Cryptosporidium parvum: Radiation-induced alteration of the oocyst proteome

Cryptosporidium parvum is a waterborne protozoan parasite that is found intracellularly in host animals, including humans, and causes severe diarrhea, which can lead to the death of an immunocompromised individual. Previously, we found that this organism is highly radioresistant as it can productively infect mice after exposure to a 10-kGy dose of γ-radiation.To understand how C. parvum avoids radiation damage, we characterized its protein expression patterns 6, 24, and 48 h after a 10-kGy dose of γ-radiation using two-dimensional PAGE. The gels showed 10 silver-stained spots that increased or decreased in size following γ-irradiation. Five proteins contained in these spots were identified using MALDI-TOF MS peptide fingerprinting, and two of these showed an increase in expression after γ-irradiation. These proteins were identified by LC–MS/MS as proteasome subunit alpha type 4 (NTN hydrolase fold) and thioredoxin peroxidase-like protein. The roles of these two upregulated proteins as related to the radioresistance of C. parvum remain to be evaluated.     

Recombinant thioredoxin peroxidase from Cryptosporidium parvum has more powerful antioxidant activity than that from Cryptosporidium muris

Cryptosporidium parvum can survive exposure to harsh environmental conditions, various disinfectants, and high doses of γ-irradiation. In an animal study, more than 25kGy of γ-irradiation was necessary to eliminate C. parvum infectivity from mice. In contrast, Cryptosporidium muris (murine Cryptosporidium), which lives in stomach epithelium, lost its infectivity in mice with 1kGy of γ-irradiation. Recently, it was found that thioredoxin peroxidase was highly expressed in C. parvum oocysts irradiated with high doses of γ-irradiation. Therefore we hypothesize that antioxidant activity of the thioredoxin peroxidase is involved in the radioresistance of C. parvum. To verify this, thioredoxin peroxidases of C. parvum (CpTPx) and C. muris (CmTPx) were expressed in Escherichia coli cells, and their antioxidant activities were compared. Both CpTPx and CmTPx belong to the 2-Cys family of peroxiredoxins. Hydrogen peroxide consumption was approximately 2- to 12-fold greater in recombinant CpTPx (rCpTPx) than in recombinant CmTPx (rCmTPx) in the presence of 0.2mM dithioerythritol or glutathione (GSH), respectively. The peroxidase activity of rCpTPx was highly enhanced by GSH, but that of rCmTPx was not. The minimum dose of rCpTPx required to protect supercoiled plasmid DNA from damage by metal-catalyzed oxidation was only 12% of that required with rCmTPx. The results showed that rCpTPx has more powerful antioxidant activity than rCmTPx. Further investigations on the role of CpTPx in the radioresistance of C. parvum are warranted.     

Effect of various environmental factors on the viability of Cryptosporidium parvum oocysts

Aims: To evaluate individual and combined effects of temperature (4, 18 and 25°C), pH (7 and 10), ammonia (5 and 50 mg l^?1) and exposure time (1, 2, 4 and 6 days) on the viability of Cryptosporidium parvum oocysts in water. Methods and Results: The viability of oocysts was evaluated using the fluorogenic vital dyes assay (4′,6‐diamidino‐2‐phenylindole and propidium iodide). All the factors analysed (temperature, pH, ammonia and exposure time) and their interaction were statistically significant (P < 0·005). Exposure of oocysts to pH 10 for 6 days at 25°C reduced oocyst viability from ～80% to 51%. Similarly, the exposure of C. parvum oocysts to 5 mg NH₃ l^?1 and 50 mg NH₃ l^?1 for 4 days reduced their viability from between ～80% to 41·5% and 14·8%, respectively. Conclusions: The interaction between pH, temperature and exposure time may have adverse effects on the survival of C. parvum oocysts in water. Low concentrations of ammonia, as commonly found in alga‐based wastewater systems, over a long period of time can produce high C. parvum oocyst inactivation rates. Significance and Impact of the Study: This study provides relevant data on the inactivation of C. parvum oocysts in alga‐based wastewater‐treatment systems in the northwest of Spain.     

