Effect of the three-dimensional structure on the deamidation reaction of ribonuclease A.

Kinetic data on the deamidation reaction of Asn67 in RNase A and of Asn3 in the two peptides Ac-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cys-NH2 and Ac-Cys(Me)-Lys-Asn-Gly-Gln-Thr-Asn-Cys(Me)-NH2, whose sequences are similar to that of the deamidation site in the enzyme, have been determined in a wide range of pH and buffer concentrations. The values of the observed rate constant (k) for the enzyme are markedly lower than those for the peptides. However, the k dependence on pH and buffers is similar for all three substrates, indicating a similar reaction mechanism. The lower k-values for the enzyme have been quantitatively related to the thermal stability and the three-dimensional structure of the enzyme.     

Determination of new 2,3-benzodiazepines in rat plasma using high-performance liquid chromatography with ultraviolet detection

A method for the analysis of [1-(4-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one] (CFM-2) and its analogues CFM-3, CFM-4 and CFM-5 in rat plasma was developed. The 2,3-benzodiazepines (2,3-BZs) were extracted by liquid–liquid extraction and analyzed using high-performance liquid chromatography (HPLC) with ultraviolet detection (UV) at 240 nm. The method exhibited a large linear range from 0.05 to 2 μg/ml with an intra-assay accuracy for all studied compounds ranging from 92 to 105.5%; whereas the intra-assay precision ranged from 0.59 to 8.16% in rat plasma. The inter-assay accuracy of CFM-2, CFM-4 and their 3-methyl derivatives, CFM-3 and CFM-5 ranged from 92.2 to 107% and the inter-assay precision ranged from 2.17 to 11.9% in rat plasma. The lower limit of detection was 5.5 ng/ml for CFM-2, 6.5 ng/ml for CFM-3, 7 ng/ml for CFM-4 and 8.5 ng/ml for CFM-5 in rat plasma. The pharmacokinetic study demonstrated that 2,3-BZs achieved a peak plasma concentration between 45 and 75 min after drug administration. Moreover, we observed that plasma chromatograms of rats treated with CFM-3, CFM-4 and CFM-5, respectively, showed a peak consistent with CFM-2. Our study suggests that CFM-4, CFM-5 and CFM-3 are prodrugs of CFM-2, in which they are biotransformed in vivo via different metabolic pathways. In particular, CFM-2 has been proven to possess anticonvulsant activity in various models of seizures, acting as α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist.     

Characterization of experimental cattle populations from Argentina with a low-density SNP genotyping panel

The objectives of the present experiment were to evaluate a low-density SNP array designed for the molecular characterisation of gene banks and to assess the genetic diversity and population structure of beef cattle herds from an Argentinean research station. Forty-nine animals from three breeds (Angus, Hereford, and Argentinean Creole) were genotyped using the multi-species IMAGE001 60-K SNP array (10 K for cattle). Genotypes of other 19 cattle populations from Argentina, other American countries, and Europe were included in the study. Of special interest was the characterization of the Argentinean Creole, the only autochthonous cattle breed in the country. Due to the merging of different datasets, approximately 5 K SNPs were effectively used. Genetic differentiation (F_ST), principal component analysis, neighbour-joining tree of Reynolds distances and ancestry analysis showed that autochthonous American breeds are clearly differentiated, but all have genetic influences of Iberian cattle. The analysed herds of Argentinean Creole showed no evidence of recent admixture and represent a unique genetic pool within local American breeds. An experimental herd and the local commercial Hereford population have also diverged, probably due to the influence of current selection objectives in the breed. Our results illustrate the utility of using low-cost, low density SNP arrays in the evaluation of animal genetic resources. This type of panels could become a very useful resource in developing countries, where most endangered cattle breeds are located. The results also reinforce the importance of experimental herds as reservoir of genetic diversity, particularly in the case of local breeds under-represented in traditional production systems.     

Is carbonic anhydrase inhibition useful as a complementary therapy of Covid-19 infection?

The ongoing Covid-19 is a contagious disease, and it is characterised by different symptoms such as fever, cough, and shortness of breath. Rising concerns about Covid-19 have severely affected the healthcare system in all countries as the Covid-19 outbreak has developed at a rapid rate all around the globe. Intriguing, a clinically used drug, acetazolamide (a specific inhibitor of carbonic anhydrase, CA, EC 4.2.1.1), is used to treat high-altitude pulmonary oedema (HAPE), showing a high degree of clinical similarities with the pulmonary disease caused by Covid-19. In this context, this preliminary study aims to provide insights into some factors affecting the Covid-19 patients, such as hypoxaemia, hypoxia as well as the blood CA activity. We hypothesise that patients with Covid-19 problems could show a dysregulated acid–base status influenced by CA activity. These preliminary results suggest that the use of CA inhibitors as a pharmacological treatment for Covid-19 may be beneficial.     

