A BAC-based transgenic mouse specifically expresses an inducible Cre in the urothelium

Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC)-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported.     

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

N-Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface.

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wild-type strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm(-1) region were measured for films of the intact and in situ trypsin-degraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm(-1)), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference ('wet' minus 'dry') spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.     

The last 50 years of climate‐induced melting of the Maliy Aktru glacier (Altai Mountains,Russia) revealed in a primary ecological succession

In this article, we report and discuss the results obtained from a survey of plants, microorganisms (bacteria and fungi), and soil elements along a chronosequence in the first 600 m of the Maliy Aktru glacier's forefront (Altai Mountains, Russia). Many glaciers of the world show effects of climate change. Nonetheless, except for some local reports, the ecological effects of deglaciation have been poorly studied and have not been quantitatively assessed in the Altai Mountains. Here, we studied the ecological changes of plants, fungi, bacteria, and soil elements that take the form of a primary ecological succession and that took place over the deglaciated soil of the Maliy Aktru glacier during the last 50 year. According to our measurements, the glacier lost about 12 m per year during the last 50 years. Plant succession shows clear signs of changes along the incremental distance from the glacier forefront. The analysis of the plant α‐ and β‐diversity confirmed an expected increase of them with increasing distance from the glacier forefront. Moreover, the analysis of β‐diversity confirmed the hypothesis of the presence of three main stages of the plant succession: (a) initial (pioneer species) from 30 to 100 m; (b) intermediate (r‐selected species) from 110 to 120–150 m; and (c) final (K‐selected species) from 150 to 550. Our study also shows that saprotrophic communities of fungi are widely distributed in the glacier retreating area with higher relative abundances of saprotroph ascomycetes at early successional stages. The evolution of a primary succession is also evident for bacteria, soil elements, and CO₂ emission and respiration. The development of biological communities and the variation in geochemical parameters represent an irrefutable proof that climate change is altering soils that have been long covered by ice.     

A spectrophotometric assay for nitrate in an excess of nitrite.

Miranda et al. have developed a method for simultaneous evaluation of nitrate and nitrite concentrations using reduction of nitrate by vanadium(III) combined with detection by the acidic Griess reaction [K.M. Miranda, M.G. Espey, D.A. Wink, A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite, Nitric Oxide 5 (2001) 62-71]. The sensitivity of the nitrate assay decline if the mixture analyzed contains a large excess of nitrite relative to nitrate, for instance, in the case of oxidation products of nitric oxide (NO) in aerated solutions, or in sweat. By this reason nitrite should be removed before the nitrate assay, if [NO2-]>[NO3-]. Here we lay out an improved method allowing the above limitation to be erased, using sulfamic acid for nitrite removal. We also describe some modifications that enhance the reproducibility of the assay.     

Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells

Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.     

A micro-fluidic study of whole blood behaviour on PMMA topographical nanostructures

Background  

Polymers are attractive materials for both biomedical engineering and cardiovascular applications. Although nano-topography has been found to influence cell behaviour, no established method exists to understand and evaluate the effects of nano-topography on polymer-blood interaction.