Tumor necrosis factor α1 decreases mucosal immune and antioxidant response in the midgut of hybrid fish (white crucian carp ♀ × red crucian carp ♂).

Tumor necrosis factor α1 (TNFα) is a pleiotropic cytokine involved in immune regulation and cellular homeostasis, but the crucial role of TNFα in fish gut remained unclear. The current study aimed to evaluate the immunoregulatory function of TNFα1 on gut barrier in a novel hybrid fish (WR), which was produced by crossing white crucian carp (Carassius cuvieri, ♀) with red crucian carp (Carassius auratus red var, ♂). In this study, WR-tnfα1 sequence was identified, and a high-level expression was detected in the intestine. Elevated levels of WR-tnfα1 expressions were detected in immune-related tissues and cultured fish cells on stimulation. The appearance of vacuolization and submucosal rupture was observed in TNFα1-treated midgut of WR, along with elevated levels of goblet cell atrophy, whereas no significant changes were detected in most expressions of tight-junction genes and mucin genes. In contrast, WR receiving gut perfusion with WR-TNFα1 showed a remarkable decrease in antioxidant status in midgut, whereas the expression levels of apoptotic genes and redox responsive genes increased sharply. These results suggested that TNFα1 could exhibit a detrimental effect on antioxidant defense and immune regulation in the midgut of WR.

Comparative analysis on the immunoregulatory roles of ferritin M in hybrid fish (Carassius cuvieri ♀ × Carassius auratus red var ♂) and its parental species after bacterial infection.

Ferritin M is involved in the regulation of fish immunity. In this study, open reading frame (ORF) sequences of ferritin M from hybrid fish and its parental species were 534 bp. Tissue-specific analysis indicated that the highest level of ferritin M from red crucian carp was observed in kidney, while peaked expressions of ferritin M from white crucian carp and hybrid carp were observed in gill. Elevated levels of ferritin M from hybrid carp and its parental species were detected in immune-related tissues following Aeromonas hydrophila infection or in cultured fish cell lines after lipopolysaccharide (LPS) challenge. Ferritin M overexpression could attenuate NF-κB and TNFα promoter activity in their respective fish cells. Purified ferritin M fusion proteins elicited in vitro binding activity to A. hydrophila and Edwardsiella tarda, lowered bacterial dissemination to tissues and alleviated inflammatory response. Furthermore, treatment with ferritin M fusion proteins could mitigate bacteria-induced liver damage and rescue antioxidant activity. These results suggested that ferritin M in hybrid fish showed a similar immune defense against bacteria infection in comparison with those of its parental species.

A novel ferritin L (FerL) in hybrid crucian carp could participate in host defense against Aeromonas hydrophila infection and diminish inflammatory signals.

FerL, a multifunctional iron-storage polypeptide, not only exhibited a regulatory role in iron metabolism, but also participated in the regulation of fish immunity. In this study, ORF sequence of WR-FerL was 522 bp, encoding 173 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-FerL was detected in spleen. A. hydrophila challenge and LPS stimulation could sharply enhance WR-FerL mRNA expression in tissues and fish cells, respectively. Purified WR-FerL fusion peptide exhibited in vitro binding activity to A. hydrophila and endotoxin, limited bacterial dissemination to tissues as well as attenuated A. hydrophila-induced production of pro-inflammatory cytokines. Moreover, WR-FerL overexpression could abrogate NF-κB and TNFα promoter activity in fish cells. These results indicated that WR-FerL could play an important role in host defense against A. hydrophila infection.

Transcriptome analysis and co-expression network reveal the mechanism linking mitochondrial function to immune regulation in red crucian carp (Carassius auratus red var) after Aeromonas hydrophila challenge.

Aeromonas hydrophila is a common pathogen of freshwater fish. In this study, A. hydrophila infection was shown to cause tissue damage, trigger physiological changes as well as alter the expression profiles of immune- and metabolic-related genes in immune tissues of red crucian carp (RCC). Transcriptome analysis revealed that acute A. hydrophila infection exerted a profound effect on mitochondrial oxidative phosphorylation linking metabolic regulation to immune response. In addition, we further identified cellular senescence, apoptosis, necrosis and mitogen-activated protein kinase signal pathways as crucial signal pathways in the kidney of RCC subjected to A. hydrophila infection. These findings may have important implications for understanding modulation of immunometabolic response to bacterial infection.

