Effects of pH, light and temperature on (1→3,1→4)-β-glucanase stability in wheat leaves

Changes in (1→3,1→4)-β-D-glucan endohydrolase (EC 3.2.1.73) protein levels were investigated in segments from second leaves of wheat (Triticum aestivum L.). The abundance of the enzyme protein markedly increased when leaf segments were incubated in the dark whereas the enzyme rapidly disappeared when dark-incubated segments were illuminated or fed with sucrose. Addition of cycloheximide (CHI) to the incubation medium led to the disappearance of previously synthesized (1→3,1→4)-β-glucanase and suppressed the dark-induced accumulation indicating that the enzyme was rather unstable. The degradation of (1→3,1→4)-β-glucanase was analyzed without the interference of de-novo synthesis in intercellular washing fluid (IWF). The loss of the enzyme protein during incubation of IWF (containing naturally present peptide hydrolases) indicated that the stability increased from pH 4 to pH 7 and that an increase in the temperature from 25 to 35 °C considerably decreased the stability. Chelating divalent cations in the IWF with o-phenanthroline also resulted in a lowered stability of the enzyme. A strong temperature effect in the range from 25 to 35 °C was also observed in wheat leaf segments. Diurnal changes in (1→3,1→4)-β-glucanase activity were followed in intact second leaves from young wheat plants. At the end of the dark period, the activity was high but constantly decreased during the light phase and remained low if the light period was extended. Activity returned to the initial level during a 10-h dark phase. During a diurnal cycle, changes in (1→3,1→4)-β-glucanase activity were associated with reciprocal changes in soluble carbohydrates. The results suggest that the synthesis and the proteolytic degradation of an apoplastic enzyme may rapidly respond to changing environmental conditions.

Vapor phase composition as an indicator of thermal stability of the Sm, Eu, and Yb chlorides

The Sm, Eu, and Yb tri- and dichlorides were investigated by Knudsen effusion mass spectrometry. It was found out by the analysis of mass spectra and ionization efficiency curves that the vapor composition is complex due to the partial high temperature decomposition/disproportionation of the samples. Up to five vapor species were identified for both LnCl3 (LnCl3, LnCl2, Ln2Cl4, Ln2Cl5, and Ln2Cl6) and LnCl2 (LnCl3, LnCl2, LnCl, Ln, and Ln2Cl4). The quantitative evaluation of vapor composition was made. It indicates that the disproportionation of SmCl2 and EuCl2 is negligible in the temperature range studied whereas that of YbCl2 and the decomposition of SmCl3 and YbCl3 cannot be neglected.

High-precision confocal reflection measurement for two dimensional refractive index mapping of optical fibers

We introduce a new fiber-optical approach for reflection based refractive index mapping. Our approach leads to improved stability and reliability over existing free-space confocal instruments and significantly cuts alignment efforts and reduces the number of components needed. Other than properly cleaved fiber end-faces, this setup requires no additional sample preparation. The instrument is calibrated by means of a set of samples with known refractive indices. The index steps of commercially available fibers are measured accurately down to < 10⁻³. The precision limit of the instrument is currently of the order of 10⁻⁴.

Attitudes towards a maintenance (-agonist) treatment approach in high-dose benzodiazepine-dependent patients: a qualitative study

BACKGROUND High-dose benzodiazepine dependence constitutes a major clinical concern. Although withdrawal treatment is recommended, it is unsuccessful for a significant proportion of affected patients. More recently, a benzodiazepine maintenance approach has been suggested as an alternative for patients' failing discontinuation treatment. While there is some data supporting its effectiveness, patients' perceptions of such an intervention have not been investigated. METHODS An exploratory qualitative study was conducted among a sample of 41 high-dose benzodiazepine (BZD)-dependent patients, with long-term use defined as doses equivalent to more than 40 mg diazepam per day and/or otherwise problematic use, such as mixing substances, dose escalation, recreational use, or obtainment by illegal means. A qualitative content analysis approach was used to evaluate findings. RESULTS Participants generally favored a treatment discontinuation approach with abstinence from BZD as its ultimate aim, despite repeated failed attempts at withdrawal. A maintenance treatment approach with continued prescription of a slow-onset, long-acting agonist was viewed ambivalently, with responses ranging from positive and welcoming to rejection. Three overlapping themes of maintenance treatment were identified: "Only if I can try to discontinue…and please don't call it that," "More stability and less criminal activity…and that is why I would try it," and "No cure, no brain and no flash…and thus, just for everybody else!" CONCLUSIONS Some patients experienced slow-onset, long-acting BZDs as having stabilized their symptoms and viewed these BZDs as having helped avoid uncontrolled withdrawal and abstain from criminal activity. We therefore encourage clinicians to consider treatment alternatives if discontinuation strategies fail.

Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis

Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

The activation peptide of coagulation factor XIII is vital for its expression and stability.

BACKGROUND The human activation peptide of factor XIII (AP-FXIII) comprises the first 37 amino acids of the N-terminus and holds the FXIII in an inactive state. FXIII is activated either proteolytically by cleavage of AP-FXIII by thrombin, or non-proteolytically by high calcium concentrations. OBJECTIVE To investigate the role of AP-FXIII in the expression and stability of FXIII. METHODS We cloned 13 FXIII variants with progressive truncations of AP-FXIII from the N-terminus (delN-FXIII-A), expressed them in mammalian cells, and measured their thermostability, activation, and transglutaminase activity. We also used in silico calculations to analyze the stability of hypothetical delN-FXIII dimers and to identify crucial motifs within AP-FXIII. RESULTS Variants with deletions longer than the first 10 amino acids and an R11Q point mutant were not expressed as proteins. In silico calculations indicated that the sequence (8) FGGR(12) R plays a substantial role in intersubunit interactions in FXIII-A2 homodimers. In agreement with this prediction, the temperature stability of delN-FXIII variants decreased with increasing length of deletion. These results may suggest a role of the N-terminus of AP-FXIII in dimer stability. Substantial sequence homology was found among activation peptides of vertebrate and even invertebrate (crustacean) FXIII-A orthologs, which further supports our conclusion. CONCLUSIONS We conclude that deletion of 11 or more N-terminal amino acids disrupts intersubunit interactions, which may prevent FXIII-A2 homodimer formation. Therefore, AP-FXIII plays an important role in the stability of the FXIII-A2 dimer.

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.