Repigmentation by outer-root-sheath-derived melanocytes: proof of concept in vitiligo and leucoderma

BACKGROUND: Treatment of depigmented skin is an unmet medical need. OBJECTIVE: Melanocytes or stem cells thereof residing in the outer root sheath (ORS) of hair follicles might be used to repigment skin. METHODS: After de-epidermisation, autologous ORS cell solutions were applied to 5 patients with vitiligo and 1 with leucoderma. RESULTS: Stable repigmentation in a variable percentage was documented in all the patients. CONCLUSION: Applying ORS-derived melanocytes is a promising technology to improve autologous melanocyte transplantation.

Primary splenic peripheral nerve sheath tumour in a dog

An 8-year-old crossbred dog was presented with a one-month history of progressive weakness, respiratory impairment and abdominal distension. Surgical exploration revealed the presence of a splenic mass that infiltrated the mesentery and was adherent to the stomach and pancreas. The mass was composed of highly cellular areas of spindle-shaped cells arranged in interlacing bundles, streams, whorls and storiform patterns (Antoni A pattern) and less cellular areas with more loosely arranged spindle to oval cells (Antoni B pattern). The majority of neoplastic cells expressed vimentin, S-100 and glial fibrillary acidic protein (GFAP), but did not express desmin, alpha-smooth muscle actin or factor VIII. These morphological and immunohistochemical findings characterized the lesion as a malignant peripheral nerve sheath tumour (PNST). Primary splenic PNST has not been documented previously in the dog.

Immunohistochemical evaluation of heat shock protein expression in normal canine nerve and peripheral nerve sheath tumours

Abnormal expression of heat shock proteins (HSPs) has been observed in many human neoplasms and such expression has prognostic, predictive and therapeutic implications. The aim of this study was to evaluate immunohistochemically the expression of HSP 27, HSP 32 and HSP 90 in normal canine peripheral nerves and in four benign and 15 malignant canine peripheral nerve sheath tumours (PNSTs). In normal nerve, all of the HSPs were detected in axons, epineurial fibroblasts and scattered Schwann cell bodies. Cytoplasmic expression of HSP 27 was more widespread and intense in benign PNSTs compared with malignant PNSTs (P <0.05). Widespread and intense nuclear expression of HSP 32 was also associated with benign tumours (P <0.01), while high HSP 90 immunoreactivity was detected in all tumours, suggesting that HSP 90 might represent a new therapeutic target.

Modelling susceptibility of grassland soil to macropore flow

Investigating preferential flow, including macropore flow, is crucial to predicting and preventing point sources of contamination in soil, for example in the vicinity of pumping wells. With a view to advancing groundwater protection, this study aimed (i) to quantify the strength of macropore flow in four representative natural grassland soils on the Swiss plateau, and (ii) to define the parameters that significantly control macropore flow in grassland soil. For each soil type we selected three measurement points on which three successive irrigation experiments were carried out, resulting in a total of 36 irrigations. The strength of macropore flow, parameterized as the cumulated water volume flowing from macropores at a depth of 1 m in response to an irrigation of 60 mm h−1 intensity and 1 h duration, was simulated using the dual-permeability MACRO model. The model calibration was based on the key soil parameters and fine measurements of water content at different depths. Modelling results indicate high performance of macropore flow in all investigated soil types except in gleysols. The volume of water that flowed from macropores and was hence expected to reach groundwater varied between 81% and 94% in brown soils, 59% and 67% in para-brown soils, 43% and 56% in acid brown soils, and 22% and 35% in gleysols. These results show that spreading pesticides and herbicides in pumping well protection zones poses a high risk of contamination and must be strictly prohibited. We also found that organic carbon content was not correlated with the strength of macropore flow, probably due to its very weak variation in our study, while saturated water content showed a negative correlation with macropore flow. The correlation between saturated hydraulic conductivity (Ks) and macropore flow was negative as well, but weak. Macropore flow appears to be controlled by the interaction between the bulk density of the uppermost topsoil layer (0–0.10 m) and the macroporosity of the soil below. This interaction also affects the variations in Ks and saturated water content. Further investigations are needed to better understand the combined effect of all these processes including the exchange between micropore and macropore domains.