Cloning, expression and functional characterization of recombinant tumor necrosis factor α1 (TNFα1) from white crucian carp in gut immune regulation.

TNFα is one of important cytokines belonging to TNF superfamily, which can exhibit a pleiotropic effect in immune modulation, homeostasis as well as pathogenesis. However, its immunoregulatory function on mucosal immunity in fish gut are still unclear. In this study, we aimed to investigated the immunoregulatory role of TNFα1 in midgut of white crucian carp (WCC). WCC-TNFα1 sequence and its deduced structure were firstly identified in WCC. Then, tissue-specific analysis revealed that high-level WCC-TNFα1 expression was detected in gill. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulated, increased trends of WCC-TNFα1 expressions were detected in immune-related tissues and cultured fish cells, respectively. WCC anal-intubated with WCC-TNFα1 fusion protein showed the increased levels of edema and fuzzy appearance in impaired villi, along with atrophy and reduction of goblet cells (GC). Moreover, the expression levels of tight junction (TJ) genes and mucin genes were consistently lower than those of the control (P < 0.05). WCC-TNFα1 treatment could sharply decrease antioxidant status in midgut, while the expression levels of caspase (CASP) genes, unfolded protein response (UPR) genes and redox response genes increased dramatically. Our results suggested that WCC-TNFα1 could exhibit a detrimental effect on antioxidant and mucosal immune regulation in midgut of WCC.

Multiomics Analyses Explore the Immunometabolic Interplay in the Liver of White Crucian Carp (Carassius cuvieri) After Aeromonas veronii Challenge.

Aeromonas veronii is one of the predominant pathogenic species that can imperil the survival of farmed fish. However, the interactive networks of immune regulation and metabolic response in A. veronii-infected fish are still unclear. In this investigation, we aimed to explore immunometabolic interplay in white crucian carp (WCC) after the A. veronii challenge. Elevated levels of immune-related genes were observed in various tissues after A. veronii infection, along with the sharp alteration of disease-related enzymatic activities. Besides, decreased levels of antioxidant status were observed in the liver, but most metabolic gene expressions increased dramatically. Multiomics analyses revealed that metabolic products of amino acids, such as formiminoglutamic acid (FIGLU), L-glutamate (L-Glu), and 4-hydroxyhippuric acid, were considered the crucial liver biomarkers in A. veronii-infected WCC. In addition, A. veronii infection may dysregulate endoplasmic reticulum (ER) function to affect the metabolic process of lipids, carbohydrates, and amino acids in the liver of WCC. These results may have a comprehensive implication for understanding immunometabolic response in WCC upon A. veronii infection.

Identification of the Genetic Requirements for Zinc Tolerance and Toxicity in Saccharomyces cerevisiae.

Zinc is essential for almost all living organisms, since it serves as a crucial cofactor for transcription factors and enzymes. However, it is toxic to cell growth when present in excess. The present work aims to investigate the toxicity mechanisms induced by zinc stress in yeast cells. To this end, 108 yeast single-gene deletion mutants were identified sensitive to 6 mM ZnCl2 through a genome-wide screen. These genes were predominantly related to the biological processes of vacuolar acidification and transport, polyphosphate metabolic process, cytosolic transport, the process utilizing autophagic mechanism. A result from the measurement of intracellular zinc content showed that 64 mutants accumulated higher intracellular zinc under zinc stress than the wild-type cells. We further measured the intracellular ROS (reactive oxygen species) levels of 108 zinc-sensitive mutants treated with 3 mM ZnCl2 We showed that the intracellular ROS levels in 51 mutants were increased by high zinc stress, suggesting their possible involvement in regulating ROS homeostasis in response to high zinc. The results also revealed that excess zinc could generate oxidative damage and then activate the expression of several antioxidant defenses genes. Taken together, the data obtained indicated that excess zinc toxicity might be mainly due to the high intracellular zinc levels and ROS levels induced by zinc stress in yeast cells. Our current findings would provide a basis to understand the molecular mechanisms of zinc toxicity in yeast cells.

