Spin-Trapping and Chemiluminescence Studies of Neutrophils from a Holstein-Friesian Calf with Bovine Leukocyte Adhesion Deficiency

The ability of neutrophils from a Holstein-Friesian calf with bovine leukocyte adhesion deficiency (the proband with a genetic deficiency of the Mac-1 (CD11b/CD18) glycoprotein corresponding to the receptor of complement iC3b) to generate oxygen radicals was examined using electron spin resonance spectrometry (ESR) combined with a spin-trapping technique and luminol-dependent chemiluminescence spectrometry. When the neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), an ESR spectrum confirming the generation of superoxide anions (O₂^-) was clearly observed in both healthy and diseased calves. However, when the neutrophils were stimulated by opsonized zymosan, appearance of the ESR spectrum was recognized in the healthy calves but not in the diseased calf. Similar results were obtained from chemiluminescence experiments.     

Enhanced Expression of Fc Receptors on Neutrophils from Calves with Leukocyte Adhesion Deficiency

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).     

Inhibitory Effect of Bifemelane on Superoxide Generation by Activated Neutrophils Measured Using a Simple Chemiluminescence Method

We evaluated the effect of 4-(2-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane hydrochloride) on superoxide production by human neutrophils using an MCLA-dependent chemiluminescence assay. Bifemelane hydrochloride dose-dependently inhibited superoxide production by neutrophils stimulated with phorbol myristate acetate, opsonized zymosan, or N-formyl-methionyl-leucyl-pheny-lalanine, while it had no effect on superoxide production by a hypoxanthine-xanthine oxidase system. These results indicate that bifemelane hydrochloride does not have a scavenging effect, but has an inhibitory effect on superoxide generation by neutrophils. Although this drug is commonly used for treating chronic cerebral infarction, it may also have a protective effect on acute ischemia/reperfusion injury.     

甲状腺癌组织中ALCAM 的表达及其与临床病理参数及预后的关系

目的:探讨甲状腺癌组织中活化白细胞黏附分子(activated leukocyte cell adhesion molecule,ALCAM)的表达及其与临床病理参数及预后的关系。方法:选择2011年1月到2013年1月在我院确诊为甲状腺癌的患者60例作为研究对象。收集所有患者甲状腺癌组织及其癌旁正常组织标本,采用免疫组织化学法检测不同组织中ALCAM蛋白表达,分析其与甲状腺癌患者临床病理参数的相关性,并对所有患者随访3年以上,并记录3年生存率。结果:ALCAM蛋白在甲状腺癌组织中的总阳性率为71.67%,明显高于癌旁正常组织的31.67%(P0.05)。ALCAM蛋白在高分化型甲状腺癌组织中的表达率明显高于低分化及无分化型癌组织(P0.05)。ALCAM蛋白高表达的甲状腺癌患者的3年生存率为81.45%,明显高于ALCAM蛋白低表达者的51.32%(P0.05)。结论:组织中ALCAM蛋白表达与甲状腺癌的发生、发展及患者预后密切相关。     

Adhesiveness for Extracellular Matrices and Lysosomal Enzyme Release from Normal and β2 Integrin-Deficient Bovine Neutrophils

The adhesiveness of control and CD18-deficient bovine neutrophils on culture plates precoated with collagen I, collagen IV, fibronectin and laminin was measured to evaluate the possible factors for adherence to extracellular matrices. The release of N-acetyl-β-D -glucosaminidase (NAG_ase) from control and CD18-deficient neutrophils stimulated with complement receptor type 3 (CR3) or Fc receptor dependent stimuli was also evaluated. The adhesive activities of CD18-deficient neutrophils to collagen I, collagen IV and fibronectin were significantly diminished (P < 0.05); however, similar adhesion to laminin was observed in CD18-deficient neutrophils and control neutrophils. The adhesive activity of control neutrophils on uncoated plates increased 2.5 times (P < 0.05) with the presence of PMA. The mean activities for NAG_ase release from CD18-deficient neutrophils stimulated with opsonized zymosan and aggregated bovine immunoglobulin G (Agg-IgG) were 46.7 and 82.7% that of the control neutrophils, respectively. The Agg-IgG-induced NAG_ase release from control and CD18-deficient neutrophils was eliminated by H7, a protein kinase C inhibitor. These results support that an association between CR3 and Fc receptors on neutrophils appears to play an essential role in neutrophil functions.     

