Heterogeneous phenotype of human glioblastoma: In vitro study

Glioblastomas (GBMs) are the most lethal primary brain tumours. Increasing evidence shows that brain tumours contain the population of stem cells, so‐called cancer stem cells (CSCs). Stem cell marker CD133 was reported to identify CSC population in GBM. Further studies have indicated that CD133 negative cells exhibiting similar properties and are able to initiate the tumour, self‐renew and undergo multilineage differentiation. GBM is a highly heterogeneous tumour and may contain different stem cell populations with different functional properties. We characterized five GBM cell lines, established from surgical samples, according to the marker expression, proliferation and differentiation potential. CD133 positive cell lines showed increased proliferation rate in neurosphere condition and marked differentiation potential towards neuronal lineages. Whereas two cell lines low‐expressing CD133 marker showed mesenchymal properties in vitro, that is high proliferation rate in serum condition and differentiation in mesenchymal cell types. Further, we compared therapy resistance capacity of GBM cell lines treated with hydroxyurea. Our results suggest that CSC concept is more complex than it was believed before, and CD133 could not define entire stem cell population within GBM. At least two different subtypes of GBM CSCs exist, which may have different biological characteristics and imply different therapeutic strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed germination

Enzymatic hydrolysis of glucosinolates, a class of compounds found in Brassica species, results in a number of products with potential to inhibit seed germination. To investigate the impact of both volatile and water-soluble allelochemicals, germination bioassays using Lactuca sativa seeds were conducted with root and combined leaf and stem tissues of Brassica napus. Tissues in which glucosinolates were hydrolyzed to remove volatile glucosinolate degradation products were compared with intact tissues and water controls. Only tissues containing glucosinolates produced volatiles that inhibited germination. Volatiles were trapped and identified using GC-MS. Volatiles produced in greater quanitity from intact tissues than from tissues without glucosinolates were almost exclusively glucosinolate hydrolysis products. Water-soluble components also inhibited germination. Chemical analysis of extracts confirmed the presence of glucosinolate hydrolysis products, but indicated the involvement of additional allelochemicals, especially in leaf and stem tissues. Results support the proposal that glucosinolate-containing plant tissues may contribute to reductions in synthetic pesticide use if weed seeds are targeted.Abbreviations ITC isothiocyanates - CN organic cyanides - OZT oxazolidinethione - iRoot intact root tissue - iL&S intact leaf and stem tissue - hRoot hydrolyzed root tissue - hL&S hydrolyzed leaf and stem tissue     

Pharmacological profile of the tachykinin receptor involved in the stimulation of corticosteroid secretion in the frog Rana ridibunda

It has recently been shown that the adrenal gland of the frog Rana ridibunda is densely innervated by a network of fibers containing two novel tachykinins, i.e. ranakinin (the counterpart of substance P) and [Leu³, Ile⁷]neurokinin A. Both ranakinin and [Leu³, Ile⁷]neurokinin A stimulate corticosteroid secretion from frog adrenal glands in vitro. In the present study, we have investigated the pharmacological profile of the receptors involved in the stimulatory action of ranakinin on perifused frog adrenal slices. The selective NK-1 receptor antagonists [ -Pro⁴, -Trp^7,9]substance P 4–11 and CP-96,345, did not affect the stimulatory action of ranakinin. The selective NK-1 agonist substance P 6–11 had no effect on corticosteroid secretion. The non-peptidic NK-1 receptor antagonist RP 67580 significantly reduced the stimulatory effect of ranakinin on corticosterone and aldosterone secretion by 57 and 55%, respectively. In addition, the dual NK-1/NK-2 receptor antagonist FK-224 significantly inhibited the effect of ranakinin on corticosterone (−80%) and aldosterone secretion (−95%). Finally, the amphiphilic analogue of substance P, [ -Pro², -Phe⁷, -Trp⁹]substance P, had no effect on corticosteroid secretion. These data suggest that in the frog adrenal gland the stimulatory action of ranakinin on steroid secretion is mediated by a novel type of receptor which differs substantially from the mammalian NK-1 receptor subtype.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

The biological markers of non-Hodgkin's lymphomas: their role in diagnosis, prognostic assessment and therapeutic strategy

