TGF-β1 receptor blockade attenuates unilateral ureteral obstruction-induced renal fibrosis in C57BL/6 mice through attenuating Smad and MAPK pathways

Journal of Molecular Histology - Renal fibrosis is characterized by accumulation of extracellular matrix components and collagen deposition. TGF-β1 acts as a master switch promoting renal...     

Insights into the Renal Pathogenesis in Schimke Immuno-Osseous Dysplasia: A Renal Histological Characterization and Expression Analysis

Schimke immuno-osseous dysplasia (SIOD) is a pleiotropic disorder caused by mutations in the SWI/SNF2-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like-1 (SMARCAL1) gene, with multiple clinical features, notably end-stage renal disease. Here we characterize the renal pathology in SIOD patients. Our analysis of SIOD patient renal biopsies demonstrates the tip and collapsing variants of focal segmental glomerulosclerosis (FSGS). Additionally, electron microscopy revealed numerous glomerular abnormalities most notably in the podocyte and Bowman’s capsule. To better understand the role of SMARCAL1 in the pathogenesis of FSGS, we defined SMARCAL1 expression in the developing and mature kidney. In the developing fetal kidney, SMARCAL1 is expressed in the ureteric epithelium, stroma, metanephric mesenchyme, and in all stages of the developing nephron, including the maturing glomerulus. In postnatal kidneys, SMARCAL1 expression is localized to epithelial tubules of the nephron, collecting ducts, and glomerulus (podocytes and endothelial cells). Interestingly, not all cells within the same lineage expressed SMARCAL1. In renal biopsies from SIOD patients, TUNEL analysis detected marked increases in DNA fragmentation. Our results highlight the cells that may contribute to the renal pathogenesis in SIOD. Further, we suggest that disruptions in genomic integrity during fetal kidney development contribute to the pathogenesis of FSGS in SIOD patients.     

Functional hair cell mechanotransducer channels are required for aminoglycoside ototoxicity

Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.     

LIV-1 ZIP Ectodomain Shedding in Prion-Infected Mice Resembles Cellular Response to Transition Metal Starvation

We recently documented the co-purification of members of the LIV-1 subfamily of ZIP (Zrt-, Irt-like Protein) zinc transporters (LZTs) with the cellular prion protein (PrP(C)) and, subsequently, established that the prion gene family descended from an ancestral LZT gene. Here, we begin to address whether the study of LZTs can shed light on the biology of prion proteins in health and disease. Starting from an observation of an abnormal LZT immunoreactive band in prion-infected mice, subsequent cell biological analyses uncovered a surprisingly coordinated biology of ZIP10 (an LZT member) and prion proteins that involves alterations to N-glycosylation and endoproteolysis in response to manipulations to the extracellular divalent cation milieu. Starving cells of manganese or zinc, but not copper, causes shedding of the N1 fragment of PrP(C) and of the ectodomain of ZIP10. For ZIP10, this posttranslational biology is influenced by an interaction between its PrP-like ectodomain and a conserved metal coordination site within its C-terminal multi-spanning transmembrane domain. The transition metal starvation-induced cleavage of ZIP10 can be differentiated by an immature N-glycosylation signature from a constitutive cleavage targeting the same site. Data from this work provide a first glimpse into a hitherto neglected molecular biology that ties PrP to its LZT cousins and suggest that manganese or zinc starvation may contribute to the etiology of prion disease in mice.     

Mechanics and deformation of the nucleus in micropipette aspiration experiment

Robust biomechanical models are essential for the study of nuclear mechanics and deformation and can help shed light on the underlying mechanisms of stress transition in nuclear elements. Here, we develop a computational model for an isolated nucleus undergoing micropipette aspiration. Our model includes distinct components representing the nucleoplasm and nuclear envelope. The nuclear envelope itself comprises three layers: inner and outer nuclear membranes and one thicker layer representing the nuclear lamina. The nucleoplasm is modeled as a viscoelastic Maxwell material with a single time constant, while a modified Maxwell model, equivalent to a spring and a dashpot in series and both in parallel with a spring, is adopted for the inner and outer nuclear membranes. The nuclear envelope layer is taken as a linear elastic material. The proposed computational model, validated using experimental observations of Guilak et al. [2000. Viscoelastic properties of the cell nucleus. Biochemical and Biophysical Research Communications 269, 781-786] and Deguchi et al. [2005, Flow-induced hardening of endothelial nucleus as an intracellular stress-bearing organelle. Journal of Biomechanics 38, 1751-1759], is employed to study nuclear mechanics and deformation in micropipette aspiration and to shed light on the contribution of individual nuclear components on the response. The results indicate that the overall response of an isolated nucleus in micropipette aspiration is highly sensitive to the apparent stiffness of the nuclear lamina. This observation suggests that micropipette aspiration is an effective technique for examining the influence of various kinds of alteration in the nuclear lamina, such as mutations in the gene encoding lamin A, and also structural remodeling due to mechanical perturbation.     

