Short-term responses of soil enzyme activities and stoichiometry to litter input in Castanopsis carlesii and Cunninghamia lanceolata plantations.

Litter input triggers the secretion of soil extracellular enzymes and facilitates the release of carbon (C), nitrogen (N), and phosphorus (P) from decomposing litter. However, how soil extracellular enzyme activities were controlled by litter input with various substrates is not fully understood. We examined the activities and stoichiometry of five enzymes including ß-1,4-glucosidase, ß-D-cellobiosidase, ß-1,4-N-acetyl-glucosaminidase, leucine aminopeptidase and acidic phosphatase (AP) with and without litter input in 10-year-old Castanopsis carlesii and Cunninghamia lanceolata plantations monthly during April to August, in October, and in December 2021 by using an in situ microcosm experiment. The results showed that: 1) There was no significant effect of short-term litter input on soil enzyme activity, stoichiometry, and vector properties in C. carlesii plantation. In contrast, short-term litter input significantly increased the AP activity by 1.7% in May and decreased the enzymatic C/N ratio by 3.8% in August, and decreased enzymatic C/P and N/P ratios by 11.7% and 10.3%, respectively, in October in C. lanceolata plantation. Meanwhile, litter input increased the soil enzymatic vector angle to 53.8° in October in C. lanceolata plantations, suggesting a significant P limitation for soil microorganisms. 2) Results from partial least squares regression analyses showed that soil dissolved organic matter and microbial biomass C and N were the primary factors in explaining the responses of soil enzymatic activity to short-term litter input in both plantations. Overall, input of low-quality (high C/N) litter stimulates the secretion of soil extracellular enzymes and accelerates litter decomposition. There is a P limitation for soil microorganisms in the study area.

Local thiamet-G delivery by a thermosensitive hydrogel confers ischemic cardiac repair via myeloid M2-like activation in a STAT6 O-GlcNAcylation-dependent manner.

Infarct healing requires a dynamic and orchestrated inflammatory reaction following myocardial infarction (MI). While an uncontrolled excessive inflammatory response exaggerates ischemic injury post-MI, M2-like reparative macrophages may facilitate inflammation regression and promote myocardial healing. However, how protein post-translational modification regulates post-MI cardiac repair and dynamic myeloid activation remains unknown. Here we show that M2-like reparative, but not M1-like inflammatory activation, is enhanced by pharmacologically-induced hyper-O-GlcNAcylation. Mechanistically, myeloid knockdown of O-GlcNAc hydrolase O-GlcNAcase (Oga), which also results in hyper-O-GlcNAcylation, positively regulates M2-like activation in a STAT6-dependent fashion, which is controlled by O-GlcNAcylation of STAT6. Of note, both systemic and local supplementation of thiamet-G (TMG), an Oga inhibitor, effectively facilitates cardiac recovery in mice by elevating the accumulation of M2-like macrophages in infarcted hearts. Our study provides a novel clue for monocyte/macrophage modulating therapies aimed at reducing post-MI hyperinflammation in ischemic myocardium.

A fluorescent probe for selective detection of lysosomal ß-hexosaminidase in live cells.

Determining the activity of lysosomal ß-hexosaminidase in cells is of great importance for understanding the roles that these enzymes play in pathophysiological events. Herein, we designed the new fluorescent probe, ßGalNAc-Rhod-CM(NEt2), which consisted of a ßGalNAc-linked rhodol unit serving as a ß-hexosaminidase reactive fluorogenic moiety and a N,N'-diethylaminocoumarin (CM(NEt2)) group acting as a fluorescence marker for determining the degree of cell permeabilization. Treatment of ßGalNAc-Rhod-CM(NEt2) with ß-hexosaminidase promoted generation of Rhod-CM(NEt2), thereby leading to an increase in the intensity of fluorescence of Rhod. However, this probe did not respond to the functionally related glycosidase, O-GlcNAcase. The detection limit of ßGalNAc-Rhod-CM(NEt2) for ß-hexosaminidase was determined to be 0.52 nM, indicating that it has high sensitivity for this enzyme. Furthermore, the probe functioned as an excellent fluorogenic substrate for ß-hexosaminidase with kcat and Km values of 17 sec-1 and 22 µM, respectively. The results of cell studies using ßGalNAc-Rhod-CM(NEt2) showed that levels of ß-hexosaminidase activity in cells can be determined by measuring the intensity of fluorescence arising from Rhod and that the intensity of fluorescence of CM(NEt2) can be employed to determine the degree of cell permeabilization of the probe. Utilizing the new probe, we assessed ß-hexosaminidase activities in several types of cells and evaluated the effect of glucose concentrations in culture media on the activity of this enzyme.

