Deciphering the “Fuzzy” Interaction of FG Nucleoporins and Transport Factors Using Small-Angle Neutron Scattering

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Using selective media to assess aerosolization damage and ultraviolet germicidal irradiation susceptibility of Serratia marcescens

This study determined whether selective media, McConkey agar (MC) and minimal salt glucose agar (MA) are suitable for monitoring aerosolization damage of airborne Serratia marcescens in our laboratory aerosol exposure system and assessed the relationship between bacterial culturability in these media and ultraviolet germicidal irradiation (UVGI) susceptibility of the bacteria. Two types of bacterial cultures were prepared. The first culture was taken from bacteria growing on Tryptic soy agar (TSA) as complete medium (fresh culture), which provided nearly 100% of MC/MA tolerant bacteria, while the second one was prepared from freezing the fresh culture (frozen culture), which produced 55 and 81% of MC and MA tolerant bacteria, respectively. We monitored bacterial culturability in TSA, MC and MA from these cultures in the nebulizer reservoir and bioaerosls collected on a six-stage Andersen cascade bio-impactor. The results indicated that both concentration and percentage of MC/MA tolerant bacteria maintained at a similar level during nebulization. For the bioaerosols, although the concentration recovered in the media from the fresh culture was higher than that from the frozen culture, the percentage of MC/MA tolerant bacteria was similar to that before aerosolization. We concluded that MC and MA are not suitable for monitoring aerosolization damage of the bacteria. Moreover, culturability of the bacteria in MC and MA has no effect on their survival after aerosolization. With respect to the bacterial susceptibility to UVGI, MC/MA sensitive and tolerant population as well as the fresh and frozen cultures showed the same susceptibility.     

Effect of process parameters on the production and drying of Leuconostoc mesenteroides cultures

Leuconostoc mesenteroides BLAC was grown on MRS broth or on a carrot juice medium, and the effects of sugar concentration, type of pH control, aeration and fermentor size on viable counts were examined. The effect on viability of the type of centrifuge used to concentrate the bacterial culture was also examined. When the MRS broth had the traditional 110 mM glucose, pH control did not increase the final population. However, using a zone pH control mode, increasing the glucose content of MRS both from 110 to 220 mM almost doubled the population. In MRS broth, the amount of acetic acid produced was the same for all treatments, and was proportional to the amount of citrate consumed. There was a significantly lower cell yield in the carrot juice medium when the pH was not regulated. In the carrot juice medium, pH had a more pronounced effect on the final population level than did aeration, even though the quantity of viable cells was greater when the culture was aerated. In MRS broth, glucose was completely consumed during fermentation, but this was not the case in carrot juice medium. Aeration resulted in increased acetic acid content of the fermented medium. Viable counts were not affected by scaling the volume of the fermentation from 2 to 15 l ,or by the type of centrifuge used to concentrate the cells. Cells were concentrated by a factor of 10, but in both centrifuge types, viable counts showed only an eightfold average increase. However, freeze-dried powders obtained from the continuous pilot-plant-centrifuged cultures had, on the average, 33% lower populations than those obtained from the laboratory unit. Journal of Industrial Microbiology & Biotechnology (2002) 28, 291–296 DOI: 10.1038/sj/jim/7000245 Received 09 July 2001/ Accepted in revised form 25 January 2002     

Correlation of structure to antitumor activities of five derivatives of a beta-glucan from Poria cocos sclerotium

A water-insoluble (1-->3)-beta-D-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses (Mw) and intrinsic viscosities ([eta]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native beta-glucan and the five derivatives, were tested in vitro and in vivo. The Mw values of the five derivatives in PBS were determined to be 3.8 x 10(4), 18.9 x 10(4), 16.0 x 10(4), 76.8 x 10(4), and 224.3 x 10(4), respectively. The high Mw values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true Mw values obtained in dimethyl sulfoxide were 20.1 x 10(4) and 19.1 x 10(4). The sulfated and carboxymethylated derivatives having DS of 1.0-1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native beta-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.     

Mutual structural effect of bilirubin and model membranes by vibrational circular dichroism

In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1 × 10^− 4 M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes—a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15–70 °C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.     

Ligand‐induced formation of transient dimers of mammalian 12/15‐lipoxygenase: A key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.     

Interaction of detergents with bovine lens α-crystallin: evidence for an oligomeric structure based on amphiphilic interactions

We have studied the quaternary structure of α-crystallin in the presence of increasing concentrations of amphiphilic and neutral detergents using gel filtration, light-scattering, boundary and equilibrium sedimentation. We observed a continuous reduction of the molar mass of the polymeric α-crystallin on increasing the concentration of sodium dodecyl sulphate from 0.1 mM to 5 mM, ending up with the monomeric peptides. Dodecyltrimethylammonium bromide also disrupts the oligomeric structure of α-crystallin but the interaction appears to be cooperative: in the sharp transition region (for a 1 mg/ml protein solution) from 3 to 8 mM of the detergent, only the native protein and a mixture of monomeric and dimeric peptide-DTAB complexes can be observed. Concomitant studies of the circular dichroism in the far UV revealed a substantial decrease of the β-sheet and increase of the α-helix secondary structure. The latter can be related to the presence of amphiphilic polypeptide sequences in the constituent αA and αB peptides. These studies reveal for the first time a direct relation between changes in the secondary structure of the αA and αB peptides and the formation of the oligomeric α-crystallin structure: the binding of the amphiphilic detergent reduces the β-sheet content, induces the formation of α-helix secondary structure and reduces the tendency of the peptide to form large aggregates. The different mechanisms for reducing the oligomeric size by anionic and cationic detergents with identical apolar parts stresses the importance of charge interactions. Our findings support some aspects of the micelle model of α-crystallin and can be related to its chaperone activity. Accepted: 18 October 1996     

Effect of conformational states on protein dynamical transition

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.     

Structure and Function of N-Terminal Zinc Finger Domain of SARS-CoV-2 NSP2

Virologica Sinica - SARS-CoV-2 has become a global pandemic threatening human health and safety. It is urgent to find effective therapeutic agents and targets with the continuous emergence of novel...