Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry

Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1–5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This data provides a large-scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates.     

Structure determination of channel and transport proteins by high-resolution microscopy techniques

High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.     

Rescue of photoreceptor degeneration by curcumin in transgenic rats with P23H rhodopsin mutation

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.     

Insect feeding deterrent and growth inhibitory activities of scopoletin isolated from Artemisia annua against Spilarctia obliqua (Lepidoptera: Noctuidae)

Abstract Artemisia annua (Asteraceae) is well known for its antimalarial activities due to presence of the compound artemisinin. We isolated a methoxy coumarin from the stem part of A. annua and confirmed its identity as scopoletin through mass spectral data. The structure was established from ¹H‐nuclear magnetic resonance (NMR), ¹³C‐NMR. The compound scopoletin was evaluated for its feeding deterrence and growth inhibitory potential against a noxious lepidopteran insect, Spilartctia obliqua Walker. Scopoletin gave FD₅₀ (feeding deterrence of 50%) value of 96.7 μg/g diet when mixed into artificial diet. S. obliqua larvae (12‐day‐old) exposed to the highest concentration (250 μg/g diet) of scopoletin showed 77.1% feeding‐deterrence. In a growth inhibitory assay, scopoletin provided 116.9% growth inhibition at the highest dose of 250 μg/g diet with a GI₅₀ (growth inhibition of 50%) value of 20.9 μg/g diet. Statistical analysis showed a concentration‐dependent dose response relationship toward both feeding deterrent and growth inhibitory activities. Artemisinin is found mainly in the leaves of A. annua and not in the stems, which are typically discarded as waste. Therefore identification of scopoletin in stems of A. annua may be important as a source of this material for pest control.     

2-deoxy-D-glucose combined with ferulic acid enhances radiation response in non-small cell lung carcinoma cells

The present study was undertaken to investigate the radiosensitizing effects of 2-deoxy-D-glucose (2DG), a glycolytic inhibitor, and ferulic acid (FA), a phenolic prooxidant, in relatively radioresistant human non-small cell lung carcinoma cells (NCI-H460). NCI-H460 cells were treated with 4 mM 2DG and/or 53.8 μM FA for 24 h and then exposed to 2 Gy irradiation. Compared to cells that were 2 Gy-irradiated alone (50%), FA and 2DG with radiation (FA+2DG+IR) showed additional decrease in cell viability (15%). This has been further validated by decreased (86%) colony formation in 2DG+FA+IR group compared to 2DG (29%), FA (24%) and IR (37%) group alone. Increased apoptotic cells (84%) in 2DG+FA+IR group further confirm the radiosensitizing property of 2DG or FA. In NCI-H460 cells 2DG decreased NADPH levels (10%) and FA increased ROS levels leading to enhanced oxidative damage in the 2DG+FA+IR group. This was reflected as altered mitochondrial membrane potential, increased lipid peroxidative markers (TBARS), DNA damage and decreased intracellular glutathione (GSH) levels in combined treatment groups when compared to radiation or 2DG or FA treatment alone. The present study suggests that FA and 2DG act by increasing oxidative damage in NCI-H460 cells.     

Thyroid hormones stimulate Na+–Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na⁺–P_i transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na⁺–P_i transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na⁺–P_i transport in response to thyroid hormone status of animals. (Mol Cell Biochem 268: 75–82, 2005)     

Increased expression of calcineurin in human colorectal adenocarcinomas

Colorectal cancer (CRC) is the third most common cause of cancer death in the Western world. Calcineurin (CaN), a Ca2+/calmodulin (CaM)-dependent protein phosphatase, is important for Ca2+-mediated signal transduction. The main objective of this study is to examine the potential role of Ca2+/CaM-dependent protein phosphatase in both normal and in invasive tumor components of human samples. In this study, we carried out 45 cases of CaN activity, 13 cases of CaN protein expression by Western blot analysis, and 6 cases for immunohistochemical analysis in both normal and invasive tumor components of human samples. Immunohistochemical analysis revealed that strong cytoplasmic staining of varying intensity was observed in colon tumors of all patients compared to normal mucosa. In addition, Western blot analysis revealed a prominent overexpressed immunoreactive band with an apparent molecular mass of 60 kDa catalytic alpha subunit (CaN A) as well as CaN Aalpha and beta in colon tumor samples. Elevated CaN protein expression appears to be a possible link between Ca2+ signaling and oncogenic processes.     

