Evolution of the dorsal-ventral patterning network in the mosquito, Anopheles gambiae

The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. We wish to understand how changes in this network produce evolutionary diversity in insect gastrulation. The present study focuses on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene.     

Distinct Requirements for HIV-Cell Fusion and HIV-mediated Cell-Cell Fusion

Whether HIV-1 enters cells by fusing with the plasma membrane or with endosomes is a subject of active debate. The ability of HIV-1 to mediate fusion between adjacent cells, a process referred to as “fusion-from-without” (FFWO), shows that this virus can fuse with the plasma membrane. To compare FFWO occurring at the cell surface with HIV-cell fusion through a conventional entry route, we designed an experimental approach that enabled the measurements of both processes in the same sample. The following key differences were observed. First, a very small fraction of viruses fusing with target cells participated in FFWO. Second, whereas HIV-1 fusion with adherent cells was insensitive to actin inhibitors, post-CD4/coreceptor binding steps during FFWO were abrogated. A partial dependence of HIV-cell fusion on actin remodeling was observed in CD4⁺ T cells, but this effect appeared to be due to the actin dependence of virus uptake. Third, deletion of the cytoplasmic tail of HIV-1 gp41 dramatically enhanced the ability of the virus to promote FFWO, while having a modest effect on virus-cell fusion. Distinct efficiencies and actin dependences of FFWO versus HIV-cell fusion are consistent with the notion that, except for a minor fraction of particles that mediate fusion between the plasma membranes of adjacent cells, HIV-1 enters through an endocytic pathway. We surmise, however, that cell-cell contacts enabling HIV-1 fusion with the plasma membrane could be favored at the sites of high density of target cells, such as lymph nodes.     

Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory

Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h^−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h^−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h^−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system.     

Synthesis and evaluation of novel pyrimidine-based dual EGFR/Her-2 inhibitors

A structure-activity relationship study of 4-anilinopyrimidines for dual EGFR/Her-2 inhibitor has resulted in the identification of 4-anilino-5-alkenyl or 5-alkynyl-6-methylpyrimidine derivatives that have exhibited effective inhibitory activity against both enzymes. The presence of 5-alkenyl or 5-alkynyl moiety bearing terminal hydrophilic group played important role for inhibition of these enzymes. Selected compounds in the series demonstrated some activity against Her-2 dependent cell line (BT474).     

KF-1 Ubiquitin Ligase: Anxiety Suppressor Model

Anxiety disorders are the most popular psychiatric disease in any human societies irrespective of nation, culture, religion, economics or politics. Anxiety expression mediated by the amygdala may be suppressed by signals transmitted from the prefrontal cortex and hippocampus. KF-1 is an endoplasmic reticulum (ER)-based E3-ubiquitin (Ub) ligase with a RING-H2 finger motif at the C-terminus. The kf-1 gene expression is up-regulated in the frontal cortex and hippocampus in rats after anti-depressant treatments. The kf-1 null mice show no apparent abnormalities, but exhibit selectively pronounced anxiety-like behaviors or increased timidity-like responses. The kf-1 orthologous genes had been generated after the Poriferan emergence, and are found widely in all animals except insects, arachnids and threadworms such as Drosophila, Ixodes and Caenorhabditis, respectively. This suggests that the kf-1 gene may be relevant to some biological functions characteristic to animals. Based on these observations, the Anxiety Suppressor Model has been proposed, which assumes that KF-1 Ub ligase may suppress the amygdala-mediated anxiety by degrading some anxiety promoting protein(s), such as a neurotransmitter receptor, through the ER-associated degradation pathway in the frontal cortex and hippocampus. According to this model, the emotional sensitivity to environmental stresses may be regulated by the cellular protein level of KF-1 relative to that of the putative anxiety promoter. The kf-1 null mice should be useful in elucidating the molecular mechanisms of the anxiety regulation and for screening novel anxiolytic compounds, which may block the putative anxiety promoter.     

