Inactivation of Max-interacting Protein 1 Induces Renal Cilia Disassembly through Reduction in Levels of Intraflagellar Transport 20 in Polycystic Kidney

Cilia in ciliated cells consist of protruding structures that sense mechanical and chemical signals from the extracellular environment. Cilia are assembled with variety molecules via a process known as intraflagellar transport (IFT). What controls the length of cilia in ciliated cells is critical to understand ciliary disease such as autosomal dominant polycystic kidney disease, which involves abnormally short cilia. But this control mechanism is not well understood. Previously, multiple tubular cysts have been observed in the kidneys of max-interacting protein 1 (Mxi1)-deficient mice aged 6 months or more. Here, we clarified the relationship between Mxi1 inactivation and cilia disassembly. Cilia phenotypes were observed in kidneys of Mxi1-deficient mice using scanning electron microscopy to elucidate the effect of Mxi1 on renal cilia phenotype, and cilia disassembly was observed in Mxi1-deficient kidney. In addition, genes related to cilia were validated in vitro and in vivo using quantitative PCR, and Ift20 was selected as a candidate gene in this study. The length of cilium decreased, and p-ERK level induced by a cilia defect increased in kidneys of Mxi1-deficient mice. Ciliogenesis of Mxi1-deficient mouse embryonic fibroblasts (MEFs) decreased, and this abnormality was restored by Mxi1 transfection in Mxi1-deficient MEFs. We confirmed that ciliogenesis and Ift20 expression were regulated by Mxi1 in vitro. We also determined that Mxi1 regulates Ift20 promoter activity via Ets-1 binding to the Ift20 promoter. These results indicate that inactivating Mxi1 induces ciliary defects in polycystic kidney.     

Combinatorial biosynthesis and antibacterial evaluation of glycosylated derivatives of 12-membered macrolide antibiotic YC-17

Expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into Streptomyces venezuelae YJ003 mutant strain bearing a deletion of a desosamine biosynthetic (des) gene cluster. The resulting recombinants produced macrolide antibiotic YC-17 analogs possessing unnatural sugars replacing native d-desosamine. These metabolites were isolated and further purified using chromatographic techniques and their structures were determined as d-quinovosyl-10-deoxymethynolide, l-rhamnosyl-10-deoxymethynolide, l-olivosyl-10-deoxymethynolide, and d-boivinosyl-10-deoxymethynolide on the basis of 1D and 2D NMR and MS analyses and the stereochemistry of sugars was confirmed using coupling constant values and NOE correlations. Their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with l-rhamnose displayed better antibacterial activity than parent compound YC-17 containing native sugar d-desosamine. The present study on relationships between chemical structures and antibacterial activities could be useful in generation of novel advanced antibiotics utilizing combinatorial biosynthesis approach.     

Nitrogen, organic carbon and sulphur cycling in terrestrial ecosystems: linking nitrogen saturation to carbon limitation of soil microbial processes

Elevated and chronic nitrogen (N) deposition to N-limited terrestrial ecosystems can lead to ‘N saturation’, with resultant ecosystem damage and leaching of nitrate (NO₃ ^?) to surface waters. Present-day N deposition, however, is often a poor predictor of NO₃ ^? leaching, and the pathway of the ecosystem transition from N-limited to N-saturated remains incompletely understood. The dynamics of N cycling are intimately linked to the associated carbon (C) and sulphur (S) cycles. We hypothesize that N saturation is associated with shifts in the microbial community, manifest by a decrease in the fungi-to-bacteria ratio and a transition from N to C limitation. Three mechanisms could lead to lower amount of bioavailable dissolved organic C (DOC) for the microbial community and to C limitation of N-rich systems: (1) Increased abundance of N for plant uptake, causing lower C allocation to plant roots; (2) chemical suppression of DOC solubility by soil acidification; and (3) enhanced mineralisation of DOC due to increased abundance of electron acceptors in the form of ${{\text{SO}}_{ 4}}^{ 2-}$ SO 4 2 ? and NO₃ ^? in anoxic soil micro-sites. Here we consider each of these mechanisms, the extent to which their hypothesised impacts are consistent with observations from intensively-monitored sites, and the potential to improve biogeochemical models by incorporating mechanistic links to the C and S cycles.     

