Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Chemical composition and larvicidal activity of essential oils of three Artemisia species

Aedes aegypti L. (Diptera: Culicidae) is a vector for serious diseases in tropical regions. This pest is mainly controlled by commercial larvicides but the application of such products has led to environmental problems. Essential oils (EO) have been consistently reported as molecules with insecticidal activity and can be used to produce more environmentally friendly larvicides in the control of A. aegypti. In this study, the larvicidal effect of essential oils (EO) from the leaves of three Artemisia species was evaluated against A. aegypti. The oils were obtained from steam distillation and their chemical composition was determined by gas chromatography–mass spectrometry. The EO of Artemisia camphorata was the most active in the screening bioassay and presented LC₅₀ and LC₉₅ of 64.95 and 74.18 μg ml⁻¹, respectively. In addition, we found that germacrene D-4-ol was the constituent responsible for the toxicity of this EO. Artemisia camphorata EO and its major constituent, germacrene D-4-ol, are promising for the development of natural larvicides against A. aegypti.     

COVID-19 plasma proteome reveals novel temporal and cell-specific signatures for disease severity and high-precision disease management

Coronavirus disease 2019 (COVID-19) is a systemic inflammatory condition with high mortality that may benefit from personalized medicine and high-precision approaches. COVID-19 patient plasma was analysed with targeted proteomics of 1161 proteins. Patients were monitored from Days 1 to 10 of their intensive care unit (ICU) stay. Age- and gender-matched COVID-19-negative sepsis ICU patients and healthy subjects were examined as controls. Proteomic data were resolved using both cell-specific annotation and deep-analysis for functional enrichment. COVID-19 caused extensive remodelling of the plasma microenvironment associated with a relative immunosuppressive milieu between ICU Days 3–7, and characterized by extensive organ damage. COVID-19 resulted in (1) reduced antigen presentation and B/T-cell function, (2) increased repurposed neutrophils and M1-type macrophages, (3) relatively immature or disrupted endothelia and fibroblasts with a defined secretome, and (4) reactive myeloid lines. Extracellular matrix changes identified in COVID-19 plasma could represent impaired immune cell homing and programmed cell death. The major functional modules disrupted in COVID-19 were exaggerated in patients with fatal outcome. Taken together, these findings provide systems-level insight into the mechanisms of COVID-19 inflammation and identify potential prognostic biomarkers. Therapeutic strategies could be tailored to the immune response of severely ill patients.     

Litter quality and nutrient cycling affected by grazing-induced species replacements along a precipitation gradient

One of the potential mechanisms for the impact of herbivores on nutrient cycling is the effect of selective grazing on litter quality through changes in species composition. However, the scarce evidence collected on this mechanism is controversial and seemingly influenced by site-specific variables. In this paper, we explored the consequences of grazing-induced changes in species composition on litter quality and nitrogen cycling with a regional perspective. Along a 900-mm of mean annual rainfall gradient, we selected species promoted and diminished by grazing from three natural rangelands of Argentina, analyzed their litter quality, and determined their decomposition and nutrient release kinetics under common greenhouse conditions. Litter quality and decomposition rates were strongly associated with plant response to grazing. However, the magnitude and direction of these differences depended on the ecosystem considered. In the wettest site, the species promoted by grazing (forbs) had higher nitrogen and phosphorus contents, faster decomposition rates, and higher release of nitrogen to the soil than species diminished by grazing (C₃ and C₄ grasses). In the intermediate and dry sites, species promoted by grazing had lower nitrogen and phosphorus contents, and slower decomposition rates than those diminished by grazing (C₃ grasses in both cases). Decomposition of the entire group of species was not correlated with mean annual rainfall, but when litter of the species diminished by grazing was analyzed, it was negatively correlated with precipitation. Nitrogen was immobilized more often than mineralized, even after one year of incubation. Immobilization was negatively correlated with precipitation. All these results indicate that grazing may significantly alter nutrient cycling by affecting litter quality through changes in species composition. These effects seem to be larger when species replacements induced by grazing either involve functional groups, as it was the case in our wettest site, or change root to shoot ratios. Therefore, the functional groups involved in the replacement of species as well as shifts between belowground and aboveground allocation should play a key role in grazing-induced changes on nitrogen cycling.     

