High Leucine Aminopeptidase Activity Research Articles

Effects of Chitosan-Coated Microdiet on Dietary Physical Properties, Growth Performance, Digestive Enzyme Activities, Antioxidant Capacity, and Inflammation Response of Large Yellow Croaker (Larimichthys crocea) Larvae.

A 30-day feeding trial was designed to investigate the physical properties of chitosan-coated microdiet (CCD) and the effect of CCD on survival, growth performance, activities of digestive enzymes, intestinal development, antioxidant capacity, and inflammatory response of large yellow croaker larvae (initial weight: 3.81 ± 0.20 mg). Four isonitrogenous (50% crude protein) and isolipidic (20% crude lipid) microdiets were prepared with different concentrations of chitosan wall material by spray drying method (0.00%, 0.30%, 0.60%, and 0.90%, weight (chitosan) : volume (acetic acid)). Results showed that the lipid encapsulation efficiency (control: 60.52%, Diet1: 84.63%, Diet2: 88.06%, Diet3: 88.65%) and nitrogen retention efficiency (control: 63.76%, Diet1: 76.14%, Diet2: 79.52%, Diet3: 84.68%) correlated positively with the concentration of wall material (P < 0.05). Furthermore, the loss rate of CCD was significantly lower than the uncoated diet. Larvae fed the diet with 0.60% CCD had significantly higher specific growth rate (13.52 and 9.95%/day) and survival rate (14.73 and 12.58%) compared to the control group (P < 0.05). Larvae fed the diet with 0.30% CCD had significantly higher trypsin activity in pancreatic segments than the control group (4.47 and 3.05 U/mg protein) (P < 0.05). Larvae fed the diet with 0.60% CCD had significantly higher activity of leucine aminopeptidase (7.29 and 4.77 mU/mg protein) and alkaline phosphatase (83.37 and 46.09 U/mg protein) in the brush border membrane than those of the control group (P < 0.05). The intestinal epithelial proliferation- and differentiation-related factors (zo-1, zo-2, and pcna) in larvae fed the diet with 0.30% CCD had higher expression than those of the control group (P < 0.05). When the concentration of wall material reached 0.90%, the larvae had significantly higher superoxide dismutase activity than that of the control group (27.27 and 13.72 U/mg protein) (P < 0.05). Meanwhile, malondialdehyde contents were significantly lower in larvae fed the diet with 0.90% CCD than that of the control group (8.79 and 6.79 nmol/mg protein) (P < 0.05). 0.30%~0.60% CCD significantly increased the activity of total nitric oxide synthase (2.31, 2.60, and 2.05 mU/mg protein) and inducible nitric oxide synthase (1.91, 2.01, and 1.63 mU/mg protein) and had significantly higher transcriptional levels of inflammatory factor genes (il-1β, tnf-α, and il-6) than those of the control group (P < 0.05). The results indicated chitosan-coated microdiet had great potential in feeding large yellow croaker larvae in addition to reducing nutrition loss.

Community composition and functional genes explain different ecological roles of heterotrophic bacteria attached to two bloom-forming cyanobacterial genera

Eutrophication leads to frequent outbreaks of cyanobacterial blooms, however, the effect of heterotrophic bacteria attached to cyanobacterial cells is unclear. Field investigations were carried out to gain a deeper understanding of the community composition and functional role of heterotrophic bacteria attached to Dolichospermum and Microcystins cells. The significantly positive relationships between Dolichospermum density and total nitrogen (TN) and between Microcystins density and particle nitrogen (PN) indicated the strong nitrogen (N) demand of these two species. The lack of functional genes that mediate the nitrification process in bacteria attached to both Microcystins and Dolichospermum cells indicated that these two genera preferred ammonium (NH4+-N). Dolichospermum cells obtained more available N through N2 fixation, which was expressed by high nitrogenase gene abundance. Bacteria attached to Microcystins cells showed a higher activity of leucine aminopeptidase and a significantly higher abundance of functional genes that mediate dissimilatory nitrate reduction to ammonium (DNRA) than those attached to Dolichospermum cells. The significantly higher abundance of carbon degradation genes and β-glucosidase activity of bacteria attached to Microcystins cells compared with those of bacteria attached to Dolichospermum cells suggested that abundant organic carbon was bound to Microcystins cells, which is a prerequisite for DNRA. In addition, Microcystins cells exhibited a great advantage in soluble reactive phosphorus (SRP) production through high levels of organic phosphorus (P) hydrolysis associated with high levels of phosphatase genes of attached bacteria. In conclusion, bacteria attached to Microcystins cells performed more important functions on NH4+-N and SRP production through ammonification and DNRA, as well as phosphatase hydrolysis respectively, compared to those attached to Dolichospermum. Thus, algal growth is the result of different variables such as nutrient concentration, their ratio and the microbial ability.

