Immunoregulatory effects of a glucogalactan from the root of Panax quinquefolium L.

In this research we purified one homogeneous glucogalactan from the roots of Panax quinquefolium, with a molecular weight of 54 kDa estimated by high-performance gel permeation chromatography (HPGPC). The monosaccharide composition of PPQ was composed of Glc (glucose) and Gal (galactose) in a molar ratio of 2.1:1, as determined by gas chromatography (GC). In order to evaluate the anti-lung carcinoma and immunoregulatory effects of this glucogalactan in mice, we established a Lewis lung carcinoma model in C57BL/6 mouse. The results showed that PPQ not only could inhibit the growth of lung tumor, but also enhance the thymus and spleen indices, as well as the level of IFN-γ, IL-10 and IL-2, indicating PPQ could have a possible cancer therapeutic potential.     

Preparation of Immunoglobulin Y (IgY) Against Lipopolysaccharide using Gel Chromatography from the Yolks of Eggs Laid by Immunized Hens

The objective is to prevent and treat injuries caused by lipopolysaccharide (LPS) from gram negative bacteria in animals and humans, we produced antibodies against LPS from egg yolk. LPS from E. coli (O111:B4) mixed with Freund’s Adjuvant was used as the immunogen to immunize Roman hens. Immunized eggs were collected, and immunoglobulin Y (IgY) was purified using a water solution, salt precipitation and gel chromatography. The molecular weight and purity were determined by SDS–PAGE, the antibody titer by noncompetitive enzyme-linked immunosorbent assay (ELISA) and antibody activity against LPS by the mortality of mice intraperitoneally injected with LPS or LPS-IgY solutions. IgY against LPS showed two protein bands at 68 and 26 kDa on the gel; the antibody titer was almost 1:25,600. After incubation with LPS, IgY decreased the mortality of mice challenged with LPS. This study provided an efficient way to produce high-titer egg yolk antibodies, which could attenuate lethal effects of LPS, by immunizing hens. Furthermore, the LPS antibody was purified well using a water solution, salting-out and gel chromatography.     

Purification and Some Properties of Thiosulfate Dehydrogenase from Acidithiobacillus ferrooxidans

Abstract

Thiosulfate dehydrogenase was purified from Acidithiobacillus ferrooxidans using three purification steps. The purification procedure involved ammonium sulfate fractionation, ion‐exchange chromatography, and gel permeation chromatography. Specific activity of the purified enzyme (after IEC) was 3.26 nkat/mg, and yield of the enzyme was 78%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is a tetramer composed of four probably identical subunits of relative molecular weight 45,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 3.0. The isoelectric point of the enzyme was 8.3. Enzyme activity was found to be particularly sensitive to the histidine‐selective reagent diethylpyrocarbonate. Reagents selective for arginine, cysteine, and tryptophane had no effect on enzyme activity.     

Plasmodium chabaudi chabaudi(AS): Inflammatory Cytokines and Pathology in an Erythrocytic-Stage Infection in Mice

Cross, C. E., and Longhorne J. 1998.Plasmodium chabaudi chabaudi(AS): Inflammatory cytokines and pathology in an erythrocytic-stage infection in mice.Experimental Parasitology90220–229. We have sought to characterizePlasmodium chabaudi chabaudiinfection in mice for use as a model for malaria pathology. Different mouse strains vary in their susceptibility to the erythrocytic stages of this parasite and this is manifested not only in the outcome of infection (survival versus death) but also by differences in the numbers of circulating parasites at the peak of infection. We have shown that regardless of final outcome, both resistant and susceptible mice exhibit other parameters of disease such as loss in body weight and anemia. By contrast, other parameters such as hypothermia appear more severe in susceptible mice. The severe symptoms coincide with high levels of inflammatory cytokines in the circulation of susceptible mice, not seen in H-2-matched resistant mice. However, levels of mRNA for the same cytokines, measured in the spleen of the same mice was not significantly different between the two strains. Neutralization of IFN-γin vivoled to an increase in parasitemia, in both susceptible and resistant mice, but did not affect the final outcome of disease. Indeed, symptoms were exacerbated in the absence of IFN-γ, presumably because of larger numbers of circulating parasites. These data suggest that IFN-γ does not directly contribute to the lethal outcome of infection in susceptible strains of mice.     

