Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Human M-proinsulin was cleaved by trypsin at the R³¹R³²–E³³ and K⁶⁴R⁶⁵–G⁶⁶ bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K⁵⁹R⁶⁰–G⁶¹ bond but at the B/C junction cleavage occurred at the R³¹R³²–E³³ as well as the R³¹–R³²E³³ bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k_cat./K_m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 10⁵ s^− 1 M^− 1) and the cleavage of the K²⁹–T³⁰ bond of M-insulin-RR (1.3 ± 0.07 × 10⁵ s^− 1 M^− 1). However, the K²⁹–T³⁰ bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k_cat./K_m values around 1000 s^− 1 M^− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K²⁹–T³⁰ bond becomes shielded, exposed then shielded again respectively.     

Evaluation of structural features in fungal cytochromes P450 predicted to rule catalytic diversification

Fungi belong to the large kingdom of lower eukaryotic organisms encompassing yeasts along with filamentous and dimorphic members. Microbial P450 enzymes have contributed to exploration of and adaptation to diverse ecological niches such as conversion of lipophilic compounds to more hydrophilic derivatives or degradation of a vast array of environmental toxicants. To better understand diversification of the catalytic behavior of fungal P450s, detailed insight into the molecular machinery steering oxidative attack on the distinctly structured endogenous and xenobiotic substrates is of preeminent interest. Based on a general, CYP102A1-related template the bulk of predicted substrate/inhibitor-binding determinants were shown to cluster near the distal heme face within the six known substrate recognition sites (SRSs) made up by the α-helical B′/F/G/I tetrad, the B′–C interhelical loop and strands of the β6-sheet, population density being highest in the structurally flexible SRS-1 and SRS-4 domains, showing a low degree of conservation. Reactivity toward ligands favorably coincides with the lipophilicity/hydrophilicity profile and bulkiness of critical amino acids acting as selective filters. Some decisive elements may also serve in maintenance of catalytic competence via their action as gatekeepers directing substrate access/positioning or stabilizers of the heme environment enabling dioxygen activation. Non-SRS residues seem to control spin state equilibria and attract redox partners by electrostatic forces. Of note, the inhibitory potency of azole-type fungicides is likely to arise from perturbation of the complex interplay of the mechanistic principles addressed above. Knowledge-supported exploitation of the topological data will be helpful in the manufacture of commodity/specialty chemicals as well as therapeutic agents. Also, engineered fungal P450s may be used to improve pollutant-specific bioremediation of contaminated soils.     

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.     

钢渣组分特征及其用于土壤改良的可行性初步研究

通过测定钢渣的化学组成分析得出其组分特征,进一步通过在有降解碱剂HPMA参与的重（轻）度盐碱土壤及钢渣中栽种不同植物种子（玉米、茄子、水稻和长春花）,观察植物的长势,探究钢渣是否可以用于土壤改良。结果发现钢渣的化学成分与土壤基本相同,但部分成分如CaO、Fe₂O₃、MgO、MnO₂等显著高于土壤,而另外一些组分TiO₂、K₂O、Na₂O、P₂O₅在钢渣中未检测出来。对比重（轻）度盐碱土壤,钢渣与HPMA共同作用时,玉米、茄子、水稻和长春花等发芽率及生长速率明显提高;与常用的蛭石、珍珠岩改良剂对比实验中,不同植物存在类似的发芽率和生长速率,说明钢渣可以作为温室土壤结构改良剂;与钢渣产地丰富的树皮土资源配比形成复方改良剂,发现其具有类似花土（泥炭土）的生长速率和发芽率。初步研究表明,钢渣可以用于盐碱地改良,而且对于设施农业的土壤结构改良具有功效,与当地树皮土资源结合,具有形成复方土壤改良剂的潜力。     

Predicted future distribution of the African skimmer in response to a changing climate,land cover and distance from water in the mid-Zambezi Valley

Climate models project a hot and dry future for Southern Africa. In this research, Maximum Entropy was used to model the extent to which climate change, land cover and distance from water edges may influence current and future distribution of the African skimmer in the mid-Zambezi Valley. Global Biodiversity Information Facility data collected between the years 2000–2019 were used to develop the models. Three models were built: one for current distribution and two for future distribution under Representative Concentration Pathways (RCPs) 2.6 and 6.0. Results revealed that annual precipitation and distance from water edges were the most important predictors of habitat suitability for the African skimmer under current and future climate. Temperature and land cover were least important in explaining current and future distribution of the species. The RCP 2.6 predicted future decrease in suitable habitat for the African skimmer in the mid-Zambezi Valley, while RCP 6.0 predicted future increase in suitable habitat for the species. This research conclusively revealed that precipitation and distance from water edges were consistently key predictors of suitable habitat for the African skimmer.     

