Isolation and functional analysis of cotton universal stress protein promoter in response to phytohormones and abiotic stresses

The 949 bp promoter fragment upstream from the translation initiation site of the GUSP gene encoding a universal stress protein was isolated from the genomic DNA of Gossypium arboreum. Some putative cis-acting elements involved in stress responses including E-box, ABRE, DPBF-box, and MYB-core elements were found in the promoter region. In an Agrobacterium-mediated transient expression assay, strong activation of the GUSP full promoter region occurred in tobacco leaves following dehydration, abscisic acid, salt, heavy metal, gibberellic acid and dark treatments. Deletion analysis of the promoter revealed that the dehydration, abscisic acid and salt responses were affected by the deletion between −208 and −949 bp and showed 2–4-fold induction. However, in response to dark, gibberellic acid and heavy metals the induction was only 2-fold. These findings further our understanding of the regulation of GUSP expression. This is an important study as no report of this universal stress protein promoter is available in literature.     

Induction of apoptosis in HuH-7 cancer cells by monoterpene and β-carboline indole alkaloids isolated from the leaves of Tabernaemontana elegans

Three known (1–3) and a novel (4) monoterpene indole alkaloids have been isolated from the methanol extract of leaves of Tabernaemontana elegans and their structures were elucidated by a series of spectroscopic experiments, involving NMR, MS, UV, and IR techniques. The isolated monoterpene indole alkaloids along with previously described β-carbolines (5–7) from the same specimen were studied for their apoptosis induction activity in human hepatoma HuH-7 cells. Methodology for apoptosis induction studies included cell viability assays, nuclear morphology assessments, and general caspase-3-like activity assays. The monoterpene indole alkaloids, tabernaemontanine (1) and vobasine (3) showed the most promising apoptosis induction profile in HuH-7 cells.     

Combination mode of antimalarial drug mefloquine and human serum albumin: Insights from spectroscopic and docking approaches

The interaction between mefloquine (MEF), the antimalarial drug, and human serum albumin (HSA), the main carrier protein in blood circulation, was explored using fluorescence, absorption, and circular dichroism spectroscopic techniques. Quenching of HSA fluorescence with MEF was characterized as static quenching and thus confirmed the complex formation between MEF and HSA. Association constant values for MEF-HSA interaction were found to fall within the range of 3.79-5.73 × 10⁴ M^˗1 at various temperatures (288, 298, and 308 K), which revealed moderate binding affinity. Hydrogen bonds and hydrophobic interactions were predicted to connect MEF and HSA together in the MEF-HSA complex, as deduced from the thermodynamic data (ΔS = +133.52 J mol⁻¹ K⁻¹ and ΔH = +13.09 kJ mol⁻¹) of the binding reaction and molecular docking analysis. Three-dimensional fluorescence spectral analysis pointed out alterations in the microenvironment around aromatic amino acid (tryptophan and tyrosine) residues of HSA consequent to the addition of MEF. Circular dichroic spectra of HSA in the wavelength ranges of 200-250 and 250-300 nm hinted smaller changes in the protein's secondary and tertiary structures, respectively, induced by MEF binding. Noncovalent conjugation of MEF to HSA bettered protein thermostability. Site marker competitive drug displacement results suggested HSA Sudlow's site I as the MEF binding site, which was also supported by molecular docking analysis.     

Gossypium arboreum GHSP26 enhances drought tolerance in Gossypium hirsutum

Heat‐shock proteins (HSP) are molecular chaperones for protein molecules. These proteins play an important role in protein–protein interactions such as, folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. A small HSP gene GHSP26 present in Gossypium arboreum responds to dehydration. In the present study, an attempt was made to overcome the problem of drought stress in cotton. A cDNA of GHSP26 was isolated from G. arboreum, cloned in plant expression vector, pCAMBIA‐1301 driven by the cauliflower mosaic virus 35S promoter and introduced into Gossypium hirsutum. The integration and expression studies of putative transgenic plants were performed through GUS assay; PCR from genomic DNA, and quantitative real‐time PCR analysis. Transgenic cotton plants showed an enhanced drought tolerance, suggesting that GHSP26 may play a role in plant responsiveness to drought. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Probing the Interaction of a Therapeutic Flavonoid,Pinostrobin with Human Serum Albumin: Multiple Spectroscopic and Molecular Modeling Investigations