Effects of dietary Astragalus polysaccharides (APS) on survival, growth performance, activities of digestive enzyme, antioxidant responses and intestinal development of large yellow croaker (Larimichthys crocea) larvae

A 30-day feeding trial was conducted to investigate the effects of dietary Astragalus polysaccharides (APS) on survival, growth performance, activities of digestive enzyme, antioxidant responses and intestinal development of large yellow croaker (Larimichthys crocea) larvae. Five isonitrogenous (53% crude protein) and isolipidic (18% crude lipid) diets were formulated with graded levels of APS (0.00%, 0.05%, 0.10%, 0.15% and 0.20%). Results showed that larvae fed diets with 0.10% and 0.15% APS had significantly higher specific growth rate and body crude protein content. Meanwhile, larvae fed diets with 0.10% and 0.20% APS had significantly higher trypsin activity in intestinal segments (IS) at 45 days after hatch (DAH). Supplementation with 0.10% APS significantly improved the activity of alkaline phosphatase both in brush border membrane and IS of larvae at 45 DAH. Similarly, larvae fed the diet with 0.10% APS had significantly higher activity of leucine aminopeptidase in IS at 45 DAH than the control group. Also, larvae fed the diet with 0.10% APS showed significantly higher activity of superoxide dismutase in IS than the control group. Total antioxidant capacity in pancreatic segments (PS) were significantly higher in larvae fed diets with 0.15% and 0.20% than the control group. Malondialdehyde contents in PS were significantly lower in larvae fed the diet with 0.10% APS than the control group. Furthermore, supplementation of 0.10% APS significantly increased the length of intestinal microvillus and the height of enterocyte. The selected markers for intestinal epithelial proliferation and differentiation were higher expressed in larvae fed diets with APS than the control group, such as tight junction protein-2, proliferating cell nuclear antigen and sucrase-isomaltase. In conclusion, appropriate supplementation of APS (0.05% - 0.10%) could promote growth performance of large yellow croaker larvae through increasing the activities of digestive enzyme and antioxidant enzyme, improving intestinal morphology and promoting the intestinal development.

Long‐Term Manure Application Effects on Nutrients and Selected Enzymes Involved in Their Cycling

Core Ideas We assessed long‐term manure rates and application times on soil nutrients and enzymes at two sites. Increasing manure rate enhanced soil fertility but hydrolase enzymes showed a quadratic trend. Manure application prior to period of heavy rainfall (spring and fall) was associated with phosphorus losses. Peroxidase and phenol oxidase activities declined with increasing manure application rates. Oxidative enzyme activities were good indices of long‐term soil organic carbon storage. We investigated how soil nutrients (soil organic carbon [SOC], nitrogen [N], and Mehlich‐3‐extractable phosphorus [P‐M3]) and enzymes involved in their cycling were impacted by 10 yr of beef cattle manure application at two sites in Nova Scotia, Canada: a dikeland site (silty clay loam) and an upland site (sandy loam site), under timothy (Phleum pratense L.) cover. Manure was applied at four rates (from 0 to 300 kg total N ha−1) at different times (spring, summer, early fall, and late fall). Activities of hydrolytic enzymes: β‐glucosidase (BG), cellobiohydrolase (CB), leucine aminopeptidase (LAP), and phosphomonoesterase (PME) and oxidative enzymes: phenol oxidase (PO), peroxidase (PP), were quantified. We hypothesize that manure application will increase soil nutrients and BG, CB, LAP, and PME activities and will decrease PO and PP activities. After 10 yr of manure application, compared to a control, soil pH, SOC, N, and P‐M3 increased linearly by 41, 45, and 125%, respectively, with increasing manure rate, while BG, CB, PME, and LAP activities showed a quadratic response. Conversely, enzymes degrading less labile components (PP and PO) declined with increasing manure rate. Higher SOC and P‐M3 contents and higher LAP activity were found at the upland site, whereas higher PME, PP, and PO activities were found at the dikeland site. Inverse relationships between PME activity and P‐M3 content and between PP activity and SOC or N content were found, whereas a positive correlation between N content and LAP activity was observed. Regular manure application is a good way to sustain soil fertility, with the extent of soil C storage controlled by soil type. The optimal rate and timing of manure application is essential, since application prior to periods of heavy rainfall is associated with phosphorus losses. Our results demonstrated that oxidative enzymes (PP and PO) are good indices of long‐term SOC storage. The lower their activity, the higher is the SOC sequestration.

