Novel features of the rabbit transverse tubular system revealed by quantitative analysis of three-dimensional reconstructions from confocal images

With scanning confocal microscopy we obtained three-dimensional (3D) reconstructions of the transverse tubular system (t-system) of rabbit ventricular cells. We accomplished this by labeling the t-system with dextran linked to fluorescein or, alternatively, wheat-germ agglutinin conjugated to an Alexa fluor dye. Image processing and visualization techniques allowed us to reconstruct the t-system in three dimensions. In a myocyte lying flat on a coverslip, t-tubules typically progressed from its upper and lower surfaces. 3D reconstructions of the t-tubules also suggested that some of them progressed from the sides of the cell. The analysis of single t-tubules revealed novel morphological features. The average diameter of single t-tubules from six cells was estimated to 448 ± 172 nm (mean ± SD, number of t-tubules 348, number of cross sections 5323). From reconstructions we were able to identify constrictions occurring every 1.87 ± 1.09 μm along the principal axis of the tubule. The cross-sectional area of these constrictions was reduced to an average of 57.7 ± 27.5% (number of constrictions 170) of the adjacent local maximal areas. Principal component analysis revealed flattening of t-tubular cross sections, confirming findings that we obtained from electron micrographs. Dextran- and wheat-germ agglutinin-associated signals were correlated in the t-system and are therefore equally good markers. The 3D structure of the t-system in rabbit ventricular myocytes seems to be less complex than that found in rat. Moreover, we found that t-tubules in rabbit have approximately twice the diameter of those in rat. We speculate that the constrictions (or regions between them) are sites of dyadic clefts and therefore can provide geometric markers for colocalizing dyadic proteins. In consideration of the resolution of the imaging system, we suggest that our methods permit us to obtain spatially resolved 3D reconstructions of the t-system in rabbit cells. We also propose that our methods allow us to characterize pathological defects of the t-system, e.g., its remodeling as a result of heart failure.     

Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome

To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPgammaS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)-depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPgammaS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of alpha-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.     

Generation of functional neutrophils from a mouse model of X-linked chronic granulomatous disorder using induced pluripotent stem cells

Murine models of human genetic disorders provide a valuable tool for investigating the scope for application of induced pluripotent stem cells (iPSC). Here we present a proof-of-concept study to demonstrate generation of iPSC from a mouse model of X-linked chronic granulomatous disease (X-CGD), and their successful differentiation into haematopoietic progenitors of the myeloid lineage. We further demonstrate that additive gene transfer using lentiviral vectors encoding gp91(phox) is capable of restoring NADPH-oxidase activity in mature neutrophils derived from X-CGD iPSC. In the longer term, correction of iPSC from human patients with CGD has therapeutic potential not only through generation of transplantable haematopoietic stem cells, but also through production of large numbers of autologous functional neutrophils.     

Risk management in optimal foragers: the effect of sightlines and predator type on patch use,time allocation,and vigilance in gerbils

In the foraging game between gerbils and their predators, gerbils manage risk of predation using the tools of time allocation (where, when and for how long to forage) and vigilance. The optimal level of a forager's vigilance should be affected by its encounter rate with predators and the effectiveness of its vigilance in reducing mortality risk. The physical structure of the environment can alter the effectiveness of its vigilance and therefore alter its foraging behaviour. We tested this for gerbils at risk of predation from barn owls or foxes in a large vivarium. In particular, we reduced the effectiveness of vigilance by placing obstructions around feeding trays that blocked sight lines along either the vertical (vigilance directed against owls) or horizontal axis (vigilance directed against foxes), thereby changing the physical structure of the environment. In addition, we manipulated the presence of foxes and owls. In general, gerbils harvested fewer seeds, allocated less time to foraging in dangerous patches, and used more vigilance while foraging where and when risks were higher (i.e. in the presence of predators and in bright moonlight). Vertical and horizontal sightline treatments interacted synergistically to further raise perceived risk. These results imply that blocking sight lines reduces the effectiveness of vigilance, causing gerbils to use it less. Moreover, in the presence of a predator, the gerbils’ response to the blocked sightlines was more severe – harvesting less food and spending less time and vigilance – in the patches with the increased risk. This was especially so in the presence of the predator that was expected to most benefit from blocking that particular type of sight line: cover that blocked vertical sight lines was riskiest in the presence of owls, and cover that blocked horizontal sight lines was riskiest in the presence of foxes. These results strongly indicate the importance of sightlines and landscape features such as bushes in the risk management and forging decisions of gerbils, demonstrating that bush cover provides mixed blessing to gerbils by providing cover, but making vigilance ineffective.     

Steroids and related products. 39. The functionalization of the 17-methyl group of 17 -methyl steroids. II. The synthesis of 12 ,17 1 -epoxy-17 -methylprogesterone and of 20-oxygenated 17 -hydroxymethyl steroids

The functionalization of the 17-methyl group of 17α-methylated 20-oxygenated steroids is reported. Two methods were employed for that purpose: the Jeger reaction with lead tetraacetate, applied to a 12α-hydroxy 17α-methyletianic ester, leading to a 12,17¹-epoxide and the photolytic Barton reaction, applied to the corresponding 12-nitrite, and leading to a 17¹-nitroso derivative which was transformed into a 17β-methoxycarbonyl lactone of a 12α-hydroxy 17α-etianic acid. The 17β-ester group of this compound can be selectively hydrolyzed and the lactone group can be reduced to a 12α-hydroxy 17α-hydroxymethyl steroid. The synthesis of 12,17¹-epoxy-17α-methylprogesterone is also described.