Intra-isolate Heterogeneity and Reproducibility of PCR-based Genotyping of Cryptosporidium parvum Using the β-tubulin Gene

Cryptosporidium parvum is a common contaminant in surface waters and presents significant problems for the water industry, public health and agriculture. Consequently, ascertaining the contaminating source of waterborne oocysts is of paramount importance. Based on currently available information, isolates of C. parvum can be differentiated into at least two genotypes using polymorphic genetic markers: genotype 1, to date isolated almost exclusively from humans, and genotype 2 isolates from humans and many other animals. Differentiation into these two genotypes has been based on either restriction fragment length polymorphisms or sequencing of PCR amplified gene fragments. The objective of this study was to evaluate the reproducibility of genotyping methods using a single isolate of C. parvum. A 620 bp fragment of the C. parvum -tubulin gene, generated by PCR from multiple aliquots of a single preparation of oocysts of the Iowa isolate, was sequenced. Significant sequence heterogeneity was detected within this single isolate; there was more sequence variation between clones originating from the Iowa isolate (up to 0.9 %) than between individual clones originating from different isolates of C. parvum. Over 6 % of the -tubulin gene sequence positions (38 out of 620 bp) were variable when comparing multiple clones from the one isolate. The results indicated that while the various procedures used for genotyping isolates may introduce some sequence errors, the Iowa isolate used for this investigation appeared to be composed of multiple sub-genotypes. While none of the sequence variations resulted in clones of the Iowa isolate (genotype 2) being mis-identified as genotype 1, the results have important implications if minor sequence variations are to be used for subtyping isolates and drawing conclusions regarding the origin of, or relationships between, C. parvum oocysts in water and the community.     

Serological changes in adjuvant-treated mice,their specificity and relevance to tumor immunity

Summary The effects of a variety of adjuvant protocols on immunoglobulin levels in normal and tumor bearing CBA mice have been investigated together with their ability to elicit immunoglobulin which bind to tumor cells in vitro and inhibit the growth of a transplanted syngeneic MC-induced fibrosarcoma.A marked increase in serum levels of certain immunoglobulins (especially IgG _2a , IgG _2b and IgM) and immunoglobulin interacting with tumor cells in vitro was noted in normal and tumor bearing mice following the administration of C. parvum (strain No. CN6134 and 10387), P. freudenreichii (strain No. 10470) and B. pertussis while a modest increase in some of these accompanied BCG injection. The Freund's complete and incomplete adjuvant protocols adopted had little effect on any of these parameters. The C. parvum protocols alone inhibited tumor growth.The immunoglobulins evoked by C. parvum strain No. 6134 which bound to tumor cells in vitro were extremely heterogeneous, activity being detected in all Ig classes and subclasses. This organism also evoked immunoglobulins which interacted with syngeneic embryonic fibroblasts, and adult syngeneic kidney and spleen cells.Tumor cells which had been preincubated in sera rich in immunoglobulins binding to tumor cells in vitro (i.e. from C. parvum-treated mice) did not exhibit reduced growth following i.v. or s.c. transplantation to syngeneic recipients.     

Effects of managanese salts on the AIDS-related pathogen,Cryptosporidium parvum in vitro and in vivo

The authors examined the effects of manganese salts on the interaction of the AIDS-related pathogen,Cryptosporidium parvum, with human ileoadenocarcinoma (HCT-8) cells in vitro. Manganese (Mn) inhibited binding ofC. parvum sporozoite membrane antigens to intact, fixed HCT-8 cells in a dose-dependent fashion, whereas Ca⁺⁺, Mg⁺⁺, and Zn⁺⁺ salts had no effect. Manganese was also found to affect sporozoite penetration of live HCT-8 cells, which resulted in a dose-dependent inhibition of parasite development. However, the levels of Mn⁺⁺ needed in the live cell assays was approx 10-fold greater than in the fixed-cell assays. This inhibition of parasite development was not reversible when Ca⁺⁺ or Mg⁺⁺ were used as competitors. Oral supplementation of suckling mice infected withC. parvum with MnSO₄ resulted in significant reductions and, in some cases, elimination of intestinally derived oocysts.     