Isolation of Milk-Clotting Enzyme from Transgenic Sheep Milk and Its Comparison with Calf Chymosin

Technology for preparation of chymosin from milk of transgenic sheep has been elaborated.Purification of the preparation by ion-exchange chromatography on aminosilochrom and biospecific chromatography on bacitracin-Sepharose yielded homogeneous active enzyme. Hydrolysis of protein substrates (hemoglobin, BSA, and sodium caseinate) by the transgenic sheep chymosin and stability of the enzyme at various values of pH were studied. Judging by the amino acid composition, the N-terminal sequence involving six amino acid residues, molecular mass, stability at various pH values, and the cat alytic activity against the protein substrates, the transgenic sheep chymosin is identical to calf chymosin.     

Effect of sulphite on the oxidative metabolism of human neutrophils: Studies with lucigenin- and luminol-dependent chemiluminescence

To assess the effect of sulphite on the oxidative metabolism of human neutrophils, chemiluminescence (CL) measurements were performed using lucigenin and luminol as chemiluminigenic probes. Lucigenin-dependent CL was used for measuring superoxide anion (O) production, and luminol-dependent CL was used for determination of myeloperoxidase (MPO)-connected processes. With sulphite concentrations of 0.01 to 1 mmol/L, resting neutrophils showed an up to sixfold increase of lucigenin-dependent CL, but only a 1.9-fold increase of luminol-dependent CL. Subsequent stimulation of sulphite-treated neutrophils with phorbol myristate acetate (PMA) (soluble stimulant) or zymosan (particulate stimulant) resulted in an additional significant increase of lucigenin-dependent CL compared to stimulated control cells, whereas luminol-dependent CL increased slightly by 0.01 mmol/L sulphite and decreased then continuously. Sulphite concentrations above 1 mmol/L decreased both lucigenin- and luminol-dependent CL of resting and PMA- or zymosan-stimulated neutrophils. Lucigenin-dependent CL of sulphite-treated and subsequently stimulated neutrophils was strongly inhibited by extracellularly added superoxide dismutase, whereas luminol-dependent CL was markedly reduced by the MPO inhibitor azide. The intracellular activity of MPO in neutrophils stimulated with PMA in the presence of sulphite (2 mmol/L) was reduced by 55%. Sulphite (0.1 mmol/L) also inhibited strongly the activity of MPO in a cell-free system. These results indicate that micromolar concentrations of sulphite exert a stimulating effect on the O production of neutrophils extracellularly, but have an inhibitory effect on MPO-catalysed reactions intracellularly.     

Isolation and Structure of Cotylenin E

²-Casein was modified by trypsin, chymosin or phosphoprotein phosphatase. Each modified ²-casein was fortified to the test-milk which was pretreated at 10°C by immobilized thermolysin to curdle non-enzymatically at 35°C. Fortification with chymosin-modified ²-casein, devoid of the hydrophobic moiety near the C-terminus, increased the curd tension as did native ²-casein. The test-milk fortified with trypsin-modified ²-casein, which has been deleted some part of the hydrophilic region near the N-terminus, had similar curd tension to unfortified test-milk. The test-milk fortified with dephosphorylated ²-casein, formed a softer curd than that with native ²-casein. Trypsin-modified ²-casein or dephosphorylated ²-casein retained its associative ability, but chymosin-modified ²-casein revealed negligible ability to associate.

These results led to the conclusion that the phosphoryl group on the hydrophilic moiety of ²-casein contributes to the increase in curd tension, while the hydrophobic moiety is responsible for the association of ²-casein.     

Scavenging of Superoxide Anion by Phosphorylethanolamine: Studies in Human Neutrophils and in a Cell Free System

On the basis of previous observations, we attempted to characterize the effects of various products of phospholipid hydrolysis on neutrophil (PMN) respiratory burst activity. We studied the effects of phos- phorylcholine (PC) and phosphorylethanoline (PE) on superoxide anion production in PMN and in a cell free system. We found that PE but not PC inhibited measured superoxide anion, but that this was not due to inhibition of cellular superoxide generation but to scavenging of generated superoxide anion. Further, utilizing a system based upon the photo-oxidation of O-dianisidine sensitized by riboflavin, we were able to determine that the scavenging effect of PE was not superoxide dismutase (SOD)-like but rather a general scavenging or glutathione (GSH)-like effect. These data underscore the importance of identifying the mechanism of inhibition of superoxide generation by putative inhibitors as being due to a direct cellular effect or to a scavenging property.     