Enhanced thermal and ultrasonic stability of a fungal protease encapsulated within biomimetically generated silicate nanospheres

Background

Dendrimers are highly branched synthetic macromolecules with a globular shape. They have been successfully used for generation of nanospheres at mild conditions via biomimetic silicification. Encapsulation of enzyme molecules within these nanospheres during their synthesis is a promising method for rapid and efficient entrapment of several enzymes. However, encapsulation of proteolytic enzymes has been rarely done via biomimetic silicification. As well, the operational stability of encapsulated enzyme has not been systematically reported.

Methods

A proteolytic enzyme, either α-Chymotrypsin or a fungal protease from Aspergilus Oryzea was encapsulated along with iron oxide nanoparticles within particles yielded via biomimetic silicification of different generations of polyamidoamine (PAMAM) dendrimers. Stability of encapsulated enzyme was compared to that of free enzyme during storage at room temperature. As well, their thermal and ultrasonic stabilities were measured. Scanning electron microscopy, transmission electron microscopy and optical microscopy were used to investigate the morphology of nanospheres.

Results

Determination of encapsulation efficiency revealed that ∼ 85% of fungal protease with concentration 1.4 mg mL^− 1 stock solution was immobilized within particles yielded by generation 0. Based on microscopic images the generated particles interconnected with each other and had spherical morphologies independent of generation. Kinetic analysis of encapsulated fungal protease demonstrated that Mechaelis-Menten constant (K_m) slightly increased.

Conclusion

PAMAM dendrimer generation 0 could be effectively used for rapid encapsulation of a fungal protease from Aspegilus Oryzae.

General significance

Encapsulation significantly enhances the thermal and ultrasonic stabilities of enzymes, suggesting a range of diverse applications for them.     

D-4F decreases brain arteriole inflammation and improves cognitive performance in LDL receptor-null mice on a Western diet

LDL receptor-null mice on a Western diet (WD) have inflammation in large arteries and endothelial dysfunction in small arteries, which are improved with the apolipoprotein A-I mimetic D-4F. The role of hyperlipidemia in causing inflammation of very small vessels such as brain arterioles has not previously been studied. A WD caused a marked increase in the percent of brain arterioles with associated macrophages (microglia) (P < 0.01), which was reduced by oral D-4F but not by scrambled D-4F (ScD-4F; P < 0.01). D-4F (but not ScD-4F) reduced the percent of brain arterioles associated with CCL3/macrophage inflammatory protein-1alpha (P < 0.01) and CCL2/monocyte chemoattractant protein-1 (P < 0.001). A WD increased (P < 0.001) brain arteriole wall thickness and smooth muscle alpha-actin, which was reduced by D-4F but not by ScD-4F (P < 0.0001). There was no difference in plasma lipid levels, blood pressure, or arteriole lumen diameter with D-4F treatment. Cognitive performance in the T-maze continuous alternation task and in the Morris Water Maze was impaired by a WD and was significantly improved with D-4F but not ScD-4F (P < 0.05). We conclude that a WD induces brain arteriole inflammation and cognitive impairment that is ameliorated by oral D-4F without altering plasma lipids, blood pressure, or arteriole lumen size.     

Regiocontrolled synthesis and HIV inhibitory activity of unsymmetrical binaphthoquinone and trimeric naphthoquinone derivatives of conocurvone

Unsymmetrical biquinone and trimeric quinone derivatives were synthesized using halotriflate-biselectrophilic naphthoquinones through stepwise regioselective quinone substitution chemistry and evaluated for their ability to inhibit the cytopathogenic effects of HIV-1 using an MTT colorimetric assay. Compounds were also screened for their ability to inhibit the activity of HIV-1 integrase in vitro. Pyranylated trimeric quinones and biquinones exhibited both antiviral activity and integrase inhibitory activity. Conocurvone 1 and trimeric quinone 21 were the most potent HIV integrase inhibitors in the series. All of the biquinones showed HIV inhibitory activity. Simple methoxy substituted biquinones did not inhibit HIV-1 integrase.