Location of F1 ATPase-like Genes on the Physical Map of the Bacillus subtilis 168 Chromosome

A water-soluble polysaccharide, FI, extracted from the mycelium of Granoderma tsugae, was fractionated and purified by ion-exchange chromatography, gel filtration, and affinity chromatography. Sixteen polysaccharides obtained were examined for antitumor effects on Sarcoma 180 in mice.

The three active polysaccharides obtained were as follows:

FI₀-a: A glycan-protein complex containing 9.3% protein, and having a hetero-glyco-chain of mannose and xylose.

FI₀-b-α: Molecular weight 10,000, glucan-protein complex containing 25.8% protein. The inhibition ratio was 61.8% against the solid cancer Sarcoma 180/mice; the survival ratio was more than 194% of the control group (100).

FA-1-b-α: Molcular weight 16,000, a complex of glycan: protein = 42:58 w/w, consisting of glucose as a main component, and associated with arabinose, mannose, xylose, and galactose. This had a tumor inhibition ratio of 56% and a survival ratio of more than 182%.

Comparison of active glycan with the fruiting body and mycelium: Among water-soluble polysaccharides of fruiting body, FI₀-a and FA-1, with antitumor activity, were both glucogalactan-protein complexes of molecular weight 10,000, but that of mycelium was a homoglucan protein complex in FI₀-b-α and heteroglucan protein in both FA-l-a and FA-l-b-α. The heteroglucan had a low tumor inhibition ratio, but caused a high survival ratio in mice.     

Enzymatic Dehalogenation of Pentachlorophenol by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> of the Microbial Community from Tannery Effluent

Four different bacterial isolates obtained from a stable bacterial consortium were capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in the chemostat. The degradation of PCP by bacterial strain was preceded through an oxidative route as indicated by accumulation of tetrachloro-ρ-hydroquinone and dichlorohydroquinone as determined by high performance liquid chromatography (HPLC). Among the four isolates, Pseudomonas fluorescens exhibited maximum degradation capability and enzyme production. PCP-monooxygenase enzyme was extracted from culture extract and fractionated by DEAE-cellulose ion exchange chromatography. The molecular weight of the enzyme, purified from Pseudomonas fluorescens, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography was found to be 24,000 Da. Received: 22 July 2002 / Accepted: 23 September 2002     

A polysaccharide from Sargassum fusiforme protects against immunosuppression in cyclophosphamide-treated mice

A water-soluble polysaccharide (SFPS) isolated from Sargassum fusiforme was purified by DEAE-52 cellulose anion-exchange and Sephadex G-200 gel filtration chromatography. The high performance gel permeation chromatography (HPGPC) analysis showed that the average molecular weight (Mw) of SFPS was 299kDa. The SFPS was composed of d-fucose, l-xylose, d-mannose and d-galactose in a molar ratio of 5.9:1.0:2.3:2.2. The results showed that SFPS stimulated proliferation and the cytokines (IL-2, IL-6 and IFN-γ) secretion of splenic lymphocytes in cyclophosphamide-induced immunosuppressed mice. SFPS markedly increased the phagocytic rates and cytokines (IL-2, IL-6 and TNF-α) secretion of peritoneal macrophages. Administration of SFPS significantly raised spleen index. It could act as an efficacious adjacent immunopotentiating therapy or an alternative means in lessening chemotherapy-induced immunosuppression, and also can be utilized as immunostimulants for food and pharmaceutical industries.     

