Isolation and Identification of l-Menthyl-β-d-glucoside from Shubi

Quinoxalines derived from d-galactose with o-phenylenediamine (OPD) in acidic media under reflux were studied by using GLC and NMR measurements. Four quinoxaline derivatives were obtained from the reaction mixture, and were identical with those derived from d-glucose. The yields of 2-(D-lyxo-tetrahydroxybutyl)quinoxaline (GA-III), and the stereoisomeric derivative of GA-III, i.e., 2-(D-arabino-tetrahydroxybutyl)quinoxaline (ATBQ), were 13.2 and 5.3–, respectively. The ratio of GA-III to ATBQ derived from d-galactose was reciprocally coincident with that from d-glucose. Some proposals are made on the relationship between the isomerization of these sugars and the formation of quinoxaline derivatives.     

Gordonan,an Acidic Polysaccharide with Cell Aggregation-Inducing Activity in Insect BM-N4 Cells,Produced by Gordonia sp.

An acidic polysaccharide, termed gordonan, was isolated from the culture medium of Gordonia sp. as an inducer of cell aggregation in an insect cell line, BM-N4. Gordonan had an average molecular weight of 5×10⁶ and its structure was identified as →3)-4-O-(1-carboxyethyl)-β-D-Manp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→ mainly by acid hydrolysis experiments and NMR analysis. It induces cell aggregation at the concentration of 4 μg/ml. A partially hydrolyzed polysaccharide derived from gordonan with a molecular weight of 5×10⁵ showed weak activity, while any fragment molecules with lower molecular weights prepared from gordonan showed no activity.     

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.     

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.     

A hidden aggregation‐prone structure in the heart of hypoxia inducible factor prolyl hydroxylase

Prolyl hydroxylase domain‐containing protein 2 (PHD2), as one of the most important regulators of angiogenesis and metastasis of cancer cells, is a promising target for cancer therapy drug design. Progressive studies imply that abnormality in PHD2 function may be due to misfolding. Therefore, study of the PHD2 unfolding pathway paves the way for a better understanding of the influence of PHD2 mutations and cancer cell metabolites on the protein folding pathway. We study the unfolding of the PHD2 catalytic domain using differential scanning calorimetry (DSC), fluorescence spectroscopy, and discrete molecular dynamics simulations (DMD). Using computational and experimental techniques, we find that PHD2 undergoes four transitions along the thermal unfolding pathway. To illustrate PHD2 unfolding events in atomic detail, we utilize DMD simulations. Analysis of computational results indicates an intermediate species in the PHD2 unfolding pathway that may enhance aggregation propensity, explaining mutation‐independent PHD2 malfunction. Proteins 2016; 84:611–623. © 2016 Wiley Periodicals, Inc.     

The cell aggregating propensity of probiotic actinobacterial isolates: isolation and characterization of the aggregation inducing peptide pheromone

The auto-aggregating ability of a probiotic is a prerequisite for colonization and protection of the gastrointestinal tract, whereas co-aggregation provides a close interaction with pathogenic bacteria. Peptide pheromone mediated signaling has been studied in several systems. However, it has not yet been explored in prokaryotes, especially actinobacteria. Hence, in the present study, the diffusible aggregation promoting factor was purified from the culture supernatant of a potent actinobacterial probiont and characterized using 20 different actinobacterial cultures isolated from the gut region of chicken and goat. The results showed that the pheromone-like compound induces the aggregation propensity of treated isolates. The factor was found to be a heat stable, acidic pH resistant, low molecular weight peptide which enhances the biofilm forming ability of other actinobacterial isolates. The aggregation promoting factor represents a bacterial sex factor (pheromone) and its characterization confirms its usage in the probiotic formulation     

An ethanol extract of Ramulus mori improves blood circulation by inhibiting platelet aggregation

Inappropriate platelet aggregation can cause blood coagulation and thrombosis. In this study, the effect of an ethanol extract of Ramulus mori (ERM) on blood circulation was investigated. The antithrombotic activity of ERM on rat carotid arterial thrombosis was evaluated in vivo, and the effect of ERM on platelet aggregation and blood coagulation time was evaluated ex vivo. To evaluate the safety of ERM, its cytotoxicity to platelets and its effect on tail bleeding time were assessed; ERM was not toxic to rat platelets and did not prolong bleeding time. Moreover, administering ERM to rats had a significant preventive effect on carotid arterial thrombosis in vivo, and significantly inhibited adenosine diphosphate- and collagen-induced platelet aggregation ex vivo, whereas it did not prolong coagulation periods, such as prothrombin time and activated partial thromboplastin time. The results suggest that ERM is effective in improving blood circulation via antiplatelet activity rather than anticoagulation activity.     

Naturally occurring biflavonoids with amyloid β aggregation inhibitory activity for development of anti-Alzheimer agents

Amyloid β (Aβ) aggregation plays an essential role in promoting the progression of Alzheimer’s disease (AD). Therefore, the inhibition of Aβ aggregation is a potential therapeutic approach for AD. Herein, twenty-seven biflavonoids with different inter-flavonyl linkages and methoxy substitution patterns were isolated from several plants, and their Aβ₄₀ aggregation inhibitory activity was evaluated by the thioflavin-T fluorescence assay. Amentoflavone (1) and its monomethoxy derivatives (2, 3, and 5) exhibited the most potent inhibitory activity, with IC₅₀ values of approximately 5 μM. It was clarified that increasing the number of methoxy substituents on the biflavonoid structures attenuated the inhibitory activity. Moreover, the linkage and the methoxy substitution pattern had a marked influence on the inhibitory activity. Our investigation strongly supports that biflavonoids can be considered a new type of anti-Alzheimer agents that may be successfully developed for AD patients.     

Suppression,disaggregation, and modulation of γ‐Synuclein fibrillation pathway by green tea polyphenol EGCG

Oligomerization of γ‐Synuclein is known to have implications for both neurodegeneration and cancer. Although it is known to co‐exist with the fibrillar deposits of α‐Synuclein (Lewy bodies), a hallmark in Parkinson's disease (PD), the effect of potential therapeutic modulators on the fibrillation pathway of γ‐Syn remains unexplored. By a combined use of various biophysical tools and cytotoxicity assays we demonstrate that the flavonoid epigallocatechin‐3‐gallate (EGCG) significantly suppresses γ‐Syn fibrillation by affecting its nucleation and binds with the unstructured, nucleus forming oligomers of γ‐Syn to modulate the pathway to form α‐helical containing higher‐order oligomers (~158 kDa and ~ 670 kDa) that are SDS‐resistant and conformationally restrained in nature. Seeding studies reveal that these oligomers although “on‐pathway” in nature, are kinetically retarded and rate‐limiting species that slows down fibril elongation. We observe that EGCG also disaggregates the protofibrils and mature γ‐Syn fibrils into similar SDS‐resistant oligomers. Steady‐state and time‐resolved fluorescence spectroscopy and isothermal titration calorimetry (ITC) reveal a weak non‐covalent interaction between EGCG and γ‐Syn with the dissociation constant in the mM range (K_d ~ 2–10 mM). Interestingly, while EGCG‐generated oligomers completely rescue the breast cancer (MCF‐7) cells from γ‐Syn toxicity, it reduces the viability of neuroblastoma (SH‐SY5Y) cells. However, the disaggregated oligomers of γ‐Syn are more toxic than the disaggregated fibrils for MCF‐7cells. These findings throw light on EGCG‐mediated modulation of γ‐Syn fibrillation and suggest that investigation on the effects of such modulators on γ‐Syn fibrillation is critical in identifying effective therapeutic strategies using small molecule modulators of synucleopathies.