Possibility of reducing the luteolytic dose of cloprostenol in cyclic dairy cows

The administration of cloprostenol by intravulvosubmucous (i.v.s.m.) injection at 1 2 and 1 4 of the dose usually given by intramuscular (i.m.) injection, was tested in dairy cows for luteolysis and estrus synchronization. The i.m. injection was used in ten adult cows at the usual dose of 500 mug/animal. Eleven adult cows and 11 heifers were treated i.v.s.m. with a dose equivalent to 250 mug/animal and 125 mug/animal, respectively. Two injections of cloprostenol were administered 11 days apart to the cows not detected in oestrus after a single injection. Forty-three out of the total 46 animals were detected to be in dioestrus at the time of at least one of the injections, as reflected by the plasma progesterone concentrations at the time of treatments. Three out of the 43 animals injected during dioestrus were refractory to the luteolytic effect of cloprostenol; this appeared to be independent of the dosage and the route of administration (refractory cows were: one adult cow treated i.m. and two treated i.v.s.m. with 125 mug of cloprostenol). The mean time interval from injection to the onset of heat was 82.8 hours with a confidence limit for 95% of probability between 67.9 hours and 92.7 hours. The difference between treatments is not significant. The results suggest that in heifers and adult cows cloprostenol can be given i.v.s.m. route at a reduced dose of 1 4 of the usual 500 mug i.m. dosage without affecting the luteolytic effect of the drug or fertility.     

Antiproliferative activity of Pterodon pubescens Benth. seed oil and its active principle on human melanoma cells

On a preliminary screening, relevant in vitro antiproliferative activity was observed to the crude ethanolic extract of Pterodon pubescens seed oil against the human melanoma cell line SK MEL 37. The diethyl ether fraction from crude ethanolic extract which exhibited stronger activity was submitted to fractionation by gradient elution with hexane/ethyl acetate. Subfraction A, eluted by hexane/ethyl acetate (80:20), was essentially the most active between all the assayed subfractions with an IC₅₀ of 37 μg/ml calculated by the MTT colorimetric method. At this concentration, subfraction A caused morphological features and internucleosomal DNA fragmentation pattern of apoptosis. Through chromatographic separation, the furane diterpene 1 was isolated from this active subfraction and identified by spectral techniques. Compound 1 showed an IC₅₀ value of 32 μM and fluorescence staining with DAPI revealed some typical nuclear changes which are characteristic of apoptosis. These findings support a role for diterpenoids vouacapan-type skeleton as a model to develop new anticancer agents.     

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution,new validation tools,and implications for the future

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) “Resolution Revolution” made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.     

High dietary salt decreases antioxidant defenses in the liver of fructose-fed insulin-resistant rats

In this study we investigated the hypothesis that a high-salt diet to hyperinsulinemic rats might impair antioxidant defense owing to its involvement in the activation of sodium reabsorption to lead to higher oxidative stress. Rats were fed a standard (CON), a high-salt (HS), or a high-fructose (HF) diet for 10 weeks after which, 50% of the animals belonging to the HF group were switched to a regimen of high-fructose and high-salt diet (HFS) for 10 more weeks, while the other groups were fed with their respective diets. Animals were then euthanized and their blood and liver were examined. Fasting plasma glucose was found to be significantly higher (approximately 50%) in fructose-fed rats than in the control and HS rats, whereas fat liver also differed in these animals, producing steatosis. Feeding fructose-fed rats with the high-salt diet triggered hyperinsulinemia and lowered insulin sensitivity, which led to increased levels of serum sodium compared to the HS group. This resulted in membrane perturbation, which in the presence of steatosis potentially enhanced hepatic lipid peroxidation, thereby decreasing the level of antioxidant defenses, as shown by GSH/GSSG ratio (HFS rats, 7.098±2.1 versus CON rats, 13.2±6.1) and superoxide dismutase (HFS rats, 2.1±0.05 versus CON rats, 2.3±0.1%), and catalase (HFS rats, 526.6±88.6 versus CON rats, 745.8±228.7 U/mg ptn) activities. Our results indicate that consumption of a salt-rich diet by insulin-resistant rats may lead to regulation of sodium reabsorption, worsening hepatic lipid peroxidation associated with impaired antioxidant defenses.     

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W., Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525–532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, V.I., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66, 331–341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1' in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high β sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae.     

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S,and G2 Phases of the Cell Cycle in Trypanosomatids

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU‐ and EdU‐incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and “click” chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re‐estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.     

Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.