Partial purification of the aminopeptidase from the midgut of the human body louse, Pediculus humanus humanus

The midgut of the human body louse Pediculus humanus humanus contains a thermally stable leucine aminopeptidase, which was detected by agarose gel electrophoresis using l ‐amino oxidase. Midgut extracts were homogenized in saline or in 1% Triton X‐100 and the aminopeptidase was purified by Superose 6 gel filtration chromatography. A peak with enzyme activity that was extracted with or without Triton X‐100 was eluted at a molecular weight 67–69 kDa. Non‐denaturing polyacrylamide gel electrophoresis resolved one band of molecular weight of 69 kDa for samples that were extracted in a saline buffer. Two closely linked bands of molecular weight 67 kDa and 69 kDa were observed in samples that were extracted in 1% Triton X‐100.     

State of aggregation of recombinant hirudin in solution under physiological conditions

The state of aggregation of recombinant desulfatohirudin (r-HV1) in solution under physiological conditions (pH 7.5, 0.15N NaCl) was investigated by sedimentation equilibrium. The weight-average molecular weight ¯M _w determined by sedimentation equilibrium was found to be 6914±76 Da compared to 6964 Da expected from the amino acid sequence. The ¯M _z /¯M _w ratio was found to be 1.03, which demonstrates that under the conditions studied hirudin exists in solution as a monomer. This result is in agreement with the relative molecular weight (M _r ) of recombinant hirudin variant 3 reported by Otto and Seckler [(1991),Eur. J. Biochem. 202, 67–73], who also used equilibrium ultracentrifugation, but not with the molecular weight estimated from gel permeation chromatography of natural hirudin (51,300 Da) [Konnoet al. (1988),Arch. Biochem. Biophys. 267, 158–166]. Knowledge of the state of aggregation is essential for understanding the mechanism of interaction of thrombin and hirudin under physiological conditions.Abbreviations ¯M _w weight-average molecular weight - ¯M _z Z-average molecular weight - M _r relative molecular weight - NTSB 2-nitro-5-thiosulfobenzoic acid - Tris Tris(hydroxymethyl)aminomethane - r-HV1 recombinant desulfatohirudin - _M molar extinction coefficient     

Purification of malic enzyme from bovine heart mitochondria by affinity chromatography

A simple two-step method for the purification of malic enzyme from bovine heart mitochondria in high yield is described. It consists of successive affinity chromatography steps on immobilized C⁸-(aminohexyl)-NADP and N⁶-(aminohexyl)-ADP. The molecular weight estimated by gel filtration of the homogeneous enzyme is 250,000 and the subunit molecular weight by SDS-polyacrylamide gel electrophoresis is 59,000.     

Immunogenic fractions of Cryptococcus neoformans

Cryptococcus neoformans cell and culture supernatant extracts were fractionated by ion exchange and gel filtration column chromatography. Various fractions were used to immunize mice, and to assess the release of migration inhibition factor, delayed type hypersensitivity, and protective immunity after challenge with C. neoformans. Results suggest that the C. neoformans fractions, which protect mice, contain a high molecular weight, predominantly carbohydrate antigen that can be distinguished from the capsular polysaccharide.     

嗜热链球菌MGB80-7所产胞外多糖的表型结构及其抗氧化活性

【背景】胞外多糖（exopolysaccharide,EPS）是乳酸菌生长代谢过程中所产生的一种次级代谢产物,除了可以改善产品质构和品质外,其生理功能也是近年来研究人员追捧的热点。【目的】探究乳酸菌EPS的表征特性和分子结构,揭示其与EPS益生特性之间的联系。【方法】以产EPS的嗜热链球菌（Streptococcus thermophilus,S. thermophilus） MGB80-7为研究对象,利用苯酚-硫酸法测定菌株EPS产量。采用离子交换柱层析和凝胶分子筛层析对该菌株所产EPS进行分离纯化,结合凝胶色谱、红外光谱及高效液相色谱对EPS表型结构进行剖析。此外,为确定EPS表型特征对其抗氧化活性的影响,测定了EPS对超氧阴离子、羟自由基及DPPH自由基等的清除能力。【结果】S. thermophilus MGB80-7在M17培养基中EPS产量较高,为（268.25±5.36） mg/mL,分离纯化后共得到2种多糖组分,其中中性多糖（WPS-807）分子量为1.028×10⁵ Da,主要由葡萄糖、半乳糖和甘露糖组成,并含有少量的鼠李糖和阿拉伯糖,酸性多糖（...     