Near-Infrared Fluorescent Probe with Remarkable Large Stokes Shift and Favorable Water Solubility for Real-Time Tracking Leucine Aminopeptidase in Living Cells and In Vivo.

Leucine aminopeptidase (LAP) is a kind of proteolytic enzymes and associated closely with pathogenesis of cancer and liver injury. Accurate detection of LAP activity with high sensitivity and selectivity is imperative to detect its distribution and dynamic changes for understanding LAP's function and early diagnosing the disease states. However, fluorescent detection of LAP in living systems is challenging. To date, rarely fluorescent probes have been reported for imaging LAP in vivo. In this study, a novel probe (TMN-Leu) was developed by conjugating a near-infrared dicyanoisophorone derivative fluorophore with LAP activatable l-leucine amide moiety for the first time. TMN-Leu featured large Stokes shift (198 nm), favorable water solubility, ultrasensitive sensitivity (detection limit of ∼0.38 ng/mL), good specificity, excellent cell membrane permeability, low toxicity, and a prominent near-infrared emission (658 nm) in response to LAP. TMN-Leu has been successfully applied to track LAP of cancer cells and normal cells, monitor LAP changes in different disease models, and rapidly evaluate LAP inhibitor in cell-based assay. Notably, this probe firstly revealed that HCT116 cells with higher LAP activity were more invasive than LAP siRNA transfected HCT116 cells, suggesting that LAP might serve as an indicator reflecting the intrinsic invasion ability of cancer cells. Finally, TMN-Leu was also employed for in vivo real-time imaging LAP in living tumor-bearing nude mice with low background interference. All together, our probe possesses potential value as a promising tool for diagnostic application, cell-based screening inhibitors and in vivo real-time tracking enzymatic activity in preclinical applications.

The effects of fructo-oligosaccharides or whole wheat on the performance and digestive tract of broiler chickens

1. The objective of this experiment was to study two feeding methods, which could potentially act on the gut microflora, the structure and/or the function of the digestive tract and thereby improve the performance of broilers. 2. Four dietary treatments were studied: a negative control (wheat based) with no additives (C), a positive control with 0·01 g/kg avilamycin (AV), a treatment with 0·6 g/kg fructo-oligosaccharides (FOS) and a treatment with the same composition as treatment C but in which a part or all (400 g/kg) of the wheat was given as whole wheat and a concentrate supplement (WW). The measurements were: the performance from 0 to 6 weeks, the bacterial counts at 3 and 6 weeks, the digestive tract morphology and the activity of some intestinal enzymes at 3 weeks. 3. The birds given AV had better daily live weight gain (DLWG) and feed conversion ratio (FCR) compared to treatment C. The birds given FOS had a lower feed intake and a lower DLWG than the birds on treatment C but their FCR was significantly improved. WW resulted in a numerically lower feed intake and a significantly lower DLWG than treatment C. With AV, the number of aerobic mesophilic bacteria in the caeca was reduced at 3 weeks. With WW, gizzard and pancreas weights were greater and the surfaces of the ileal crypts were larger. An increased activity of leucine aminopeptidase (LAP) in the duodenum was found for treatments AV, FOS and WW. 4. In conclusion, in this study, treatments WW and FOS reduced DLWG, which may have been due to a lower feed intake during the whole period. With WW, the FCR was not affected, maybe because of both positive and negative effects on digestive tract (higher gizzard and pancreas development and LAP activity; larger crypts). However, FOS improved FCR, which may be partly explained by the higher LAP activity.