Genetic analysis of oxidative and endoplasmic reticulum stress responses induced by cobalt toxicity in budding yeast.

BACKGROUND: Cobalt is an important metal cofactor of many living cells. However, excessive cobalt is toxic and can cause cell death and even several diseases in humans. Saccharomyces cerevisiae is a useful tool for studying metal homeostasis and many of the genes and pathways are highly conserved in higher eukaryotes including humans. METHODS: The intracellular cobalt and reactive oxygen species (ROS) levels were measured by an atomic absorption spectrometer and DHE staining method, respectively. The expression of genes involved in scavenging oxidative stress was tested by qPCR method, while the expression of UPRE-lacZ report gene was analyzed via ß-galactosidase activity assay. RESULTS: Using a genome-scale genetic screen, 153 cobalt-sensitive and 37 cobalt-tolerant gene deletion mutants were identified from Saccharomyces cerevisiae. We showed that 101 of the cobalt-sensitive mutants accumulated higher intracellular cobalt compared to wild-type. The intracellular ROS levels in 112 of the mutants were induced by cobalt, which might be caused by the decreased expression of genes involved in scavenging oxidative stress in response to cobalt. Moreover, more than one-third of the cobalt-sensitive mutants were also sensitive to tunicamycin, and cobalt stress might induce the unfolded protein response (UPR) through serine/threonine kinase and endoribonuclease Ire1. CONCLUSIONS: This study reinforced the fact that cobalt toxicity might be due to the high intracellular cobalt and ROS levels, and the endoplasmic reticulum stress responses induced by cobalt. GENERAL SIGNIFICANCE: Elucidating the toxicity mechanisms of cobalt stress response will help reveal new routes for the treatment of the diseases induced by cobalt.

Ultrastructure of the liver microcirculation influences hepatic and systemic insulin activity and provides a mechanism for age-related insulin resistance.

While age-related insulin resistance and hyperinsulinemia are usually considered to be secondary to changes in muscle, the liver also plays a key role in whole-body insulin handling and its role in age-related changes in insulin homeostasis is largely unknown. Here, we show that patent pores called 'fenestrations' are essential for insulin transfer across the liver sinusoidal endothelium and that age-related loss of fenestrations causes an impaired insulin clearance and hyperinsulinemia, induces hepatic insulin resistance, impairs hepatic insulin signaling, and deranges glucose homeostasis. To further define the role of fenestrations in hepatic insulin signaling without any of the long-term adaptive responses that occur with aging, we induced acute defenestration using poloxamer 407 (P407), and this replicated many of the age-related changes in hepatic glucose and insulin handling. Loss of fenestrations in the liver sinusoidal endothelium is a hallmark of aging that has previously been shown to cause deficits in hepatic drug and lipoprotein metabolism and now insulin. Liver defenestration thus provides a new mechanism that potentially contributes to age-related insulin resistance.

Oxidized Raffinose Intramolecular Cross-linking of Porcine Hemoglobin Effects on Its Structure and Function.

Periodate-oxidized raffinose is utilized to modify porcine oxyhemoglobin and deoxyhemoglobin, respectively. These modified protein derivatives showed special characteristics that it can resist the dissociation of alpha(2)beta(2) tetramer in comparison with natural proteins. According to the analysis in SDS-PAGE and reverse phase HPLC, the two porcine hemoglobin derivatives are all intramolecular linked proteins and the cross-linking sites lie between the two beta subunits. No eminent differences were found in the UV-VIS absorption spectrum between natural and modified proteins. However, after calibration for protein concentration, the emission of modified porcine deoxyhemoglobin is much weaker than the modified oxyhemoglobin and the natural protein. Furthermore, the porcine hemoglobin derivatives' characteristics of the oxygen affinity and Auto-oxidized rate also show significant differences with natural protein. The possible application of these porcine hemoglobin derivatives in the preparation of hemoglobin-based blood substitutes is discussed.

