Accumulation of a GPI-anchored protein at the cell surface requires sorting at multiple intracellular levels

Proteins modified by glycosylphosphatidylinositol membrane anchors have become popular for investigating the role of membrane lipid microdomains in cellular sorting processes. To this end, trypanosomatids offer the advantage that they express these molecules in high abundance. The parasitic protozoan Trypanosoma brucei is covered by a dense and nearly homogeneous coat composed of a glycosylphosphatidylinositol-anchored protein, the variant surface glycoprotein, which is essential for survival of the parasite in the mammalian blood. Therefore, T. brucei must possess mechanisms to selectively and efficiently deliver variant surface glycoprotein to the cell surface. In this study, we have quantified the steady-state distribution of variant surface glycoprotein by differential biotinylation, by fluorescence microscopy and by immunoelectron microscopy on high-pressure frozen and freeze-substituted samples. These three techniques provide very similar estimates of the fraction of variant surface glycoprotein located on the cell surface, on average 89.4%. The intracellular variant surface glycoprotein (10.6%) is predominantly located in the endosomal compartment (75%), while 25% are associated with the endoplasmic reticulum, Golgi apparatus and lysosomes. The density of variant surface glycoprotein in the plasma membrane including the membrane of the flagellar pocket, the only site for endo- and exocytosis in this organism, is 48-52 times higher than the density in endoplasmic reticulum membranes. The relative densities of the Golgi complex and of the endosomes are 2.7 and 10.8, respectively, compared to the endoplasmic reticulum. This data set provides the basis for an analysis of the dynamics of sorting. Depending on the intracellular itinerary of newly formed variant surface glycoprotein, the high surface density is achieved in two (endoplasmic reticulum --> Golgi complex --> cell surface) or three enrichment steps (endoplasmic reticulum --> Golgi complex --> endosomes --> cell surface), suggesting sorting between several membrane compartments.     

Renal cortical remodelling by NO-synthesis blockers in rats is prevented by angiotensin-converting enzyme inhibitor and calcium channel blocker

The cortical remodelling was studied when chronically nitric oxide synthesis (NOs) blockade (L-NAME-induced) hypertensive rats are simultaneously treated, or not, with angiotensin-converting enzyme inhibitor or calcium channel blocker. Four groups of eight rats each were studied as follows: Control (C), L-NAME (L), L-NAME+Enalapril (L+E) and L-NAME+Verapamil (L+V). The systolic blood pressure (SBP) was weekly recorded. The cortex of the left kidneys was analysed according to the vertical section design. The volume-weighted mean glomerular volume (VWGV) was made through the "point-sampled intercepts" method. Enalapril and verapamil were efficient in reducing the SBP in rats submitted to NOs blockade. Glomeruli had considerable alterations in L group rats (glomerular hypertrophy or sclerosis) and tubular atrophy. The VWGV was 100% greater in L group rats than in the C group rats, while it was 30% smaller in L+E and L+V groups than in L group. The tubular volume was 30–50% greater, while the tubular lenght was 20–30% smaller in L group than in the other groups. The renal cortical region showed glomerular sclerosis/hypertrophy and tubular remodelling in rats with NOs blockade that was efficiently prevented with the simultaneous treatment with enalapril or verapamil.     

Statistical evaluation of colocalization patterns in immunogold labeling experiments

The ultrastructural localization of various antigens in a cell using antibodies conjugated to gold particles is a powerful instrument in biological research. However, statistical or stereological tools for testing the observed patterns for significant clustering or colocalization are missing. The paper presents a method for the quantitative analysis of single or multiple immunogold labeling patterns using interpoint distances and tests the method using experimental data. The clustering or colocalization of gold particles was detected using various characteristics of the distribution of distances between them. Pair correlation and cross-correlation functions were used for exploratory analysis; second order reduced K (or cross-K) functions were used for testing the statistical significance of observed events. Confidence intervals of function values were estimated by Monte Carlo simulations of the Poisson process for independent particles, and results were visualized in histograms. Furthermore, a suitability of K functions modified by censoring or weighting was tested. The reliability of the method was assessed by evaluating the labeling patterns of nascent DNA and several nuclear proteins with known functions in replication foci of HeLa cells. The results demonstrate that the method is a powerful tool in biological investigations for testing the statistical significance of observed clustering or colocalization patterns in immunogold labeling experiments.     