The aglomerular kidney of the deep-sea gulper eel <Emphasis Type="Italic">Saccopharynx ampullaceus</Emphasis> (Saccopharyngiformes: Saccopharyngidae)

We report the presence of an aglomerular kidney in the pelagic deep-sea fish Saccopharynx ampullaceus (Saccopharyngiformes: Saccopharyngidae). The thin kidney is unpaired and ribbon-like rostrally, while it is thicker caudally with a rod-like shape. Light microscopic observation of serial sections revealed no glomeruli at all. The kidney is composed of renal tubules, sinusoids and capillaries of the renal portal system and extensive interstitial lymphoid tissues. Each renal tubule is surrounded by well-developed renal portal sinusoids, and the tubules are well separated from each other. There is a large space dorsal to the vertebrae, similar to the situation in the closely related Eurypharynx pelecanoides. We consider that S. ampullaceus possesses an aglomerular kidney to gain neutral buoyancy. The urinary bladder of S. ampullaceus is a distinct vesicular structure, unlike that of E. pelecanoides.     

Probiotic-derived polyphosphate enhances the epithelial barrier function and maintains intestinal homeostasis through integrin-p38 MAPK pathway

Probiotics exhibit beneficial effects on human health, particularly in the maintenance of intestinal homeostasis in a complex manner notwithstanding the diversity of an intestinal flora between individuals. Thus, it is highly probable that some common molecules secreted by probiotic and/or commensal bacteria contribute to the maintenance of intestinal homeostasis and protect the intestinal epithelium from injurious stimuli. To address this question, we aimed to isolate the cytoprotective compound from a lactobacillus strain, Lactobacillus brevis SBC8803 which possess the ability to induce cytoprotective heat shock proteins in mouse small intestine. L. brevis was incubated in MRS broth and the supernatant was passed through with a 0.2-μm filter. Caco2/bbe cells were treated with the culture supernatant, and HSP27 expression was evaluated by Western blotting. HSP27-inducible components were separated by ammonium sulfate precipitation, DEAE anion exchange chromatography, gel filtration, and HPLC. Finally, we identified that the HSP27-inducible fraction was polyphosphate (poly P), a simple repeated structure of phosphates, which is a common product of lactobacilli and other bacteria associated with intestinal microflora without any definitive physiological functions. Then, poly P was synthesized by poly P-synthesizing enzyme polyphosphate kinase. The synthesized poly P significantly induced HSP27 from Caco2/BBE cells. In addition, Poly P suppressed the oxidant-induced intestinal permeability in the mouse small intestine and pharmacological inhibitors of p38 MAPK and integrins counteract its protective effect. Daily intrarectal administration of poly P (10 μg) improved the inflammation grade and survival rate in 4% sodium dextran sulfate-administered mice. This study, for the first time, demonstrated that poly P is the molecule responsible for maintaining intestinal barrier actions which are mediated through the intestinal integrin β1-p38 MAPK.     

Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

Background

Dendritic cells (DCs) are among the first cells to encounter HIV-1 and play important roles in viral transmission and pathogenesis. Immature DCs allow productive HIV-1 replication and long-term viral dissemination. The pro-inflammatory factor lipopolysaccharide (LPS) induces DC maturation and enhances the efficiency of DC-mediated HIV-1 transmission. Type I interferon (IFN) partially inhibits HIV-1 replication and cell-cell transmission in CD4⁺ T cells and macrophages. Tetherin is a type I IFN-inducible restriction factor that blocks HIV-1 release and modulates CD4⁺ T cell-mediated cell-to-cell transmission of HIV-1. However, the role of type I IFN and tetherin in HIV-1 infection of DCs and DC-mediated viral transmission remains unknown.

Results

We demonstrated that IFN-alpha (IFNα)-induced mature DCs restricted HIV-1 replication and trans-infection of CD4⁺ T cells. Tetherin expression in monocyte-derived immature DCs was undetectable or very low. High levels of tetherin were transiently expressed in LPS- and IFNα-induced mature DCs, while HIV-1 localized into distinct patches in these DCs. Knockdown of induced tetherin in LPS- or IFNα-matured DCs modestly enhanced HIV-1 transmission to CD4⁺ T cells, but had no significant effect on wild-type HIV-1 replication in mature DCs. Intriguingly, we found that HIV-1 replication in immature DCs induced significant tetherin expression in a Nef-dependent manner.

Conclusions

The restriction of HIV-1 replication and transmission in IFNα-induced mature DCs indicates a potent anti-HIV-1 response; however, high levels of tetherin induced in mature DCs cannot significantly restrict wild-type HIV-1 release and DC-mediated HIV-1 transmission. Nef-dependent tetherin induction in HIV-1-infected immature DCs suggests an innate immune response of DCs to HIV-1 infection.