovaries cells and evaluation of the relationship between ghrelin occurrence and cells proliferation. METHODS: In the present work we have compared ten polycystic ovaries with ovaries without pathology as the control group. We used immunohistochemical method to detect ghrelin. The cells proliferation was evaluated by Ki 67 proliferation index. RESULTS: Ghrelin immunostaining was demonstrated in cytoplasm of ovarian secondary interstitial cells and in atretic corpus luteum. The cell nuclei were ghrelin positive in granulosa, theca layers of follicular cyst in both groups as well as in luteal cells of young corpus luteum in healthy ovaries. Ki 67 immunostaining was observed in granulosa and theca layers of follicular cyst in polycystic and healthy ovaries. CONCLUSIONS: It is possible that local ghrelin expression plays an important role in the direct control of ovarian development and function and ghrelin may participate in patomechanism of PCOS.     

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus.     

The cell surface receptor DC-SIGN discriminates between Mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan

Interactions between dendritic cells (DCs) and Mycobacterium tuberculosis, the etiological agent of tuberculosis, most likely play a key role in anti-mycobacterial immunity. We have recently shown that M. tuberculosis binds to and infects DCs through ligation of the DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and that M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) inhibits binding of the bacilli to the lectin, suggesting that ManLAM might be a key DC-SIGN ligand. In the present study, we investigated the molecular basis of DC-SIGN ligation by LAM. Contrary to what was found for slow growing mycobacteria, such as M. tuberculosis and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin, our data demonstrate that the fast growing saprophytic species Mycobacterium smegmatis hardly binds to DC-SIGN. Consistent with the former finding, we show that M. smegmatis-derived lipoarabinomannan, which is capped by phosphoinositide residues (PILAM), exhibits a limited ability to inhibit M. tuberculosis binding to DC-SIGN. Moreover, using enzymatically demannosylated and chemically deacylated ManLAM molecules, we demonstrate that both the acyl chains on the ManLAM mannosylphosphatidylinositol anchor and the mannooligosaccharide caps play a critical role in DC-SIGN-ManLAM interaction. Finally, we report that DC-SIGN binds poorly to the PILAM and uncapped AraLAM-containing species Mycobacterium fortuitum and Mycobacterium chelonae, respectively. Interestingly, smooth colony-forming Mycobacterium avium, in which ManLAM is capped with single mannose residues, was also poorly recognized by the lectin. Altogether, our results provide molecular insight into the mechanisms of mycobacteria-DC-SIGN interaction, and suggest that DC-SIGN may act as a pattern recognition receptor and discriminate between Mycobacterium species through selective recognition of the mannose caps on LAM molecules.     

Mn3O4 nanoparticles: Synthesis,characterization and their antimicrobial and anticancer activity against A549 and MCF-7 cell lines

Due to their inexpensive and eco-friendly nature, and existence of manganese in various oxidation states and their natural abundance have attained significant attention for the formation of Mn₃O₄ nanoparticles (Mn₃O₄ NPs). Herein, we report the preparation of Mn₃O₄ nanoparticles using manganese nitrate as a precursor material by utilization of a precipitation technique. The as-prepared Mn₃O₄ nanoparticles (Mn₃O₄ NPs) were characterized by using X-ray powder diffraction (XRD), UV–Visible spectroscopy (UV–Vis), High-Resolution Transmission electron microscopy (HRTEM), Field emission scanning electron microscopy (FESEM), Thermal gravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FT-IR). The antimicrobial properties of the as-synthesized Mn₃O₄ nanoparticles were investigated against numerous bacterial and fungal strains including S. aureus, E. coli, B. subtilis, P. aeruginosa, A. flavus and C. albicans. The Mn₃O₄ NPs inhibited the growth of S. aureus with a minimum inhibitory concentration (MIC) of 40 μg/ml and C. albicans with a MIC of 15 μg/ml. Furthermore, the Mn₃O₄ NPs anti-cancer activity was examined using MTT essay against A549 lung and MCF-7 breast cancer cell lines. The Mn₃O₄ NPs revealed significant activity against the examined cancer cell lines A549 and MCF-7. The IC₅₀ values of Mn₃O₄ NPs with A549 cell line was found at concentration of 98 µg/mL and MCF-7 cell line was found at concentration of 25 µg/mL.