Ligand-switchable Substrates for a Ubiquitin-Proteasome System

Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates.     

Nanomedicine Scale-up Technologies: Feasibilities and Challenges

Nanomedicine refers to biomedical and pharmaceutical applications of nanosized cargos of drugs/vaccine/DNA therapeutics including nanoparticles, nanoclusters, and nanospheres. Such particles have unique characteristics related to their size, surface, drug loading, and targeting potential. They are widely used to combat disease by controlled delivery of bioactive(s) or for diagnosis of life-threatening problems in their very early stage. The bioactive agent can be combined with a diagnostic agent in a nanodevice for theragnostic applications. However, the formulation scientist faces numerous challenges related to their development, scale-up feasibilities, regulatory aspects, and commercialization. This article reviews recent progress in the method of development of nanoparticles with a focus on polymeric and lipid nanoparticles, their scale-up techniques, and challenges in their commercialization.KEY WORDS: lipid nanoparticles, nanomedicine, polymeric nanoparticles, scale-up production     

EpCAM Knockdown Alters MicroRNA Expression in Retinoblastoma- Functional Implication of EpCAM Regulated MiRNA in Tumor Progression

The co-ordinated regulation of oncogenes along with miRNAs play crucial role in carcinogenesis. In retinoblastoma (RB), several miRNAs are known to be differentially expressed. Epithelial cell adhesion molecule (EpCAM) gene is involved in many epithelial cancers including, retinoblastoma (RB) tumorigenesis. EpCAM silencing effectively reduces the oncogenic miR-17-92 cluster. In order to investigate whether EpCAM has wider effect as an inducer or silencer of miRNAs, we performed a global microRNA expression profile in EpCAM siRNA knockdown Y79 cells. MicroRNA profiling in EpCAM silenced Y79 cells showed seventy-three significantly up regulated and thirty-six down regulated miRNAs. A subset of these miRNAs was also validated in tumors. Functional studies on Y79 and WERI-Rb-1 cells transfected with antagomirs against two miRNAs of miR-181c and miR-130b showed striking changes in tumor cell properties in RB cells. Treatment with anti-miR-181c and miR-130b showed significant decrease in cell viability and cell invasion. Increase in caspase-3 level was noticed in antagomir transfected cell lines indicating the induction of apoptosis. Possible genes altered by EpCAM influenced microRNAs were predicted by bioinformatic tools. Many of these belong to pathways implicated in cancer. The study shows significant influence of EpCAM on global microRNA expression. EpCAM regulated miR-181c and miR-130b may play significant roles in RB progression. EpCAM based targeted therapies may reduce carcinogenesis through several miRNAs and target genes.     

Bayesian-based selection of metabolic objective functions

MOTIVATION: A critical component of in silico analysis of underdetermined metabolic systems is the identification of the appropriate objective function. A common assumption is that the objective of the cell is to maximize growth. This objective function has been shown to be consistent in a few limited experimental cases, but may not be universally appropriate. Here a method is presented to quantitatively determine the most probable objective function. RESULTS: The genome-scale metabolism of Escherichia coli growing on succinate was used as a case-study for analysis. Five different objective functions, including maximization of growth rate, were chosen based on biological plausibility. A combination of flux balance analysis and linear programming was used to simulate cellular metabolism, which was then compared to independent experimental data using a Bayesian objective function discrimination technique. After comparing rates of oxygen uptake and acetate production, minimization of the production rate of redox potential was determined to be the most probable objective function. Given the appropriate reaction network and experimental data, the discrimination technique can be applied to any bacterium to test a variety of different possible objective functions. SUPPLEMENTARY INFORMATION: Additional files, code and a program for carrying out model discrimination are available at http://www.engr.uconn.edu/~srivasta/modisc.html.     

