Expression from symbiotic promoters ofRhizobium meliloti inAzotobacter vinelandii andAzospirillum brasilense

InRhizobium meliloti, the promoter P1 of thenif HDK operon, and also the promoter P2, have earlier been shown to be active in the bacteria present in alfalfa root nodules, but not in the bacteria grown aerobically in culture. Here we have looked at the expression from P1 and P2 in two non-symbiotic nitrogen-fixing bacteria,Azotobacter vinelandii andAzospirillum brasilense, using constructions in which the promoters are fused upstream of theβ-galactosidase gene. The promoter P1, but not P2, is active inA. vinelandii, while neither P1 nor P2 is active inAzospirillum brasilense.     

Development of Novel Docetaxel Phospholipid Nanoparticles for Intravenous Administration: Quality by Design Approach

The objective of this study was to develop novel docetaxel phospholipid nanoparticles (NDPNs) for intravenous administration. Modified solvent diffusion-evaporation method was adopted in the NDPN preparation. Central composite design (CCD) was employed in the optimization of the critical formulation factor (drug content) and process variable (stirring rate) to obtain NDPNs with 215.53 ± 1.9-nm particle size, 0.329 ± 0.02 polydispersity index (PDI), and 75.41 ± 4.81% entrapment efficiency. The morphological examination by transmission electron microscopy revealed spherical structure composed of a drug core stabilized within the phospholipid shell. Enhanced cell uptake of coumarin-6-loaded phospholipid nanoparticles by MCF-7 cell line indicated NDPN-efficient cell uptake. In vitro hemolysis test confirmed the safety of the phospholipid nanoparticles. NDPNs exhibited increased area under the curve (AUC) and mean residence time (MRT) by 3.0- and 3.3-fold, respectively, in comparison with the existing docetaxel parenteral formulation (Taxotere®), indicating a potential for sustained action. Thus, the novel NDPNs exhibit an ability to be an intravenous docetaxel formulation with enhanced uptake, decreased toxicity, and prolonged activity.KEY WORDS: cancer, delivery vehicle, docetaxel phospholipid nanoparticles, nanoparticles, pharmacokinetics     

A γ-Secretase Independent Role for Presenilin in Calcium Homeostasis Impacts Mitochondrial Function and Morphology in Caenorhabditis elegans

Mutations in the presenilin (PSEN) encoding genes (PSEN1 and PSEN2) occur in most early onset familial Alzheimer’s Disease. Despite the identification of the involvement of PSEN in Alzheimer’s Disease (AD) ∼20 years ago, the underlying role of PSEN in AD is not fully understood. To gain insight into the biological function of PSEN, we investigated the role of the PSEN homolog SEL-12 in Caenorhabditis elegans. Using genetic, cell biological, and pharmacological approaches, we demonstrate that mutations in sel-12 result in defects in calcium homeostasis, leading to mitochondrial dysfunction. Moreover, consistent with mammalian PSEN, we provide evidence that SEL-12 has a critical role in mediating endoplasmic reticulum (ER) calcium release. Furthermore, we found that in SEL-12-deficient animals, calcium transfer from the ER to the mitochondria leads to fragmentation of the mitochondria and mitochondrial dysfunction. Additionally, we show that the impact that SEL-12 has on mitochondrial function is independent of its role in Notch signaling, γ-secretase proteolytic activity, and amyloid plaques. Our results reveal a critical role for PSEN in mediating mitochondrial function by regulating calcium transfer from the ER to the mitochondria.     

Effect of Cyclodextrin Complexation of Curcumin on its Solubility and Antiangiogenic and Anti-inflammatory Activity in Rat Colitis Model

The purpose of the study was to prepare and evaluate the anti-inflammatory activity of cyclodextrin (CD) complex of curcumin for the treatment of inflammatory bowel disease (IBD) in colitis-induced rat model. Inclusion complexes of curcumin were prepared by common solvent and kneading methods. These complexes were further evaluated for increase in solubility of poorly soluble curcumin. The inclusion complexes were characterized for enhancement in solubility, in vitro dissolution, surface morphology, infrared, differential scanning calorimetry, and X-ray studies. Solubility, phase solubility, and in vitro dissolution studies showed that curcumin has higher affinity for hydroxypropyl-β-CD (HPβCD) than other CDs. HPβCD complex of curcumin was further investigated for its antiangiogenic and anti-inflammatory activity using chick embryo and rat colitis model. HPβCD complex of curcumin proved to be a potent angioinhibitory compound, as demonstrated by inhibition of angiogenesis in chorioallantoic membrane assay. Curcumin- and HPβCD-treated rats showed a faster weight gain compared to dextran sulfate solution (DSS) controls. Whole colon length appeared to be significantly longer in HPβCD-treated rats than pure curcumin and DSS controls. An additional finding in the DSS-treated rats was the predominance of eosinophils in the chronic cell infiltrate. Decreased mast cell numbers in the mucosa of the colon of CD of curcumin- and pure-curcumin-treated rats was observed. This study concluded that the degree of colitis caused by administration of DSS was significantly attenuated by CD of curcumin. Being a nontoxic natural dietary product, curcumin could be useful in the therapeutic strategy for IBD patients.     

