Toxoplasma gondii: Ultrastructure study of the entry of tachyzoites into mammalian cells

Toxoplama gondii (Apicomplexa: Coccidia), an obligatory intracellular parasite with a unique capacity to invade virtually all nucleated cell type from warm-blooded vertebrate hosts. Despite the efficiency with which Toxoplasma enters its host cell, it remains unresolved if invasion occurs by direct penetration of the parasite or through phagocytosis. In the present work, electron microscopic study was designed to examine the entry process of Toxoplasma (RH strain) into macrophages and non phagocytic-host cells (Hela cells) and to observe the ultrastructure changes associated with intracellular parasitism. The results showed that both active invasion and phagocytosis were occurred and revealed that invasion is an ordered process that initiates with binding of the parasite at its apical end followed by tight-fitting invagination of the host cell membrane and a prominent constriction in the parasite at the site of penetration. The process ended by the professional parasitophorous vacuole that is distinct at the outset from those formed by phagocytosis in which once Toxoplasma triggered, phagocytic uptake can proceed by capture of the parasite within a loose fitting vacuole formed by localized membrane ruffling. The cytopathic effects of the parasite on macrophages and Hela cells were demonstrated within 5–15 h post-inoculation in the form of degenerative mitochondria, swelling Golgi apparatus and widening of endoplasmic reticulum indicating intracellular oedema. These changes were exaggerated and several cells were found dead after 48–72 h.     

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via,mitochondrial mediated caspase-dependent mechanism

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.     

Mutation discovery in mice by whole exome sequencing

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.     

Production of Levansucrase from Bacillus subtilis NRC 33a and Enzymic Synthesis of Levan and Fructo-Oligosaccharides

Bacillus subtilis NRC33a was able to produce both inducible and constitutive extracellular levansucrase, respectively, using sucrose and glucose as carbon source. The optimal production of the levansucrase was at 30°C. The effect of different nitrogen sources showed that baker’s yeast with 2% concentration gave the highest levansucrase activity. Addition of 0.15 g/L MgSO₄ was the most favorable for levansucrase production. The enzymic synthesis of levan was studied using 60% acetone fraction. The results indicated that high enzyme concentrations produced increasing amounts of levan, and hence conversion of fructose to levan reached 84% using 1000 μg/ml enzyme protein. Sucrose concentration was the most effective factor controlling the molecular weight of the synthesized levan. The conversion of fructose to levan was maximal at 30°C. The time of reaction clearly affected the conversion of fructose to levan, which reached its maximum productivity at 18 hours (92%). Identification of levan indicated that fructose was the building unit of levan.     

Oestrogen receptor β is present in both muscle fibres and endothelial cells within human skeletal muscle tissue

Oestrogen receptor β (ERβ) is expressed in human skeletal muscle tissue. In the present study, we have developed an immunohistochemical method to reveal if ERβ is located within the muscle fibres as well as within capillaries. Skeletal muscle biopsies were obtained from m. quadriceps femoris vastus lateralis in four healthy young subjects. Immunohistochemical triple staining was applied to transverse sections of paraffin-wax-embedded tissue. The basement membrane of muscle fibres and capillaries was identified by using an antibody to collagen IV, endothelial cells using an antibody to CD34 and ERβ using a corresponding antibody. The ERβ-positive (ERβ+) nuclei were located within the muscle fibre defined by the localisation of collagen IV. ERβ+ nuclei were also, for the first time, found in endothelial cells of capillaries in skeletal muscle tissue. Quantification was performed on transverse cryostat sections after performing a double staining (collagen IV and ERβ). It was shown that 24% of the ERβ+ nuclei were located within capillaries, and 76% were located within muscle fibres. In conclusion, ERβ in human skeletal muscle tissue is expressed not only in the muscle fibres themselves, but also within the capillary endothelial cells. This observation might improve understanding of the physiological role of oestrogen and its receptor.