The Predominance of Alternatively Activated Macrophages Following Challenge with Cell Wall Peptide-Polysaccharide After Prior Infection with Sporothrix schenckii

Sporotrichosis is a subcutaneous mycosis that is caused by the dimorphic fungus Sporothrix schenckii. This disease generally occurs within the skin and subcutaneous tissues, causing lesions that can spread through adjacent lymphatic vessels and sometimes leading to systemic diseases in immunocompromised patients. Macrophages are crucial for proper immune responses against a variety of pathogens. Furthermore, macrophages can play different roles in response to different microorganisms and forms of activation, and they can be divided into “classic” or “alternatively” activated populations, as also known as M1 and M2 macrophages. M1 cells can lead to tissue injury and contribute to pathogenesis, whereas M2 cells promote angiogenesis, tissue remodeling, and repair. The aim of this study was to investigate the roles of M1 and M2 macrophages in a sporotrichosis model. Toward this end, we performed phenotyping of peritoneal exudate cells and evaluated the concomitant production of several immunomediators, including IL-12, IL-10, TGF-β, nitric oxide, and arginase-I activity, which were stimulated ex vivo with cell wall peptide-polysaccharide. Our results showed the predominance of the M2 macrophage population, indicated by peaks of arginase-I activity as well as IL-10 and TGF-β production during the 6th and 8th weeks after infection. These results were consistent with cellular phenotyping that revealed increases in CD206-positive cells over this period. This is the first report of the participation of M2 macrophages in sporotrichosis infections.     

PxdA interacts with the DipA phosphatase to regulate peroxisome hitchhiking on early endosomes

In canonical microtubule-based transport, adaptor proteins link cargoes to dynein and kinesin motors. Recently, an alternative mode of transport known as “hitchhiking” was discovered, where cargoes achieve motility by hitching a ride on already-motile cargoes, rather than attaching to a motor protein. Hitchhiking has been best studied in two filamentous fungi, Aspergillus nidulans and Ustilago maydis. In U. maydis, ribonucleoprotein complexes, peroxisomes, lipid droplets (LDs), and endoplasmic reticulum hitchhike on early endosomes (EEs). In A. nidulans, peroxisomes hitchhike using a putative molecular linker, peroxisome distribution mutant A (PxdA), which associates with EEs. However, whether other organelles use PxdA to hitchhike on EEs is unclear, as are the molecular mechanisms that regulate hitchhiking. Here we find that the proper distribution of LDs, mitochondria, and preautophagosomes do not require PxdA, suggesting that PxdA is a peroxisome-specific molecular linker. We identify two new pxdA alleles, including a point mutation (R2044P) that disrupts PxdA’s ability to associate with EEs and reduces peroxisome movement. We also identify a novel regulator of peroxisome hitchhiking, the phosphatase DipA. DipA colocalizes with EEs and its association with EEs relies on PxdA. Together, our data suggest that PxdA and the DipA phosphatase are specific regulators of peroxisome hitchhiking on EEs.     

Evidence for the presence of β-subunit of hexosaminidase in a case of Sandhoff disease using a blotting technique

Hexosaminidases, lysosomal enzymes whose deficiency is responsible for several genetic disorders, exist as two major forms: form A, containing two types of subunits alpha and beta; and form B, containing only beta subunits. We have used a technique involving successively electrophoresis of denatured proteins, transfer (blotting) onto nitrocellulose, and labelling by appropriate antibodies raised against the dissociated forms of hexosaminidases A and B. This technique allows the detection of alpha and beta subunits in crude extracts of normal tissues. The presence of beta chains was demonstrated in the liver of a fetus affected with Sandhoff's disease, deficient in functional hexosaminidases A and B.     

Global phosphorylation at Ser16 of the 32-residue cytoplasmic domain of phospholamban: Comparison of di-t-butyl- and dibenzyl-N,N-diisopropylphosphoramidites

Summary Phospholamban (PLB), a 52-residue integral membrane protein of the cardiac sarcoplasmic reticulum, is known as the regulator of the Ca²⁺-ATPase pump via its phosphorylation-dephosphorylation at Ser¹⁶. In order to investigate the structural effects of the phosphorylation on the cytoplasmic domain of PLB, we synthesized PLB 2–33 in its nonphosphorylated and phosphorylated forms using Fmoc chemistry. PLB 2–33 was phosphorylated using the global phosphorylation method. Phosphorylation with di-t-butyl-N,N-diisopropylphosphoramidite led to an incomplete reaction (nonphosphorylated peptide was recovered) and to the formation of an H-phosphonate. In contrast, the use of dibenzyl-N,N-diisopropylphosphoramidite yielded the desired phosphorylated peptide quantitatively and did not give rise to the H-phosphonate. Our results show the effectiveness of the dibenzyl-protected phosphoramidite when the site to be phosphorylated is particularly hindered.     

Short Communication: Urinary excretion of glutamine transaminase K as an early index of mercuric chloride-induced nephrotoxicity

The possibili that urinary glutamine transaminase K activity might be a marker of a proximal tubule segment-specific response to mercuric chloride was investigated in male rats after a single i.p. injection in time-course and dose-response experiments. Urinary total proteins and angiotensin converting enzyme activity were determined simultaneously. Urinary indices showed an early increase (within 5 h of treatment) of total proteins and angiotensin converting enzyme, whereas glubmine transaminase K increased 10 h after treatment. The peak of all these indices was observed 24 h after mercuric chloride injection. The lowest dose that induced a significant increase in proteins and enzymes was 0.25 mg kg^-1; in addition, a dose-response effect was observed. Glutamine transaminase K appeared to be an early and sensitive index of response of mercuric chloride effects, similar to total proteins and angiotensin converting enzyme. It is suggested that this enzyme is mainly localized in the 'pars recta' of the proximal tubule. Therefore glutamine transaminase K might be a segment-specific marker for the detection of damage localized in this portion of the proximal tubule.     

Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii

Biotechnology advances have allowed bacteria, yeasts, plants, mammalian and insect cells to function as heterologous protein expression systems. Recently, microalgae have gained attention as an innovative platform for recombinant protein production, due to low culture media cost, compared to traditional systems, as well as the fact that microalgae such as Chlamydomonas reinhardtii are considered safe (GRAS) by the Food and Drug Administration (FDA). Previous studies showed that recombinant protein production in traditional platforms by semicontinuous process increased biomass and bio product productivity, when compared to batch process. As there is a lack of studies on semicontinuous process for recombinant protein production in microalgae, the production of recombinant mCherry fluorescent protein was evaluated by semicontinuous cultivation of Chlamydomonas reinhardtii in bubble column photobioreactor. This semicontinuous cultivation process was evaluated in the following conditions: 20%, 40%, and 60% culture portion withdrawal. The highest culture withdrawal percentage (60%) provided the best results, as an up to 161% increase in mCherry productivity (454.5 RFU h⁻¹ – Relative Fluorescence Unit h⁻¹), in comparison to batch cultivation (174.0 RFU h⁻¹) of the same strain. All cultivations were carried out for 13 days, at pH 7, temperature 25°C and, by semicontinuous process, two culture withdrawals were taken during the cultivations. Throughout the production cycles, it was possible to obtain biomass concentration up to 1.36 g L⁻¹.