Interaction of a pharmacologically important flavonoid, pinostrobin (PS) with the major transport protein of human blood circulation, human serum albumin (HSA) has been examined using a multitude of spectroscopic techniques and molecular docking studies. Analysis of the fluorescence quenching data showed a moderate binding affinity (1.03 × 10⁵ M⁻¹ at 25°C) between PS and HSA with a 1∶1 stoichiometry. Thermodynamic analysis of the binding data (ΔS = +44.06 J mol⁻¹ K⁻¹ and ΔH = −15.48 kJ mol⁻¹) and molecular simulation results suggested the involvement of hydrophobic and van der Waals forces, as well as hydrogen bonding in the complex formation. Both secondary and tertiary structural perturbations in HSA were observed upon PS binding, as revealed by intrinsic, synchronous, and three-dimensional fluorescence results. Far-UV circular dichroism data revealed increased thermal stability of the protein upon complexation with PS. Competitive drug displacement results suggested the binding site of PS on HSA as Sudlow’s site I, located at subdomain IIA, and was well supported by the molecular modelling data.     

Sucrose Synthase (SuSy) Gene Expression: An Indicator for Cotton Fiber Initiation and Early Development

Russian Journal of Plant Physiology - Cotton (Gossypium hirsutum L.) fiber initiation from ovule epidermal cells happen from 0 to 5 DPA, invertase (INV) and sucrose synthase (SuSy) are...     

Liposome-based vascular endothelial growth factor-165 transfection with skeletal myoblast for treatment of ischaemic limb disease

The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (VEGF(165)) gene transfer into skeletal myoblasts (SkMs) for treatment of acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid-VEGF(165) (CD-pVEGF(165)) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non-transfected SkM (group 2) or CD-pVEGF(165) transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF(165) up to day 18 with peak at day 2. SkMs were observed in all cell-transplanted groups, as visualized with 6-diamidino-2-phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 +/- 2.0) compared with group 2 (21.6 +/- 1.1%, P < 0.001) and group 1 (16.9 +/- 1.1%, P < 0.001). Immunostaining for CD31 showed significantly increased capillary density in group 3 (14.88 +/- 0.9) compared with group 2 (8.5 +/- 0.49, P < 0.001) and group 1 (5.69 +/- 0.3, P < 0.001). Improved blood flow (ml/min./g) was achieved in animal group 3 (0.173 +/- 0.04) as compared with animal group 2 (0.122 +/- 0.016; P= 0.047) and group 1 (0.062 +/- 0.012; P < 0.001). In conclusion, CD liposome mediated VEGF(165) gene transfer with SkMs effectively induced neovascularization in the ischaemic hind limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.     

The autosomal recessive nonsyndromic deafness locus DFNB72 is located on chromosome 19p13.3

We ascertained three consanguineous Pakistani families (PKDF291, PKDF335 and PKDF793) segregating nonsyndromic recessive hearing loss. The hearing loss segregating in PKDF335 and PKDF793 is moderate to severe, whereas it is profound in PKDF291. The maximum two-point LOD scores are 3.01 (D19S1034), 3.85 (D19S894) and 3.71 (D19S894) for PKDF291, PKDF335 and PKDF793, respectively. Haplotype analyses of the three families define a 1.16 Mb region of overlap of the homozygous linkage intervals bounded by markers D19S216 (20.01 cM) and D19S1034 (20.75 cM). These results define a novel locus, DFNB72, on chromosome 19p13.3. There are at least 22 genes in the 1.16 Mb interval, including PTPRS, ZNRF4 and CAPS. We identified no pathogenic variants in the exons and flanking intronic sequences of these three genes in affected members of the DFNB72 families. DFNB72 is telomeric to DFNB68, the only other known deafness locus with statistically significant support for linkage to chromosome 19p.     

Bromophenol blue binding as a probe to study urea and guanidine hydrochloride denaturation of bovine serum albumin

Urea and guanidine hydrochloride (GdnHCl) denaturation of bovine serum albumin (BSA) were investigated using bromophenol blue (BPB) binding as a probe. Addition of BPB to BSA produced an absorption difference spectrum in the wavelength range, 525-675 nm with a minimum at 587 nm and a maximum at 619 nm. The magnitude of absorption difference (DeltaAbs.) at 619 nm decreased on increasing urea/GdnHCl concentration and followed the denaturation curve. The denaturation was found to be a two-state, single-step transition. The transitions started at 1.75 and 0.875 M and completed at 6.5 and 3.25 M with the mid point occurring around 4.0 and 1.5 M urea and GdnHCl concentrations, respectively. The value of free energy of stabilization, DeltaGDH2O as determined from urea and GdnHCl denaturation curves was found to be 4041 and 4602 cal/mol, respectively. Taken together, these results suggest that BPB binding can be used as a probe to study urea and GdnHCl denaturation of BSA.