Resting Stages of Skeletonema marinoi Assimilate Nitrogen From the Ambient Environment Under Dark,Anoxic Conditions

The planktonic marine diatom Skeletonema marinoi forms resting stages, which can survive for decades buried in aphotic, anoxic sediments and resume growth when re-exposed to light, oxygen, and nutrients. The mechanisms by which they maintain cell viability during dormancy are poorly known. Here, we investigated cell-specific nitrogen (N) and carbon (C) assimilation and survival rate in resting stages of three S. marinoi strains. Resting stages were incubated with stable isotopes of dissolved inorganic N (DIN), in the form of ¹⁵N-ammonium (NH₄⁺) or -nitrate (NO₃⁻) and dissolved inorganic C (DIC) as ¹³C-bicarbonate (HCO₃⁻) under dark and anoxic conditions for 2 months. Particulate C and N concentration remained close to the Redfield ratio (6.6) during the experiment, indicating viable diatoms. However, survival varied between <0.1% and 47.6% among the three different S. marinoi strains, and overall survival was higher when NO₃⁻ was available. One strain did not survive in the NH₄⁺ treatment. Using secondary ion mass spectrometry (SIMS), we quantified assimilation of labeled DIC and DIN from the ambient environment within the resting stages. Dark fixation of DIC was insignificant across all strains. Significant assimilation of ¹⁵N-NO₃⁻ and ¹⁵N-NH₄⁺ occurred in all S. marinoi strains at rates that would double the nitrogenous biomass over 77–380 years depending on strain and treatment. Hence, resting stages of S. marinoi assimilate N from the ambient environment at slow rates during darkness and anoxia. This activity may explain their well-documented long survival and swift resumption of vegetative growth after dormancy in dark and anoxic sediments.     

Mimicking SIV chimpanzee viral evolution toward HIV-1 during cross-species transmission

HIV-1 evolved from SIV during cross-species transmission events, though viral genetic changes are not well understood. Here, we studied the evolution of SIVcpzLB715 into HIV-1 Group M using humanized mice. High viral loads, rapid CD4⁺ T-cell decline, and non-synonymous substitutions were identified throughout the viral genome suggesting viral adaptation.     

New insights into deleterious impacts of in vivo glycation on albumin antioxidant activities

Background

Albumin constitutes the most abundant circulating antioxidant and prevents oxidative damages. However, in diabetes, this plasmatic protein is exposed to several oxidative modifications, which impact on albumin antioxidant properties.

Methods

Most studies dealing on albumin antioxidant activities were conducted on in vitro modified protein. Here we tried to decipher whether reduced antioxidant properties of albumin could be evidenced in vivo. For this, we compared the antioxidant properties of albumin purified from diabetic patients to in vitro models of glycated albumin.

Results

Both in vivo and in vitro glycated albumins displayed impaired antioxidant activities in the free radical-induced hemolysis test. Surprisingly, the ORAC method (Oxygen Radical Antioxidant Capacity) showed an enhanced antioxidant activity for glycated albumin. Faced with this paradox, we investigated antioxidant and anti-inflammatory activities of our albumin preparations on cultured cells (macrophages and adipocytes). Reduced cellular metabolism and enhanced intracellular oxidative stress were measured in cells treated with albumin from diabetics. NF-kB –mediated gene induction was higher in macrophages treated with both type of glycated albumin compared with cells treated with native albumin. Anti inflammatory activity of native albumin is significantly impaired after in vitro glycation and albumin purified from diabetics significantly enhanced IL6 secretion by adipocytes. Expression of receptor for advanced glycation products is significantly enhanced in glycated albumin-treated cells.

Conclusions and general significance

Our results bring new evidences on the deleterious impairments of albumin important functions after glycation and emphasize the importance of in vivo model of glycation in studies relied to diabetes pathology.     

Antagonistic and plant growth promoting activities of endophytic and soil actinomycetes

The objective of this study was the isolation and screening of actinomycete isolates for antagonistic potential and plant growth promoting activities. A total of 321 isolates were recovered from different plants, their rhizospheric soils and non-rhizospheric soils of Punjab and Himachal Pradesh regions. Out of these, 62 were endophytic, 156 were rhizospheric and 103 were non-rhizospheric isolates. In primary screening (dual culture assay), 83 isolates antagonised one or more test phytopathogenic fungi. From these active isolates, 20 were found to be antagonistic in well diffusion assay (secondary screening) and most of them demonstrated broad spectrum inhibitory activity against five to six test fungi. Studies on plant growth promoting activities revealed that 12 showed abilities to produce indole acetic acid, 10 produced siderophores and 12 showed ammonia production. Phosphate solubilisation was observed in five isolates and four fixed atmospheric N₂. In addition, production of hydrolytic enzymes such as chitinase, amylase, cellulase and protease was demonstrated by five, twenty, eleven and eleven isolates, respectively. The results of this study indicate that these isolates may be used as biocontrol and plant growth promoting agents. Morphological and chemotaxonomic studies revealed that all the active isolates belonged to the genus Streptomyces     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.