A Phylogenetic Framework for the Aquaporin Family in Eukaryotes

A comprehensive evolutionary analysis of aquaporins, a family of intrinsic membrane proteins that function as water channels, was conducted to establish groups of homology (i.e., to identify orthologues and paralogues) within the family and to gain insights into the functional constraints acting on the structure of the aquaporin molecule structure. Aquaporins are present in all living organisms, and therefore, they provide an excellent opportunity to further our understanding of the broader biological significance of molecular evolution by gene duplication followed by functional and structural specialization. Based on the resulting phylogeny, the 153 channel proteins analyzed were classified into six major paralogous groups: (1) GLPs, or glycerol-transporting channel proteins, which include mammalian AQP3, AQP7, and AQP9, several nematode paralogues, a yeast paralogue, and Escherichia coli GLP; (2) AQPs, or aquaporins, which include metazoan AQP0, AQP1, AQP2, AQP4, AQP5, and AQP6; (3) PIPs, or plasma membrane intrinsic proteins of plants, which include PIP1 and PIP2; (4) TIPs, or tonoplast intrinsic proteins of plants, which include alphaTIP, gammaTIP, and deltaTIP; (5) NODs, or nodulins of plants; and (6) AQP8s, or metazoan aquaporin 8 proteins. Of these groups, AQPs, PIPs, and TIPs cluster together. According to the results, the capacity to transport glycerol shown by several members of the family was acquired only early in the history of the family. The new phylogeny reveals that several water channel proteins are misclassified and require reassignment, whereas several previously undetermined ones can now be classified with confidence. The deduced phylogenetic framework was used to characterize the molecular features of water channel proteins. Three motifs are common to all family members: AEF (Ala-Glu-Phe), which is located in the N-terminal domain; and two NPA (Asp-Pro-Ala) boxes, which are located in the center and C-terminal domains, respectively. Other residues are found to be conserved within the major groups but not among them. Overall, the PIP subfamily showed the least variation. In general, no radical amino acid replacements affecting tertiary structure were identified, with the exception of Ala-->Ser in the TIP subfamily. Constancy of rates of evolution was demonstrated within the different paralogues but rejected among several of them (GLP and NOD).     

Acinetobacter baumannii RecA protein in repair of DNA damage, antimicrobial resistance, general stress response, and virulence

RecA is the major enzyme involved in homologous recombination and plays a central role in SOS mutagenesis. In Acinetobacter spp., including Acinetobacter baumannii , a multidrug-resistant bacterium responsible for nosocomial infections worldwide, DNA repair responses differ in many ways from those of other bacterial species. In this work, the function of A. baumannii RecA was examined by constructing a recA mutant. Alteration of this single gene had a pleiotropic effect, showing the involvement of RecA in DNA damage repair and consequently in cellular protection against stresses induced by DNA damaging agents, several classes of antibiotics, and oxidative agents. In addition, the absence of RecA decreased survival in response to both heat shock and desiccation. Virulence assays in vitro (with macrophages) and in vivo (using a mouse model) similarly implicated RecA in the pathogenicity of A. baumannii . Thus, the data strongly suggest a protective role for RecA in the bacterium and indicate that inactivation of the protein can contribute to a combined therapeutic approach to controlling A. baumannii infections.     

APETALA1 and SEPALLATA3 interact to promote flower development

In Arabidopsis, the closely related APETALA1 (AP1) and CAULIFLOWER (CAL) MADS-box genes share overlapping roles in promoting flower meristem identity. Later in flower development, the AP1 gene is required for normal development of sepals and petals. Studies of MADS-domain proteins in diverse species have shown that they often function as heterodimers or in larger ternary complexes, suggesting that additional proteins may interact with AP1 and CAL during flower development. To identify proteins that may interact with AP1 and CAL, we used the yeast two-hybrid assay. Among the five MADS-box genes identified in this screen, the SEPALLATA3 (SEP3) gene was chosen for further study. Mutations in the SEP3 gene, as well as SEP3 antisense plants that have a reduction in SEP3 RNA, display phenotypes that closely resemble intermediate alleles of AP1. Furthermore, the early flowering phenotype of plants constitutively expressing AP1 is significantly enhanced by constitutive SEP3 expression. Taken together, these studies suggest that SEP3 interacts with AP1 to promote normal flower development.     

RAB6 and dynein drive post‐Golgi apical transport to prevent neuronal progenitor delamination

Radial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that aRG delamination is regulated by the post‐Golgi secretory pathway. Using in situ subcellular live imaging, we show that post‐Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A'' and RAB6B impairs apical localization of CRB3 and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knockout of the dynein activator LIS1. Overall, our results identify a RAB6‐dynein‐LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity.     

