Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher''s forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.     

Correlates of delayed disease progression in HIV-1-infected Kenyan children

Without treatment most HIV-1-infected children in Africa die before their third birthday (>89%) and long-term nonprogressors are rare. The mechanisms underlying nonprogression in HIV-1-infected children are not well understood. In the present study, we examined potential correlates of delayed HIV disease progression in 51 HIV-1-infected African children. Children were assigned to progression subgroups based on clinical characterization. HIV-1-specific immune responses were studied using a combination of ELISPOT assays, tetramer staining, and FACS analysis to characterize the magnitude, specificity, and functional phenotype of HIV-1-specific CD8(+) and CD4(+) T cells. Host genetic factors were examined by genotyping with sequence-specific primers. HIV-1 nef gene sequences from infecting isolates from the children were examined for potential attenuating deletions. Thymic output was measured by T cell rearrangement excision circle assays. HIV-1-specific CD8(+) T cell responses were detected in all progression groups. The most striking attribute of long-term survivor nonprogressors was the detection of HIV-1-specific CD4(+) Th responses in this group at a magnitude substantially greater than previously observed in adult long-term nonprogressors. Although long-term survivor nonprogressors had a significantly higher percentage of CD45RA(+)CD4(+) T cells, nonprogression was not associated with higher thymic output. No protective genotypes for known coreceptor polymorphisms or large sequence deletions in the nef gene associated with delayed disease progression were identified. In the absence of host genotypes and attenuating mutations in HIV-1 nef, long-term surviving children generated strong CD4(+) T cell responses to HIV-1. As HIV-1-specific helper cells support anti-HIV-1 effector responses in active disease, their presence may be important in delaying disease progression.     

Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.     

Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome $\text{c}$ reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Assembly with the Cul4A-DDB1DCAF1 ubiquitin ligase protects HIV-1 Vpr from proteasomal degradation

Many viruses subvert the host ubiquitin-proteasome system to optimize their life cycle. We recently documented such a mechanism for the human immunodeficiency virus type 1 Vpr protein, which promotes cell cycle arrest by recruiting the DCAF1 adaptor of the Cul4A-DDB1 ubiquitin ligase, a finding now confirmed by several groups. Here we examined the impact of Cul4A-DDB1(DCAF1) on Vpr stability. We show that the Vpr(Q65R) mutant, which is defective in DCAF1 binding, undergoes proteasome-mediated degradation at a higher rate than wild-type Vpr. DCAF1 overexpression stabilizes wild-type Vpr and leads to its cytoplasmic accumulation, whereas it has no effect on the Vpr(Q65R) mutant. Conversely, small interfering RNA-mediated silencing of DCAF1 decreases the steady state amount of the viral protein. Stabilization by DCAF1, which is conserved by Vpr species from human immunodeficiency virus type 2 and the SIVmac strain, results in increased G(2) arrest and requires the presence of DDB1, indicating that it occurs through assembly of Vpr with a functional Cul4A-DDB1(DCAF1) complex. Furthermore, in human immunodeficiency virus type 1-infected cells, the Vpr protein, issued from the incoming viral particle, is destabilized under DCAF1 or DDB1 silencing. Together with our previous findings, our data suggest that Cul4A-DDB1(DCAF1) acts at a dual level by providing Vpr with the equipment for the degradation of specific host proteins and by counter-acting its proteasome targeting by another cellular E3 ubiquitin ligase. This protection mechanism may represent an efficient way to optimize the activity of Vpr molecules that are delivered by the incoming virus before neosynthesis takes place. Targeting the Vpr-DCAF1 interaction might therefore present therapeutic interest.     

Uptake of phosphate from soils and fertilizers as affected by soil P availability and solubility of phosphorus fertilizers

Fertilizers labelled with ³²P were used to measure amounts of phosphorus, P_s and P_F, taken up by Lolium perenne from available soil P and from P fertilizer respectively, when applied at a rate of 66 mg P·(kg soil^–1) in greenhouse experiments. The quantity P_s of phosphorus taken up from soil in the presence of P fertilizer was compared to the quantity P_o taken up from soil without P fertilizer. The quantity (P_s–P_o) is positive for low P_o values, i.e. in soils poor in available phosphorus, but is negative for high P_o values indicating that an input of P fertilizer can induce a decrease in the utilization of available soil phosphorus. Moreover, for a given soil, the quantity (P_s–P_o) depends on the chemical form of the fertilizer. The standard method of evaluation of P fertilizer efficiency is based on the assumption that P_s=P_o, but P_s can differ from P_o. This result can explain the contradictory data published from field experiments about the efficiency of the various P fertilizers.     

The superoxide-generating oxidase of phagocytic cells. Physiological, molecular and pathological aspects.

Professional phagocytes (neutrophils, eosinophils, monocytes and macrophages) possess an enzymatic complex, the NADPH oxidase, which is able to catalyze the one-electron reduction of molecular oxygen to superoxide, O2-. The NADPH oxidase is dormant in non-activated phagocytes. It is suddenly activated upon exposure of phagocytes to the appropriate stimuli and thereby contributes to the microbicidal activity of these cells. Oxidase activation in phagocytes involves the assembly, in the plasma membrane, of membrane-bound and cytosolic components of the oxidase complex, which were diassembled in the resting state. One of the membrane-bound components in resting phagocytes has been identified as a low-potential b-type cytochrome, a heterodimer composed of two subunits of 22-kDa and 91-kDa. The link between NADPH and cytochrome b is probably a flavoprotein whose subcellular localization in resting phagocytes remains to be determined. Genetic defects in the cytochrome b subunits and in the cytosolic factors have been shown to be the molecular basis of chronic granulomatous disease, a group of inherited disorders in the host defense, characterized by severe, recurrent bacterial and fungal infections in which phagocytic cells fail to generate O2- upon stimulation. The present review is focused on recent data concerning the signaling pathway which leads to oxidase activation, including specific receptors, the production of second messengers, the organization of the oxidase complex and the molecular defects responsible for granulomatous disease.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminals triggers a Ca-dependent acetylcholine release

The venom glands of the annelid Glycera convoluta contain a neurotoxin which triggers ACh release from frog motor terminals and Torpedo synaptosomes. This neurotoxin binds to presynaptic, but not postsynaptic plasma membranes prepared from Torpedo electric organ. The binding site is an ectocellularly oriented protein. The binding does not require Ca. It is inhibited by pretreatment of the membrane by Concanavalin A. The toxin induced ACh release is Ca-dependent and inhibited by D 600.     

Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex

Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, ?B and ?C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.