The Role of Different Species of Bacteria in the Hydrolysis of Protein in the Rumen

Proteolytic strains of Bacteroides amylophilus, Bacteroides ruminicola, Butyrivibrio fibrisolvens, Butyriribrio alactacidigens, Selenomonas ruminantium var. Ruminantium, Se. ruminantium var. lactilytica and of the genera Eubacterium, Fusobacterium and Clostridium were isolated from the rumen of sheep. The location of the proteolytic enzymens, their activity against various substrates and their sensitivity to inhibitors were compared with the same properties of mixed bacteria prepared from strained rumen fluid, in order to assess the relative importance of the different species in the hydrolysis of protein in the rumen. Bacteroides ruminicola was judged by these criteria to be the most important of these proteolytic isolates in vivo. Other isolates of higher specific activity had enzymes with properties different from rumen fluid. Species of Butyrivibrio, Selenomonas and Clostridium of lower specific activity may also be important if present in sufficiently large numbers, as their activity was of the same type as the mixed population. Streptococcus bovis had a low proteolytic activity as determined by the conversion of 14C-labelled casein to TCA-soluble products, but was able to grow on casein, probably by virtue of its exceptionally high leucine aminopeptidase activity.

Comparative studies on the leucine aminopeptidase activity of different types of leukocytes

1. 1. The distribution of the leucine aminopeptidase activity among different types of leukocytes in a few animals was examined. 2. 2. In guinea-pig, the enzyme activity was detected in all cell types examined but its activity per cell was diverse among leukocytes, i.e. neutrophils and monocyte-macrophage cell lines, so-called “professional” phagocytes, showed higher enzyme activity than other types of leukocytes, lymphocytes and eosinophils. 3. 3. The same distribution pattern of leucine aminopeptidase activity as that in guinea-pig was also observed among leukocytes in human, rat and mouse. 4. 4. When leukocytes were modified with a poorly permeant reagent, diazotized sulfanilic acid to examine the subcellular localization of leucine aminopeptidase in leukocytes, the leucine aminopeptidase activity of the cells was inhibited by 50% without the inhibition of lactate dehydrogenase, a soluble cytoplasmic enzyme, in various types of leukocytes from all animals used here. 5. 5. The possibility was suggested from these observations that higher leucine aminopeptidase activity is distributed on the cell surface of “professional” phagocytes.

Tumor-associated macrophages of mouse mammary tumors. II. Differential distribution of macrophages from metastatic and nonmetastatic tumors.

Macrophage subpopulations were isolated by counterflow centrifugal elutriation from a number of closely related mammary tumors originally derived from a single spontaneous tumor in a BALB/cfC3H mouse. Each tumor line had its own distinct characteristics with regard to tumor cell distribution, macrophage size (density), macrophage content, and macrophage distribution. Although there is no correlation with the metastatic potential of the tumors for the first three characteristics listed, distinct differences are apparent between metastatic and nonmetastatic tumors in macrophage distribution into the elutriated fractions. The distribution of the tumor-associated macrophages was shifted toward the later fractions in the metastatic tumors (lines 66 and 410.4), with approximately 40% more macrophages in fractions 3 and 4 than in those fractions from the nonmetastatic tumors (lines 67 and 168). The levels of three ectoenzymes, leucine aminopeptidase (LAP), alkaline phosphodiesterase I (APD), and 5' nucleotidase (5'N) were determined for macrophages from the elutriated fractions as well as for the total tumor digest. No significant differences were observed in absolute levels of either LAP or APD between most of the fractions from the different tumor lines. Differences were seen between the fractions in levels of 5'N among the lines and in ADP levels between lines 66 and 168 from the total tumor digest. Striking differences in ectoenzyme levels were apparent when the activity was normalized to macrophage surface area: macrophages from line 66 tumors were 3- to 17-fold higher in their LAP concentrations than were macrophages from either 67 or 168 tumors. Each tumor line had a unique and reproducible pattern of macrophage-associated prostaglandin E (PGE) synthesis. The three nonmetastatic tumor lines showed peak PGE levels in fraction three with lower levels in fraction 2, whereas the highly metastatic tumor yielded more active macrophages in fraction 2 vs fraction 3. Changes in PGE levels were associated with differing ectoenzyme levels. Macrophages in fraction 3 from metastatic tumors were enriched for populations with high LAP activity and low PGE levels. Conversely, macrophages from nonmetastatic tumors exhibited lower LAP activity in fraction 3 vs 2 and increasing PGE levels. Thus, differences between metastatic and nonmetastatic mammary tumors were seen in 1) distribution of macrophages in the elutriation fractions, 2) size of infiltrating macrophages, 3) macrophage ectoenzyme concentrations, and 4) macrophage-associated PGE levels.