Myelopoietic Efficacy of Orlistat in Murine Hosts Bearing T Cell Lymphoma: Implication in Macrophage Differentiation and Activation

Orlistat, an inhibitor of fatty acid synthase (FASN), acts as an antitumor agent by blocking de novo fatty acid synthesis of tumor cells. Although, myelopoiesis also depends on de novo fatty acid synthesis, the effect of orlistat on differentiation of macrophages, which play a central role in host’s antitumor defence, remains unexplored in a tumor-bearing host. Therefore, the present investigation was undertaken to examine the effect of orlistat administration on macrophage differentiation in a T cell lymphoma bearing host. Administration of orlistat (240 mg/kg/day/mice) to tumor-bearing mice resulted in a decline of tumor load accompanied by an augmentation of bone marrow cellularity and survival of bone marrow cells (BMC). The expression of apoptosis regulatory caspase-3, Bax and Bcl2 was modulated in the BMC of orlistat-administered tumor-bearing mice. Orlistat administration also resulted in an increase in serum level of IFN-γ along with decreased TGF-β and IL-10. BMC of orlistat-administered tumor-bearing mice showed augmented differentiation into macrophages accompanied by enhanced expression of macrophage colony stimulating factor (M-CSF) and its receptor (M-CSFR). The macrophages differentiated from BMC of orlistat-administered mice showed characteristic features of M₁ macrophage phenotype confirmed by expression of CD11c, TLR-2, generation of reactive oxygen species, phagocytosis, tumor cell cytotoxicity, production of IL-1,TNF-α and nitric oxide. These novel findings indicate that orlistat could be useful to support myelopoesis in a tumor-bearing host.     

Differential effects of ret and TRKB on axonal branching and survival of parasympathetic neurons

Interactions between neurons and their targets of innervation influence many aspects of neural development. To examine how synaptic activity interacts with neurotrophic signaling, we determined the effects of blocking neuromuscular transmission on survival and axonal outgrowth of ciliary neurons from the embryonic chicken ciliary ganglion. Ciliary neurons undergo a period of cell loss due to programmed cell death between embryonic Days (E) 8 and 14 and they innervate the striated muscle of the iris. The nicotinic antagonist d‐tubocurarine (dTC) induces an increase in branching measured by counting neurofilament‐positive voxels (NF‐VU) in the iris between E14‐17 while reducing ciliary neuron survival. Blocking ganglionic transmission with dihyro‐β‐erythroidin and α‐methyllycacontine does not mimic dTC. At E8, many trophic factors stimulate neurite outgrowth and branching of neurons placed in cell culture; however, at E13, only GDNF stimulates branching selectively in cultured ciliary neurons. The GDNF‐induced branching at E13 could be inhibited by BDNF. Blocking ret signaling in vivo with a dominant negative (dn)ret decreases survival of ciliary and choroid neurons at E14 and prevents dTC induced increases in NF‐VU in the iris at E17. Blocking TRKB signaling with dn TRKB increases NF‐VU in the iris at E17 and decreases neuronal survival at E17, but not at E14. Thus, RET promotes survival during programmed cell death in the ciliary ganglion and contributes to promoting branching when synaptic transmission is blocked while TRKB inhibits branching and promotes maintenance of neuronal survival. These studies highlight the multifunctional nature of trophic molecule function during neuronal development. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Structure-function analysis of the EF-hand protein centrin-2 for its intracellular localization and nucleotide excision repair

Centrin-2 is an evolutionarily conserved, calmodulin-related protein, which is involved in multiple cellular functions including centrosome regulation and nucleotide excision repair (NER) of DNA. Particularly to exert the latter function, complex formation with the XPC protein, the pivotal NER damage recognition factor, is crucial. Here, we show that the C-terminal half of centrin-2, containing two calcium-binding EF-hand motifs, is necessary and sufficient for both its localization to the centrosome and interaction with XPC. In XPC-deficient cells, nuclear localization of overexpressed centrin-2 largely depends on co-overexpression of XPC, and mutational analyses of the C-terminal domain suggest that XPC and the major binding partner in the centrosome share a common binding surface on the centrin-2 molecule. On the other hand, the N-terminal domain of centrin-2 also contains two EF-hand motifs but shows only low-binding affinity for calcium ions. Although the N-terminal domain is dispensable for enhancement of the DNA damage recognition activity of XPC, it contributes to augmenting rather weak physical interaction between XPC and XPA, another key factor involved in NER. These results suggest that centrin-2 may have evolved to bridge two protein factors, one with high affinity and the other with low affinity, thereby allowing delicate regulation of various biological processes.     