In vivo and in silico toxicity assessment of four common liquid crystal monomers to Daphnia magna: Novel endocrine disrupting chemicals in crustaceans?

Liquid crystal monomers (LCMs) are widely used in liquid crystal displays (LCDs) and are proposed to be a new generation of environmentally persistent, bioaccumulative and toxic (PBT) substances that are increasingly detected in rivers and seas. However, there is a lack of in vivo data that characterize adverse responses and toxic mechanisms of LCMs on aquatic organisms. The aim of this study was to comprehensively investigate the effect of four typical LCMs on the lethality, growth, molting, and reproductive capacity of Daphnia magna (D. magna), a highly studied aquatic species in environmental toxicology. Whole body and enzymatic biomarkers (i.e., body length, chitobiase, acetylcholinesterase, antioxidant defense) were measured to assess the toxicity of LCMs. The 48 h mortality rate and observations of disrupted thorax development and inhibition of ecdysis indicate that D. magna are sensitive to LCMs exposure. Oxidative stress, impaired neurotransmission, and disruptions in molting were observed in short-term biomarker tests using LCMs. A 21 day exposure of D. magna to LCMs resulted in reduced growth, reproduction, and population intrinsic growth rate. In addition, chitobiase and 20-hydroxyecdysone, enzymes important for the molting process, were altered at 7, 14 and 21 d. This is hypothesized to be related to endocrine imbalance resulting from LCM exposure. Based on molecular docking simulations, there is evidence that LCMs bind directly to ecdysteroid receptors; this may explain the observed endocrine disrupting effects of LCMs. These data support the hypothesis that LCMs are endocrine disrupting chemicals in aquatic species, impacting the process of molting. This may subsequently lead to lower reproduction and unbalanced population dynamics.

Cryo-EM structure of human O-GlcNAcylation enzyme pair OGT-OGA complex.

O-GlcNAcylation is a conserved post-translational modification that attaches N-acetyl glucosamine (GlcNAc) to myriad cellular proteins. In response to nutritional and hormonal signals, O-GlcNAcylation regulates diverse cellular processes by modulating the stability, structure, and function of target proteins. Dysregulation of O-GlcNAcylation has been implicated in the pathogenesis of cancer, diabetes, and neurodegeneration. A single pair of enzymes, the O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), catalyzes the addition and removal of O-GlcNAc on over 3,000 proteins in the human proteome. However, how OGT selects its native substrates and maintains the homeostatic control of O-GlcNAcylation of so many substrates against OGA is not fully understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of human OGT and the OGT-OGA complex. Our studies reveal that OGT forms a functionally important scissor-shaped dimer. Within the OGT-OGA complex structure, a long flexible OGA segment occupies the extended substrate-binding groove of OGT and positions a serine for O-GlcNAcylation, thus preventing OGT from modifying other substrates. Conversely, OGT disrupts the functional dimerization of OGA and occludes its active site, resulting in the blocking of access by other substrates. This mutual inhibition between OGT and OGA may limit the futile O-GlcNAcylation cycles and help to maintain O-GlcNAc homeostasis.

Exploiting O-GlcNAc transferase promiscuity to dissect site-specific O-GlcNAcylation.

Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). How site-specific O-GlcNAcylation modulates a diverse range of cellular processes is largely unknown. A limiting factor in studying this is the lack of accessible techniques capable of producing homogeneously O-GlcNAcylated proteins, in high yield, for in vitro studies. Here, we exploit the tolerance of OGT for cysteine instead of serine, combined with a co-expressed OGA to achieve site-specific, highly homogeneous mono-glycosylation. Applying this to DDX3X, TAB1, and CK2α, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2α solubility and enables production of mono-S-GlcNAcylated TAB1 crystals, albeit with limited diffraction. Taken together, this work provides a new approach for functional dissection of protein O-GlcNAcylation.

Growing and dividing: how O-GlcNAcylation leads the way.

Cell cycle errors can lead to mutations, chromosomal instability, or death; thus, the precise control of cell cycle progression is essential for viability. The nutrient-sensing posttranslational modification, O-GlcNAc, regulates the cell cycle allowing one central control point directing progression of the cell cycle. O-GlcNAc is a single N-acetylglucosamine sugar modification to intracellular proteins that is dynamically added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. These enzymes act as a rheostat to fine-tune protein function in response to a plethora of stimuli from nutrients to hormones. O-GlcNAc modulates mitogenic growth signaling, senses nutrient flux through the hexosamine biosynthetic pathway, and coordinates with other nutrient-sensing enzymes to progress cells through Gap phase 1 (G1). At the G1/S transition, O-GlcNAc modulates checkpoint control, while in S Phase, O-GlcNAcylation coordinates the replication fork. DNA replication errors activate O-GlcNAcylation to control the function of the tumor-suppressor p53 at Gap Phase 2 (G2). Finally, in mitosis (M phase), O-GlcNAc controls M phase progression and the organization of the mitotic spindle and midbody. Critical for M phase control is the interplay between OGT and OGA with mitotic kinases. Importantly, disruptions in OGT and OGA activity induce M phase defects and aneuploidy. These data point to an essential role for the O-GlcNAc rheostat in regulating cell division. In this review, we highlight O-GlcNAc nutrient sensing regulating G1, O-GlcNAc control of DNA replication and repair, and finally, O-GlcNAc organization of mitotic progression and spindle dynamics.

A novel endo-type chitinase possessing chitobiase activity derived from the chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T.

One of the chitinases (ChiG) derived from the chitinolytic bacterium Chitiniphilus shinanonensis SAY3T exhibited chitobiase activity cleaving dimers of N-acetyl-D-glucosamine (GlcNAc) into monomers, which is not detected in typical endo-type chitinases. Analysis of the reaction products for GlcNAc hexamers revealed that all the five internal glycosidic bonds were cleaved at the initial stage. The overall reaction catalyzed by chitobiases toward GlcNAc dimers was similar to that catalyzed by N-acetyl-D-glucosaminidases (NAGs). SAY3 possesses two NAGs (ChiI and ChiT) that are thought to be important in chitin catabolism. Unexpectedly, a triple gene-disrupted mutant (ΔchiIΔchiTΔchiG) was still able to grow on synthetic medium containing GlcNAc dimers or powdered chitin, similar to the wild-type SAY3, although it exhibited only 3% of total cellular NAG activity compared to the wild-type. This indicates the presence of unidentified enzyme(s) capable of supporting normal bacterial growth on the chitin medium by NAG activity compensation.

Mass Spectrometry-Based Methods to Determine the Substrate Specificities and Kinetics of N-Linked Glycan Hydrolysis by Endo-ß-N-Acetylglucosaminidases.

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer. Carbohydrate-active enzymes (CAZymes) are produced in bacteria, fungi, and humans and are enzymes which modify glycans via the addition or subtraction of individual or multiple saccharides from glycans. One of the hurdles in studying these enzymes is determining the types of substrates each enzyme is specific for and the kinetics of enzymatic activity. In this chapter, we discuss methods which are currently used to study the substrate specificity and kinetics of CAZymes and introduce a novel mass spectrometry-based technique which enables the specificity and kinetics of CAZymes to be determined accurately and efficiently.

GH20 and GH84 ß-N-acetylglucosaminidases with different linkage specificities underpin mucin O-glycan breakdown capability of Bifidobacterium bifidum.