Brief Report: Estimating the size and number of autophagic bodies by electron microscopy

Much recent and ongoing research is focused on understanding the mechanisms and regulation of autophagy, a cellular self-degradation pathway with many links to human health. Although many assays exist to measure the total magnitude of autophagy, electron microscopy remains the tool of choice for the determination of the size and the number of autophagosomes formed in a given mutant or under given induction conditions. Here we present a detailed protocol for measuring autophagic bodies in the yeast Saccharomyces cerevisiae by electron microscopy. Furthermore, we present an improved mathematical method for estimating body size and a new method for estimating body number. Finally, we include a discussion of the merits and limitations of these methods and an example of their application to autophagic bodies formed in the ume6∆ strain.     

Examination of Age‐dependent effects of fetal ethanol exposure on behavior,hippocampal cell counts,and doublecortin immunoreactivity in rats

Ethanol is known as a potent teratogen having adverse effects on brain and behavior. However, some of the behavioral deficits caused by fetal alcohol exposure and well expressed in juveniles ameliorate with maturation may suggest some kind of functional recovery occurring during postnatal development. The aim of this study was to reexamine age‐dependent behavioral impairments in fetal‐alcohol rats and to investigate the changes in neurogenesis and gross morphology of the hippocampus during a protracted postnatal period searching for developmental deficits and/or delays that would correlate with behavioral impairments in juveniles and for potential compensatory processes responsible for their amelioration in adults. Ethanol was delivered to the pregnant dams by intragastric intubation throughout 7–21 gestation days at daily dose of 6 g/kg. Isocaloric intubation and intact control groups were included. Locomotor activity, anxiety, and spatial learning tasks were applied to juvenile and young‐adult rats from all groups. Unbiased stereological estimates of hippocampal volumes, the total number of pyramidal and granular cells, and double cortin expressing neurons were carried out for postnatal days (PDs) PD1, PD10, PD30, and PD60. Alcohol insult during second trimester equivalent caused significant deficits in the spatial learning in juvenile rats; however, its effect on hippocampal morphology was limited to a marginally lower number of granular cells in dentate gyrus (DG) on PD30. Thus, initial behavioral deficits and the following functional recovery in fetal‐alcohol subjects may be due to more subtle plastic changes within the hippocampal formation but also in other structures of the extended hippocampal circuit. Further investigation is required. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 498–513, 2014     

A calcium channel blocker flunarizine attenuates the neurotoxic effects of iron

Iron is a metal highly concentrated in liver and brain tissue, and known to induce neuronal hyperactivity and oxidative stress. It has been established that iron levels rise in the brain in some neurodegenerative diseases such as Parkinson's and Alzheimer's diseases (AD). A body of evidence indicates a link between neuronal death and intracellular excessive calcium accumulation. The aim of the present study was to investigate the effects of a calcium antagonist, flunarizine, on neurotoxicity induced by intracerebroventricular (i.c.v.) iron injection. For this reason rats were divided into three groups as control, iron and iron+flunarizine groups. Animals in iron and iron+flunarizine groups received i.c.v. FeCl₃ injection (200 mM, 2.5 μl), while control rats received the same amount of saline into the cerebral ventricles. Rats in iron+flunarizine group also received i.c.v. flunarizine (1 μM, 2 μl) following FeCl₃ injection. All animals were kept alive for ten days following the operation and animals in iron+flunarizine group received intraperitoneal (i.p.) flunarizine injections once a day (10 mg/kg/day) during this period. After ten days, rats were sacrificed. The total numbers of neurons in hippocampus of all rats were estimated with the latest, unbiased stereological techniques. Findings of the present study suggest that flunarizine may attenuate the neurotoxic effects of iron injection by inhibiting the cellular influx of excessive calcium ions.     