Evaluation of the co-agglutination test in diagnosis of experimental microsporidiosis

Microsporidiosis is an emerging and opportunistic infection associated with wide range of clinical syndromes in humans. Confirmation of the presence of microsporidia in different samples is laborious, costly and often difficult. The present study was designed to evaluate the utility of the Co-agglutination test (Co-A test) for detection of urinary, fecal and circulating microsporidial antigens in experimentally infected mice. One hundred and twenty male Swiss albino mice were divided into non infected control and infected experimental groups which were further subdivided into two equal subgroups; immunosuppressed and immunocompetent. Microsporidial spores were isolated from human stools and identified to be Encephalitozoon intestinalis by the molecular methods. They were used to infect each subgroup of mice, then their urine, stools and sera were collected at the 1st, 3rd, 5th, 7th and 9th days post-infection (PI). Co-A test, using prepared hyperimmune serum, was used to detect antigens in all samples collected. The cross reactivity of microsporidial hyperimmune sera with antigens of Cyclospora cyatenensis and Cryptosporidium parvum was investigated by Co-A test. The results showed that Co-A test was effective in detecting microsporidial antigen in stool of immunosuppressed infected mice from the 1st day PI, and in urine and serum from the 3rd day PI till the end of the study. In the immunocompetent subgroup, Co-A test detected microsporidial antigens in stool, serum and urine of mice from the 1st day, 3rd day and the 5th day PI, respectively till the end of the study, without cross reactivity with C. cyatenensis or C. parvum in both subgroups. Co-A test proved to be simple and suitable tool for detecting microsporidial antigen in different specimens and did not need sophisticated equipment. It is very practical under field or rural conditions and in poorly equipped clinical laboratories.     

The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro

The protozoan parasites Giardia duodenalis and Cryptosporidium parvum are common causes of diarrhoea, worldwide. Effective drug treatment is available for G. duodenalis, but with anecdotal evidence of resistance or reduced compliance. There is no effective specific chemotherapeutic intervention for Cryptosporidium. Recently, there has been renewed interest in the antimicrobial properties of berries and their phenolic compounds but little work has been done on their antiparasitic actions. The effect of various preparations of blueberry (Vaccinium myrtillus) extract on G. duodenalis trophozoites and C. parvum oocysts were investigated. Pressed blueberry extract, a polyphenolic-rich blueberry extract, and a commercially produced blueberry drink (Bouvrage) all demonstrated antigiardial activity. The polyphenol-rich blueberry extract reduced trophozoite viability in a dose dependent manner. At 167 μg ml⁻¹, this extract performed as well as all dilutions of pressed blueberry extract and the Bouvrage beverage (9.6 ± 2.8% live trophozoites remaining after 24 h incubation). The lowest dilution of blueberry extract tested (12.5% v/v) contained >167 μg ml⁻¹ of polyphenolic compounds suggesting that polyphenols are responsible for the reduced survival of G. duodenalis trophozoites. The pressed blueberry extract, Bouvrage beverage and the polyphenolic-rich blueberry extract increased the spontaneous excystation of C. parvum oocysts at 37 °C, compared to controls, but only at a dilution of 50% Bouvrage beverage, equivalent to 213 μg ml⁻¹ gallic acid equivalents in the polyphenolic-rich blueberry extract. Above this level, spontaneous excystation is decreased. We conclude that water soluble extracts of blueberries can kill G. duodenalis trophozoites and modify the morphology of G. duodenalis and C. parvum.     

Methanocorpusculaceae fam. nov., represented by Methanocorpusculum parvum,Methanocorpusculum sinense spec. nov. and Methanocorpusculum bavaricum spec. nov.