Linear oligopeptides: 65′. Conformational analysis of the N-protected aromatic α-amino acid N-tert-butyloxycarbonyl-l-phenylalanine by X-ray diffraction and infrared absorption

The X-ray diffraction and i.r. absorption conformational analysis of $\text{N}\text{-tert-butyloxycarbonyl-}\text{l}\text{-phenylalanine}$ has showed the absence of intramolecularly hydrogen-bonded peptide conformations in the solid state. The molecules are held together in rows of ‘cyclic dimer’ motifs through intermolecular NH … OC (acid) and OH … OC {urethane} hydrogen bonds, the secondary amide-like group of the urethane moiety being in the unusual cis conformation, whereas the carboxylic acid group in the common syn conformation. The two molecules in the unit cell present a centrosymmetric set of ?, ψ¹, and ψ² values. In polar solvents solvated species largely predominate. In saturated hydrocarbon solution non-associated and associated (mostly involving the carboxylic acid CO as the proton acceptor) species simultaneously occur. The extent of association decreases with dilution. The amount of intramolecularly hydrogen-bonded oxy-C₇ and C₅ forms if any, should be extremely small. The type of association at saturation seems to differ from that found in the crystalline compound obtained by precipitation with saturated aliphatic hydrocarbons (from a diethyl ether solution).     

Two isoaccepting seryl tRNAs coded by separate mitochondrial genes in yeast

Summary In S. cerevisiae four isoacceptor mitochondrial tRNAs for serine have been separated by reversed phase chromatography. At least two of these species are products of different genes. In this work the deletion mapping technique has been used to locate two genes for tRNA^ser. The gene for tRNA^ser previously localized in the oli I region of the mitochondrial genome has been found to code for tRNA _ser ² , and another gene coding for tRNA _ser ¹ has been detected in the region where most of other tRNA genes are found. Results of fine mapping experiments allowed to localize this gene in the proximity of the gene for tRNA^arg.     

Positive regulation in a eukaryote, a study of the uaY gene of Aspergillus nidulans

Summary In this publication we report the identification of a protein likely to be coded by uaY, a regulatory gene in the ascomycete Aspergillus nidulans. uaY is a positive control gene necessary for the expression of at least eight unlinked structural genes involved in purine uptake and degradation (Scazzocchio and Gorton 1977). The physiological effector of the uaY system is uric acid, while some of its thioanalogs serve as gratuitous inducers. Effector binding proteins were detected by binding to 2-thiouric acid after phosphocellulose column chromatography, or as uric acid binding fractions after DNA-cellulose column chromatography. Two binding peaks are present in mycelial extracts purified by either method. These are missing in a putative small deletion of the uaY gene. A leaky mutation, uaY 109 described in detail elsewhere (Scazzocchio et al. 1980) shows only one peak. The wild type peaks are eluted at 55 mM NaCl and at 720 mM NaCl while the peak present in uaY109 is eluted at 120 mM NaCl. This implies that at least one peak represents a protein coded by the uaY gene. The major peak was analysed by equilibrium dialysis experiments. These establish a K_diss.2×10^-7 and a minimum number of binding sites of 3×10^-14 moles/mg of soluble protein in a crude extract derived from protoplast lysis. An extract from a strain carrying the uaY207 deletion, purified blind, lacks any binding activity in the equilibrium dialysis cell.     

Immunoreactivity for substance P in the gasserian ganglion, ophthalmic nerve and anterior segment of the rabbit eye

Summary The distribution of substance P (SP) immunofluorescence was investigated in the Gasserian ganglion, ophthalmic nerve and in the anterior segment of the rabbit eye. About one third of the nerve cell bodies in the Gasserian ganglion exhibited SP immunofluorescence, which was also observed in some nerve fibres of the ophthalmic nerve. In the cornea, some SP-positive iris contained numerous nerve fibres with SP immunofluorescence. In the sphincter area such fibres were circular, while the orientation of the SP fibres was radial in the dilator muscle. Both in the iris and in the ciliary body, the largest vessels were surrounded by nerves exhibiting SP immunofluorescence. A few nerve fibres also appeared in the stroma of the ciliary processes.