Preparation of Inositol Tetrakisphosphate and Its Application in Modification of Porcine Hemoglobin.

Modification of hemoglobin using inositol tetrakisphosphate (IP(4)) can improve the oxygen affinity of hemoglubin. Phytase was extracted from wheat bran and purified by ammonium sulphate fractionation, followed by Sephadex G-50 gel filtration and Mono Q chromatography. The purified phytase was used for hydrolysis of phytic acid under controled conditions. IP(4), as a major composition of the hydrolysate, was further purified on resin 714 column. The purified IP(4) was oxidized by periodate to obtain dialdehyde-IP(4). By the reaction between the aldehyde group of IP(4) derivative and the amino group of porcine hemoglobin (pHb), pHb-IP(4) conjugate was formed and was found to have better ability of O(2) binding and releasing than the native hemoglobin.

Oxygen-binding Properties of Complexes between Dextran and Pyridoxal 5-Phosphate-Modified Porcine Hemoglobin.

It was demonstrated by oxygen equilibrium curve that the pyridoxal 5-phosphate(PLP) modified porcine deoxyhemoglobin(pHbbeta) had lower oxygen affinity than that of stroma-free porcine hemoglobin(pHb). Accourding to analysis of SDS-PAGE, the porcine hemoglobin derivatives were not cross-linked between its subunits. A conjugate was synthesized between pHbbeta and p-toluenesolfonyl chloride-actived dextran. The oxygen affinity of Dx-pHbbeta was high than that of pHbbeta, but still lower than that of pHb. Judged by cellulose acetate film electrophoresis, the mobility of Dx-pHbbeta was apparently different from that of pHbbeta. Dx-pHbbeta has characterized absorbance peak in UV spectrum, which can be used to analysis the binding ratio between Dx and pHbbeta.

High Expression and Purification of Recombinant Human Serum Albumin from Pichia pastoris.

Recombinant human serum albumin (rHSA) was produced in methylotrophic yeast Pichia pastoris. By optimization of expression, about 150 mg/L of rHSA was obtained from broth of Pichia pastoris GS115/HAS (his+Mut(S)) supernatant. The rHSA was isolated and purified by hollow-fiber ultrafiltration, Phenyl-Sepharose hydrophobic chromatography and antibody-immunoadsorbent chromatography. Finally, rHSA was purified to electrophoretic purity.

PEG and DBBF modified porcine hemoglobin and their oxygen-carrying capacity.

Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge molecule size of proteins, to prolong their retention time in the circulation as well as blunt immune or allergic reactions. Hemoglobin cross-linked with small molecular modifiers turns out to be more stable and to have better oxygen carrying capacity. In the present study, four derivatives of PEG with different activation groups, and several PEGs with different molecular weights were covalently bound to porcine hemoglobin (pHb). PEG-pHbs exhibited a variety of differences in their properties depending on the molecular weights of the used PEGs, the amounts of bound PEGs and the presence or absence of allosteric cofactors. The optimal modification conditions for bis (3, 5-dibromosalicyl) fumarate (DBBF) as well as the physical features and oxygen carrying capacity of DBBF-modified pHb were evaluated. Furthermore, both PEG and DBBF were used simultaneously to modify pHb. The results indicate that the pHbs modified with PEG and DBBF have more stable tetrameric conformations with a molecular weight of 107 kD. Their oxygen half-saturation pressure (P(50)) is around 3.33 kPa which approximates the physiological P(50) of human red blood cells.

[The pH-dependent catalytic reaction of penicillin G acylase and its mutants].

The pH-dependence in the catalytic reaction of recombinant penicillin G acylase and its mutants from B.megaterium has been studied by using kinetic methods. pK(1) and pK(2)of the residues of the wild type penicillin G a cylase, involved in the catalyzed reaction, were 5.50-5.87 and 10.73, respectively, from the curves of logV(m) and log(V(m)/K(m)) versus pH. Results showed tha t the pK(1) and pK(2) values of these residues of the mutants were similar to that of the wild type. pK(1) of 5.64-5.86 for mutant A and 5.69-6.96 for mutant B were obtained, while pK(2) was 10.61 and 10.48 for mutant A and B, respectively. At the same time, pK values at different temperatures were investigated. The ionization enthalpies(deltaH) were 44.38-59.03 kJ/mol and 147.37 kJ/mol, respectively, from th e curve of pK versus temperature. It was presumed according to the results mentioned above that the ionizing residues, involved in the reaction, wer e histidine and lysine that are localized around the active site.