Two new methanogenic bacteria, Methanocorpusculum sinense spec. nov. strain DSM 4274 from a pilot plant for treatment of distillery wastewater in Chengdu (Province Sichuan, China), and Methanocorpusculum bavaricum spec. nov. strain DSM 4179, from a wastewater pond of the sugar factory in Regensburg (Bavaria, FRG) are described. Methanocorpusculum strains are weakly motile and form irregularly coccoid cells, about 1 μm in diameter. The cell envelope consists of a cytoplasmic membrane and a S-layer, composed of hexagonally arranged glycoprotein subunits with molecular weights of 90000 (Methanocorpusculum parvum), 92000 (M. sinense), and 94000 (M. bavaricum). The center-to-center spacings are 14.3 nm, 15.8 nm and 16.0 nm, respectively. Optimal growth of strains is obtained in the mesophilic temperature range and at a pH around 7. Methane is produced from H₂/CO₂, formate, 2-propanol/CO₂ and 2-butanol/CO₂ by M. parvum and M. bavaricum, whereas M. sinense can only utilize H₂/CO₂ and formate. Growth of M. sinense and M. bavaricum is dependent on the presence of clarified rumen fluid. The G+C content of the DNA of the three strains is ranging from 47.7–53.6 mol% as determined by different methods. A similar, but distinct polar lipid pattern indicates a close relationship between the three Methanocorpusculum species. The polyamine patterns of M. parvum, M. sinense and M. bavaricum are similar, but distinct from those of other methanogens and are characterized by a high concentration of the otherwise rare 1,3-diaminopropane. Quantitative comparison of the antigenic fingerprint of members of Methanocorpusculum revealed no antigenic relationship with any one of the reference methanogens tested. On the basis of the distant phylogenetic position of M. parvum and the data presented in this paper a new family, the Methanocorpusculaceae fam. nov., is defined.     

High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc-(His)₆ tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested post-translational modifications may contribute to the size difference. The NH₂-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.     

Purification and characterization of lignin peroxidases from <Emphasis Type="Italic">Penicillium decumbens</Emphasis> P6

Summary Peroxidases are essential enzymes in biodegradation of lignin and lignite which have been investigated intensively in the white-rot fungi. This is the first report of purification and characterization of lignin peroxidase from Penicillium sp. P6 as lignite degradation fungus. The results indicated that the lignin peroxidase of Penicillium decumbens P6 had physical and chemical properties and a N-terminal amino acid sequence different from the lignin peroxidases of white-rot fungi. The lignin peroxidase was isolated from a liquid culture of P. decumbens P6. This enzyme had a molecular weight of 46.3 KDa in SDS-PAGE and exhibited greater activity, temperature stability and wider pH range than those previously reported. The isolation procedure involved (NH₄)₂SO₄ precipitation, ion-exchange chromatography on DEAE-cellulose and CM-cellulose, gel filtration on Sephadex G-100, and non-denaturing, discontinuous polyacrylamide gel electrophoresis. The K _m and V _max values of this enzyme using veratryl alcohol as substrate were 0.565 mmol L ⁻¹ and 0.088 mmol (mg protein) ⁻¹ min ⁻¹ respectively. The optimum pH of P6 lignin peroxidase was 4.0, and 70.6 of the relative activity was remained at pH 9.0. The optimum temperature of the enzyme was 45 °C.     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.     

Measurement of OH radical CT for inactivating Cryptosporidium parvum using photo/ferrioxalate and photo/TiO2 systems

Aim: This study investigates the inactivation of Cryptosporidium parvum using the OH radical and reports the OH radical CT (OH radical concentration × contact time) values for C. parvum inactivation. Methods and Results: Although a wealth of information has demonstrated the efficacy of the microbial inactivation activity of the OH radical, no studies have performed a quantitative estimation of the OH radical for C. parvum inactivation. The CT value of the OH radical required for 2 log C. parvum inactivation was measured with two OH radical‐generating systems, photo/ferrioxalate and photo/TiO₂. The OH radical was approx. 10⁴–10⁷‐fold more effective for microbial inactivation than other popular chemical disinfectants such as ozone, chlorine dioxide and free chlorine. Conclusions: The OH radical appears to be suitable for microbial inactivation with a calculated CT value required for 2 log C. parvum inactivation of 9·3 × 10^?5 mg min l^?1. Significance and Impact of the Study: This study is the first report of an investigation on the role of the OH radical in the photo/ferrioxalate and photo/TiO₂ systems and on the OH radical CT required for C. parvum inactivation.     

