Effect of a single point mutation on the stability,residual structure and dynamics in the denatured state of GED: Relevance to self-assembly

The GTPase effector domain (GED) of dynamin forms large soluble oligomers in vitro, while its mutant – I697A – lacks this property at low concentrations. With a view to understand the intrinsic structural characteristics of the polypeptide chain, the global unfolding characteristics of GED wild type (WT) and I697A were compared using biophysical techniques. Quantitative analysis of the CD and fluorescence denaturation profiles revealed that unfolding occurred by a two-state process and the mutant was less stable than the WT. Even in the denatured state, the mutation caused chemical shift perturbations and significant differences were observed in the ¹⁵N transverse relaxation rates (R₂), not only at the mutation site but all around. These results demonstrate that the hydrophobic change associated with the mutation perturbs the structural and motional preferences locally, which are then relayed via different folding pathways along the chain and the property of oligomerization in the native state is affected.     

Diabetic nephropathy: mechanisms of renal disease progression

Diabetic nephropathy is characterized by excessive amassing of extracellular matrix (ECM) with thickening of glomerular and tubular basement membranes and increased amount of mesangial matrix, which ultimately progress to glomerulosclerosis and tubulo-interstitial fibrosis. In view of this outcome, it would mean that all the kidney cellular elements, i.e., glomerular endothelia, mesangial cells, podocytes, and tubular epithelia, are targets of hyperglycemic injury. Conceivably, high glucose activates various pathways via similar mechanisms in different cell types of the kidney except for minor exceptions that are related to the selective expression of a given molecule in a particular renal compartment. To begin with, there is an obligatory excessive channeling of glucose intermediaries into various metabolic pathways with generation of advanced glycation products (AGEs), activation of protein kinase C (PKC), increased expression of transforming growth factor-beta (TGF-beta), GTP-binding proteins, and generation of reactive oxygen species (ROS). The ROS seem to be the common denominator in various pathways and are central to the pathogenesis of hyperglycemic injury. In addition, there are marked alterations in intraglomerular hemodynamics, i.e., hyperfiltration, and this along with metabolic derangements adversely compounds the hyperglycemia-induced injury. Here, the information compiled under various subtitles of this article is derived from an enormous amount of data summarized in several excellent literature reviews, and thus their further reading is suggested to gain in-depth knowledge of each of the subject matter.     

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat(2), mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell-penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active beta-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat(2)-mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat(2)-mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat(2)-GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat(2)-plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat(2)-plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine 2000 to the Tat(2)-plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.     

Design, synthesis and activity of novel derivatives of oxybutynin and tolterodine

Novel derivatives of Tolterodine (1) and Oxybutynin (2) have been designed using conformationally restricted azabicyclics as replacement for open-chain amines. The synthesis and structure-activity relationships are presented.     

Recombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen

A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris. Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates. Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs). Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybrid protein. Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybrid protein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data.     

Juxtaglomerular apparatus tumor: a rare, surgically correctable cause of hypertension

Although uncommon, presentation of juxtaglomerular cell tumor is distinct and should allow a correct preoperative diagnosis in most patients. Typical clinical presentations include headaches, polyuria, or isolated, asymptomatic, severe hypertension. The diagnosis of a juxtaglomerular apparatus (JGA) tumor typically results from identification of plasma renin levels two- to sevenfold greater than the normal value. Although JGA tumors are considered benign, with no reports of metastases or recurrence, they are potentially lethal if left untreated. Surgical excision is curative.     