EVOLUTION OF SEX DETERMINATION SYSTEMS WITH HETEROGAMETIC MALES AND FEMALES IN SILENE

The plant genus Silene has become a model for evolutionary studies of sex chromosomes and sex‐determining mechanisms. A recent study performed in Silene colpophylla showed that dioecy and the sex chromosomes in this species evolved independently from those in Silene latifolia, the most widely studied dioecious Silene species. The results of this study show that the sex‐determining system in Silene otites, a species related to S. colpophylla, is based on female heterogamety, a sex determination system that is unique among the Silene species studied to date. Our phylogenetic data support the placing of S. otites and S. colpophylla in the subsection Otites and the analysis of ancestral states suggests that the most recent common ancestor of S. otites and S. colpophylla was most probably dioecious. These observations imply that a switch from XX/XY sex determination to a ZZ/ZW system (or vice versa) occurred in the subsection Otites. This is the first report of two different types of heterogamety within one plant genus of this mostly nondioecious plant family.     

Dual Gating Mechanism and Function of P2X7 Receptor Channels

The ATP-gated P2X7 receptor channel (P2X7R) operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. To address this question, we have combined electrophysiological, imaging, and mathematical studies using wild-type and mutant rat P2X7Rs. Activation of naïve (not previously stimulated) receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high-amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov-state model that included, in addition to naïve and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of naïve receptors favored a slow transition to desensitized states, whereas occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions.     

Vhc1, a novel transporter belonging to the family of electroneutral cation–Cl− cotransporters,participates in the regulation of cation content and morphology of Saccharomyces cerevisiae vacuoles

Cation–Cl^? cotransporters (CCCs) are integral membrane proteins which catalyze the coordinated symport of Cl^? with Na⁺ and/or K⁺ ions in plant and mammalian cells. Here we describe the first Saccharomyces cerevisiae CCC protein, encoded by the YBR235w open reading frame. Subcellular localization studies showed that this yeast CCC is targeted to the vacuolar membrane. Deletion of the YBR235w gene in a salt-sensitive strain (lacking the plasma-membrane cation exporters) resulted in an increased sensitivity to high KCl, altered vacuolar morphology control and decreased survival upon hyperosmotic shock. In addition, deletion of the YBR235w gene in a mutant strain deficient in K⁺ uptake produced a significant growth advantage over the parental strain under K⁺-limiting conditions, and a hypersensitivity to the exogenous K⁺/H⁺ exchanger nigericin. These results strongly suggest that we have identified a novel yeast vacuolar ion transporter mediating a K⁺–Cl^? cotransport and playing a role in vacuolar osmoregulation. Considering its identified function, we propose to refer to the yeast YBR235w gene as VHC1 (vacuolar protein homologous to CCC family 1).     

The first case of fatal pneumonia caused by Panton–Valentine leukocidin-producing Staphylococcus aureus in an infant in the Czech Republic

Panton–Valentine leukocidin-producing strains of Staphylococcus aureus can cause severe skin and soft tissue infections and necrotizing pneumonia with a high mortality rate. This is a report on the first case of fatal pneumonia with mediastinitis in an infant in the Czech Republic. The causative agent was a methicillin-susceptible S. aureus strain with pronounced production of the PVL toxin and hyperproduction of enterotoxin A.     

The ameliorating effects of 2,3-dihydroxy-4-methoxyacetophenone on scopolamine-induced memory impairment in mice and its neuroprotective activity

We isolated 2,3-dihydroxy-4-methoxyacetophenone, a neuroprotective compound from Cynenchum paniculatum in our previous study.The present study was conducted to investigate the possible neuroprotective effect of 2,3-dihydroxy-4-methoxyacetophenone that has been previously isolated from Cynenchum paniculatum on hippocampal neuronal cell line, HT22 cells and its possible cognitive-enhancing effect on scopolamine-induced amnesia in mice.Neuroprotective effect against glutamate-induced neurotoxicity in HT22 cells was evaluated by MTT assay. Also, cognitive enhancing effect against scopolamine (1 mg/kg, ip) induced learning and memory deficit was measured by Morris water maze test. Oral administered of 2,3-dihydroxy-4-methoxyacetophenone (1, 10, 20, 40 and 50 mg/kg) to amnesic mice induced by scopolamine. In Morris water maze test, 2,3-dihydroxy-4-methoxyacetophenone (50 mg/kg) improved the impairment of spatial memory induced by scopolamine. 2,3-Dihydroxy-4-methoxyacetophenone protect HT22 cells on glutamate induced cell-death in a dose-dependent manner (EC₅₀ value: 10.94 μM). Furthermore, 2,3-dihydroxy-4-methoxyacetophenone was found to inhibit [Ca²⁺] accumulation in HT22 cells and had antioxidantive activity. The results showed that 2,3-dihydroxy-4-methoxyacetophenone exert neuroprotective and cognitive-enhancing activities through its antioxidant activity. We suggest that 2,3-dihydroxy-4-methoxyacetophenone improves cognitive function and may be helpful for the treatment of Alzheimer’s disease.     

The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.