Rapid screening procedures for identification of succinic acid producers

Succinic acid, an intermediate of tricarboxylic acid cycle, is produced and accumulated by anaerobic microorganisms. The long-standing interest in the production of this organic acid is because it is a key compound in producing more than 30 commercially important products. The detection of succinic acid is generally carried out by gas chromatography (GC), enzymatic assays, ion-exclusion chromatography (IEC) or by high performance liquid chromatography (HPLC). However, these methods are time consuming, require sophisticated instrumentation and are expensive. In the present investigation we are reporting two rapid, cost effective screening methods for the detection of this important organic acid. These methods can be utilized to screen a large number of microbes producing succinic acid in a very short span of time.     

The SH2 domain containing inositol polyphosphate 5-phosphatase-2: SHIP2

Phosphoinositides are membrane-bound signaling molecules that recruit, activate and localize target effectors to intracellular membranes regulating apoptosis, cell proliferation, insulin signaling and membrane trafficking. The SH2 domain containing inositol polyphosphate 5-phosphatase-2 (SHIP2) hydrolyzes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generating phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Overexpression of SHIP2 inhibits insulin-stimulated phosphoinositide 3-kinase (PI3K) dependent signaling events. Analysis of diabetic human subjects has revealed an association between SHIP2 gene polymorphisms and type 2 diabetes mellitus. Genetic ablation of SHIP2 in mice has generated conflicting results. SHIP2 knockout mice were originally reported to show lethal neonatal hypoglycemia resulting from insulin hypersensitivity, but in addition to inactivating the SHIP2 gene, the Phox2a gene was also inadvertently deleted. Another SHIP2 knockout mouse has now been generated which inactivates the SHIP2 gene but leaves Phox2a intact. These animals show normal insulin and glucose tolerance but are highly resistant to weight gain on high fat diets, exhibiting an obesity-resistant phenotype. Therefore, SHIP2 remains a significant therapeutic target for the treatment of both obesity and type 2 diabetes.     

Chemoprevention of chemically-induced mammary and colon carcinogenesis by 1alpha-hydroxyvitamin D5

Epidemiological data as well as experimental models yield evidence for a protective effect of vitamin D against the genesis of several types of cancers. Given its toxic properties at effective concentrations, numerous analogs of vitamin D have been developed. We synthesized an analog of vitamin D(5), 1alpha-hydroxy-24-ethylcholechalciferol (1alpha(OH)D(5)) and previously reported on its anti-proliferative activities against several cancer cell lines. To further examine its chemopreventive potential, experiments were conducted to investigate the in vivo effects of 1alpha(OH)D(5) using the N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis model. Results showed that 1alpha(OH)D(5) (25 and 50microg/kg diet) decreased the incidence and multiplicity of mammary tumors in female Sprague-Dawley rats. In a subsequent study, the stage specific inhibition was investigated using the 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis model. While supplementation with of 1alpha(OH)D(5) (40microg/kg diet) showed no significant effects during the initiation phase, tumor incidence during the promotional stage was significantly (p<0.05) decreased by 37.5%. In the colon, 1alpha(OH)D(5) (25microg/kg diet) was highly effective (p<0.001) in inhibiting the development of azoxymethane (AOM)-induced Aberrant crypt foci (ACF) in CF-1 mice. Studies on the stage specific inhibitory effects of 1alpha(OH)D(5) in the colon demonstrated that animals receiving 1alpha(OH)D(5) (25microg/kg diet) during the initiation, promotion, and entire period had a reduction in ACF number of 71, 80 and 82%, respectively. Immunohistochemistry studies comparing the colons of animals receiving control versus 1alpha(OH)D(5) supplemented diets showed that 1alpha(OH)D(5) partly mediates its effects by regulating members of the oncogenic beta-catenin pathway. 1Alpha(OH)D(5) inhibited expression of beta-catenin and peroxisome proliferator-activated receptor beta, a beta-catenin-TCF-4 responsive gene, whereas it induced expression of VDR. Cumulatively, these studies support the chemopreventive properties of 1alpha(OH)D(5) against the development of breast and colon cancers.