Squalene epoxidase as hypocholesterolemic drug target revisited

Therapeutic success of statins has distinctly established inhibition of de novo hepatic cholesterol synthesis as an effective approach to lower plasma LDL-cholesterol, the major risk factor for atherosclerosis and coronary heart disease. Statins inhibit HMG CoA reductase, a rate limiting enzyme which catalyses conversion of HMG CoA to mevalonic acid. However, in this process statins also inhibit the synthesis of several non-sterols e.g. dolichols and ubiquinone, which are implicated in side effects observed with statins. This prompted many major pharmaceutical companies in 1990s to target selective cholesterol synthesis beyond farnesyl pyrophosphate. The enzymes squalene synthetase, squalene epoxidase and oxidosqualene cyclase were identified as potential targets. Though inhibitors of these enzymes have been developed, till date no compound has been reported to have entered clinical trials. We evaluated the literature to understand merits and demerits of pursuing squalene epoxidase as a target for hypocholesterolemic drug development. Squalene epoxidase catalyses the conversion of squalene to 2,3-oxidosqualene. Although it has been extensively exploited for antifungal drug development, it has received little attention as a target for hypocholesterolemic drug design. This enzyme though recognized in the early 1970s was cloned 25 years later. This enzyme is an attractive step for pharmacotherapeutic intervention as it is the secondary rate limiting enzyme and blocking cholesterol synthesis at this step may result in accumulation of only squalene which is known to be stable and non toxic. Synthesis of several potent, orally bioavailable inhibitors of squalene epoxidase has been reported from Yamonuchi, Pierre Fabre and Banyu pharmaceuticals. Preclinical studies with these inhibitors have clearly demonstrated the potential of squalene epoxidase inhibitors as hypocholesterolemic agents. Hypochloesterolemic therapy is intended for prolonged duration and safety is an important determinant in clinical success. Lack of clinical trials, despite demonstrated preclinical efficacy by oral route, prompted us to evaluate safety concerns with squalene epoxidase inhibitors. In dogs, NB-598, a potent competitive squalene epoxidase inhibitor has been reported to exhibit signs of dermatitis like toxicity which has been attributed by some reviewers to accumulation of squalene in skin cells. Tellurium, a non-competitive inhibitor of squalene epoxidase has been associated with neuropathy in weanling rats. On the other hand, increased plasma levels of squalene in animals and humans (such as occurring subsequent to dietary olive oil or squalene administration) are safe and associated with beneficial effect such as chemoprevention and hypocholesterolemic activity. In our view, high circulating levels of squalene epoxidase inhibitor may be responsible for dermatitis and neuropathy. Competitive inhibition and pharmacokinetic profile minimizing circulating plasma levels (e.g. by hepatic sequestration and high first pass metabolism) could be important determinants in circumventing safety concerns of squalene epoxidase inhibitors. Recently, cholesterol-lowering effect of green tea has been attributed to potent squalene epoxidase inhibition, which can be consumed in much higher doses without toxicological effect. These facts strengthen optimism for developing clinically safe squalene epoxidase inhibitors. Put in perspective squalene epoxidase appears to be undervalued target which merits attention for development of better hypocholesterolemic drugs.     

Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum dicoccum Schuble)

The present study establishes a regeneration protocol and optimizes conditions for Agrobacterium-mediated transformation of the tetraploid emmer wheat, Triticum dicoccum. Regeneration from mature and immature embryos was accomplished as a two-step process involving callus induction in the presence of 2,4-D followed by regeneration on a 2,4-D free, cytokinin-containing medium (RM1). Higher concentrations of 2,4-D (4 mg/l) though conducive for callusing (89.39% in mature embryos and 96% in immature embryos) proved detrimental for further regeneration. At lower 2,4-D (1 mg/ml) although callusing was suboptimal, (56.8% and 84% from mature and immature embryos, respectively) the regeneration response was the highest on RM1 medium (64.4% and 56.6% from mature and immature embryos, respectively). Overall, the regeneration response of immature embryos was lower than the mature embryos by 10-12%. Due to the ease of availability of mature embryos the mature embryo-derived calli were chosen as the target tissue for Agrobacterium-mediated transformation in the two Indian varieties DDK1001 and DDK1009. Histochemical GUS expression revealed the suitability of the mature embryo-derived calli for such investigations. Of the CaMV35S and Act1 promoters employed, the monocot promoter Act1 displayed higher GUS gene activity in the mature embryo derived calli when co-cultivated with LBA4404 (pBI101::Act1).