Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus

A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the K_m value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca²⁺ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.     

The chimeric cytokine Hyper-IL-6 enhances the efficiency of lentiviral gene transfer in hepatocytes both in vitro and in vivo

Lentiviral vectors have been used for gene transfer into the liver but their ability to efficiently transduce quiescent hepatocytes remains controversial. Lentivirus-mediated gene transfer is more efficient in cycling cells. We determine the effect of H-IL6 in the lentiviral transduction. The lentiviral vector was used to transduce HepG2 cells and mice liver cells, previously treated with H-IL6. The highest transduction level was observed in HepG2 cells treated with 30 ng/mL H-IL6 and in the mice that received 4 μg H-IL6. Our results suggest that H-IL6 is an inducer of lentiviral gene transfer into the liver cells without any toxicity.     

A set of fluorescent protein‐based markers expressed from constitutive and arbuscular mycorrhiza‐inducible promoters to label organelles,membranes and cytoskeletal elements in Medicago truncatula

Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans‐Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis‐specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub‐cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens‐shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology.     

The book reopened on infectious diseases

Emerging infectious diseases represent a major challenge to human health worldwide. The risk of evolving new infectious pathogens has been intensifying due to urbanization, demographic changes, air travel, inappropriate use of antibiotics, and climate change. These pathogens can affect humans from urban centers to the remotest corners of the globe. Far from being a scourge of the past, infectious diseases are relevant for the world today.     

Exploring macrophage cell therapy on Diabetic Kidney Disease

Alternatively activated macrophages (M2) have regenerative properties and shown promise as cell therapy in chronic kidney disease. However, M2 plasticity is one of the major hurdles to overcome. Our previous studies showed that genetically modified macrophages stabilized by neutrophil gelatinase‐associated lipocalin (NGAL) were able to preserve their M2 phenotype. Nowadays, little is known about M2 macrophage effects in diabetic kidney disease (DKD). The aim of the study was to investigate the therapeutic effect of both bone marrow‐derived M2 (BM‐фM2) and ф‐NGAL macrophages in the db/db mice. Seventeen‐week‐old mice with established DKD were divided into five treatment groups with their controls: D+BM‐фM2; D+ф‐BM; D+ф‐NGAL; D+ф‐RAW; D+SHAM and non‐diabetic (ND) (db/‐ and C57bl/6J) animals. We infused 1 × 10⁶ macrophages twice, at baseline and 2 weeks thereafter. BM‐фM2 did not show any therapeutic effect whereas ф‐NGAL significantly reduced albuminuria and renal fibrosis. The ф‐NGAL therapy increased the anti‐inflammatory IL‐10 and reduced some pro‐inflammatory cytokines, reduced the proportion of M1 glomerular macrophages and podocyte loss and was associated with a significant decrease of renal TGF‐β1. Overall, our study provides evidence that ф‐NGAL macrophage cell therapy has a therapeutic effect on DKD probably by modulation of the renal inflammatory response caused by the diabetic milieu.     

Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.     

Differentiation-related changes of cytokeratin expression in cultured keratinocytes and in fetal, newborn, and adult epidermis

Cytokeratin expression in differentiating cultured foreskin keratinocytes was studied using chain-specific anti-cytokeratin monoclonal antibodies directed against cytokeratins 4, 8, 10, 13, 18, and 19, respectively. Keratinocytes were cultured at low Ca2+ concentration (0.06 mM) to repress differentiation. At confluency, the cells were switched to high Ca2+ concentration (1.6 mM) to induce differentiation. Cells were harvested 0, 3, 8, 16, 24, 48, and 72 h after the switch. Keratinocytes cultured throughout at high Ca2+ concentration were also harvested. Immunoblots of cytokeratin preparations isolated from these cultures showed that cytokeratins 4, 13, and 19 were not present in nondifferentiating keratinocytes but could be detected from about 16 h after the Ca2+ switch. Immunohistochemical studies were performed on frozen sections of cell sheets incubated with anti-cytokeratin and anti-vimentin. Expression of cytokeratins 4, 13, and 19 was seen in superficial cells. Cytokeratin 10 was locally present in suprabasal and superficial cells. Vimentin was present in 40-70% of the basal cells and in only a few differentiating keratinocytes. Expression of cytokeratins 8 and 18 could not be detected. The same antibodies were also used to stain sections from fetal (15, 20, and 29 weeks), newborn (40 weeks), and mature (5 and 75 years) epidermis. In the 15-week-old epidermis, basal cells were positive for cytokeratins 8 and 19 and locally for cytokeratin 4; intermediate cells expressed cytokeratins 4, 10, 13, and 19; and the periderm contained cytokeratins 4, 8, 13, 18, and 19. In the 20-week-old epidermis, cytokeratin 4 had disappeared from the basal cell layer and cytokeratin 19 was present only locally; in the intermediate cell layer, cytokeratins 4 and 19 had disappeared; and in the periderm, the expression of the cytokeratins studied was the same as that in the 15-week-old epidermis. The basal cells of the 29-week-old fetal epidermis, the newborn epidermis, and the mature epidermis are negative with all antibodies tested, except for some scattered cells in the fetal and newborn skin, presumably Merkel cells, that were positive for cytokeratins 8, 18, and 19. Suprabasal cells in all specimens were positive only for cytokeratin 10. With respect to the cytokeratins studied, our results show that cultured differentiating keratinocytes resemble the suprabasal cells of early fetal epidermis. Basal cells of cultured keratinocytes resemble the basal cells of late fetal, newborn, and adult epidermis and therefore support previous observations.