High Expression of Penicillin G Acylase Gene from Bacillus megaterium in Bacillus subtilis.

The penicillin G acylase gene (pga) amplified by PCR from Bacillus megaterium was subcloned into an expressing vector pPZW103 (P43 as promoter). The recombinant plasmid was transferred into Bacillus subtilis DB104. Penicillin G acylase production in the B. subtilis transformant was 3-6 u/ml, higher than that of published recombinant strains. Penicillin G acylase production was induced by phenylacetic acid in B. megaterium, whereas the enzyme was produced constitutively in the B. subtilis transformant carrying B. megaterium pga. The recombinant strain showed high stability in antibiotic-free medium for 10 days. Enzyme in crude broth was purified by Al(2)O(3) chromatography and phenyl-Sepharose CL-4B hydrophobic chromatography and the total yield is 79%. The purified enzyme with specific activity of 52 u/mg can be directly immobilized for use.

Enhancing Penicillin G Acylase Stability by Site-directed Mutagenesis.

To improve the stability of penicillin G acylase(PGA) from Bacillus megaterium, a three-dimensional model of B.megaterium PGA was constructed based on crystal structure of penicillin G acylase from E.coli using PMODELING program. The mutation of Lys at beta427 and 430 to Ala was predicted to enhance the stability of PGA in acidic or organic solvent environment. The results showed that 2 mutant PGA had similar specific activity and Km as the parent PGA. Their optimum pH dropped 0.5 pH units. The stability of Lys430Ala was enhanced obviously at pH 5.2. The half lives of Lys427Ala and Lys430Ala were improved by 60 % and 166 %, respectively, in comparison with the parent PGA.

Preliminary Study on Gene Therapy of PD Monkey Using Microcapsulated Rat Transgenetic Myoblasts.

Rat myoblast cells transfected with tyrosine hydroxylase gene were microcapsulated using the ALG/PLL system. These microcapsulated cells could survive grow and produce tyrosine hydroxylase(TH)for at least two months in vitro. After the implantation of these microcapsules into monkey striatum microcapsulated cells still survived one month more. No obvious gliosis was seen around implanted microcapsules. Then the microcapsules were transplanted into the striatum of PD monkeys and their typical PD rotation numbers were significantly decreased.

Working memory and the identification of facial expression in patients with left frontal glioma.

Patients with brain tumors may have cognitive dysfunctions including memory deterioration, such as working memory, that affect quality of life. This study was to explore the presence of defects in working memory and the identification of facial expressions in patients with left frontal glioma. This case-control study recruited 11 matched pairs of patients and healthy control subjects (mean age ± standard deviation, 37.00 ± 10.96 years vs 36.73 ± 11.20 years; 7 male and 4 female) from March through December 2011. The psychological tests contained tests that estimate verbal/visual-spatial working memory, executive function, and the identification of facial expressions. According to the paired samples analysis, there were no differences in the anxiety and depression scores or in the intelligence quotients between the 2 groups (P > .05). All indices of the Digit Span Test were significantly worse in patients than in control subjects (P < .05), but the Tapping Test scores did not differ between patient and control groups. Of all 7 Wisconsin Card Sorting Test (WCST) indexes, only the Preservative Response was significantly different between patients and control subjects (P < .05). Patients were significantly less accurate in detecting angry facial expressions than were control subjects (30.3% vs 57.6%; P < .05) but showed no deficits in the identification of other expressions. The backward indexes of the Digit Span Test were associated with emotion scores and tumor size and grade (P < .05). Patients with left frontal glioma had deficits in verbal working memory and the ability to identify anger. These may have resulted from damage to functional frontal cortex regions, in which roles in these 2 capabilities have not been confirmed. However, verbal working memory performance might be affected by emotional and tumor-related factors.