Burkholderia pseudomallei γ-carbonic anhydrase is strongly activated by amino acids and amines

Activation of the γ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei (BpsγCA) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines has been investigated. The best BpsγCA activators were d-His, l-DOPA, d-Trp, 4-amino-l-Phe, dopamine, 2-(2-aminoethyl)pyridine, 2-aminoethyl-piparazine/morpholine and l-adrenaline, which showed activation constants ranging between 9 and 86 nM. The least effective activators were l-His, l-Phe and 2-pyridyl-methylamine, with K_As in the range of 1.73–24.7 μM. As little is known about the role of γ-CAs in the lifecycle and virulence of this saprophytic bacterium, this study may shed some light on such phenomena. This is the first CA activation study of a γ-CA from a pathogenic bacterium, the only other such study being on the enzyme discovered in the archaeon Methanosarcina thermophila, Cam.     

Changes of the hepatic proteome in hepatitis B-infected mouse model at early stages of fibrosis

Liver fibrosis (LF) is the accumulation of extracellular matrix (ECM) proteins due to chronic liver injury. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) to perform a comparative analysis of cytosolic and nuclear protein patterns of nontransgenic (NTg) and HBV transgenic (Tg) mice livers at early stages of fibrosis. We identified several candidate proteins, involved in a variety of pathways, which could be used as putative biomarkers for LF early detection.     

Exposure to 900 MHz radiofrequency radiation induces caspase 3 activation in proliferating human lymphocytes

Palumbo, R., Brescia, F., Capasso, D., Sannino, A., Sarti, M., Capri, M., Grassilli, E. and Scarfì, M. R. Exposure to 900 MHz Radiofrequency Radiation Induces Caspase 3 Activation in Proliferating Human Lymphocytes. Radiat. Res. 170, 327- 334 (2008).In this study, the induction of apoptosis after exposure to 900 MHz radiofrequency radiation (GSM signal) was investigated by assessing caspase 3 activation in exponentially growing Jurkat cells and in quiescent and proliferating human peripheral blood lymphocytes (PBLs). The exposure was carried out at an average specific absorption rate of 1.35 W/kg in a dual wire patch cell exposure system where the temperature of cell cultures was accurately controlled. After 1 h exposure to the radiofrequency field, a slight but statistically significant increase in caspase 3 activity, measured 6 h after exposure, was observed in Jurkat cells (32.4%) and in proliferating human PBLs (22%). In contrast, no effect was detected in quiescent human PBLs. In the same experimental conditions, apoptosis was also evaluated in Jurkat cells by Western blot analysis and in both cell types by flow cytometry. To evaluate late effects due to caspase 3 activity, flow cytometry was also employed to assess apoptosis and viability 24 h after radiofrequency-radiation exposure in both cell types. Neither the former nor the latter was affected. Since in recent years it has been reported that caspases are also involved in processes other than apoptosis, additional cell cycle studies were carried out on proliferating T cells exposed to radiofrequency radiation; however, we found no differences between sham-exposed and exposed cultures. Further studies are warranted to investigate the biological significance of our findings of a dose-response increase in caspase 3 activity after exposure to radiofrequency radiation.     

New 7,8-ethylenedioxy-2,3-benzodiazepines as noncompetitive AMPA receptor antagonists

A series of 1-aryl-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-ones 2a-f, were synthesized and screened as anticonvulsant agents in DBA/2 mice against sound-induced seizures. The new compounds display anticonvulsant properties although the ED(50) values are higher than those of prototypes 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (1) and GYKI 52466, well-known noncompetitive AMPA receptor antagonists. Functional tests were performed to evaluate the antagonistic activity at the AMPA and kainate receptors.     

Basal and Fasting/Refeeding‐regulated Tissue Levels of Endogenous PPAR‐α Ligands in Zucker Rats

N‐oleoylethanolamine (OEA) and N‐palmitoylethanolamine (PEA) are endogenous lipids that activate peroxisome proliferator–activated receptor‐α with high and intermediate potency, and exert anorectic and anti‐inflammatory actions in rats, respectively. We investigated OEA and PEA tissue level regulation by the nutritional status in lean and obese rats. OEA and PEA levels in the brainstem, duodenum, liver, pancreas, and visceral (VAT) or subcutaneous (SAT) adipose tissues of 7‐week‐old wild‐type (WT) and Zucker rats, fed ad libitum or following overnight food deprivation, with and without refeeding, were measured by liquid chromatography–mass spectrometry. In WT rats, duodenal OEA, but not PEA, levels were reduced by food deprivation and restored by refeeding, whereas the opposite was observed for OEA in the pancreas, and for both mediators in the liver and SAT. In ad lib fed Zucker rats, PEA and OEA levels were up to tenfold higher in the duodenum, slightly higher in the brainstem, and lower in the other tissues. Fasting/refeeding‐induced changes in OEA levels were maintained in the duodenum, liver, and SAT, and lost in the pancreas, whereas fasting upregulated this compound also in the VAT. The observed changes in OEA levels in WT rats are relevant to the actions of this mediator on satiety, hepatic and adipocyte metabolism, and insulin release. OEA dysregulation in Zucker rats might counteract hyperphagia in the duodenum, but contribute to hyperinsulinemia in the pancreas, and to fat accumulation in adipose tissues and liver. Changes in PEA levels might be relevant to the inflammatory state of Zucker rats.