Binding and release of cholesterol in the Osh4 protein of yeast

Sterols have been shown experimentally to bind to the Osh4 protein (a homolog of the oxysterol binding proteins) of Saccharomyces cerevisiae within a binding tunnel, which consists of antiparallel beta-sheets that resemble a beta-barrel and three alpha-helices of the N-terminus. This and other Osh proteins are essential for intracellular transport of sterols and ultimately cell life. Molecular dynamics (MD) simulations are used to study the binding of cholesterol to Osh4 at the atomic level. The structure of the protein is stable during the course of all MD simulations and has little deviation from the experimental crystal structure. The conformational stability of cholesterol within the binding tunnel is aided in part by direct or water-mediated interactions between the 3-hydroxyl (3-OH) group of cholesterol and Trp(46), Gln(96), Tyr(97), Asn(165), and/or Gln(181) as well as dispersive interactions with Phe(42), Leu(24), Leu(39), Ile(167), and Ile(203). These residues along with other nonpolar residues in the binding tunnel and lid contribute nearly 75% to the total binding energy. The strongest and most populated interaction is between Gln(96) and 3-OH with a cholesterol/Gln(96) interaction energy of -4.5 +/- 1.0 kcal/mol. Phe(42) has a similar level of attraction to cholesterol with -4.1 +/- 0.3 kcal/mol. A MD simulation without the N-terminus lid that covers the binding tunnel resulted in similar binding conformations and binding energies when compared with simulations with the full-length protein. Steered MD was used to determine details of the mechanism used by Osh4 to release cholesterol to the cytoplasm. Phe(42), Gln(96), Asn(165), Gln(181), Pro(211), and Ile(206) are found to direct the cholesterol as it exits the binding tunnel as well as Lys(109). The mechanism of sterol release is conceptualized as a molecular ladder with the rungs being amino acids or water-mediated amino acids that interact with 3-OH.     

Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung

Leukotriene A₄ epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A₄ (LTA₄) to leukotriene B₄ (LTB₄) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 μM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA₄ to LTB₄. This method was used to produce 700 mg of LTB₄ from LTA₄ methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca²⁺, Mn⁺, Fe²⁺, Zn⁺² and Cu⁺² were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 μM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 μM. From these inhibition studies, it can be theorized that the epoxide hydrolae has at least one hydrophobic and one hydrophilic binding site.     

Tackling <Emphasis Type="Italic">Salmonella</Emphasis> Persister Cells by Antibiotic–Nisin Combination via Mannitol

Bacterial persisters (defined as dormant, non-dividing cells with globally reduced metabolism) are the major cause of recurrent infections. As they neither grow nor die in presence of antibiotics, it is difficult to eradicate these cells using antibiotics, even at higher concentrations. Reports of metabolites (which help in waking up of these inactive cells) enabled eradication of bacterial persistence by aminoglycosides, suggest the new potential strategy to improve antibiotic therapy. Here we propose, mannitol enabled elimination of Salmonella persister cells by the nisin–antibiotic combination. For this, persister cells were developed and characterized for their typical properties such as non-replicative state and metabolic dormancy. Different carbon sources viz. glucose, glycerol, and mannitol were used, each as an adjunct to ampicillin for the eradication of persister cells. The maximum (but not complete) killing was observed with mannitol–ampicillin, out of all the combinations used. However, significant elimination (about 78%) could be observed, when nisin (an antimicrobial peptide) was used with ampicillin in presence of mannitol, which might have mediated the transfer of antibiotic–nisin combination at the same time when the cells tried to grab the carbon molecule. Further, the effectiveness of the trio was confirmed by flow cytometry. Overall, our findings highlight the potential of this trio-combination for developing it as an option for tackling Salmonella persister cells.     

Formulation,Pharmacokinetic, and Efficacy Studies of Mannosylated Self-Emulsifying Solid Dispersions of Noscapine

Purpose

To formulate hydroxypropyl methylcellulose-stabilized self-emulsifying solid dispersible carriers of noscapine to enhance oral bioavailability.

Methods

Formulation of noscapine (Nos) self-emulsifying solid dispersible microparticles (SESDs) was afforded by emulsification using an optimized formula of Labrafil M1944, Tween-80, and Labrasol followed by spray-drying with hydroxypropyl methylcellulose (HPMC), with and without mannosamine (Mann-Nos_SESDs and Nos_SESDs respectively); self-microemulsifying liquid dispersions (SMEDDs) with and without mannosamine (Mann-Nos_SMEDDs and Nos_SMEDDs respectively) were also prepared. SMEDDs and SESDs were characterized for size, polydispersity, surface charge, entrapment efficiency, in vitro permeability, in vitro release kinetics, and oral pharmacokinetics in Sprague-Dawley rats (10 mg/kg p.o). The antitumor efficacy of Mann-Nos_SESDs on the basis of chemosensitization to cisplatin (2.0 mg/kg, IV) was investigated in a chemorefractory lung tumor Nu/Nu mouse model up to a maximal oral dose of 300 mg/kg.

