Laccase-catalyzed coupling of catharanthine and vindoline: an efficient approach to the bisindole alkaloid anhydrovinblastine

The efficient enzyme-catalyzed coupling of the indolic alkaloids catharanthine and vindoline was carried out by exploiting the oxidoreductases laccases and atmospheric oxygen. Following NaBH₄ reduction of the eniminium cationic intermediate, the synthetically useful dimer anhydrovinblastine (AVBL) was isolated and characterized. Several reaction parameters were investigated in detail and, under the optimized reaction conditions, AVBL was isolated in 56% yield. The practicability of this bioconversion was further confirmed through the condensation of catharanthine with the vindoline analogue 11-methoxy-dihydrotabersonine.     

A single point mutation converts a proton-pumping rhodopsin into a red-shifted,turn-on fluorescent sensor for chloride

The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride in detergent micelles and in live Escherichia coli. This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The binding of chloride tunes the pK_a of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single-cell fluorescence microscopy experiments with E. coli, expressing a GR1 fusion with a cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of changes in extracellular chloride at the membrane. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin-based fluorescent chloride sensors with improved properties for cellular applications and learn how proteins can evolve and adapt to bind anions in water.

By utilizing laboratory-guided evolution, we have converted the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride.     

Multiplicity results for Sturm–Liouville boundary value problems

Multiplicity results for Sturm–Liouville boundary value problems are obtained. Proofs are based on variational methods.     

Quantitative Characterization and Study of the Relationship between Constitutive-Facultative Skin Color and Phototype in Caucasians

In this study constitutive and facultative colorimetric values were quantified to determine the physiologic changes in Caucasian skin color and to define the correlation between skin color and phototype assessed according to the Fitzpatrick method. Our population consisted of 401 subjects ranging in age from 24 to 28 years with similar life styles. Skin color was measured with a Minolta CR-200 colorimeter on the upper medial quarter of the buttock (constitutive color) and on the cheek (facultative color). Advanced multivariate statistical analysis allowed differentiation between constitutive and facultative skin color in relation to the phototype to be quantified. Moreover, Kullback divergence showed that the probability of correctly determining a subject's phototype is high when the variables of constitutive and facultative skin color are considered together. This interesting result makes it possible, in the future, to use colorimetric values of exposed and nonexposed skin, together with determination of Fitzpatrick phototype and of other phenotypic characters, to better predict cutaneous sun reactivity.     

Effects of Vocal Constitution and Autonomic Stress-Related Reactivity on Vocal Endurance in Female Student Teachers

Several studies revealed a high percentage of voice problems in future teachers. The influence of vocal constitution on the vocal endurance is, however, still unclear. The goal of this study was to evaluate whether the increase of voice fundamental frequency (F0) during teaching is caused by (1) autonomic regulation patterns under stress, (2) anxiety as an emotional factor, or (3) limitations in voice constitution. Thirty-three subjects with either normal voice constitution (n = 15, group 1) or constitutional hypofunction (n = 18, group 2) assessed by voice range profile measurements were enrolled in this study. Furthermore, they underwent a standardized baseline test to register selected autonomic test parameters and were classified into autonomic outlet types (AOT) as proposed by Johannes et al. Later the subjects were examined during 1 hour of teaching (field study). The parameters tested included heart rate, pulse transition time, finger temperature, and voice fundamental frequency. To measure situational anxiety and general anxiety proneness, a state-trait anxiety inventory was taken. Eleven subjects per group were identified as autonomic stable (AOT 1), two per group as responding cardiovascularly (AOT 2), and two of group 1 and four of group 2, respectively, as having higher heart rate and higher blood pressure responses to stress (AOT 4). One subject had to be excluded because of missing data. However, statistical analyses showed no differences between AOT groups regarding the voice constitution groups. Increased fundamental frequencies of speaking voice after 30 and 45 minutes of teaching were found in group 2 (constitutional hypofunction). No effect of state or trait anxiety on voice endurance could be detected. Thus, the increase of fundamental frequency of voice has to be regarded as a consequence of vocal fatigue. A constitutionally weak voice seems to be a risk factor for developing a professional voice disorder.