Bare-tether sheath and current: comparison of asymptotic theory and kinetic simulations in stationary plasma

Analytical expressions for current to a cylindrical Langmuir probe at rest in unmagnetized plasma are compared with results from both steady-state Vlasov and particle-in-cell simulations. Probe bias potentials that are much greater than plasma temperature (assumed equal for ions and electrons), as of interest for bare conductive tethers, are considered. At a very high bias, both the electric potential and the attracted-species density exhibit complex radial profiles; in particular, the density exhibits a minimum well within the plasma sheath and a maximum closer to the probe. Excellent agreement is found between analytical and numerical results for values of the probe radiusR close to the maximum radius Rmax for orbital-motion-limited (OML) collection at a particular bias in the following number of profile features: the values and positions of density minimum and maximum, position of sheath boundary, and value of a radius characterizing the no-space-charge behavior of a potential near the high-bias probe. Good agreement between the theory and simulations is also found for parametric laws jointly covering the following three characteristic R ranges: sheath radius versus probe radius and bias for Rmax; density minimum versus probe bias for Rmax; and (weakly bias-dependent) current drop below the OML value versus the probe radius for R > Rmax.

Morphological Functions to Quantify Three-Dimensional Tomograms of Macropore Structure in a Vineyard Soil with Two Different Management Regimes

Soil tomography and morphological functions built over Minkowski functionals were used to describe the impact on pore structure of two soil management practices in a Mediterranean vineyard. Soil structure controls important physical and biological processes in soil–plant–microbial systems. Those processes are dominated by the geometry of soil pore structure, and a correct model of this geometry is critical for understanding them. Soil tomography has been shown to provide rich three-dimensional digital information on soil pore geometry. Recently, mathematical morphological techniques have been proposed as powerful tools to analyze and quantify the geometrical features of porous media. Minkowski functionals and morphological functions built over Minkowski functionals provide computationally efficient means to measure four fundamental geometrical features of three-dimensional geometrical objects, that is, volume, boundary surface, mean boundary surface curvature, and connectivity. We used the threshold and the dilation and erosion of three-dimensional images to generate morphological functions and explore the evolution of Minkowski functionals as the threshold and as the degree of dilation and erosion changes. We analyzed the three-dimensional geometry of soil pore space with X-ray computed tomography (CT) of intact soil columns from a Spanish Mediterranean vineyard by using two different management practices (conventional tillage versus permanent cover crop of resident vegetation). Our results suggested that morphological functions built over Minkowski functionals provide promising tools to characterize soil macropore structure and that the evolution of morphological features with dilation and erosion is more informative as an indicator of structure than moving threshold for both soil managements studied.

Sheath vaporization of a monodisperse fuel-spray jet

The group vaporization of a monodisperse fuel-spray jet discharging into a hot coflowing gaseous stream is investigated for steady flow by numerical and asymptotic methods with a two-continua formulation used for the description of the gas and liquid phases. The jet is assumed to be slender and laminar, as occurs when the Reynolds number is moderately large, so that the boundary-layer form of the conservation equations can be employed in the analysis. Two dimensionless parameters are found to control the flow structure, namely the spray dilution parameter 1, defined as the mass of liquid fuel per unit mass of gas in the spray stream, and the group vaporization parameter e, defined as the ratio of the characteristic time of spray evolution due to droplet vaporization to the characteristic diffusion time across the jet. It is observed that, for the small values of e often encountered in applications, vaporization occurs only in a thin layer separating the spray from the outer droplet-free stream. This regime of sheath vaporization, which is controlled by heat conduction, is amenable to a simplified asymptotic description, independent of ε,in which the location of the vaporization layer is determined numerically as a free boundary in a parabolic problem involving matching of the separate solutions in the external streams, with appropriate jump conditions obtained from analysis of the quasi-steady vaporization front. Separate consideration of dilute and dense sprays, corresponding, respectively, to the asymptotic limits λ<<1 and λ>>1, enables simplified descriptions to be obtained for the different flow variables, including explicit analytic expressions for the spray penetration distance.

