Synthesis of phenyl O-α-l-fucopyranosyl-(1→2)-O-β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside

phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-3-O-[4,6-O-(p-methoxybenzylidene)-β-d-alactopyranosyl]-α-d-galactopyranoside (3) was prepared from phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-3-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-galactopyranoside by zemplén deacetylation, followed by reaction with p-methoxybenzaldehyde in the presence of anhydrous zinc chloride. The selective benzoylation of 3 gave the 3′-benzoate which, on condensation with 2,3,4-tri-O-benzyl-α- l-fucopyranosyl bromide under catalysis by halide ion, afforded a crystalline trisaccharide from which the title trisaccharide was obtained by debenzoylation followed by catalytic hydrogenolysis.     

Proteomic analysis reveals USP7 as a novel regulator of palmitic acid-induced hepatocellular carcinoma cell death

Nutrient surplus and consequent free fatty acid accumulation in the liver cause hepatosteatosis. The exposure of free fatty acids to cultured hepatocyte and hepatocellular carcinoma cell lines induces cellular stress, organelle adaptation, and subsequent cell death. Despite many studies, the mechanism associated with lipotoxicity and subsequent cell death still remains poorly understood. Here, we have used the proteomics approach to circumvent the mechanism for lipotoxicity using hepatocellular carcinoma cells as a model. Our quantitative proteomics data revealed that ectopic lipids accumulation in cells severely affects the ubiquitin-proteasomal system. The palmitic acid (PA) partially lowered the expression of deubiquitinating enzyme USP7 which subsequently destabilizes p53 and promotes mitotic entry of cells. Our global phosphoproteomics analysis also provides strong evidence of an altered cell cycle checkpoint proteins’ expression that abrogates early G2/M checkpoints recovery with damaged DNA and induced mitotic catastrophe leading to hepatocyte death. We observe that palmitic acid prefers apoptosis-inducing factor (AIF) mediated cell death by depolarizing mitochondria and translocating AIF to the nucleus. In summary, the present study provides evidence of PA-induced hepatocellular death mediated by deubiquitinase USP7 downregulation and subsequent mitotic catastrophe.Subject terms: Apoptosis, Protein-protein interaction networks     

LC-MS characterization and purity assessment of a prototype bispecific antibody

Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly. A new method to assess heterodimer purity of such bispecific antibody products is needed because traditional separation-based purity assays are unable to separate or quantify homodimer impurities. This paper presents a liquid chromatography-mass spectrometry (LC-MS)-based method for evaluating heterodimeric purity of a prototype asymmetric antibody containing two different heavy chains and two identical light chains. The heterodimer and independently expressed homodimeric standards were characterized by two complementary LC-MS techniques: Intact protein mass measurement of deglycosylated antibody and peptide map analyses. Intact protein mass analysis was used to check molecular integrity and composition. LC-MS^E peptide mapping of Lys-C digests was used to verify protein sequences and characterize post-translational modifications, including C-terminal truncation species. Guided by the characterization results, a heterodimer purity assay was demonstrated by intact protein mass analysis of pure deglycosylated heterodimer spiked with each deglycosylated homodimeric standard. The assay was capable of detecting low levels (2%) of spiked homodimers in conjunction with co-eluting half antibodies and multiple mass species present in the homodimer standards and providing relative purity differences between samples. Detection of minor homodimer and half-antibody C-terminal truncation species at levels as low as 0.6% demonstrates the sensitivity of the method. This method is suitable for purity assessment of heterodimer samples during process and purification development of bispecific antibodies, e.g., clone selection.     

Cenozoic climate change and diversification on the continental shelf and slope: evolution of gastropod diversity in the family Solariellidae (Trochoidea)

Recent expeditions have revealed high levels of biodiversity in the tropical deep‐sea, yet little is known about the age or origin of this biodiversity, and large‐scale molecular studies are still few in number. In this study, we had access to the largest number of solariellid gastropods ever collected for molecular studies, including many rare and unusual taxa. We used a Bayesian chronogram of these deep‐sea gastropods (1) to test the hypothesis that deep‐water communities arose onshore, (2) to determine whether Antarctica acted as a source of diversity for deep‐water communities elsewhere and (3) to determine how factors like global climate change have affected evolution on the continental slope. We show that although fossil data suggest that solariellid gastropods likely arose in a shallow, tropical environment, interpretation of the molecular data is equivocal with respect to the origin of the group. On the other hand, the molecular data clearly show that Antarctic species sampled represent a recent invasion, rather than a relictual ancestral lineage. We also show that an abrupt period of global warming during the Palaeocene Eocene Thermal Maximum (PETM) leaves no molecular record of change in diversification rate in solariellids and that the group radiated before the PETM. Conversely, there is a substantial, although not significant increase in the rate of diversification of a major clade approximately 33.7 Mya, coinciding with a period of global cooling at the Eocene–Oligocene transition. Increased nutrients made available by contemporaneous changes to erosion, ocean circulation, tectonic events and upwelling may explain increased diversification, suggesting that food availability may have been a factor limiting exploitation of deep‐sea habitats. Tectonic events that shaped diversification in reef‐associated taxa and deep‐water squat lobsters in central Indo‐West Pacific were also probably important in the evolution of solariellids during the Oligo‐Miocene.     

Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles

Mitochondria are one of the hallmarks of eukaryotic cells, exporting ATP in exchange for cytosolic ADP using ADP/ATP carriers (AAC) located in the inner mitochondrial membrane. In contrast, several evolutionarily important anaerobic eukaryotes lack mitochondria but contain hydrogenosomes, peculiar organelles of controversial ancestry that also supply ATP but, like some fermentative bacteria, make molecular hydrogen in the process. We have now identified genes from two species of the hydrogenosome-containing fungus Neocallimastix that have three-fold sequence repeats and signature motifs that, along with phylogenetic analysis, identify them as AACs. When expressed in a mitochondrial AAC- deficient yeast strain, the hydrogenosomal protein was correctly targeted to the yeast mitochondria inner membrane and yielded mitochondria able to perform ADP/ATP exchange. Characteristic inhibitors of mitochondrial AACs blocked adenine nucleotide exchange by the Neocallimastix protein. Thus, our data demonstrate that fungal hydrogenosomes and yeast mitochondria use the same pathway for ADP/ATP exchange. These experiments provide some of the strongest evidence yet that yeast mitochondria and Neocallimastix hydrogenosomes are but two manifestations of the same fundamental organelle.     

Inheritance of mitochondrial aldehyde dehydrogenase: genotyping in Chinese,Japanese and South Korean families reveals dominance of the mutant allele

Summary Genotyping of mitochondrial aldehyde dehydrogenase (ALDH I) was performed in enzymatically amplified DNA of 20 Chinese, Japanese and South Korean families (85 individuals) and in 113 unrelated persons by employing allele-specific oligonucleotide probes and dot blot hybridization. Genotyping individuals with phenotypic deficiency of ALDH I activity always showed the presence of at least one mutant allele. The data are compatible with a model assuming dominant inheritance of the mutant allele, which we have previously suggested on the basis of a population study.     

Cytoplasmic localization of the ORF2 protein of hepatitis E virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum

Hepatitis E virus (HEV) is a positive-strand RNA virus that is prevalent in much of the developing world. ORF2 is the major capsid protein of HEV. Although ORF2 is an N-linked glycoprotein, it is abundantly located in the cytoplasm in addition to having membrane and surface localization. The mechanism by which ORF2 protein obtains access to the cytoplasm is unknown. In this report, we prove that initially all ORF2 protein is present in the endoplasmic reticulum and a fraction of it becomes retrotranslocated to the cytoplasm. The ability of ORF2 to be retrotranslocated is dependent on its glycosylation status and follows the canonical dislocation pathway. However, in contrast to general substrates of the dislocation pathway, retrotranslocated ORF2 protein is not a substrate of the 26S proteasome complex and is readily detectable in the cytoplasm in the absence of any protease inhibitor, suggesting that the retrotranslocated protein is stable in the cytoplasm. This study thus defines the pathway by which ORF2 obtains access to the cytoplasm.     

Paraoxonase 1 (PON1) polymorphisms, haplotypes and activity in predicting cad risk in North-West Indian Punjabis

Background

Human serum paraoxonase-1 (PON1) prevents oxidation of low density lipoprotein cholesterol (LDL-C) and hydrolyzes the oxidized form, therefore preventing the development of atherosclerosis. The polymorphisms of PON1 gene are known to affect the PON1 activity and thereby coronary artery disease (CAD) risk. As studies are lacking in North-West Indian Punjabi''s, a distinct ethnic group with high incidence of CAD, we determined PON1 activity, genotypes and haplotypes in this population and correlated them with the risk of CAD.

Methodology/Principal Findings

350 angiographically proven (≥70% stenosis) CAD patients and 300 healthy controls were investigated. PON1 activity was determined towards paraoxon (Paraoxonase; PONase) and phenylacetate (Arylesterase; AREase) substrates. In addition, genotyping was carried out by using multiplex PCR, allele specific oligonucleotide –PCR and PCR-RFLP methods and haplotyping was determined by PHASE software. The serum PONase and AREase activities were significantly lower in CAD patients as compared to the controls. All studied polymorphisms except L55M had significant effect on PONase activity. However AREase activity was not affected by them. In a logistic regression model, after adjustment for the conventional risk factors for CAD, QR (OR: 2.73 (1.57–4.72)) and RR (OR, 16.24 (6.41–41.14)) genotypes of Q192R polymorphism and GG (OR: 2.07 (1.02–4.21)) genotype of −162A/G polymorphism had significantly higher CAD risk. Haplotypes L-T-G-Q-C (OR: 3.25 (1.72–6.16)) and L-T-G-R-G (OR: 2.82 (1.01–7.80)) were also significantly associated with CAD.

Conclusions

In conclusion this study shows that CAD patients had lower PONase and AREase activities as compared to the controls. The coding Q192R polymorphism, promoter −162A/G polymorphism and L-T-G-Q-C and L-T-G-R-G haplotypes are all independently associated with CAD.