Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen-Deuterium Exchange.

Galectins are ß-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their affinities for lactose, a common galectin inhibitor, vary significantly. Here, we aimed to examine the molecular basis of differential lactose affinities amongst galectins using solution-based techniques. Consistent dissociation constants of lactose binding were derived from nuclear magnetic resonance (NMR) spectroscopy, intrinsic tryptophan fluorescence, isothermal titration calorimetry and bio-layer interferometry for human galectin-1 (hGal1), galectin-7 (hGal7), and the N-terminal and C-terminal domains of galectin-8 (hGal8NTD and hGal8CTD, respectively). Furthermore, the dissociation rates of lactose binding were extracted from NMR lineshape analyses. Structural mapping of chemical shift perturbations revealed long-range perturbations upon lactose binding for hGal1 and hGal8NTD. We further demonstrated using the NMR-based hydrogen-deuterium exchange (HDX) that lactose binding increases the exchange rates of residues located on the opposite side of the ligand-binding pocket for hGal1 and hGal8NTD, indicative of allostery. Additionally, lactose binding induces significant stabilisation of hGal8CTD across the entire domain. Our results suggested that lactose binding reduced the internal dynamics of hGal8CTD on a very slow timescale (minutes and slower) at the expense of reduced binding affinity due to the unfavourable loss of conformational entropy.

Construction and characterization of insect cell-derived influenza VLP: cell binding, fusion, and EGFP incorporation.

We have constructed virus-like particles (VLPs) harboring hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1) ,and proton channel protein (M2) using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP) was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

Comparative analysis of ß-hexosaminidase and acid phosphatase from Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1: Localization studies of acid phosphatase and ß-hexosaminidase from H. vulgaris Ind-Pune.

The present report describes a comprehensive study on comparative biochemical characterization of two lysosomal enzymes, acid phosphatase and ß-hexosaminidase in three different strains of Hydra; Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1 (self-feeder-1). Since morphology and habitat of Hydra effect lysosomal enzymes and their response to environmental pollutants, it would be interesting to identify them in different Hydra strains so as to use them as toxicity testing. Preliminary studies revealed a differential expression of acid phosphatase, ß-hexosaminidase and ß-glucuronidase in three Hydra strains. Expression of all three lysosomal enzymes in H. vulgaris Ind-Pune was low in comparison to H. vulgaris Naukuchiatal and H. magnipapillata sf-1, while their expression is comparable in H. vulgaris Naukuchiatal and H. magnipapillata sf-1. The Michaelis-Menten (KM) values for lysosomal ß-hexosaminidase using 4-nitrophenyl N-acetyl-ß-D-glucosaminide as substrate were found to be 1.3 mM, 1.1 mM and 0.8 mM, respectively for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1. For acid phosphatase using 4-nitrophenyl-phosphate as substrate, the KM values were 0.38 mM, 1.2 mM and 0.52 mM respectively, for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and sf-1 strains. The optimum temperature for ß-hexosaminidase was 60 °C for H. vulgaris Ind-Pune, while 50 °C was observed for H. vulgaris Naukuchiatal and sf-1 strains. The optimum pH for ß-hexosaminidase was found to be 6.0 for H. vulgaris Ind-Pune and H. vulgaris Naukuchiatal, and 5.0 for sf-1. The optimum temperature and pH of acid phosphatase was similar in all three strains, viz., 40 °C and 3.0, respectively. Preliminary localization studies using whole mount in situ hybridization revealed predominant endodermal expression of three enzymes in H. vulgaris Ind-Pune. Our results thus support the conservation of lysosomal hydrolases in Hydra.

Periodontal disease and effects of antipsychotic medications in patients newly diagnosed with schizophrenia: a population-based retrospective cohort.

AIM: Compared with the general population, individuals with schizophrenia have a higher risk of periodontal disease, which can potentially reduce their life expectancy. However, evidence for the early development of periodontal disease in schizophrenia is scant. The current study investigated risk factors for periodontal disease in patients newly diagnosed with schizophrenia. METHODS: We identified a population-based cohort of patients in Taiwan with newly diagnosed schizophrenia who developed periodontal disease within 1 year of their schizophrenia diagnosis. Treatment with antipsychotics and other medications was categorised according to medication type and duration, and the association between medication use and the treated periodontal disease was assessed through logistic regression. RESULTS: Among 3610 patients with newly diagnosed schizophrenia, 2373 (65.7%) had an incidence of treated periodontal disease during the 1-year follow-up. Female sex (adjusted odds ratios [OR] 1.40; 95% confidence interval [CI] 1.20-1.63); young age (adjusted OR 0.99; 95% CI 0.98-0.99); a 2-year history of periodontal disease (adjusted OR 2.45; 95% CI 1.84-3.26); high income level (adjusted OR 2.24; 95% CI 1.64-3.06) and exposure to first-generation (adjusted OR 1.89; 95% CI 1.54-2.32) and secondary-generation (adjusted OR 1.33; 95% CI 1.11-1.58) antipsychotics, anticholinergics (adjusted OR 1.24; 95% CI 1.03-1.50) and antihypertensives (adjusted OR 1.91; 95% CI 1.64-2.23) were independent risk factors for periodontal disease. Hyposalivation - an adverse effect of first-generation antipsychotics (FGAs) (adjusted OR 2.00; 95% CI 1.63-2.45), anticholinergics (adjusted OR 1.27; 95% CI 1.05-1.53) and antihypertensives (adjusted OR 1.90; 95% CI 1.63-2.22) - was associated with increased risk of periodontal disease. Therefore, hypersalivation due to FGA use (adjusted OR 0.72; 95% CI 0.59-0.88) was considered a protective factor. CONCLUSIONS: The current study highlights that early prevention of periodontal disease in individuals with schizophrenia is crucial. Along with paying more attention to the development of periodontal disease, assessing oral health regularly, helping with oral hygiene, and lowering consumption of sugary drinks and tobacco, emphasis should also be given by physicians to reduce the prescription of antipsychotics to the extent possible under efficacious pharmacotherapy for schizophrenia.