Non-destructive,stereological estimation of plant root lengths,branching pattern and diameter distribution

The total length of a linear structure contained in an unbounded, transparent reference space can be estimated from ‘total vertical projections’ obtained by rotating the linear structure about an arbitrary ‘vertical’ axis, and projecting the linear structure onto a plane parallel to the axis of rotation. The total number of intersections between cycloid arcs with their minor axis perpendicular to the axis of rotation and the projected linear structure then provides an unbiased estimator of the total length of the structure. In this study, a stereological procedure based on the method of ‘total vertical projections’ was used to non-destructively estimate total root length, number of branches, diameter distribution and mean root diameter of crested wheatgrass plants (Agropyron cristatum L.) growing in a transparent medium. Root lengths, diameters, and number of branching points of various orders were determined at 3-day intervals over a 4-week growing period. The length estimator was very robust and efficient with sampling coefficients of error usually less than 5% for a total of 50–150 grid intersection counts over two projection directions per plant. Biological coefficients of variance for total length were between 30–70%, and were largely related to variation in the extent of branching. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inconsistent Immunohistochemical Expression Patterns of Four Different CD133 Antibody Clones in Glioblastoma

The putative tumor stem cell marker CD133 is the marker of choice for identifying brain tumor stem cells in gliomas, but the use of different CD133 antibody clones possibly recognizing different CD133 splice variants with epitopes of different glycosylation status confuses the field. The aim was to investigate if current inconsistent CD133 observations could be a result of using different CD133 antibodies for immunohistochemical identification of CD133. Ten glioblastomas were immunohistochemically stained with four different CD133 antibody clones (AC133, W6B3C1, C24B9, and ab19898) and analyzed by quantitative stereology. Moreover, the CD133 staining pattern of each antibody clone was investigated in kidney, pancreas, and placenta tissue as well as in glioblastoma and retinoblastoma cultures and cell lines. All antibody clones revealed CD133+ niches and single cells in glioblastomas, but when using different clones, their distribution rarely corresponded. Morphology of identified single cells varied, and staining of various tissues, cultures, and cells lines was also inconsistent among the clones. In conclusion, the authors report inconsistent CD133 detection when using different primary CD133 antibody clones. Thus, direct comparison of studies using different antibody clones and conclusions based on CD133 immunohistochemistry should be performed with caution.     

Correlations between Planar Random Structures,with an Ecological Application

This paper suggests some characteristics which may help to detect and quantify “correlations” between planar geometrical structures such as point patterns/point processes, fibre systems/fibre processes and region patterns/random sets. Typical examples of “correlations” are mutual attraction or inhibition. Some of the characteristics are related to moment-measures, the definition of the others is similar to that of quantities in the mathematical morphology of MATHERON and SERRA . For all quantities unbiased estimators are given. Also the detection of orientations in random structures is discussed. An ecological example demonstrates the application of the characteristics.     

Quantification of ovarian follicles in bluefin tuna Thunnus thynnus by two stereological methods

The numbers of different types of ovarian follicles (developing, degenerating and postovulatory follicles) were estimated in bluefin tuna Thunnus thynnus using two stereological procedures: the model‐based method of Weibel & Gomez, which has become a tool of broad application in the quantification of oocytes in fishes, and the assumption‐free ‘disector’ (sic) method of Sterio. The estimates of developing follicles (follicles containing lipid‐stage, vitellogenic and migratory‐nucleus oocytes) made by the model‐based method tended to be lower than those obtained with the disector, though significant differences were not observed except for vitellogenic follicles. Counts of atretic follicles by the model‐based method were higher than those made using the disector, the differences being remarkable between both techniques, particularly in the case of β‐atresia, where the statistical analysis indicated significantly unequal estimations with the two methods. In contrast, the amount of postovulatory follicles estimated by the disector, which would stand for the realized batch fecundity, was somewhat larger than that calculated with the model‐based method.