Characterization of EST3: a metagenome-derived esterase with suitable properties for biotechnological applications

Metagenomic libraries from diverse environments have been extensive sources of many lipases and esterases; nevertheless, most of these enzymes remain biochemically uncharacterized. We previously built a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation and tested it for lipolytic activity. In the present study, we identified the PL14.H10 clone that was subcloned and sequenced, which enabled the identification of the EST3 protein. This enzyme exhibited 74 % amino acid identity with the uncharacterized alpha/beta hydrolase from Parvibaculum lavamentivorans [GenBank: WP012110575.1] and was classified into lipolytic enzyme family IV. Biochemical characterization revealed that EST3 presents high activity in a wide range of temperature with highest activity from 41 to 45 °C. Also, this thermostable esterase acts from mild acidic to alkaline conditions with an optimum pH of 6.0. The enzyme exhibited activity against p-nitrophenyl esters of different chain lengths and highest catalytic efficiency against p-nitrophenyl caprylate. The activity of the protein was increased in the presence of 0.5 mM of Mn⁺², Li⁺, EDTA, and 1 % of CTAB and exhibited half of the activity in the presence of 10 % methanol and ethanol. Moreover, the homology model of EST3 was built and compared to other esterases, revealing a substrate channel that should fit a wide range of substrates. Taken together, the data presented in this work reveal the unique and interesting characteristics of EST3 that might be explored for further use in biotechnological applications.     

Cerebral renin-like activity in different functional conditions (author's transl)]

Using the Boucher micromethod of determining renin activity in the frontoparietal cortex, brain stem, hypothalamus, hypophysis, and epiphysis rat tissue in different functional conditions, we have observed the following: 1 In rats previously treated with an I. V., injection of 1-1.5 ml 10% NaCl, an increase of renin-like activity was found in the cerebral cortex, brain stem and hypothalamus, whereas in the grandular tissue there was a significant decrease...     

Guidelines for the design and conduct of human clinical trials on ingestion-time differences – chronopharmacology and chronotherapy – of hypertension medications

ABSTRACT

Current hypertension guidelines fail to provide a recommendation on when-to-treat, thus disregarding relevant circadian rhythms that regulate blood pressure (BP) level and 24 h patterning and medication pharmacokinetics and pharmacodynamics. The ideal purpose of ingestion-time (chronopharmacology, i.e. biological rhythm-dependent effects on the kinetics and dynamics of medications, and chronotherapy, i.e. the timing of pharmaceutical and other treatments to optimize efficacy and safety) trials should be to explore the potential impact of endogenous circadian rhythms on the effects of medications. Such investigations and outcome trials mandate adherence to the basic standards of human chronobiology research. In-depth review of the more than 150 human hypertension pharmacology and therapeutic trials published since 1974 that address the differential impact of upon-waking/morning versus at-bedtime/evening schedule of treatment reveals diverse protocols of sometimes suboptimal or defective design and conduct. Many have been “time-of-day,” i.e. morning versus evening, rather than circadian-time-based, and some relied on wake-time office BP rather than around-the-clock ambulatory BP measurements (ABPM). Additionally, most past studies have been of too small sample size and thus statistically underpowered. As of yet, there has been no consensual agreement on the proper design, methods and conduct of such trials. This Position Statement recommends ingestion-time hypertension trials to follow minimum guidelines: (i) Recruitment of participants should be restricted to hypertensive individuals diagnosed according to ABPM diagnostic thresholds and of a comparable activity/sleep routine. (ii) Tested treatment-times should be selected according to internal biological time, expressed by the awakening and bed times of the sleep/wake cycle. (iii) ABPM should be the primary or sole method of BP assessment. (iv) The minimum-required features for analysis of the ABPM-determined 24 h BP pattern ought to be the asleep (not “nighttime”) BP mean and sleep-time relative BP decline, calculated in reference to the activity/rest cycle per individual. (v) ABPM-obtained BP means should be derived by the so-called adjusted calculation procedure, not by inaccurate arithmetic averages. (vi) ABPM should be performed with validated and calibrated devices at least hourly throughout two or more consecutive 24 h periods (48 h in total) to achieve the highest reproducibility of mean wake-time, sleep-time and 48 h BP values plus the reliable classification of dipping status. (vii) Calculation of minimum required sample size in adherence with proper statistical methods must be provided. (viii) Hypertension chronopharmacology and chronotherapy trials should preferably be randomized double-blind, randomized open-label with blinded-endpoint, or crossover in design, the latter with sufficient washout period between tested treatment-time regimens.     

Nonpeptide HIV protease inhibitors designed to replace a bound water

Cyclic nonpeptide molecules were designed and synthesized with the goal of displacing the conserved flap-associated water of HIV-1 protease. Several such molecules were competitive inhibitors with micromolar inhibition constants, and their structure-activity relationships were consistent with the design hypothesis.     

Synthesis of 1,2-trans-disaccharides via sugar thio-orthoesters

The reaction of sugar 1,2-thio-orthoesters in the d-gluco, d-galacto, d-manno, and l-rhamno series with primary and secondary trityl ethers of monosaccharides, in the presence of triphenylmethylium perchlorate as catalyst, affords, stereospecifically, derivatives of 1,2-trans-disaccharides in good yields. 4-Trityl ethers of benzyl 2-acetamido-3,6-di-O-acetyl-2-deoxy-α-d-glucopyranoside and methyl 2,3,6-tri-O-benzoyl-α-d-galactopyranoside exhibit low reactivity in glycosylation by thio-orthoesters. A reaction scheme for the glycosylation is discussed.