Isolation and identification of differentially expressed genes between Gossypium arboreum and Gossypium hirsutum species

Plants have evolved sophisticated molecular defense mechanisms in order to survive disease conditions. So far, a number of pathogen resistance (R) genes have been reported in plants. These R genes are thought to be involved in activating the signals that lead to disease resistance. The structural specificity of R genes products makes it possible to isolate these genes from plants by homology based techniques. Knowledge regarding molecular organization of R genes in cotton is limited and other resistant gene analogues (RGAs) still need to be identified for Cotton Leaf Curl Disease (CLCuD) resistance, particularly from Gossypium arboreum. This study has, therefore, been designed to identify the natural resistance related genes from Gossypium arboreum against CLCuV. In addition to G.arboreum (resistant to CLCuV), G. hirsutum L. var S12 (highly susceptible to CLCuV), G. hirsutum L.var. CP15/2 and LRA (partially resistant to CLCuV) were used as reference controls to facilitate identification of defense related genes. This paper describes several differentially expressed transcripts through DDRT-PCR on total RNA from G. arboreum and some other tetraploid cotton species. The nucleotide sequence of these genes, when compared to the reported database, indicated that some of the differentially expressed transcripts had homology to the reported R-genes.Key words: Pathogen resistance genes (R-genes), G. arboreum, differential display, cotton leaf curl virus (CLCuV), RT-PCR

Study of total seed storage protein in indigenous Brassica species based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Genetic diversity was studied in 234 accessions of locally collected Brassica species for total seed protein content through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These accessions were collected from different locations of Pakistan. After the study of these accessions on SDS-PAGE, 28 reproducible bands were used for cluster analysis and with the help of these bands, genetic diversity were estimated. Out of 28, four major bands were observed. Dendogram was constructed and the accessions were divided into two main groups comprising 11 clusters. The results obtained from these clusters showed minimum genetic diversity in these accessions on SDS-PAGE level. Due to low genetic diversity on SDS-PAGE level, it is suggested that two dimensional (2D)- electrophoresis can be used for protein study.Key words: Genetic variation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), protein electrophoresis, cluster analysis, brassica

GROWTH COMPARISON OF EXOTIC SPECIES FOR GREEN FORAGE

Abstract Growth of exotic fodder crops (grasses and clovers) were compared in pots at Agronomy Research Farm, Khyber Pakhtunkhuwa Agricultural University, Peshawar. Initially 20 seeds was planted on 22.10.2005 and thinned out after emergence by leaving 10 seedlings pot -1 (30 x 50 cm). Clover's seeds were soaked overnight (>14 h) before sowing. Compound fertilizer (100, 60, 30) and (30, 60, 30) kg ha -1 N, P, and K were applied to grasses and clovers, respectively after thinning. Pots were manually irrigated. Biomass of pots was periodically harvested for dry matter after taking measurements of green leaf area index (GLAI) and light interception. Crop growth rate (CGR) was derived as ratio of dry matter and time-taken as growing degree days (GDD O C). LAI was measured non-destructively using LI-2000, LI-COR, USA. Radiation use efficiency (RUE) was derived from weather data and measurements made during the crop growth. The highest dry matter (1685 g m -2 ) was observed for Lolium multiflorum, followed by Lolium perenne (791 g m -2 ) and Dactylis glomerata (631 g m -2 ). GLAI were also recorded the highest for the species Lolium multiflorum (4.07) and Lolium perenne (3.93) with non-significant difference from each other. The highest dry matter of the grasses was in agreement to higher CGR and RUE. Grass species Lolium multiflorum yielded the highest CGR (1.06 g DM O C GDD) and RUE (3.41 g DM MJ -1 PAR absorbed) with strong positive relationship (r 2 = 0.95). Lolium perenne was next to yield 0.47 g DM GDD -1 ( O C) and RUE 1.63 g DM MJ -1 PAR absorbed. Rests of the grass species were found un-comparable for any observed parameter. Among the clovers, Trifolium repense was higher in dry matter (510 g m -2 ) yielded 1.10 g DM GDD -1 and RUE 0.71 g DM MJ -1 PAR absorption. From the study, it can be concluded that ryegrasses has potential to plant as green fodder in mix cultivation with local clovers on irrigated rangelands. Moreover, slow growth of fodder on arable land in early winter months can be improved through selection of an appropriate exotic grass/clover to be sown in combination with local types for the area

