The response of murine intestinal crypts to short-range promethium-147 beta irradiation: deductions concerning clonogenic cell numbers and positions

An exteriorized loop of mouse intestine was exposed to 147Pm low-energy electrons, where the dose rate decreased by a factor of 5 from the base of the crypt to the top of the proliferative zone. A crypt survival curve was obtained, expressed in terms of exposure time. The shape of the curve was interpreted in terms of survival parameters for colony-forming cells (clonogens) derived using 137Cs gamma rays and the depth-dose curve measured for 147Pm electrons. It is concluded that the shape of the crypt survival curve using 147Pm electrons is inconsistent with the notion of either the presence of a large number of clonogens or a small number near the top of the proliferative zone. A computer fitting procedure showed that the best agreement between predicted and observed curves was achieved with 2.7 +/- 0.5 clonogens at cell position 5.6 +/- 0.6, in the putative stem-cell zone.     

Comparison of organic and inorganic amendments for enhancing soil lead phytoextraction by wheat (Triticum aestivum L.)

Phytoextraction has received increasing attention as a promising, cost-effective alternative to conventional engineering-based remediation methods for metal contaminated soils. In order to enhance the phytoremediative ability of green plants chelating agents are commonly used. Our study aims to evaluate whether, citric acid (CA) or elemental sulfur (S) should be used as an alternative to the ethylene diamine tetraacetic acid (EDTA)for chemically enhanced phytoextraction. Results showed that EDTA was more efficient than CA and S in solubilizing lead (Pb) from the soil. The application of EDTA and S increased the shoot biomass of wheat. However, application of CA at higher rates (30 mmol kg(-1)) resulted in significantly lower wheat biomass. Photosynthesis and transpiration rates increased with EDTA and S application, whereas these parameters were decreased with the application of CA. Elemental sulfur was ineffective for enhancing the concentration of Pb in wheat shoots. Although CA did not increase the Pb solubility measured at the end of experiment, however, it was more effective than EDTA in enhancing the concentration of Pb in the shoots of Triticum aestivum L. It was assumed that increase in Mn concentration to toxic levels in soil with CA addition might have resulted in unusual Pb concentration in wheat plants. The results of the present study suggest that under the conditions used in this experiment, CA at the highest dose was the best amendment for enhanced phytoextraction of Pb using wheat compared to either EDTA or S.     

Construction of Adipogenic ceRNA Network Based on lncRNA Expression Profile of Adipogenic Differentiation of Human MSC Cells

Biochemical Genetics - The Long non-coding RNA (lncRNA) expression profile data of ten samples including human Mesenchymal Stem Cell (MSC) adipogenic differentiation 0, 3, and 6 days from...     

Human Rhinovirus 14 Enters Rhabdomyosarcoma Cells Expressing ICAM-1 by a Clathrin-, Caveolin-, and Flotillin-Independent Pathway

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na⁺/H⁺ ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via α_v integrin/CD46 in HeLa cells.Human rhinoviruses (HRVs), members of the family Picornaviridae that represent a major cause of the common cold, essentially utilize two different receptor types for host cell attachment. The 12 minor group HRVs, exemplified by HRV2, bind low-density lipoprotein receptor (LDLR), LDLR-related protein (LRP) (20), and very-LDLR (VLDLR) (29) and are internalized via the well-characterized clathrin-dependent endocytic pathway (44); however, these ligands, like others, can switch to different entry portals when the clathrin-dependent pathway is blocked (4). Once the virus arrives in endosomal carrier vesicles or late endosomes, uncoating (i.e., the release of the viral RNA genome) is triggered by the acidic pH (35, 39).The 87 major group HRVs, exemplified by HRV14, bind intercellular adhesion molecule-1 (ICAM-1). Following entry, uncoating is triggered by ICAM-1 itself (3), but the low endosomal pH facilitates this process (37). Based on inhibition of infection by the dominant negative (DN) dynamin-2 mutant dyn^K44A, it was proposed that HRV14 also follows a clathrin-dependent pathway in HeLa-H1 cells (9). However, ICAM-1 lacks a clathrin localization signal and even functions as a viral receptor when its cytoplasmic tail is replaced with a glycosylphosphatidylinositol (GPI) anchor (45). Furthermore, dynamin has also been shown to be involved in pathways other than clathrin-mediated endocytosis (CME), such as caveolae- and lipid raft-dependent entry, as a function of ligand and cell type (reviewed in references 30 and 34). Additionally, dynamin might play a role in formation and closure of circular pinocytic ruffles (31). More recently, a specific entry pathway for ICAM-1 ligands into human umbilical vein endothelial cells was identified and termed “cam-mediated endocytosis”; uptake was found to be triggered upon binding of multivalent ligands, such as immunoconjugates and immunobeads, and to occur independently from clathrin and caveolin. Inhibition by amiloride, actin depolymerization, and protein kinase C inhibitors pointed to macropinocytosis (33). So far, it is not known whether these findings are relevant to the entry pathway of HRVs via ICAM-1 as the uptake kinetics was significantly dependent on particle size. For all these reasons, involvement of clathrin in HRV14 uptake is questionable. Accordingly, we explored entry of HRV14 via ICAM-1 and compared the results with the well-characterized clathrin-dependent entry pathway of HRV2 (44). Employing pharmacological compounds, specific DN inhibitors, immunofluorescence, and electron microscopy, we demonstrate that HRV14 enters rhabdomyosarcoma ICAM-1-expressing (RD-ICAM) cells via a pathway independent of clathrin, caveolin, and flotillin.     