Intestinal mucous layers mediate symbiosis and dysbiosis of host-microbe interactions. These interactions are influenced by the mucin O-glycan degrading ability of several gut microbes. The identities and prevalence of many glycoside hydrolases (GHs) involved in microbial mucin O-glycan breakdown have been previously reported; however, the exact mechanisms and extent to which these GHs are dedicated to mucin O-glycan degradation pathways warrant further research. Here, using Bifidobacterium bifidum as a model mucinolytic bacterium, we revealed that two ß-N-acetylglucosaminidases belonging to the GH20 (BbhI) and GH84 (BbhIV) families play important roles in mucin O-glycan degradation. Using substrate specificity analysis of natural oligosaccharides and O-glycomic analysis of porcine gastric mucin (PGM) incubated with purified enzymes or B. bifidum carrying bbhI and/or bbhIV mutations, we showed that BbhI and BbhIV are highly specific for ß-(1→3)- and ß-(1→6)-GlcNAc linkages of mucin core structures, respectively. Interestingly, we found that efficient hydrolysis of the ß-(1→3)-linkage by BbhI of the mucin core 4 structure [GlcNAcß1-3(GlcNAcß1-6)GalNAcα-O-Thr] required prior removal of the ß-(1→6)-GlcNAc linkage by BbhIV. Consistent with this, inactivation of bbhIV markedly decreased the ability of B. bifidum to release GlcNAc from PGM. When combined with a bbhI mutation, we observed that the growth of the strain on PGM was reduced. Finally, phylogenetic analysis suggests that GH84 members may have gained diversified functions through microbe-microbe and host-microbe horizontal gene transfer events. Taken together, these data strongly suggest the involvement of GH84 family members in host glycan breakdown.

Structure-based virtual screening for potent inhibitors of GH-20 ß-N-acetylglucosaminidase: Classical and machine learning scoring functions, and molecular dynamics simulations.

GH-20 ß-N-acetylglucosaminidases (GlcNAcases) are promising targets in the development of antimicrobial agents against Vibrio infections in humans and aquatic animals. In this study, we set up structure-based virtual screening to identify potential GH-20 GlcNAcase inhibitors from the Reaxys commercial database, using VhGlcNAcase from V. campbellii type strain ATCC® BAA 1116 as the protein target and Redoxal as the reference ligand. Using ChemPLP and RF-Score-VS machine learning scoring functions, eight lead compounds were identified and further evaluated for protein interaction preference and pharmacological properties. Protein-ligand analysis demonstrated that all selected compounds interacted exclusively at subsite - 1 with five hydrophobic residues W487, W505, W546, W582 and V544 at site S1, and with two polar residues, D437 and E438, at site 3. For subsite + 1, the most common residues were R274 and E584 at site 2 and I397 and Q398 at site 4. Based on the data obtained from binding free energy changes (ΔG°binding), pharmacological property analysis and molecular dynamic simulations, two ChemPLP compounds, 338175 and 1146525, and one RF-Score-VS compound, 337447, were considered as the likely lead compounds. The most promising compound, 1146525, could serve as a scaffold for the future design of novel antimicrobial agents against Vibrio infections.

Effects of Chemical Compounds on the Activity of the N-acetyl-ß-D-Glucosaminidase of the Marine Prawn, Palaemon serratus: Screening In Vitro.

N-acetyl-ß-D-glucosaminidase (NAGase) is important for crustaceans because the enzyme activity is necessary for the molting process. The present study aimed to assess the sensitivity of Palaemon serratus NAGase activity to a set of compounds of diverse chemical families in the context of in vitro exposures. Compounds representing different chemical families were selected according to their abundance, impact in the environment, and relevance as disruptors of the molting process. In a first step, four solvents (dimethylsulfoxide [DMSO], methanol, acetone, and ethanol) were tested to determine their suitability to dissolve hydrophobic compounds without affecting NAGase activity. Exclusively, ethanol had no effect on enzyme activity and on the integrity of the proteins present in the enzyme extract. The 18 other compounds were tested and four of these compounds, pentoxifylline, fenoxycarb, dithiocarbamate, and RH5849, showed a specific alteration on the activity of NAGase, without affecting the protein content. However, cadmium, zinc, and glyphosate showed a nonspecific alteration, affecting both the enzyme activity and the proteins, whereas ibuprofen exclusively altered the protein content. Finally, 10 of the 22 tested compounds (including DMSO, acetone, and methanol) showed a direct alteration of NAGase activity. Environ Toxicol Chem 2023;42:846-858. © 2023 SETAC.