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.     

Chemistry,antioxidant and antimicrobial potential of nutmeg (Myristica fragrans Houtt)

Antioxidant and antimicrobial activities of nutmeg (Myristica fragrans Houtt) seed extracts were evaluated. Seeds were extracted with acetone, ethanol, methanol, butanol and water. All the extracts have shown significant antioxidant and antimicrobial activities against the tested microorganisms. Among all extracts, acetone extract has shown the highest antioxidant activity. The acetone extract showed 93.12 ± 1.48 mg gallic acid equivalents (GAE)/100 g dry weight total phenolic content, DPPH scavenging activity of 63.04 ± 1.56%, chelating activity of 64.11 ± 2.21% and 74.36 ± 1.94% inhibition of β-carotene bleaching, at 1 mg/mL extract concentration. Out of all extracts, acetone extract was able to exert antimicrobial activity against all tested bacteria and fungi. Acetone extract has shown the strongest antibacterial and antifungal activity with Staphylococcus aureus (13.8 ± 0.42 mm) and Aspergillus niger (14.4 ± 0.37 mm), respectively. GC–MS analysis of acetone extract has revealed the presence of 32 compounds of extract representing 99.49%. Sabinene (28.61%) has shown the highest occurrence in the extract. β-Pinene (10.26), α-pinene (9.72), myristicin (4.30%), isoeugenol (2.72%), p-cymene (1.81%), carvacrol (1.54%), eugenol (0.89%) and β-caryophellene (0.82%) were reported as possible contributor for antioxidant and antimicrobial activity of nutmeg.     

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance,Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Background

Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress.

Methodology

The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.

Results

The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.

Conclusions

Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.     

Practical one-step glucuronidation via biotransformation

We herein report a practical one-step glucuronidation method by biotransformation using Streptomyces sp. SANK 60895. This novel direct method of biotransformation has been shown to be more practical and scalable for glucuronidation than previously reported chemical and enzymatic procedures given its simplicity, high β-selectivity, cost-effectiveness, and reproducibility. We applied the present method to the synthesis of acyl glucuronide and hydroxy-β-glucuronide of mycophenolic acid and compound 4, respectively. This method was also shown to be applicable to the N-glucuronidation of various compounds.     

The mechanism of phosphatidylcholine‐induced interference of PAP (248‐286) aggregation

Seminal amyloids are well known for their role in enhancing HIV infection. Among all the amyloidogenic peptides identified in human semen, PAP_248‐286 was found to be the most active and was termed as semen‐derived enhancer of viral infection (SEVI). Although amyloidogenic nature of the peptide is mainly linked with enhancement of the viral infection, the most active physiological conformation of the aggregated peptide remains inconclusive. Lipids are known to modulate aggregation pathway of a variety of proteins and peptides and constitute one of the most abundant biomolecules in human semen. PAP_248‐286 significantly differs from the other known amyloidogenic peptides, including Aβ and IAPP, in terms of critical concentration, surface charge, fibril morphology, and structural transition during aggregation. Hence, in the present study, we aimed to assess the effect of a lipid, 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC), on PAP_248‐286 aggregation and the consequent conformational outcomes. Our initial observation suggested that the presence of the lipid considerably influenced the aggregation of PAP_248‐286. Further, ZDOCK and MD simulation studies of peptide multimerization have suggested that the hydrophobic residues at C‐terminus are crucial for PAP_248‐286 aggregation and are anticipated to be major DOPC‐interacting partners. Therefore, we further assessed the aggregation behaviour of C‐terminal (PAP_273‐286) fragment of PAP_248‐286 and observed that DOPC possesses the ability to interfere with the aggregation behaviour of both the peptides used in the current study. Mechanistically, we propose that the presence of DOPC causes considerable inhibition of the peptide aggregation by interfering with the peptide's disordered state to β‐sheet transition.