Opsonic Activity of Sera and Blister Fluid from Severely Burned Patients Evaluated by a Chemiluminescence Method

Burn wound sepsis is the most common and severe complication in the patients with severe burn. To know the systemic and local defect in immunity of burned patients, we measured the luminol-enhanced chemiluminescence (CL) response of normal polymorphonuclear leukocytes (PMNs) upon exposure to zymosan particles, bacteria or Candida albicans that were opsonized with any of patient's serum, blister fluid of burn wound or pooled normal serum (blood type AB). Sera from patients exhibited lower opsonic activities than those of pooled normal serum in the early postburn days. The levels of serum immunoglobulins, complement components and plasma fibronectin were found to correlate well with opsonin-index (OI), which was determined based on the CL response data obtained during the course of infusion therapy with fresh frozen plasma. Furthermore, patient's blister fluid showed much lower opsonic activity against bacteria such as Pseudomonas aeruginosa than patient's own serum. These results indicate that blister fluid is also not effective to opsonize bacteria because of the marked depression of the levels of immunoglobulins and complement components. Destruction of the skin barrier by thermal injury and impairment of systemic or local humoral immunity may predispose these patients to burn wound sepsis.     

ESR Studies of a Series of Phthalocyanines. Mechanism of Phototoxicity. Comparative Quantitation of O2-using ESR Spin-Trapping and Cytochrome c Reduction Techniques

ESR experiments with 2,2,6,6-tetramethyl-4-piperi-done (4-oxo-TEMP) and the spin-trap 5,5-dimethyl pyrroline-N-oxide (DMPO) have been performed on a series of new phthalocyanines: the bis(tri-n-hexyl-siloxy) silicon phthalocyanine ([(nhex)₃SiO]₂SiPc), the hexadecachloro zinc phthalocyanine (ZnPcCl₁₆), the hexadecachloro aluminum phthalocyanine (AlPcCl₁₆), the hexadecachloro aluminum phthalocyanine sulfate (HSO₄A1PcCl₁₆), whose photocytotoxicity has been studied against various leukemic and melanotic cell lines. Type I and Type II pathways occur simultaneously in DMF although the Type II seems to be prevalent. These results are not changed when the bis(tri-n-hexylsiloxy) silicon phthalocyanine is entrapped into liposomes. By contrast, the Type I process is favored in membrane models for all the perchlori-nated phthalocyanines. This modified behavior may be accounted on a possible stacking of phthalocyanines in membranes and a preventing effect of axial ligands against aggregation in the case of the bis(tri-n-hexyl-siloxy) silicon phthalocyanine. The photodynamic action of zinc perchlorinated phthalocyanine is not dependent on singlet oxygen, phototoxicity of this molecule being essentially mediated by oxygen free radicals. Quantitation of the superoxide radical was accomplished, with good agreement, by two techniques: the cytochrome c reduction and the ESR quantitation based on the double integration of the first derivative of the ESR signal. The disproportionation of the superoxide radical or degradation of the spin-trap seem to be avoided in aprotic solvents such as DMF.     

Identification of a radical formed in the reaction mixtures of oxidized phosphatidylcholine with ferrous ions using HPLC-ESR and HPLC-ESR-MS

Identification of free radicals was performed for the reaction mixtures of autoxidized 1,2-dilinoleoylphosphatidylcholine (DLPC) with ferrous ions (or DLPC hydroperoxide with ferrous ions) and of DLPC with soybean lipoxygenase using electron spin resonance (ESR), high performance liquid chromatography (HPLC)-ESR and HPLC-ESR-mass spectrometry (MS) combined use of spin trapping technique. ESR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (a^N=1.58 mT and a^Hβ=0.26 mT). Outstanding peaks with almost same retention times (autoxidized DLPC, 36.9 min; DLPC hydroperoxide, 35.0 min; DLPC with soybean lipoxygenase, 37.1 min) were observed on the elution profile of the HPLC-ESR analyses of the reaction mixtures. HPLC-ESR-MS analyses of the reaction mixtures gave two ions at m/z 266 and 179, suggesting that 4-POBN/pentyl radical adduct forms in these reaction mixtures.     

A Neonate with Recurrent Vomiting and Generalized Hypotonia Diagnosed with a Deficiency of Dihydropyrimidine Dehydrogenase

Deficiency of dihydropyrimidine dehydrogenase (DPD) is a rare inborn error of pyrimidine metabolism. To date, only about 50 patients are known worldwide. The clinical picture is varied and is not yet fully described. Most patients are diagnosed at the age of 1–3 years. We present a patient diagnosed 8 weeks postpartum.

