Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca²⁺ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.     

MiR-3691-5p is upregulated in docosahexaenoic acid-treated vascular endothelial cell and targets serpin family E member 1

Endothelium, the inner cellular lining of blood vessels, has an important role in the regulation of physiological processes and its dysfunction may initiate cardiovascular complications. Previous investigations have revealed that dietary docosahexaenoic acid (DHA) is related to a lower possibility of cardiovascular disease and mortality. Until now, the molecular mechanisms in the biological activities of DHA remain largely unknown. MicroRNAs (miRNAs) play a vital role in regulating gene expression. Thus, we aimed to investigate whether DHA improves the dysfunction via regulating miRNAs. To understand the protective effects of DHA through modulating miR-3691-5p and its target genes for palmitic acid (PAL) induced apoptosis in endothelial cells. The present study demonstrated that DHA upregulated miR-3691-5p expression, and downregulated the expression of its target gene serpin family E member 1 (SERPINE1). MiR-mimics and inhibitors modulation results indicated that miR-3691-5p regulates endothelial apoptosis through activating antiapoptotic response which controlled by the STAT3 signaling pathway. In conclusion, we have shown that PAL-induced apoptosis could be decreased by DHA treatment through miR-3691-5/SERPINE1 pathways.     

Transforming growth factor-β promotes the function of HIV-specific CXCR5+CD8 T cells

HIV replication can be inhibited by CXCR5⁺CD8 T cells (follicular cytotoxic T cell [TFC]) which transfer into B-cell follicles where latent HIV infection persists. However, how cytokines affect TFC remain unclear. Understanding which cytokines show the ability to affect TFC could be a key strategy toward curing HIV. Similar mechanisms could be used for the growth and transfer of TFCs and follicular helper T (TFH) cells; as a result, we hypothesized that cytokines IL-6, IL-21, and transforming growth factor-β (TGF-β), which are necessary for the differentiation of TFH cells, could also dictate the development of TFCs. In this work, lymph node mononuclear cells and peripheral blood mononuclear cells from HIV-infected individuals were cocultured with IL-6, IL-21, and TGF-β. We then carried out T-cell receptor (TCR) repertoire analysis to compare the differences between CXCR5^– and CXCR5⁺CD8 T cells. Our results showed that the percentage and function of TFC can be enhanced by stimulation with TGF-β. Besides, TGF-β stimulation enhanced the diversity of TCR and complementarity-determining region 3 sequences. HIV DNA showed a negative correlation with TFC. The use of TGF-β to promote the expression of CXCR5⁺CD8 T cells could become a new treatment approach for curing HIV.     

Reverse genetics system for human rotaviruses

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.     

Integrin participates in the effect of thyroxine on plasma membrane in immature rat testis

Background

The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [¹⁴C]-MeAIB (α-(methyl-amino)isobutyric acid) accumulation stimulated by T₄ and the role of the integrin receptor in this event, and also to clarify whether the T₄ effect on MeAIB accumulation and on Ca²⁺ influx culminates in cell secretion.

Methods

We have studied the rapid and plasma membrane initiated effects of T₄ by using ⁴⁵Ca²⁺ uptake and [⁴⁵C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC.

Results

The stimulation of MeAIB accumulation by T₄ appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T₄ increases extracellular Ca²⁺ uptake and Ca²⁺ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T₄. Also, the cytoskeleton and ClC-3 chloride channel contribute to the membrane-associated responses of SC.

Conclusions

T₄ integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone.

General significance

The integrin receptor activation by T₄ may take a role in plasma membrane processes involved in the male reproductive system.     

Crystal structure of the S187F variant of human liver alanine: Aminotransferase associated with primary hyperoxaluria type I and its functional implications

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5′‐phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP‐binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT‐pyridoxamine 5′‐phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300‐ to 500‐fold decrease in both the rate constant of L‐alanine half‐transamination and the k_cat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results. Proteins 2013; 81:1457–1465. © 2013 Wiley Periodicals, Inc.     

Theoretical Calculations on Zeolites: The Aluminium Substitution in Mordenite,Ferrierite and ZSM-5

Abstract

The experimental determination of Al siting in zeolites involves the use of multiple techniques. Great effort has been made on both, experimental and theoretical approaches. The present study presents a novel methodology for calculating Al/Si replacement energies. Simple semiempirical calculations were applied on Modenite, Ferrierite and ZSM-5 zeolites, resulting in good agreement with the experimetal evidences. We have found that the favored Al substitution sites are T3 and T4 in Mordenite, while T2 and T4 are in Ferrierite, and only the T9 site is favored in ZSM-5. The method presented is based on an average of partial Al/Si replacement energies, evaluated for all rings belonging to each T site, rather than in the calculation of a total replacement energy evaluated for only one representative aggregate.     

ATDC5: An excellent in vitro model cell line for skeletal development

The ATDC5 cell line is derived from mouse teratocarcinoma cells and characterized as a chondrogenic cell line which goes through a sequential process analogy to chondrocyte differentiation. Thus, it is regarded as a promising in vitro model to study the factors that influence cell behaviors during chondrogenesis. It also provides insights in exploring signaling pathways related to skeletal development as well as interactions with innovative materials. To date, over 200 studies have utilized ATDC5 to obtain lots of significant findings. In this review, we summarized the literature of ATDC5 related studies and emphasized the application of ATDC5 in chondrogenesis. In addition, the general introduction of ATDC5 including its derivation and characterization is covered in this article. J. Cell. Biochem. 114: 1223–1229, 2013. © 2012 Wiley Periodicals, Inc.