Selection and identification of a new adhesion protein of Cryptosporidium parvum from a cDNA library by ribosome display

In this study, we described a novel display method to identify surface adhesion proteins of Cryptosporidium parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum was expressed on ribosome and selectively and specifically screened with intestinal epithelial cells (IECs) from newborn Cryptosporidium-free Holstein calves. Proteins were then enriched using a multi-step panning procedure. A new surface adherence protein of C. parvum was selected, named Cp20. Sequence analyses showed that Cp20 has a N-terminal signal peptide and four transmembrane regions. Indirect immunofluorescence assay (IFA) using an antibody specific for rCp20 demonstrated that the antibody specifically bound to the surface of sporozoites and oocysts. The recombinant plasmid pVAX1-Cp20 was constructed to examine the potential of the Cp20 gene as a target for specific preventive and therapeutic measures for cryptosporidiosis. The in vivo efficacies of the DNA vaccine was tested in BALB/c mice. The results indicated that the DNA vaccine elicited significant antibody responses and specific cellular responses when compared to control mice that received vector only or PBS. The DNA vaccine induced strong protective immune response against C. parvum and lower level of the oocysts shedding after challenge infection. This study suggested that Cp20 could serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.     

Mutational and kinetic analysis of basic amino acid transport in the cyanobacterium Synechocystis sp. PCC 6803

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (¹⁴C-synthetic lignin ¹⁴CO₂), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the otherhand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H₂O₂ in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium. The results of this study indicate that the der mutants are nitrogen-deregulated for the production of a set of secondary metabolic activities associated with lignin degradation such as lignin peroxidases, manganese peroxidases and glucose oxidase.     

Single amino acid substitutions at the acyl-CoA-binding domain interrupt 14[C]palmitoyl-CoA binding of ACBP2, an Arabidopsis acyl-CoA-binding protein with ankyrin repeats

Cytosolic acyl-CoA-binding proteins (ACBPs) are small proteins (ca. 10 kDa) that bind long-chain acyl-CoAs and are involved in the storage and intracellular transport of acyl-CoAs. Previously, we have characterized an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP, designated ACBP1, demonstrating the existence of a new form of ACBP in plants (M.-L. Chye, Plant Mol. Biol. 38 (1998) 827–838). ACBP1 likely participates in intermembrane lipid transport from the ER to the plasma membrane, where it could maintain a membrane-associated acyl pool (Chye et al., Plant J. 18 (1999) 205–214). Here we report the isolation of cDNAs encoding ACBP2 (M _r 38 479) that shows conservation in the acyl-CoA-binding domain to previously reported ACBPs, and contains ankyrin repeats at its carboxy terminus. These repeats, which likely mediate protein-protein interactions, could constitute a potential docking site in ACBP2 for an enzyme that uses acyl-CoAs as substrate. In vitro binding assays on recombinant (His)₆-ACBP2 expressed in Escherichia coli show that it binds ¹⁴[C]palmitoyl-CoA preferentially to ¹⁴[C]oleoyl-CoA. Analysis of the acyl-CoA-binding domain in ACBP2 was carried out by in vitro mutagenesis. Mutant forms of recombinant (His)₆-ACBP2 with single amino acid substitutions at conserved residues within the acyl-CoA-binding domain were less effective in binding ¹⁴[C]palmitoyl-CoA. Northern blot analysis showed that the 1.6 kb ACBP2 mRNA, like that of ACBP1, is expressed in all plant organs. Analysis of the ACBP2 promoter revealed that, like the ACBP1 promoter, it lacks a TATA box suggesting the possibility of a housekeeping function for ACBP2 in plant lipid metabolism.     