The female patient presented in the first 3 days after birth with agitation, choking, and vomiting. Six weeks later, the patient presented again with vomiting and insufficient weight gain. Metabolic screening of urine showed a strongly increased excretion of uracil and thymine, with no other abnormalities. This suggested a deficiency of DPD which was confirmed by enzyme analysis in peripheral blood mononucleair (PBM) cells (patient: activity <0.01 nmol/mg/h; controls: 9.9 ± 2.8 nmol/mg/h). The patient was homozygous for the IVS14+1G>A mutation.

MRI of the brain showed some cerebral atrophy; myelinization appeared normal. Many patients with DPD-deficiency suffer from convulsions and mental retardation, some show microcephaly, feeding difficulties, autism, and hypertonia. Our patient showed feeding difficulties and in the second half-year she developed slight motor retardation and generalized hypotonia. Further observation of the development of the patient may shed more light on the relationship between clinical symptoms and DPD deficiency. DPD deficiency may present in newborns with vomiting and hypotonia as the main symptoms.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

Adhesion and surface-aggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber

Adhesive interactions between Candida albicans and oral bacteria are generally thought to play a crucial role in the microbial colonization of denture acrylic, which may lead to denture stomatitis. This study investigated the influence of saliva on the adhesive interactions between C. albicans and Streptococcus sanguis or Actinomyces naeslundii on denture acrylic. First, bacteria were allowed to adhere to the acrylic surface from a flowing suspension, and subsequently yeasts were flowed over the acrylic surface. The organisms were assayed in the presence or absence of human whole saliva. All experiments were carried out in a parallel plate flow chamber and enumeration was done in situ with an image analysis system. In the absence of adhering bacteria, adhesion of C. albicans from buffer was more extensive than from saliva. However, in the presence of adhering bacteria, yeast adhesion from saliva was increased with respect to adhesion of yeasts from buffer, indicating that specific salivary components constitute a bridge between bacteria and yeasts. In all cases, yeast aggregates consisting of 3 to 5 yeast cells were observed adhering to the surface. A surface physico-chemical analysis of the microbial cell surfaces prior to and after bathing the microorganisms in saliva, suggests that this bridging is mediated by acid-base interactions since all strains show a major increase in electron-donating surface free energy parameters upon bathing in saliva, with no change in their zeta potentials. The surface physico-chemical analysis furthermore suggests that S. sanguis and A. naeslundii may use a different mechanism for adhesive interactions with C. albicans in saliva.     

Antioxidant activity of carotenoids: An electron-spin resonance study on β-carotene and lutein interaction with free radicals generated in a chemical system

β-Carotene is thought to be a chain-breaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of β-carotene and lutein on the radical adducts of the spin-trap PBN (N-t -butyl-α-phenylnitrone) generated by the metal-ion breakdown of different tert -butyl hydroperoxide (t BOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of t BOOH (1 M), were quenched by β-carotene or lutein, in competition with the spin-trapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low t BOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by β-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence of α-tocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of β-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 299–304, 1998     

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as alpha-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.     

Adhesion capability of first two domains at N terminus of NP_785232 protein and their interaction with a UV‐absorbing component from human mucus

Aims: This work aims to investigate the binding capability of certain domains at N terminus of the protein NP_785232 of Lactobacillus plantarum to Caco‐2 cells and to test the usage of affinity chromatography to isolate the human mucus component that interacts with them. Methods and Results: Recombinant proteins containing the first and both the first and second domains at N terminus of NP_785232 fused to a His tag were constructed and used to bind the Caco‐2 cells. The interacting molecule from human mucus was isolated by affinity chromatography through immobilizing the recombinant proteins onto a Sepharose matrix. It was found both recombinant proteins could block the adhesion of Lact. plantarum to Caco‐2 cells and bind to a human mucus component. Conclusions: The first and both the first and second domains at N terminus of the protein NP_785232 have the capability to adhere Caco‐2 cells and by affinity chromatography, an interacting UV‐absorbing component from human mucus was isolated. Significance and Impact of the Study: The protein domains characterized in this study may be displayed on probiotics to promote adhesion, and further characterization of the human mucus component might be helpful to identify host factors required for prolonging probiotics persistence in the gastrointestinal tract.