Prednisolone Targets Claudins in Mouse Brain Blood Vessels

Endothelial cells in brain capillaries are crucial for the function of the blood–brain barrier (BBB), and members of the tight junction protein family of claudins are regarded to be primarily responsible for barrier properties. Thus, the analysis of bioactive substances that can affect the BBB’s permeability is of great importance and may be useful for the development of new therapeutic strategies for brain pathologies. In our study, we tested the hypothesis that the application of the glucocorticoid prednisolone affects the murine blood–brain barrier in vivo. Isolated brain tissue of control and prednisolone-injected mice was examined by employing immunoblotting and confocal laser scanning immunofluorescence microscopy, and the physiological and behavioral effects were analyzed. The control tissue samples revealed the expression of barrier-forming tight junction proteins claudin-1, -3, and -5 and of the paracellular cation and water-channel-forming protein claudin-2. Prednisolone administration for 7 days at doses of 70 mg/kg caused physiological and behavioral effects and downregulated claudin-1 and -3 and the channel-forming claudin-2 without altering their localization in cerebral blood vessels. Changes in the expression of these claudins might have effects on the ionic and acid–base balance in brain tissue, suggesting the relevance of our findings for therapeutic options in disorders such as cerebral edema and psychiatric failure

Predation on Live and Artificial Insect Prey Shows Different Global Latitudinal Patterns

AimLong-standing theory predicts that the intensity of biotic interactions increases from high to low latitudes. Studies addressing geographic variation in predation on insect prey have often relied on prey models, which lack many characteristics of live prey. Our goals were to explore global latitudinal patterns of predator attack rates on standardised live insect prey and to compare the patterns in predation on live insects with those on plasticine prey models.LocationGlobal forested areas.Time Period2021–2023.Major TaxaArthropods, birds.MethodsWe measured predation rates in 43 forested locations distributed across five continents from 34.1° S to 69.5° N latitude. At each location, we exposed 20 sets of three bait types, one set per tree. Each set included three live fly larvae (maggots), three live fly puparia and three plasticine models of the puparia. We used glue rings to isolate half of the sets from non-flying predators.ResultsArthropod attack rates on plasticine prey decreased linearly from low to high latitudes, whereas attack rates on maggots had a U shaped distribution, with the lowest predation rates at temperate latitudes and the highest rates at tropical and boreal latitudes. This difference emerged from intensive predator attacks on live maggots, but not on plasticine models, in boreal sites. Site-specific attack rates of arthropod predators on live and plasticine prey were not correlated. In contrast, bird attack rates on live maggots and plasticine models were positively correlated, but did not show significant latitudinal changes.Main ConclusionsLatitudinal patterns in predation differ between major groups of predators and between types of prey. Poleward decreases in both arthropod and combined arthropod and bird predation on plasticine models do not mirror patterns of predation on our live prey, the latter likely reflecting real patterns of predation risk better than do patterns of attack on artificial prey

SARS-CoV-2-Induced Pathology—Relevance to COVID-19 Pathophysiology

In spite of intensive studies of different aspects of a new coronavirus infection, many issues still remain unclear. In a screening analysis of histopathology in l200 lethal cases, authors succeeded in performing a wide spectrum of immune histochemical reactions (CD2, CD 3, CD 4, CD 5, CD 7, CD 8, CD14, CD 20, CD 31, CD 34, CD 56, CD 57, CD 68, CD 163, collagen 1,3, spike protein SARS-CoV-2, caspase-3, MLCM; ACE2 receptor, occludin, and claudin-1 and -3) and electron microscopy. The results of the histological and IHC studies of deceased people with varying degrees of severity of coronavirus infection confirmed the ability of these pathogens to cause cytoproliferative changes, primarily in epithelial and endothelial cells. Lesions of various organs are possible, while the reasons for significant differences in organotropy remain unclear. Severe respiratory failure in COVID-19 in humans is associated with a very peculiar viral pneumonia. In the pathogenesis of COVID-19, the most important role is played by lesions of the microcirculatory bed, the genesis of which requires further study, but direct viral damage is most likely. Endothelial damage can be associated with both thrombosis in vessels of various calibers, leading to characteristic complications, and the development of DIC syndrome with maximal kidney damage. Such lesions can be the basis of clinically diagnosed septic shock, while usually there are no morphological data in favor of classical sepsis caused by bacteria or fungi. A massive infiltration of the lung tissue and other organs, mainly by T lymphocytes, including those with suppressor properties, makes it necessary to conduct a differential diagnosis between the morphological manifestation of the protective cellular immune response and direct viral lesions but does not exclude the hypothesis of an immunopathological component of pathogenesis. In many of the deceased, even in the absence of clear clinical symptoms, a variety of extrapulmonary lesions were also detected. The mechanism of their development probably has a complex nature: direct lesions associated with the generalization of viral infection and vascular disorders associated with endothelial damage and having an autoimmune nature. Many aspects of the pathogenesis of coronavirus infection require further comprehensive study

Constitutive and activation-dependent phosphorylation of lymphocyte phosphatase-associated phosphoprotein (LPAP)

<div><p>Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein expressed exclusively in lymphocytes. LPAP is a component of a supramolecular complex composed of the phosphatase CD45, the co-receptor CD4, and the kinase Lck. In contrast to its immunologically important partners, the function of LPAP is unknown. We hypothesized that the biological role of LPAP may be determined by analyzing LPAP phosphorylation. In the present study, we identified LPAP phosphorylation sites by site-directed mutagenesis, phospho-specific antibodies, and protein immunoprecipitation coupled to mass spectrometry analysis. Our results confirmed previous reports that Ser-99, Ser-153, and Ser-163 are phosphorylated, as well as provided evidence for the phosphorylation of Ser-172. Using various SDS-PAGE techniques, we detected and quantified a set of LPAP phosphoforms that were assigned to a combination of particular phosphorylation events. The phosphorylation of LPAP appears to be a tightly regulated process. Our results support the model: following phorbol 12-myristate 13-acetate (PMA) or TCR/CD3 activation of T cells, LPAP is rapidly dephosphorylated at Ser-99 and Ser-172 and slowly phosphorylated at Ser-163. Ser-153 exhibited a high basal level of phosphorylation in both resting and activated cells. Therefore, we suggest that LPAP may function as a co-regulator of T-cell signaling.</p></div

LPAP phosphorylation in resting CEM cells.

