The microtubule (MT) “plus end” constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called “MT plus-end tracking proteins” (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassa MicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.
The digestive process of the Pacific bluefin tuna (PBT), Thunnus orientalis, was simulated through two phases of in vitro digestion: acidic digestion with porcine pepsin, followed by alkaline digestion with pancreatic crude extract (PCE) obtained from the PBT to hydrolyze fish meal (FM) and soybean meal (SBM) as protein substrates. The crude protein from FM resulted in a lower degree of hydrolysis (73.3%) compared with SBM (79.2%). However, the resulting digested products showed that FM contained 35% more small peptides, with sizes <6.5 kDa than those from the starting material (>150 kDa). The SBM had an increase of only 1.3% in the similar peptide cut-offs found after hydrolysis. These results suggested that FM appeared to be a better source of protein according to the amount of low-molecular weight peptides. In addition, the proteolytic activity of PCE showed that 88.9% of its alkaline proteolytic activity corresponded to trypsin and 2.9% corresponded to chymotrypsin activity. The results shown here demonstrate that peptide sizes are important in identifying suitable protein sources for aquafeed production to reinforce the primary results obtained from the in vitro digestibility using the pH-Stat system. These results also contribute to a better understanding of the digestibility process in aquatic organisms.
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