Fabrication of conductive fibrous scaffold for photoreceptor differentiation of mesenchymal stem cell

Conductive nanofibrous scaffolds with that can conduct electrical current have a great potential in neural tissue engineering. The purpose of this study was to survey effects of electrical stimulation and polycaprolactone/polypyrrole/multiwall carbon nanotube (PCL/PPY/MWCNTs) fibrous scaffold on photoreceptor differentiation of trabecular meshwork mesenchymal stem cells (TM-MSCs). PCL/PPY/MWCNTs scaffold was made by electrospinning method. TM-MSCs were seeded on PCL/PPY/MWCNTs scaffold and stimulated with a potential of 115 V/m. Scanning electron microscopy, transmission electron microscopy, and FT-IR were used to evaluate the fabricated scaffold. Immunofluorescence and quantitative real-time polymerase chain reaction were used to examine differentiated cells. Scanning electron microscopy, transmitting electron microscopy, and FT-IR confirmed the creation of the composite structure of fibers. RT-qPCR analysis showed that the expression of rhodopsin and peripherin genes in electrically stimulated cells were significantly higher (5.7- and 6.23-fold, respectively; p ≤ 0.05) than those with no electrical stimulation. Collectively, it seems that the combination of PCL/PPY/MWCNTs scaffold, as a suitable conductive scaffold, and electrical stimulation could be an effective approach in the differentiation of stem cells in retinal tissue engineering.     

Oxidative Stress Enhances Neurodegeneration Markers Induced by Herpes Simplex Virus Type 1 Infection in Human Neuroblastoma Cells

Mounting evidence suggests that Herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of Alzheimer’s disease (AD). Previous work from our laboratory has shown HSV-1 infection to induce the most important pathological hallmarks of AD brains. Oxidative damage is one of the earliest events of AD and is thought to play a crucial role in the onset and development of the disease. Indeed, many studies show the biomarkers of oxidative stress to be elevated in AD brains. In the present work the combined effects of HSV-1 infection and oxidative stress on Aβ levels and autophagy (neurodegeneration markers characteristic of AD) were investigated. Oxidative stress significantly potentiated the accumulation of intracellular Aβ mediated by HSV-1 infection, and further inhibited its secretion to the extracellular medium. It also triggered the accumulation of autophagic compartments without increasing the degradation of long-lived proteins, and enhanced the inhibition of the autophagic flux induced by HSV-1. These effects of oxidative stress were not due to enhanced virus replication. Together, these results suggest that HSV-1 infection and oxidative damage interact to promote the neurodegeneration events seen in AD.     

Inactivating mutations block the tumor necrosis factor-alpha-converting enzyme in the early secretory pathway

The ectodomain of different transmembrane molecules is released by a proteolytic event known as shedding. The metalloprotease disintegrin proTNF-alpha converting enzyme (TACE) is responsible for the shedding of various proteins, including protransforming growth factor-alpha (proTGF-alpha) and amyloid-beta precursor protein (APP). Inactive TACE accumulates in the early secretory pathway of cell mutants (M1 and M2) defective in proTGF-alpha and APP shedding. Although previous evidences indicated that the component mutated in M1 and M2 cells is different from TACE, recent results show the existence of two heterozygous point mutations in TACE from M2 cells. Here, we show that wild-type TACE stably transfected in M2 cells is processed, transported to the cell surface, and rescues the proTGF-alpha and APP shedding-defective phenotype. Furthermore, M1 cells also express mutant TACE and transfection with wild-type TACE restores the wild-type phenotype. Therefore, different inactivating mutations result in the accumulation of TACE in the early secretory pathway, emphasizing the importance of the initial steps in the biosynthesis of TACE.     

Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity

This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10-20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10-20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO4.7H2O, KH2PO4, K2HPO4 and NH4NO3 exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C6H11O7Na, 7.5; MgSO4.7H2O, 0.35; K2HPO4, 0.35; NH4NO3, 0.5; KH2PO4, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30 degrees C.     

IL-12 and IL-10 expression synergize to induce the immune-mediated eradication of established colon and mammary tumors and lung metastasis

Preclinical studies demonstrated that certain cytokines are potentially useful for the induction of antitumor immune responses. However, their administration in clinical settings was only marginally useful and evoked serious toxicity. In this study, we demonstrate that the combination of autologous inactivated tumor cells expressing IL-12 and IL-10 induced tumor remission in 50-70% of mice harboring large established colon or mammary tumors and spontaneous lung metastases, with the consequent establishment of an antitumor immune memory. Mice treatment with tumor cells expressing IL-12 was only marginally effective, while expression of IL-10 was not effective at all. Administration of the combined immunotherapy stimulated the recruitment of a strong inflammatory infiltrate that correlated with local, increased expression levels of the chemokines MIP-2, MCP-1, IFN-gamma-inducible protein-10, and TCA-3 and the overexpression of IFN-gamma, but not IL-4. The combined immunotherapy was also therapeutically effective on established lung metastases from both colon and mammary tumors. The antitumor effect of the combined immunotherapy was mainly dependent on CD8+ cells although CD4+ T cells also played a role. The production of IFN-gamma and IL-4 by spleen cells and the development of tumor-specific IgG1 and IgG2a Abs indicate that each cytokine stimulated its own Th pathway and that both arms were actively engaged in the antitumor effect. This study provides the first evidence of a synergistic antitumor effect of IL-12 and IL-10 suggesting that a Th1 and a Th2 cytokine can be effectively combined as a novel rational approach for cancer immunotherapy.