Interactions in vivo between the Vif protein of HIV-1 and the precursor (Pr55GAG) of the virion nucleocapsid proteins

The abnormality of viral core structure seen in vif-defective HIV-1 grown in PBMCs has suggested a role for Vif in viral morphogenesis. Using an in vivo mammalian two-hybrid assay, the interaction between Vif and the precursor (Pr55GAG) of the virion nucleocapsid proteins has been analysed. This revealed the amino-terminal (aa 1–22) and central (aa 70–100) regions of Vif to be essential for its interaction with Pr55GAG, but deletion of the carboxy-terminal (aa 158–192) region of the protein had only a minor effect on its interaction. Initial deletion studies carried out on Pr55GAG showed that a 35-amino-acid region of the protein bridging the MA(p17)–CA(p24) junction was essential for its ability to interact with Vif. Site-directed mutagenesis of a conserved tryptophan (Trp21) near the amino terminus of Vif showed it to be important for the interaction with Pr55GAG. By contrast, mutagenesis of the highly conserved YLAL residues forming part of the BC-box motif, shown to be important in Vif promoting degradation of APOBEC3G/3F, had little or no effect on the Vif–Pr55GAG interaction

Integrin‐Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor‐Specific Delivery Achieve MYCN Silencing with Improved Survival

The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor‐targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor‐mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non‐targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide‐dependent indicating integrin‐mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN‐amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor‐targeting, with minimal clearance by the liver and so enable delivery of tumor‐targeted siRNA therapeutics

Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB

Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgammanull recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man

Influence of the human leukocyte antigen complex on the development of cutaneous fibrosis: An immunogenetic perspective

A number of aesthetically and physically distressing disorders of the skin come under the general term "cutaneous fibrosis", all sharing a common abnormal wound healing process These disorders are often incurable and effective treatments remain to be established and, as such, they present a significant burden for patients and a therapeutic challenge for clinicians The aim of this review is to investigate the evidence of either positive or negative associations of the human leukocyte antigen (HLA) system with various types of cutaneous fibrosis, focussing in particular on keloid scars, hypertrophic scars and scleroderma A standard systematic literature search was performed The strengths and limitations of studies were evaluated in terms of significance, methodology and reproducibility There is a clear association between specific HLA alleles and predilection or protection to cutaneous fibrosis Of these candidate HLA alleles, the class II loci seem to be the most promising in terms of a genetic biomarker, with the DQ and DR alleles having significant associations with abnormal wound healing and cutaneous fibrosis There is strong evidence of a significant immune component in the pathogenesis of each type of fibrotic disorder explored in this review However, the exact mechanisms remain to be elucidated, since the pathogenesis of cutaneous fibrosis and abnormal wound healing are not fully understoo

Evaluation of rapidly mutating Y-STRs in Pakistani population

Y-chromosomal Short Tandem Repeats have been widely used in forensic investigations, identification of males for criminal justice purpose and population genetics. Commercially available Y-STRs kits allow the identification of male pedigrees and has a limited application in forensic genetics because of its limitation in differentiating closely related male individuals. Recent research with the Rapidly Mutating Y-STRs (RM Y-STRs) have revealed that these loci deliver significantly higher discrimination capacity and haplotype diversity in worldwide populations when compared with the conventional Y-STRs. Although a number of RM Y-STRs have found their way in most updated commercial kits, there are still some loci that are not yet used in such kits. The aim of this study is to develop RM Y-STR haplotypes frequency database for the Pakistani population, in order to appraise the resolution power of these loci. A total of 212 unrelated males from the Pakistani population were typed with 13 RM Y-STRs which comprise DYF399S1, DYF387S1, DYS570, DYS576, DYS518, DYS526a + b, DYS626, DYS627, DYF403S1a + b, DYF404S1, DYS449, DYS547 and DYS612. 211 unique haplotypes were identified, out of which 1 haplotype was shared between two individuals, accounting for 0.9952 discrimination capacity (DC). Haplotype diversity was found to be 0.999925. Gene diversity (GD) values of all the loci were higher than 0.5, where the highest GD values were observed at DYF399S1, DYF403S1a and DYF404S1; with values of 0.99419, 0.98252 and 0.93061 respectively. Results of our study revealed that these 13 RM Y-STRs produced significantly stronger discriminatory power in Pakistani populations