Structural characterization and epitope mapping of the glutamic acid/alanine-rich protein from Trypanosoma congolense: defining assembly on the parasite cell surface

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.     

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.     

Fermentation of protopanaxadiol type ginsenosides (PD) with probiotic Bifidobacterium lactis and Lactobacillus rhamnosus

Applied Microbiology and Biotechnology - Ginsenosides are believed to be the principal components behind the pharmacological actions of ginseng, and their bioactive properties are closely related...     

Semi-rational chemical modification of endoinulinase by pyridoxal 5′-phosphate and ascorbic acid

It is important to improve the quality of the enzyme inulinase used in industrial applications without allowing the treatment to have any adverse effects on enzyme activity. We achieved preferential chemical modification of the non-catalytic domain of endoinulinase (EC 3.2.1.7) to enhance the thermostability of the enzyme. We used pyridoxal 5′-phosphate (PLP) to modify the more accessible lysine residues at the surface of endoinulinase and then performed a necessary step of reduction with ascorbate. Endoinulinase was incubated in the presence of PLP at various concentrations; this step was followed by reduction of the resulting Schiff base and dialysis. The effects of different PLP concentrations and incubation times on enzyme modification were evaluated. Enzyme deactivation was observed immediately after treatment, even at low PLP concentrations, while reactivation was observed for samples treated with low PLP concentrations after a period of time. Structural analysis revealed that the α-helix content increased from 13.60% to 17.60% after applying the modification strategy; consequently, enzyme stabilization was achieved. The melting temperature (T_m) of the modified enzyme increased from 64.1 °C to 72.2 °C, and a comparative study of thermal stability at 25 °C, 45 °C, and 50 °C for 150 min confirmed that the enzyme was stabilized because of increase in its half-life (t_1/2) after PLP modification/ascorbate reduction. The modification process was optimized to achieve the optimum mole ratio for the PLP/endoinulinase (1.37). Excess moles of the modifier are thought to be responsible for enzyme deactivation through unwanted/nonspecific and noncovalent interactions, and the optimization ensured that there was no excess modifier after the desired covalent reaction was complete.     

Superovulatory response of Sistani cattle to three different doses of FSH during winter and summer

Objective of the present study was to investigate the effect of season and dose of FSH on superovulatory responses in Iranian Bos indicus beef cattle (Sistani). Cyclic cows, in summer (n=16) and winter (n=16), were assigned randomly to three dose-treatment groups of 120 (n=10), 160 (n=12) and 200 (n=10) total mg of Folltropin-V with injections given twice daily for 4 days in decreasing doses. Estrous cycles were synchronized with two prostaglandin F2alpha injections given 14 days apart. From day 5 after the ensuing cycle, daily ovarian ultrasonography was conducted to determine emergence of the second follicular wave at which time superovulation was initiated. Relative humidity, environmental and rectal temperatures were measured at 08:00, 14:00 and 20:00 h for the 3 days before and 2 days after the estrus of superovulation. Non-surgical embryo recovery was performed on day 7 after estrus. The effects of season, dose, time of estrous expression and all two-way interactions were evaluated on superovulatory responses: total numbers of CL, unovulated follicles (10 mm), ova/embryo, transferable and non-transferable embryos. Season (summer or winter), doses of Folltropin-V (120, 160 or 200 mg NIH) and time of estrous expression (08:00, 14:00 or 20:00 h) did not affect the number of transferable embryos (3.1+/-0.58). When superovulatory estrus was detected at 08:00, a FSH dose effect was detected with the greatest numbers of CL (12.2+/-0.87) and total ova/embryos (12.2+/-1.46) occurring with 200 mg FSH (dosextime of estrous expression; P<0.01).     

Therapeutic Potential of Berberine in the Treatment of Glioma: Insights into Its Regulatory Mechanisms

Glioma is known as one of the most common primary intracranial tumors accounting for four-fifths of malignant brain tumors. There are several biological pathways that play a synergistic, pathophysiological role in glioma, including apoptosis, autophagy, oxidative stress, and cell cycle arrest. According to previous rese arches, the drugs used in the treatment of glioma have been associated with significant limitations. Therefore, improved and/or new therapeutic platforms are required. In this regard, multiple flavonoids and alkaloids have been extensively studied in the treatment of glioma. Berberine is a protoberberine alkaloid with wide range of pharmacological activities, applicable to various pathological conditions. Few studies have reported beneficial roles of berberine in glioma. Berberine exerts its pharmacological functions in glioma by controlling different molecular and cellular pathways. We reviewed the existing knowledge supporting the use of berberine in the treatment of glioma and its effects on molecular and cellular mechanisms.

     

Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines

Certain types of human papillomaviruses (HPV) are causatively associated with cervical carcinoma, the second most common cancer in women worldwide. Due to limitations in the availability of currently used virus-like particle (VLP)-based vaccines against HPV to women of developing countries, where most cases of cervical cancer occur, the development of a cost-effective second-generation vaccine is a necessity. Capsomeres have recently been demonstrated to be highly immunogenic and to have a number of advantages as a potential cost-effective alternative to VLP-based HPV vaccines. We have expressed a mutated HPV-16 L1 (L1_2xCysM) gene that retained the ability to assemble L1 protein to capsomeres in tobacco chloroplasts. The recombinant protein yielded up to 1.5% of total soluble protein. The assembly of capsomeres was examined and verified by cesium chloride density gradient centrifugation and sucrose sedimentation analysis. An antigen capture enzyme-linked immunosorbent assay confirmed the formation of capsomeres by using a conformation-specific monoclonal antibody which recognized the conformational epitopes. Transplastomic tobacco plants exhibited normal growth and morphology, but all such lines showed male sterility in the T₀, T₁ and T₂ generations. Taken together, these results indicate the possibility of producing a low-cost capsomere-based vaccine by plastids.     

Synthesis and Antiviral Evaluation of Novel 2,3-Dihydroxypropyl Nucleosides from 2- and 4-Thiouracils

Regioselective alkylation of 2-thiouracils 1a–c and 4-thiouracils 7a,b with 2,3-O-isopropylidene-2,3-dihydroxypropyl chloride (2) afforded 2-{[(2,2-Dimethyl-1,3-dioxolan-4-yl) methyl]thio}pyrimidin-4(1H)-ones 3a–c and 4-{[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl]thio} pyrimidin-2(1H)-ones 8a,b, respectively. Further alkylation with 2 and/or 2,3-O-isopropylidine-1-O-(4-toluenesulfonyl)-glycerol (4) gave the acyclo N-nucleosides 5a–c and 9a,b whose deprotection afforded 6a–c and 10a,b. 2-(Methylthio)pyrimidin-4(1H)-ones 11a–c and 4-(methylthio)pyrimidin-2(1H)-ones 14a,b were treated with 2 and/or 4 to give 12a–c and 15a,b which were deprotected to give 13a–c and 16a,b. Pyrimidine-2,4(1H,3H)-dithiones 17a–c were treated with two equivalents of 2 to give 2,4-bis{[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]thio}pyrimidines 18a–c. Deprotection of compounds 18a–c gave 2,4-bis[(2,3-dihydroxypropyl)thio]pyrimidines 19a-c. The activity of the deprotected nucleosides against Hepatitis B virus was evaluated and showed moderate inhibition activity against HBV with mild cytotoxicity.