Disease correction in mucopolysaccharidosis type IIIB mice by intraparenchymal or cisternal delivery of a capsid modified AAV8 codon-optimized NAGLU vector.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the gene that encodes the protein N-acetyl-glucosaminidase (NAGLU). Defective NAGLU activity results in aberrant retention of heparan sulfate within lysosomes leading to progressive central nervous system (CNS) degeneration. Intravenous treatment options are limited by the need to overcome the blood-brain barrier and gain successful entry into the CNS. Additionally, we have demonstrated that AAV8 provides a broader transduction area in the MPS IIIB mouse brain compared with AAV5, 9 or rh10. A triple-capsid mutant (tcm) modification of AAV8 further enhanced GFP reporter expression and distribution. Using the MPS IIIB mouse model, we performed a study using either intracranial six site or intracisterna magna injection of AAVtcm8-codon-optimized (co)-NAGLU using untreated MPS IIIB mice as controls to assess disease correction. Disease correction was evaluated based on enzyme activity, heparan sulfate storage levels, CNS lysosomal signal intensity, coordination, activity level, hearing and survival. Both histologic and enzymatic assessments show that each injection method results in supranormal levels of NAGLU expression in the brain. In this study, we have shown correction of lifespan and auditory deficits, increased CNS NAGLU activity and reduced lysosomal storage levels of heparan sulfate following AAVtcm8-coNAGLU administration and partial correction of NAGLU activity in several peripheral organs in the murine model of MPS IIIB.

Oxymatrine Alleviates Gentamicin-Induced Renal Injury in Rats.

Gentamicin is an aminoglycoside antibiotic commonly used to treat Gram-negative bacterial infections that possesses considerable nephrotoxicity. Oxymatrine is a phytochemical with the ability to counter gentamicin toxicity. We investigated the effects and protective mechanism of oxymatrine in rats. The experimental groups were as follows: Control, Oxymatrine only group (100 mg/kg/d), Gentamicin only group (100 mg/kg/d), Gentamicin (100 mg/kg/d) plus Oxymatrine (100 mg/kg/d) group (n = 10). All rats were treated for seven continuous days. The results indicated that oxymatrine alleviated gentamicin-induced kidney injury, and decreased rats' kidney indices and NAG (N-acetyl-beta-d-glucosaminidase), BUN (blood urea nitrogen) and CRE (creatine) serum levels. The oxymatrine-treated group sustained less histological damage. Oxymatrine also relived gentamicin-induced oxidative and nitrative stress, indicated by the increased SOD (superoxidase dismutase), GSH (glutathione) and CAT (catalase) activities and decreased MDA (malondialdehyde), iNOS (inducible nitric oxide synthase) and NO (nitric oxide) levels. Caspase-9 and -3 activities were also decreased in the oxymatrine-treated group. Oxymatrine exhibited a potent anti-inflammatory effect on gentamicin-induced kidney injury, down-regulated the Bcl-2ax and NF-κB mRNAs, and upregulated Bcl-2, HO-1 and Nrf2 mRNAs in the kidney tissue. Our investigation revealed the renal protective effect of oxymatrine in gentamicin-induced kidney injury for the first time. The effect was achieved through activation of the Nrf2/HO-1 pathways. The study underlines the potential clinical application of oxymatrine as a renal protectant agent for gentamicin therapy.

Insect cuticular compounds affect Conidiobolus coronatus (Entomopthorales) sporulation and the activity of enzymes involved in fungal infection.