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor

Abstract

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10?kDa, respectively, and under non-reducing conditions, 26?kDa was observed. The inhibitor was shown to inhibit bovine trypsin (K_i of 3.45?nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

Characterization of a Degraded Product Derived from Serratia piscatorum Polysaccharide by Ultrasonication

Acidic polysaccharide, PLS F–II, was prepared from Serratia piscatorum polysaccharide, PLS N–I, by a sequence of ultrasonication and gel filtration and was examined for chemical composition and biological activity.

The purified PLS F–II preparation was shown to be homogeneous by ultracentrifugation, zone electrophoresis and column chromatography. The molecular weight was estimated to be about 2 × 10⁴ by the Archibald method. PLS F–II was composed of l-rhamnose, d-galactose and d-galacturonic acid in the molar ratio of 2: 1: 1 and was partially acylated on the galacturonic acid residues.

PLS F–II was found to enhance the antibody formation in mice, although it showed no anti-inflammatory activity.     

Amelioration of age-dependent increase in oxidative stress markers in male mice by extract of Potentilla fulgens

Objective: To investigate the effect of Potentilla fulgens extract on lipid peroxidation and antioxidant status in male mice as a function of age.

Methods: Eighteen-month-old Swiss albino male mice were administered the dichloromethane-methanol extract of P. fulgens (250?mg/kg b.w.) on alternate days via intraperitoneal route for a period of 14 days. Lipid peroxidation and activities of catalase (CAT) and glutathione peroxidase (GPx1) in liver and kidney were measured and serum oxygen radical absorbance capacity (ORAC) assay was estimated. Phytochemical analysis of P. fulgens extract using high performance thin layer chromatography (HPTLC) was carried out with gallic acid, quercetin, catechin, and epicatechin as markers.

Results: Significant increase in level of thiobarbituric acid-reactive substances (TBARS), decreased GPx1, and CAT activities as well as reduction in ORAC were observed in 18-month-old mice as compared to that of 2-month-old mice. Treatment with P. fulgens extract significantly lowered TBARS level, ameliorated CAT, and GPx1 activities in liver and kidney and improved serum ORAC in aging mice. HPTLC studies revealed well resolved bands of P. fulgens extract containing epicatechin and catechin.

Discussion: This study showed that P. fulgens is a potent antioxidative agent, which can emerge as a promising candidate in alleviating the age-associated oxidative stress and related diseases.     

Two Phenolic Amides in the Seed Balls of Sugar Beet (Beta vulgaris L. var. saccharifera Alefeld)

Ferredoxin-dependent sulfite reductase (Fd-SiR) (EC 1.8.7.1) was purified about 1136-fold, with a yield of 11%, from fresh thalli of Porphyra yezoensis by a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Buty 1-Toyopearl chromatography, Sephadex G-100 gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous, as judged on polyacrylamide disc gel electrophoresis, with a specific activity of 100 units/mg of protein. The molecular weight of the enzyme was estimated to be 70 kilodaltons by gel filtration. On subunit analysis by SDS-PAGE, a single band corresponding to molecular weight of 65 kilodaltons appeared. The purified enzyme (Fd-SiR) showed 5-times higher ferredoxin-dependent activity than methyl viologen-linked activity. In the oxidized form, the enzyme exhibited absorption maxima at 278, 390 (Soret band), 586 (a band) and 714 (CT band) nm, indicating that siroheme is involved in the catalysis of sulfite reduction. The absorbance ratios, A₃₉₀: A₂₁₈ and A₅₈₆ :A₃₉₀, were 0.32 and 0.31, respectively. A plot of the substrate (sulfite) and electron donor (ferredoxin) concentrations versus enzymatic (Fd-SiR) activity yielded sigmoidal curves, giving Hill coefficients («) of 2.3 (for sulfite) and 2.7 (for ferredoxin), respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion suggested that it was specific against isolated Fd-SiR. Using the antiserum, dot immunoblotting was performed to determine the immunological similarity of Fd-SiRs from Porphyra yezoensis, Spirulina platensis, Brassica chinensis and Spinacia oleracea. The tests revealed that the four forms of assimilatory Fd-SiR have antigenic determinants in common.     