Linkage of a RAPD marker with powdery mildew resistance

The aim of this study was to investigate the inheritance of powdery mildew disease and to tag it with a DNA marker to utilize for the marker-assisted selection (MAS) breeding program. The powdery mildew resistant genotype Fallon(er) and susceptible genotype 11760-3ER were selected from 177 genotypes by heavy infestation of germplasm with Erysiphe pisi through artificial inoculation. The F1 plants of the cross Fallon/11760-3 indicated the dominance of the susceptible allele, while F2 plants segregated in 3 : 1 ratio (susceptible : resistant) that fit for goodness of fitness by chi2 (P > 0.07), indicating monogenic recessive inheritance for powdery mildew resistance in Pisum sativum. A novel RAPD marker OPB18 (5'-CCACAGCAGT-3') was linked to the er-1 gene with 83% probability with a LOD score of 4.13, and was located at a distance of 11.2 cM from the er-1 gene.     

Olive (Olea europaea L.) freezing tolerance related to antioxidant enzymes activity during cold acclimation and non acclimation

The changes in the antioxidant enzymes activity, total protein and proline content and their correlations with freezing tolerance (FT) (expressed as LT₅₀) were investigated at 11 different olive cultivars at cold-acclimation (CA, in February) and non-acclimation (NA, in August) stages. Leaf samples were collected from each cultivar and were divided into two groups. The first group was immediately frozen in liquid nitrogen for further biochemical analysis. The second ones was subjected to different freezing temperatures (?5, ?10, ?15 and ?20 °C) for 10 h, in order to determine their FT. The unfrozen control samples were kept at 4 °C. The results showed that Fishomi, Mission and Shengeh were the most freezing tolerant among other cultivars. In contrast, Zard, Manzanilla and Amigdalolia were the most sensitive ones. The cold acclimation enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), polyphenol oxidase (PPO) and total protein content. However, proline content and phenylalanine ammonia-lyase (PAL) activity did not change or even decreased slightly at CA stage, compare to those samples at NA stage. It was found that LT₅₀ to be closely correlated to POD, CAT, and PPO activity at CA and NA stages. Overall, higher leaf POD, CAT, and PPO activity could be used as important selection criteria in screening tolerant olive cultivars for cold zone climatic.     

Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture

Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.     

Molecular characterization of Ephedra species found in Pakistan

Ephedra, also known as "ma huang", is a dioecious, drought- and frost-resistant, perennial, evergreen shrub with compelling medicinal value. The genus is represented by 42 species around the world, 9 of which were provisionally reported from Pakistan. Species of the genus have a controversial taxonomy due to their overlapping morphological features. Conventional tools alone are not sufficient for characterizing the species. The objective of present study was to assess the genetic variability present in different biotypes of Ephedra growing in Pakistan using molecular markers. A total of six genotypes collected from diverse geographic zones of Pakistan were used. The DNA of all genotypes was amplified using nine randomly amplified polymorphic DNA (RAPD) primers to study genetic variability at the molecular level. The dissimilarity coefficient matrix based on the data of 9 RAPD primers was used to construct a dendrogram which was then used to group the genotypes in clusters. Based on the dendrogram and dissimilarity coefficient matrix, the RAPD markers used here revealed a moderate to high level of genetic polymorphism (6 to 49%) among the genotypes. It was found that the collection of genotype accessions from Swat Valley in northwestern Pakistan was most distantly related to the other five collections. More molecular markers including functional genes and ribosomal spacer regions are suggested to find a better estimate of the genetic diversity present in Ephedra growing in Pakistan. The information provided here is useful for identifying valuable Ephedra variants which will be used for medicinal purposes and earning foreign currency.     

First proteomic assay of Pakistani <Emphasis Type="Italic">Pisum sativum</Emphasis> L. germplasm relation to geographic pattern

Proteomic assay was carried out to asses genetic diversity in relation to geographic pattern in 97 genotypes of Pisum sativum L., collected from all over Pakistan. In total 34 bands were observed and among these, 26.7% bands were monomorphic, while 73.5% bands showed polymorphism. Based on both Province Wise Analysis (PWA) and Agro-ecological Zones (AEZ) the genotypes collected from Punjab, North West Frontier Province (NWFP) exhibited 70.6%, and 64.7% variation respectively. The germplasm collected from Azad Kashmir showed the lowest level of genetic diversity. Cluster analysis exhibited, moderate level of association, between genetic diversity and geographic pattern of the genotypes. The article is published in the original.