Mycoses are a global problem that affects humans and animals. In the present study, the entomopathogenic soil fungus Conidiobolus coronatus (Entomophthorales), infecting in tropics also humans, sheep and horses, was cultivated with the addition of insect cuticular compounds (CCs) previously detected in the cuticle of C. coronatus-resistant fly species (C10-C30 fatty alcohols, butyl oleate, butyl stearate, glycerol oleate, squalene, tocopherol acetate). Our findings indicate that CCs have diversified and complex effects on the growth and sporulation of C. coronatus and its ability to infect the larvae of Galleria mellonella (Lepidoptera). The CCs affected protein content and cuticle-degrading enzymes (CDEs) activity in the conidia. Some CCs inhibited fungal growth (0.1% C10), decreased sporulation (C12, C16, C24, C28, C30, butyl stearate, squalene), virulence (C12, C14, butyl oleate, butyl stearate) and protein content (C18). They also reduced conidial CDE activity: elastase (C24, butyl oleate, butyl stearate, squalene, tocopherol acetate), chitobiosidase (C12, C14, C20) and lipase (C12, C18, C26, squalene, tocopherol acetate). Several CCs enhanced sporulation (C14, C18, C22, C26, C30), virulence (C18, C26, squalene), conidial protein content (C16, C24, C30, squalene) and CDE activity: elastase (C10, C16, C18), NAGase (C16, C20), chitobiosidase (C16) and lipase (C10, C14, C16, C20, butyl oleate). Our findings indicate that C. coronatus colonies grown on media supplemented with CCs employ various compensation strategies: colonies grown with C16 alcohol demonstrated reduced sporulation but greater conidial protein accumulation and increased elastase, NAGase, chitobiosidase and lipase activity, thus preserving high virulence. Also, colonies supplemented with C18 alcohol demonstrated high virulence and enhanced sporulation and elastase activity but slightly decreased conidial protein content. CCs that inhibit the activity of lipases and proteases show promise in the fight against conidiobolomycosis.

Identification and characterization of a novel thermo-stable endo-ß-N-acetylglucosaminidase from Rhizomucorpusillus.

Endo-ß-N-acetylglucosaminidase (ENGase) is an enzyme that hydrolyzes the chitobiose core of N-glycans and is widely used for glycan analysis on glycoproteins and preparation of precursors for glycosylated compounds. While most of the ENGases that can hydrolyze complex-type glycans are derived from eukaryotes, their production by heterologous expression using Escherichia coli is insufficient, making the production process expensive. From an industrial perspective, there is a need for a less expensive enzyme with higher activity and stability. In this study, we identified a novel ENGase gene from a thermophilic fungus, Rhizomucor pusillus, and named it Endo-Rp. Characterization of the recombinant Endo-Rp showed that the enzyme had maximum hydrolytic activity at 60 °C and hydrolyzed high-mannose-type and biantennary complex-type glycans, but not (2,4)-branched triantennary complex-type or fucosylated glycans. Endo-Rp also hydrolyzed N-glycans attached to RNase B and human transferrin. In summary, we consider Endo-Rp to be a valuable enzyme in various scientific and industrial applications.

Activated autophagy-lysosomal pathway in dairy cows with hyperketonemia is associated with lipolysis of adipose tissues.