Aryl alcohol oxidases from the white-rot basidiomycete Pleurotus ostreatus

 Three aryl alcohol oxidases (AAOs; EC 1.1.3.7) I, II, and III from the culture filtrate of a strain of white-rot fungus Pleurotus ostreatus were purified by multistep chromatography. Each of the purified AAOs I, II, and III had the same molecular masses of 70 kDa and 72 kDa on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Their optimum temperature was 40°C, but their optimum pHs differed slightly. The N-terminal amino acid sequence of AAOs I, II, and III was determined to be Ala-Asp-Lys-Asp-Tyr-Ile-Val-Val-Gly-Ala, which showed significant similarity to those of Pleurotus eryngii (80% identity) and Pleurotus ostreatus Florida (60% identity). Received: May 30, 2002 / Accepted: July 10, 2002 Acknowledgment This work was supported in part by a Grant-in-Aid for the Encouragement of Young Scientists (no. 12760117) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Correspondence to:K. Okamoto     

Identification,Characterization, and Partial Purification of Glucoamylase from Aspergillus Niger (Syn A. Ficuum) NRRL 3135

Abstract

The crude extracellular extract of Aspergillus niger (syn A. ficuum) NRRL 3135 contains glucoamylase (exo-1,4-α-D-glucanohydrolase, EC 3.2.1.2). The enzyme, a glycoprotein, was purified 7-fold by ion-exchange chromatography, chromatofocusing, and conconavalin A affinity chromatography. The molecular weight of the enzyme was estimated to be 90 kDa by SDS-PAGE and gel permeation chromatography. The pI of the enzyme was 3.4. The temperature optimum of the enzyme was 60°C and the pH optimum was 5.0. The V_max values for soluble starch, maltose, maltotriose, maltotetraose, maltopentaose, and isomaltose were 55.2, 11.7, 32.3, 47.8, 59.2, 12.5 nKat glucose/sec, respectively and the K_m values for the same substrates were 0.09%, 0.67 mM, 0.76 mM, 0.76 mM, 0.68 mM, and 122.01 mM, respectively.     

In vitro propagation of Lilium testaceum and structural investigation of the storage β-1,4-glucomannan

Bulblet and callus cultures of Lilium testaceum were initiated in vitro from bulbscales. Continous propagation of the bulblet cultures was achieved on a modified Murashige and Skoog agar medium containing 1-naphthalene acetic acid (0.1 mg/l) and kinetin (0.1 mg/l) as phytohormones. The in vitro grown bulbs synthesized large quantities of storage ß-1,4-glucomannans (mannose: glucose = 73; molecular weight = 200 kd) with an identical structure to the glucomannans from the in vivo grown bulbs. Higher 1-naphthalene acetic acid concentrations (1 mg/l) resulted in increased callus formation. Liquid suspension cultures derived from callus exhibited only small amounts of reserve glucomannans.Abbreviations DEAE 2-(diethylamino)ethyl - GF growth factor - GM glucomannan - GPC gel permeation chromatography - IAA indole-3-acetic acid - IEC ion exchange chromatography - MS Murashige and Skoog - MW molecular weight - MWCO molecular weight cut off - NAA 1-naphthalene acetic acid - NMR nuclear magnetic resonance - PVP polyvinylpyrrolidone