Generation of ceramide in murine macrophages infected with Leishmania donovani alters macrophage signaling events and aids intracellular parasitic survival

In the present study, we examined the involvement of intracellular ceramide in host pathogen interaction of BALB/c mouse peritoneal macrophages infected with the obligate intracellular protozoan, Leishmania donovani. Our findings indicate that the level of intracellular ceramide was enhanced as a result of the in vitro infection. While the elevated ceramide was largely due to de novo synthesis, activation of the sphingomyelinases was also observed. The enhanced ceramide was responsible for the downregulation of classical PKC activity, upregulation of calcium independent atypical PKC- expression and activity of calcium independent PKC. Ceramide also impaired the phosphorylation of MAPK. Evidently, ceramide suppressed the generation of nitric oxide during leishmanial infection and also facilitated the survival of leishmanial parasites in the intramacrophageal milieu. These data present newer insight to the signaling events in leishmania-infected murine macrophages, which might offer ceramide as a new therapeutic target in the future.     

Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase

The Corynebacterium glutamicum (C. glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form. This is the first report of the production of C. glutamicum PCK overexpressed in E. coli. The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step. This one-step, easy purification method would be very useful for future mutational and structural studies. The molecular mass of the purified protein is approximately 68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme. Also, the enzyme assays revealed that C. glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+.     

Identification and partial characterization of the allergenic proteins of Ricinus communis L. pollen - a new approach

The pollen of Ricinus communis L., a potentially allergenic plant, was extracted to identify the allergenic determinants responsible for causing respiratory disorders. The soluble proteins were extracted and subjected to ammonium sulphate precipitation at 80% saturation and the total protein separated on 12% SDS-Polyacrylamide gel. In order to avoid the time consuming and expensive biochemical methods of column chromatography, each band was directly recovered from the gel by electroelution and the allergenic proteins identified directly by skin tests, without the necessity of Phadezym RAST or ELISA inhibition by reaction with serum IgE, the general procedure to identify the allergens. The fourth and the fifth band in the protein profile of R. communis pollen, RC4 (77 kD) and RC5 (66 kD) were the two major allergenic components. RC3 (91 kD) also induced a considerable amount of reactivity in sensitive patients. Contrary to the earlier reports of protein bands of R. communis ranging from 14 kD to 70 kD, 4 bands above 70 kD i.e. RC1 (123 kD), RC2 (97 kD), RC3 (91 kD) and RC4 (77 kD) are reported here for the first time. Immunodiffusion analysis with pooled sera of patients sensitive to the total extract also revealed similar results.     

Evaluation of selected antitumor agents as subversive substrate and potential inhibitor of trypanothione reductase: an alternative approach for chemotherapy of Leishmaniasis

Trypanothione reductase (TryR) is a validated drug target against Leishmaniasis. Using integrated computational and experimental approaches, the authors report doxorubicin and mitomycin C, known antitumor agents, as novel inhibitors of TryR of leishmania parasite. Interestingly, these compounds also act as subversive substrates and subvert the physiological function of enzyme by converting it from an anti-oxidant to a pro-oxidant. Possible mechanism of subversive substrate is discussed. Both doxorubicin and mitomycin C show significant effect on redox homeostasis of the parasite and high-leishmanicidal activity. The toxicity studies as well as available toxicity data in literature indicate these compounds to have acceptable toxicity in limited dose.     

Role of Ferulic Acid in the Amelioration of Ionizing Radiation Induced Inflammation: A Murine Model

Ionizing radiation is responsible for oxidative stress by generating reactive oxygen species (ROS), which alters the cellular redox potential. This change activates several redox sensitive enzymes which are crucial in activating signaling pathways at molecular level and can lead to oxidative stress induced inflammation. Therefore, the present study was intended to assess the anti-inflammatory role of ferulic acid (FA), a plant flavonoid, against radiation-induced oxidative stress with a novel mechanistic viewpoint. FA was administered (50 mg/kg body wt) to Swiss albino mice for five consecutive days prior to exposing them to a single dose of 10 Gy ⁶⁰Co γ-irradiation. The dose of FA was optimized from the survival experiment and 50 mg/kg body wt dose showed optimum effect. FA significantly ameliorated the radiation induced inflammatory response such as phosphorylation of IKKα/β and IκBα and consequent nuclear translocation of nuclear factor kappa B (NF-κB). FA also prevented the increase of cycloxygenase-2 (Cox-2) protein, inducible nitric oxide synthase-2 (iNOS-2) gene expression, lipid peroxidation in liver and the increase of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in serum. It was observed that exposure to radiation results in decreased activity of superoxide dismutase (SOD), catalase (CAT) and the pool of reduced glutathione (GSH) content. However, FA treatment prior to irradiation increased the activities of the same endogenous antioxidants. Thus, pretreatment with FA offers protection against gamma radiation induced inflammation.     

DNA,peroxidase profile,and biochemical composition changes during the treatment in vitro of pollen of Argemone mexicana L. with SO 2

The allergenic pollen of Argemone mexicana L. a common road-side weed was subjected to artificial SO 2 fumigation at a concentration of 100ppm for 24 hours, 48 hours and 72 hours and its effect on the total carbohydrate, lipid, free amino acid, DNA and RNA content as well as peroxidase isozyme and DNA profile studied. In comparison to the control (pollen exposed to charcoal-filtered air), the carbohydrate, lipid, DNA and RNA content decreased with the increase in the time of exposure to SO 2 , where as the percentage composition of free amino acid increased. Contrary, to this, the activity of peroxidase was found to increase with a change in the iso-peroxidase zymogram showing three new anionic isozyme bands. SO 2 affected the total DNA profile leading to the gradual breakdown of DNA with several bands observed on agarose gel electrophoresis with an increase in the time of fumigation.     

Lymphatic system: a vital link between metabolic syndrome and inflammation

Metabolic syndrome is defined by a cluster of different metabolic risk factors that include overall and central obesity, elevated fasting glucose levels, dyslipidemia, hypertension, and intimal atherogenesis. Metabolic syndrome leads to increased risk for the development of type 2 diabetes and cardiovascular disease (e.g., heart disease and stroke). The exacerbated progression of metabolic syndrome to cardiovascular disease has lead to intense study of the physiological ramifications of metabolic syndrome on the blood vasculature. These studies have particularly focused on the signaling and architectural alterations that manifest in hypertension and atherosclerosis. However, despite the overlap of metabolic syndrome pathology with lymphatic function, tangent effects on the lymphatic system have not been extensively documented. In this review, we discuss the current status of metabolic syndrome and provide evidence for, and the remaining challenges in studying, the connections among the lymphatic system, lipid transport, obesity, insulin resistance, and general inflammation.     

Structure of a GTP-dependent bacterial PEP-carboxykinase from Corynebacterium glutamicum

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2₁ with four molecules per asymmetric unit. The 2.3 Å resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guanine binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.