Activated autophagy-lysosomal pathway (ALP) can degrade virtually all kinds of cellular components, including intracellular lipid droplets, especially during catabolic conditions. Sustained lipolysis and increased plasma fatty acids concentrations are characteristic of dairy cows with hyperketonemia. However, the status of ALP in adipose tissue during this physiological condition is not well known. The present study aimed to ascertain whether lipolysis is associated with activation of ALP in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes. In vivo, blood and subcutaneous adipose tissue (SAT) biopsies were collected from nonhyperketonemic (nonHYK) cows [blood ß-hydroxybutyrate (BHB) concentration <1.2 mM, n = 10] and hyperketonemic (HYK) cows (blood BHB concentration 1.2-3.0 mM, n = 10) with similar days in milk (range: 3-9) and parity (range: 2-4). In vitro, calf adipocytes isolated from 5 healthy Holstein calves (1 d old, female, 30-40 kg) were differentiated and used for (1) treatment with lipolysis inducer isoproterenol (ISO, 10 µM, 3 h) or mammalian target of rapamycin inhibitor Torin1 (250 nM, 3 h), and (2) pretreatment with or without the ALP inhibitor leupeptin (10 µg/mL, 4 h) followed by ISO (10 µM, 3 h) treatment. Compared with nonHYK cows, serum concentration of free fatty acids was greater and serum glucose concentration, DMI, and milk yield were lower in HYK cows. In SAT of HYK cows, ratio of phosphorylated hormone-sensitive lipase to hormone-sensitive lipase, and protein abundance of adipose triacylglycerol lipase were greater, but protein abundance of perilipin 1 (PLIN1) and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC) was lower. In addition, mRNA abundance of autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), protein abundance of lysosome-associated membrane protein 1, and cathepsin D, and activity of ß-N-acetylglucosaminidase were greater, whereas protein abundance of sequestosome-1 (p62) was lower in SAT of HYK cows. In calf adipocytes, treatment with ISO or Torin1 decreased protein abundance of PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes, but increased glycerol content in the supernatant of calf adipocytes. Moreover, the mRNA abundance of ATG5, ATG7, and MAP1LC3B was upregulated, the protein abundance of lysosome-associated membrane protein 1, cathepsin D, and activity of ß-N-acetylglucosaminidase were increased, whereas the protein abundance of p62 was decreased in calf adipocytes treated with ISO or Torin1 compared with control group. Compared with treatment with ISO alone, the protein abundance of p62, PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes were higher, but the glycerol content in the supernatant of calf adipocytes was lower in ISO and leupeptin co-treated group. Overall, these data indicated that activated ALP is associated with increased lipolysis in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes.

Characterization of novel endo-ß-N-acetylglucosaminidase from Bacteroides nordii that hydrolyzes multi-branched complex type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

Mechanism of cooperative N-glycan processing by the multi-modular endoglycosidase EndoE.

Bacteria produce a remarkably diverse range of glycoside hydrolases to metabolize glycans from the environment as a primary source of nutrients, and to promote the colonization and infection of a host. Here we focus on EndoE, a multi-modular glycoside hydrolase secreted by Enterococcus faecalis, one of the leading causes of healthcare-associated infections. We provide X-ray crystal structures of EndoE, which show an architecture composed of four domains, including GH18 and GH20 glycoside hydrolases connected by two consecutive three α-helical bundles. We determine that the GH20 domain is an exo-ß-1,2-N-acetylglucosaminidase, whereas the GH18 domain is an endo-ß-1,4-N-acetylglucosaminidase that exclusively processes the central core of complex-type or high-mannose-type N-glycans. Both glycoside hydrolase domains act in a concerted manner to process diverse N-glycans on glycoproteins, including therapeutic IgG antibodies. EndoE combines two enzyme domains with distinct functions and glycan specificities to play a dual role in glycan metabolism and immune evasion.

Biomarkers of cadmium exposure and renal function in estuarine adult villagers.

OBJECTIVE: Assess cadmium (Cd) exposure of adults living in two estuarine communities in Aratu bay, Bahia, Brazil and its association with effects on renal function. METHODS: This cross-sectional study included 88 volunteers aged 17-55 years, living in the following two communities: Santa Luzia (SL) located more intimately in the bay and Cotegipe (CT), a bit further and closer to a ferro-manganese alloy plant. Cd in blood (CdB) and urine (CdU), along with blood lead (PbB) levels were determined by graphite furnace atomic absorption spectrometry. Renal function was evaluated by the estimated glomerular filtration rate (eGFR) and tubular cell biomarkers: retinol binding protein (RBP), ß2-microglobulin (ß2M), and N-acetyl-ß-D-glucosaminidase (NAG). RESULTS: The median CdU levels in villagers of the two communities were 0.20 and 0.44 µg/g creat. and SL vs CT, respectively. Age range (> 35 years), cigarette smoking and lower family income were significantly associated with more elevated CdU levels. Multiple linear regression analysis demonstrated a significant association between LnCdU and LnRBP levels (ß = 0.200, 95%CI 0.074-0.365) after adjusted for sex, urinary creatinine and blood lead levels. CONCLUSION: These data show consistent evidences of association between Cd exposure and elevated tubular cell biomarker excretion in estuarine villagers living close to an industrial site.