Reactive oxygen and nitrogen species are involved in sorbitol-induced apoptosis of human erithroleukaemia cells K562

In this study, we found that production of both reactive oxygen (ROS) and nitrogen (RNS) species is a very early event related to treatment with hyperosmotic concentration of sorbitol. The production of nitric oxide (NO) was paralleled by the increase of the mRNA and protein level of the inducible form of the nitric oxide synthase (iNOS). ROS and RNS enhancement, process concomitant to the failure of mitochondrial trans-membrane potential (ΔΨ), was necessary for the induction of apoptosis as demonstrated by the protection against sorbitol-mediated toxicity observed after treatment with ROS scavengers or NOS inhibitors. The synergistic action of ROS and RNS was finally demonstrated by pre-treatment with rosmarinic acid that, by powerfully buffering both these species, prevents impairment of ΔΨ and cell death. Overall results suggest that the occurrence of apoptosis upon sorbitol treatment is an event mediated by oxidative/nitrosative stress rather than a canonical hyperosmotic shock.     

The molecular evolution of avian ultraviolet- and violet-sensitive visual pigments

The shortwave-sensitive SWS1 class of vertebrate visual pigments range in lambda(max) from the violet (385-445 nm) to the ultraviolet (UV) (365-355 nm), with UV-sensitivity almost certainly ancestral. In birds, however, the UV-sensitive pigments present in a number of species have evolved secondarily from an avian violet-sensitive (VS) pigment. All avian VS pigments expressed in vitro to date encode Ser86 whereas Phe86 is present in all non-avian ultraviolet sensitive (UVS) pigments. In this paper, we show by site directed mutagenesis of avian VS pigments that Ser86 is required in an avian VS pigment to maintain violet-sensitivity and therefore underlies the evolution of avian VS pigments. The major mechanism for the evolution of avian UVS pigments from an ancestral avian VS pigment is undoubtedly a Ser90Cys substitution. However, Phe86, as found in the Blue-crowned trogon, will also short-wave shift the pigeon VS pigment into the UV whereas Ala86 and Cys86 which are also found in natural avian pigments do not generate short-wave shifts when substituted into the pigeon pigment. From available data on avian SWS1 pigments, it would appear that UVS pigments have evolved on at least 5 separate occasions and utilize 2 different mechanisms for the short-wave shift.     

Choline Kinase β Mutant Mice Exhibit Reduced Phosphocholine,Elevated Osteoclast Activity,and Low Bone Mass

The maintenance of bone homeostasis requires tight coupling between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the precise molecular mechanism(s) underlying the differentiation and activities of these specialized cells are still largely unknown. Here, we identify choline kinase β (CHKB), a kinase involved in the biosynthesis of phosphatidylcholine, as a novel regulator of bone homeostasis. Choline kinase β mutant mice (flp/flp) exhibit a systemic low bone mass phenotype. Consistently, osteoclast numbers and activity are elevated in flp/flp mice. Interestingly, osteoclasts derived from flp/flp mice exhibit reduced sensitivity to excessive levels of extracellular calcium, which could account for the increased bone resorption. Conversely, supplementation of cytidine 5′-diphosphocholine in vivo and in vitro, a regimen that bypasses CHKB deficiency, restores osteoclast numbers to physiological levels. Finally, we demonstrate that, in addition to modulating osteoclast formation and function, loss of CHKB corresponds with a reduction in bone formation by osteoblasts. Taken together, these data posit CHKB as a new modulator of bone homeostasis.     

Antiplasmodial and cytotoxicity evaluation of 3-functionalized 2-azetidinone derivatives

3-Azido-, 3-amino- and 3-(1,2,3-triazol-1-yl)-β-lactams were synthesized and evaluated for their antiplasmodial activity against four strains of Plasmodium falciparum and KB cells for their cytotoxicity profiles. The presence of a cyclohexyl substituent at N-1 and a phenyl group on the triazole ring markedly improved the activity profiles of triazole-tethered β-lactam exhibiting IC₅₀ values of 1.13, 1.21 and 1.00 μM against 3D7, K1 and W2 strains respectively.     

Purification, and biochemical and biophysical characterization of cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha- helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-β-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.     

Autosomal recessive dilated cardiomyopathy due to DOLK mutations results from abnormal dystroglycan O-mannosylation

Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5-13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations.     

Membrane curvature response in autophagy

Membrane trafficking is key for signal transduction, cargo transportation, and in the case of autophagy, delivering cytoplasmic substrates to the lysosome for degradation. Autophagy requires the formation of a unique double membrane vesicle, the autophagosome. However, the mechanism by which the autophagosome forms is unknown. Our recent study focused on the role of Barkor/Atg14(L) in targeting the autophagy-specific class III phosphatidylinositol-3-kinase (PtdIns3KC3) complex to the early autophagosome has implicated this complex in autophagosome formation. This study found that the BATS domain of Barkor targets the PtdIns3KC3 complex to early autophagic structures and senses highly curved membranes enriched in phosphatidylinositol-3-phosphate (PtdIns(3)P). Consequently, this study uncovered an exciting new role for the PtdIns3KC3 complex as a potential inducer of autophagosome formation.     

The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella

Eimeria spp. are a highly successful group of intracellular protozoan parasites that develop within intestinal epithelial cells of poultry, causing coccidiosis. As a result of resistance against anticoccidial drugs and the expense of manufacturing live vaccines, it is necessary to understand the relationship between Eimeria and its host more deeply, with a view to developing recombinant vaccines. Eimeria possesses a family of microneme lectins (MICs) that contain microneme adhesive repeat regions (MARR). We show that the major MARR protein from Eimeria tenella, EtMIC3, is deployed at the parasite-host interface during the early stages of invasion. EtMIC3 consists of seven tandem MAR1-type domains, which possess a high specificity for sialylated glycans as shown by cell-based assays and carbohydrate microarray analyses. The restricted tissue staining pattern observed for EtMIC3 in the chicken caecal epithelium indicates that EtMIC3 contributes to guiding the parasite to the site of invasion in the chicken gut. The microarray analyses also reveal a lack of recognition of glycan sequences terminating in the N-glycolyl form of sialic acid by EtMIC3. Thus the parasite is well adapted to the avian host which lacks N-glycolyl neuraminic acid. We provide new structural insight into the MAR1 family of domains and reveal the atomic resolution basis for the sialic acid-based carbohydrate recognition. Finally, a preliminary chicken immunization trial provides evidence that recombinant EtMIC3 protein and EtMIC3 DNA are effective vaccine candidates.