Therapeutic Potential of Clove Essential Oil in Diabetes: Modulation of Pro-Inflammatory Mediators,Oxidative Stress and Metabolic Enzyme Activities

Type 1 diabetes is characterized by insulin deficiency due to the destruction of pancreatic β cells, leading to hyperglycemia, which in turn induces vascular complications. In the current study, we investigated the effect of intraperitoneal administration of clove essential oil (CEO: 20 mg/kg body weight) on certain oxidative stress and glucose metabolism enzymes, as well as the expression of proinflammatory mediators. Administration of CEO to diabetic rats showed a significant decline in blood glucose levels, total cholesterol, and xanthine oxidase, compared to the streptozotocin group. Furthermore, these treated rats elicited a notable attenuation in the levels of lipid peroxides, and thiols groups in both liver and brain tissues. The activities of antioxidant and metabolic enzymes were reverted to normality in diabetic upon CEO administration. In addition to its protective effects on red blood cell hemolysis, CEO is a potent α-amylase inhibitor with an IC₅₀=298.0±2.75 μg/mL. Also, treatment of diabetic rats with CEO significantly reduced the iNOS expression in the spleen. Our data showed that CEO has potential beneficial effects on diabetes, which can possibly prevent the pathogenesis of diabetic micro- and macrovascular complications.     

Characterization of CD200 Ectodomain Shedding

We have previously reported the existence of a soluble form of CD200 (sCD200) in human plasma, and found sCD200 to be elevated in the plasma of Chronic Lymphocytic Leukemia (CLL) patients. CLL cells release CD200 at a constitutive level, which could be attenuated partially by ADAM28 silencing. In this study, we further explored mechanisms of CD200 shedding beyond that of ADAM28, and performed biochemical analysis of sCD200 using materials derived from purified CLL cells and Hek293 cells stably transfected with CD200, and antibodies generated specifically against either the extracellular or cytoplasmic regions of CD200. CD200 shedding was enhanced by PMA stimulation, and the loss of cell surface CD200 could be monitored as a reduction in CD200 cell surface expression by flow cytometry, in parallel with an increase in the detection of sCD200 in the supernatant. Western blot analyses and functional studies using CD200R1 expressing Hek293 cells showed that the shed CD200 detected in CLL and Hek293-hCD200 supernatants lacked the cytoplasmic domain of CD200 but retained the functional extracellular domain required for binding to, and phosphorylation of, CD200R. These data confirms that a functionally active CD200 extracellular moiety can be cleaved from the surface of CD200 expressing cells following ectodomain shedding.     

Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action. Focused SAR around the pyridine heterocycle (Ar) in 12a (Tables 1 and 2) resulted into four shortlisted compounds, (?)-12a, (?)-12i, (?)-12l–m. The required (?)-enantiomers were obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15. Based on comparative profile of FAAH potency, metabolic stability in liver microsome, liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two SAR optimized compounds, (?)-12l and (?)-12m, were selected for efficacy study in rat model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds exhibited dose related antihyperalgesic effects, when treated with 3–30?mg/kg po for 7?days. The effects at 30?mg/kg are comparable to that of PF-04457845 (10?mg/kg) and Tramadol (40?mg/kg).     

Reserpine Is the New Addition into the Repertoire of AcrB Efflux Pump Inhibitors

Molecular Biology - Acriflavine resistance protein B (AcrB) serves as prototype for multidrug resistance (MDR) efflux transporters of resistance nodulation division (RND) superfamily. AcrB has been...     

Topology-informed strategies for the overexpression and purification of membrane proteins

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.     

Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

Background  

The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysis     

Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component

Background

A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.

Methodology/Principal Findings

NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.

Significance

The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00392015","term_id":"NCT00392015"}}NCT00392015