<p>LPAP-deficient CEM cells stably transfected with LPAP mutants were lysed in TX-100. Immunoprecipitated LPAP was either treated (+) or untreated (-) with the phosphatase CIP as indicated and subjected to 2D-DIGE or phosphate-affinity SDS-PAGE. For 2D-DIGE analysis LPAP was Cy3-labeled (green, CIP-treated) or Cy5-labeled (red, CIP-untreated). (A) 2D-DIGE analysis of LPAP Δ163–206 and Δ153–206 deletion mutants. Arrows indicate the positions of Δ163–206 and Δ153–206 deletion mutants, which were run on the same gel. (B) phosphorylation of S99A LPAP mutant (first column), S153A (second column), triple mutant S99A/S153A/S172A (third column), and triple mutant S99A/S155A/S172A (fourth column) detected by 2D-DIGE. (C) LPAP phosphorylation detected by Phos-tag SDS-PAGE. LPAP point mutants S99A (lanes 3–4), S153A (lanes 5–6, 9–10), S172A (lanes 7–8) or LPAP from parental CEM (lanes 1–2) were subjected to Phos-tag SDS-PAGE and blotted with anti-LPAP antibody. Lanes 9–10 were exposed for longer time to show minor bands (right panel). LPAP bands were named P0, P1, P2, P3, and P4 according to their relative mobility from fast to slow and assigned to the phosphorylation sites. The phosphorylation site(s) of each band were assigned as described on the left side: P0, unphosphorylated; P1, Ser-153; P2, Ser-99/Ser-153 and Ser-99; P3, Ser-172/Ser-153 and Ser-172; P4, Ser-99/Ser172/Ser-153 and Ser-99/Ser-172. (D) graph of band intensities ± s.d. (n = 5) in Phos-tag SDS-PAGE for resting CEM cells.</p

Generation and characterization of LPAP-deficient CEM cells stably transfected with mutant LPAP.

<p>(A) schematic of human LPAP gene targeting using Cas9 double-nicking strategy. Target regions of each sgRNA are labeled in blue, and PAM sequences are highlighted in red. (B) comparison of LPAP expression in parental CEM cells, LPAP-deficient clone CEM98, and LPAP-deficient cells stably transfected with LPAP mutants by Western blotting with anti-LPAP (upper panel), and by immunofluorescent staining of permeabilized cells (lower panel). Tubulin served as a loading control in Western blotting. Gray-filled histogram, isotype control in flow cytometry. C, 2D-DIGE comparison of LPAP proteoforms from parental CEM (green) and LPAP-deficient CEM98 cells stably transfected with wild-type LPAP (red).</p

LPAP mutations that affect phosphorylation-dependent mobility shift.

<p>LPAP-deficient CEM cells stably transfected with LPAP were lysed in TX-100. Cell lysates were immunoprecipitated with anti-LPAP antibody. Samples were either treated (+) or untreated with the phosphatase CIP (-) as indicated and subjected to 18% SDS-PAGE or 2D-PAGE. (A) Western blot analysis of LPAP alanine point mutants. LPAP from parental CEM cells (lanes 1–2) was included for comparison. (B) 2D-DIGE analysis of S172A LPAP mutant. Cy3-labeled LPAP (green) was dephosporylated (indicated as +CIP), whereas Cy5-labeled LPAP (red) was left untreated (-CIP).</p

Schematic of LPAP structure with phosphorylation site prediction and representation of LPAP deletion and point mutants.

<p>(A) schematic representation of LPAP domains: SP, signal peptide; EX, extracellular; TR, transmembrane; Lck, Lck binding domain; LCR, low complexity region domain; a.a., amino acids; CL3, CL7, epitopes of anti-LPAP mAbs. Numbers indicate the position of amino acids. (B) phosphosites predicted by NetPhos 2.0. C, point and deletion LPAP mutants used in this work.</p

LPAP phosphorylation in PMA-activated PBMCs.

<p>PBMCs were activated with PMA (10 ng/ml) for 30 min or left untreated, lysed and immunoprecipitated with anti-LPAP antibody. (A) samples from PMA-activated Cy3-labeled cells and resting Cy5-labeled cells were mixed, run on 2D-PAGE and visualized with a fluorescent gel scanner. (B) samples from resting or PMA-activated PBMCs were either treated (+) or untreated with the phosphatase CIP (-) and analyzed by Phos-tag SDS-PAGE followed by Western blotting using monoclonal anti-LPAP. LPAP from CEM cells is presented for comparison. Arrows indicate the band, which appears after PMA activation. (C) PBMC and purified CD4⁺ or CD8⁺ cells were activated with PMA (10 ng/ml) for 30 min or left untreated, lysed, resolved on 18% SDS-PAGE and blotted with anti-LPAP or p153 antibodies.</p