Targeting of a Germ Cell-specific Type 1 Hexokinase Lacking a Porin-binding Domain to the Mitochondria as Well as to the Head and Fibrous Sheath of Murine Spermatozoa

Multiple isoforms of type 1 hexokinase (HK1) are transcribed during spermatogenesis in the mouse, including at least three that are presumably germ cell specific: HK1-sa, HK1-sb, and HK1-sc. Each of these predicted proteins contains a common, germ cell-specific sequence that replaces the porin-binding domain found in somatic HK1. Although HK1 protein is present in mature sperm and is tyrosine phosphorylated, it is not known whether the various potential isoforms are differentially translated and localized within the developing germ cells and mature sperm. Using antipeptide antisera against unique regions of HK1-sa and HK1-sb, it was demonstrated that these isoforms were not found in pachytene spermatocytes, round spermatids, condensing spermatids, or sperm, suggesting that HK1-sa and HK1-sb are not translated during spermatogenesis. Immunoreactivity was detected in protein from round spermatids, condensing spermatids, and mature sperm using an antipeptide antiserum against the common, germ cell-specific region, suggesting that HK1-sc was the only germ cell-specific isoform present in these cells. Two-dimensional SDS-PAGE suggested that all of the sperm HK1-sc was tyrosine phosphorylated, and that the somatic HK1 isoform was not present. Immunoelectron microscopy revealed that HK1-sc was associated with the mitochondria and with the fibrous sheath of the flagellum and was found in discrete clusters in the region of the membranes of the sperm head. The unusual distribution of HK1-sc in sperm suggests novel functions, such as extramitochondrial energy production, and also demonstrates that a hexokinase without a classical porin-binding domain can localize to mitochondria.

Complementary DNA Cloning and Characterization of Ferredoxin Localized in Bundle-Sheath Cells of Maize Leaves1

In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells. A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe. The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry. RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively. The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds. Fd I and Fd II had similar redox potentials of −423 and −406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I. Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase. Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I. These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.

Carbohydrates in Individual Cells of Epidermis, Mesophyll, and Bundle Sheath in Barley Leaves with Changed Export or Photosynthetic Rate1

Carbohydrate metabolism of barley (Hordeum vulgare) leaves induced to accumulate sucrose (Suc) and fructans was investigated at the single-cell level using single-cell sampling and analysis. Cooling of the root and shoot apical meristem of barley plants led to the accumulation of Suc and fructan in leaf tissue. Suc and fructan accumulated in both mesophyll and parenchymatous bundle-sheath (PBS) cells because of the reduced export of sugars from leaves under cooling and to increased photosynthesis under high photon fluence rates. The general trends of Suc and fructan accumulation were similar for mesophyll and PBS cells. The fructan-to-Suc ratio was higher for PBS cells than for mesophyll cells, suggesting that the threshold Suc concentration needed for the initiation of fructan synthesis was lower for PBS cells. Epidermal cells contained very low concentrations of sugar throughout the cooling experiment. The difference in Suc concentration between control and treated plants was much less if compared at the single-cell level rather than the whole-tissue level, suggesting that the vascular tissue contains a significant proportion of total leaf Suc. We discuss the importance of analyzing complex tissues at the resolution of individual cells to assign molecular mechanisms to phenomena observed at the whole-plant level.

A Functional Calvin Cycle Is Not Indispensable for the Light Activation of C4 Phosphoenolpyruvate Carboxylase Kinase and Its Target Enzyme in the Maize Mutant bundle sheath defective2-mutable11

We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) by its Ca2+-insensitive protein-serine/threonine kinase (PEPC kinase) in the C4 mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 μmol m−2 s−1 photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll- and bundle-sheath-specific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO2-exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.