Results

The oil/surfactant/co-surfactant mixture of Labrafil M1944, Tween-80, and Labrasol optimized at weight ratios of 62.8:9.30:27.90% produced stable self-microemulsifying dispersions (SMEDDs) at a SMEDD to water ratio of 1–3:7–9 parts by weight. SMEDDs had hydrodynamic diameters between 231 and 246 nm; surface charges ranged from -16.50 to -18.7 mV; and entrapment efficiencies were between 32 and 35%. SESDs ranged in size between 5.84 and 6.60 μm with surface charges from -10.62 to -12.40 mV and entrapment efficiencies of 30.96±4.66 and 32.05±3.72% (Nos_SESDs and Mann-Nos_SESDs respectively). Mann-Nos_SESDs exhibited saturating uptake across Caco-2 monolayers (P_app = 4.94±0.18 × 10⁻⁶ cm/s), with controlled release of 50% of Nos in 6 hr at pH 6.8 following Higuchi kinetics. Mann-Nos_ SESDs was 40% more bioavailable compared to Nos_SESDs; and was effective in sensitizing H1650 SP cells to Cisplatin in vitro and in an orthotopic lung tumor model of H1650 SP origin.

Conclusions

Mannosylated noscapine self-emulsifying solid dispersions (Mann-Nos_SESDs) are bioavailable and potentiate the antineoplastic effect of cisplatin-based chemotherapy in cisplatin-resistant NSCLC.     

Cost-effective production of cellulose hydrolysing enzymes from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. RCK65 under SSF and its evaluation in saccharification of cellulosic substrates

Trichoderma sp. is a potential cellulase producing mesophilic fungi which grow under mild acidic condition. In this study, growth and nutritional conditions were manipulated for the maximum and cost-effective production of cellulase using lab strain Trichoderma sp. RCK65 and checked for its efficiency in hydrolysis of Prosopis juliflora (a woody substrate). Preliminary studies suggested that when 48 h old secondary fungal culture (20 % v/w) was inoculated in wheat bran moistened with mineral salt solution (pH 4.5 and 1:3 solid to moisture ratio), incubated at 30 °C and after 72 h, it produced maximum cellulase (CMCase 145 U/gds, FPase 38 U/gds and β-glucosidase 105 U/gds). However, using statistical approach a S:L ratio (1:1) was surprisingly found to be optimum that improved cellulase that is CMCase activity by 6.21 %, FPase activity by 23.68 % and β-glucosidase activity by 37.28 %. The estimated cost of crude enzyme (Rs. 5.311/1000 FPase units) seems to be economically feasible which may be due to high enzyme titre, less cultivation time and low media cost. Moreover, when the crude enzyme was used to saccharify pretreated Prosopis juliflora (a woody substrate), it resulted up to 83 % (w/w) saccharification.     

Relationship of serum guanase levels with economic characters in zebu purebred and temperate × zebu crossbred dairy cattle

Serum guanase levels of Indian Zebu (Hariana) cattle and its crosses with different exotic levels of temperate breeds of dairy cattle were estimated in 527 animals, and their associations with economic characters were determined. The overall mean value of serum guanase level was 13.46±0.45 IU/ml of serum. An inverse relationship between the concentration of the enzyme and the frequency of cattle falling within a definite range was observed, as over 75% of the animals possessed 1 to 20 IU/ml of serum guanase. An analysis of variance revealed significant differences (P<0.01) in enzyme levels among different genetic groups under study. Hariana (Zebu) purebreds had significantly higher enzyme levels (19.60±1.85) than any of the crossbred groups, whose enzyme levels ranged from 8.99±1.41 in Red Dane F₁ half-breds to 14.30±1.11 in Brown Swiss F₁ half-breds. The correlation and regression coefficients were significantly positive for reproductive traits such as age at first service and age at first calving, and significantly negative for production traits such as lactation yield and lactation length. These results suggest that selection of dairy cattle based on low serum guanase levels might result in correlated improvements in dairy cattle productivity.