The biological markers of non-Hodgkin's lymphomas (NHL) are distinguished in three categories: serological, immunophenotypic, and molecular markers. The clinical importance of biological markers in NHL is based on their support of morphologic diagnosis, their role in staging and prognostic assessment, and their contribution to monitoring minimal residual disease (MRD). The most important serological markers reflect the tumor load (beta-2 microglobulin, beta 2-M), proliferative activity (lactic dehydrogenase, LDH), and invasive potential of lymphomas (CA 125). LDH and beta 2-M are included as important prognostic parameters in widely used staging systems. Immunophenotypic analysis identifies specific markers of lineage (B or T-cells), maturation level, cell proliferation, and clonality. Results of immunophenotyping are particularly useful in low to intermediate-grade NHLs to support the morphologic diagnosis and facilitate the detection of MRD after treatment. The molecular markers are genetic lesions involved in the pathogenesis of some categories of NHL. Their use as markers for diagnosis is justified by the selective association with specific lymphoma categories: follicular, mantle cell, diffuse large cell, and anaplastic large cell lymphomas. Molecular lesions are the most specific and sensitive markers for evaluating MRD. Today the biological markers of NHL are widely employed for diagnosis, staging, and prognostic assessment. Their systematic use may complement clinical parameters in the stratification of NHL patients, who may thus become candidates for treatments of different intensity. The detection of MRD after first-line treatment identifies patients at high risk of relapse who require additional therapy to cure their disease.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions

Polystyrene Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of two different enzymatically modified hairy regions (HRs) from pectin containing, for example, rhamnogalacturonan-I and xylogalacturonan structural elements. The two polysaccharide preparations share the same structural elements of apple pectin, but the relative amounts and lengths of the neutral side chains present differ. Surface analysis by X-ray photoelectron spectroscopy, contact angle measurement, and atomic force microscope (AFM) force-separation curves was used to characterize the effects on surface chemistry and interfacial forces of the surface modification process. Cell adhesion experiments using continuous L-929 fibroblasts and primary aortic smooth muscle cells were performed to evaluate the effect of the polysaccharide nature on cell adhesion. Results show that immobilization of the HR affects the interfacial field of forces and the cell behavior: "equilibrium" contact angles, obtained by a recently introduced vibrational approach, decrease after HR immobilization reaching a value close to 20 degrees . AFM force-separation curves show a more extended (or softer) interface in the case of the HR bearing longer side chains. Accordingly, depending on the HR preparation, cells shifted from spread morphology and adhesion behavior quantitatively comparable to that observed on conventional tissue culture polystyrene to rounded morphology and significantly lower adhesion. These data show that engineering of plant pectins can be a valuable tool to prepare novel and finely tuned polysaccharides having different chemico-physical and biological properties, to be used in the surface modification of medical devices and materials.     

Novel plant-microbe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant (Pisum sativum)

A previously undescribed plant-microbe interaction between a root-colonizing Streptomyces species, S. lydicus WYEC108, and the legume Pisum sativum is described. The interaction is potentially of great importance to the health and growth in nature of this nodulating legume. The root-colonizing soil actinomycete S. lydicus WYEC108 influences pea root nodulation by increasing root nodulation frequency, possibly at the level of infection by Rhizobium spp. S. lydicus also colonizes and then sporulates within the surface cell layers of the nodules. Colonization leads to an increase in the average size of the nodules that form and improves the vigor of bacteroids within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients. Bacteroid accumulation of the carbon storage polymer, poly-beta-hydroxybutyrate, is reduced in colonized nodules. Root nodules of peas taken from agricultural fields in the Palouse hills of northern Idaho were also found to be colonized by actinomycete hyphae. We hypothesize that root and nodule colonization is one of several mechanisms by which Streptomyces acts as a naturally occurring plant growth-promoting bacterium in pea and possibly other leguminous plants.     

Regional Distribution and Evolution of Gray Matter Damage in Different Populations of Multiple Sclerosis Patients

Background

Both gray-matter (GM) atrophy and lesions occur from the earliest stages of Multiple Sclerosis (MS) and are one of the major determinants of long-term clinical outcomes. Nevertheless, the relationship between focal and diffuse GM damage has not been clarified yet. Here we investigate the regional distribution and temporal evolution of cortical thinning and how it is influenced by the local appearance of new GM lesions at different stages of the disease in different populations of MS patients.

Methods

We studied twenty MS patients with clinically isolated syndrome (CIS), 27 with early relapsing-remitting MS (RRMS, disease duration <5 years), 29 with late RRMS (disease duration ≥ 5 years) and 20 with secondary-progressive MS (SPMS). The distribution and evolution of regional cortical thickness and GM lesions were assessed during 5-year follow-up.

Results

The results showed that new lesions appeared more frequently in hippocampus and parahippocampal gyri (9.1%), insula (8.9%), cingulate cortex (8.3%), superior frontal gyrus (8.1%), and cerebellum (6.5%). The aforementioned regions showed the greatest reduction in thickness/volume, although (several) differences were observed across subgroups. The correlation between the appearance of new cortical lesions and cortical thinning was stronger in CIS (r² = 50.0, p<0.001) and in early RRMS (r² = 52.3, p<0.001), compared to late RRMS (r² = 25.5, p<0.001) and SPMS (r² = 6.3, p = 0.133).

Conclusions

We conclude that GM atrophy and lesions appear to be different signatures of cortical disease in MS having in common overlapping spatio-temporal distribution patterns. However, the correlation between focal and diffuse damage is only moderate and more evident in the early phase of the disease.