<Emphasis Type="Italic">In Vitro</Emphasis>-<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Novel Co-spray Dried Rifampicin Phospholipid Lipospheres for Oral Delivery

The objective of this study comprises of developing novel co-spray dried rifampicin phospholipid lipospheres (SDRPL) to investigate its influence on rifampicin solubility and oral bioavailability. Solid-state techniques were employed to characterize the liposphere formulation. SDRPL solubility was determined in distilled water. BACTEC 460TB System was employed to evaluate SDRPL antimycobacterial activity. The oral bioavailability of the lipospheres was evaluated in Sprague Dawley rats. Lipospheres exhibited amorphous, smooth spherical morphology with a significant increase (p?<?0.001) in solubility of SDRPL (2:1), 350.9?±?23 versus 105.1?±?12 μg/ml and SDRPL (1:1) 306.4?±?20 versus 105.1?±?12 μg/ml in comparison to rifampicin (RMP). SDRPL exhibited enhanced activity against Mycobacterium tuberculosis, H37Rv strain, with over twofolds less minimum inhibitory concentration (MIC) than the free drug. Lipospheres exhibited higher peak plasma concentration (109.92?±?25 versus 54.31?±?18 μg/ml), faster T _max (two versus four hours), and enhanced area under the curve (AUC_0–∞) (406.92?±?18 versus 147.72?±?15 μg h/L) in comparison to pure RMP. Thus, SDRPL represents a promising carrier system exhibiting enhanced antimycobacterial activity and oral bioavailability of rifampicin.     

Increased mitochondrial calcium uptake and concomitant mitochondrial activity by presenilin loss promotes mTORC1 signaling to drive neurodegeneration

Metabolic dysfunction and protein aggregation are common characteristics that occur in age‐related neurodegenerative disease. However, the mechanisms underlying these abnormalities remain poorly understood. We have found that mutations in the gene encoding presenilin in Caenorhabditis elegans, sel‐12, results in elevated mitochondrial activity that drives oxidative stress and neuronal dysfunction. Mutations in the human presenilin genes are the primary cause of familial Alzheimer''s disease. Here, we demonstrate that loss of SEL‐12/presenilin results in the hyperactivation of the mTORC1 pathway. This hyperactivation is caused by elevated mitochondrial calcium influx and, likely, the associated increase in mitochondrial activity. Reducing mTORC1 activity improves proteostasis defects and neurodegenerative phenotypes associated with loss of SEL‐12 function. Consistent with high mTORC1 activity, we find that SEL‐12 loss reduces autophagosome formation, and this reduction is prevented by limiting mitochondrial calcium uptake. Moreover, the improvements of proteostasis and neuronal defects in sel‐12 mutants due to mTORC1 inhibition require the induction of autophagy. These results indicate that mTORC1 hyperactivation exacerbates the defects in proteostasis and neuronal function in sel‐12 mutants and demonstrate a critical role of presenilin in promoting neuronal health.     

X-ray Studies on Crystalline Complexes Involving Amino Acids and Peptides. XXVII. Effect of Chirality,Specific Interactions and Characteristic Aggregation Patterns in the Structures of Arginine and its Complexes with Formic Acid

Abstract

The crystal structures of DL- arginine dihydrate, DL-arginine formate dihydrate and L-arginine formate have been determined and refined using X-ray crystallographic techniques. The three structures, along with other related ones, demonstrate the conformational variability of arginine. The amino acid molecules aggregate essentially in a similar manner in DL- arginine dihydrate and in the known structure of L-arginine dihydrate; the effects arising out of the reversal of the chirality of half the amino acid molecules are absorbed by small local adjustments. However, such a reversal leads to profound differences in aggregation in DL- arginine and L- arginine formates, in contrast to the situation in the corresponding acetates. Thus the effect of chirality on biomolecular aggregation cannot be easily predicted or even rationalized. Arginine-carboxylate interactions in the complexes primarily involve the guanidyl groups and contain specific interactions. Indeed the primary mode of arginine-carboxylic acid aggregation is substantially invariant in the arginine complexes of succinic, acetic and formic acids.     

Corrupted ER‐mitochondrial calcium homeostasis promotes the collapse of proteostasis

Aging and age‐related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.