Molecular modeling and dynamics simulation of a histidine-tagged cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript>

Although an affinity tag such as six consecutive histidines, (His)₆-tag, has been widely used to obtain high quantity of recombinant proteins, little is known about its influences on heme proteins for lack of structural information. When (His)₆-tag was introduced to the N-terminus of a small heme protein, cytochrome b ₅, experimental results showed the resultant protein, (His)₆-cyt b ₅, has similar property and function to that of isolated cyt b ₅. To provide structural information for this observation, we herein performed a structural prediction of (His)₆-cyt b ₅ by molecular modeling in combination with molecular dynamics simulation. The predicted structure, as assessed by a series of criteria with good quality, reveals that the (His)₆-tag adopts a helical conformation and packs against the hydrophobic core 2 of cyt b ₅ through salt bridges, hydrogen bonding and hydrophobic interactions. The heme group, with the axial His ligands slightly rotated, was found to have similar conformation as in isolated cyt b ₅, which indicates that the N-terminal (His)₆-tag does not alter the heme active site, resulting in similar dynamics properties for core 1. This study provides valuable information of interactions between (His)₆-tag and the rest of the protein, aiding in rational design and application of functional His-tagged proteins.     

Endothelial heparan sulphate: Compositional analysis and comparison of chains from different proteoglycan populations

From cultures of human umbilical vein endothelial cells incubated with³H-glucosamine or³⁵S-sulphate, we have purified three heparan sulphate proteoglycans: 1) a low density (1.31 g/ml) proteoglycan from the cell extract, 2) a low density proteoglycan from the medium, and 3) a high density (>1.4 g/ml) proteoglycan from the medium. The disaccharide composition of heparan sulphate chains from the low density proteoglycan of the medium was examined, using specific chemical and enzymic degradations followed by gel chromatography and strong anion exchange HPLC. Chains released from each of the different proteoglycan populations were then compared by gel chromatography and gradient polyacrylamide gel electrophoresis before and after various specific degradations. The results indicate that heparan sulphate from human endothelial cells are large polymers (MW>50,000) of low overall sulphation (32–35%N-sulphated glucosamine and an N/O-linked sulphate ratio of 2.0) with rare and solitary heparin-like disaccharides. Heparan sulphate from the different proteoglycan populations appeared to have similar structure except that chains from the high density fraction were larger polymers.Abbreviations HSPG heparan sulphate proteoglycan - DSPG dermatan sulphate proteoglycan - GlcNAc(6S) N-acetylglucosamine 6-sulphate - GlcNAc6R glucosamine with either-OH or-OSO₃ at C-6 - GlcNR glucosamine with either-SO₃ or-COCH₃ as N-substituent - GlcNSO₃ N-sulphated glucosamine - GlcNSO₃(3S) N-sulphated glucosamine 3-sulphate - GlcA d-glucuronic acid - IdoA l-iduronic acid - IdoA(2S) iduronic acid 2-sulphate - HexA hexuronic acid - DHexA hexuronic acid with a 4,5-double bond - Xyl xylose - SAX strong anion exchange - d.p. degree of polymerization (a disaccharide has d.p.=1 etc) - AUFS absorbance units full scale The codes used for proteoglycans denote in turn: C 2, low-density (1.35–1.28 g/ml) HSPG from the cell extract; M 1a, high density (>1.4 g/ml) HSPG fraction from the spent medium; M 2a, low-density (1.31 g/ml) HSPG from the spent medium [6].     

The effect of combination treatment with alpha-difluoromethylornithine and Corynebacterium parvum on B16 melanoma growth and tumoricidal effector cell generation in vivo

Summary The objective of the present investigation was to establish whether a known lymphoreticular-stimulating agent Corynebacterium parvum would augment the established antitumor activity of -difluoromethylornithine in vivo. Furthermore, since C. parvum is known to boost cell mediated cytotoxicity, the effect of DFMO (DL--difluoromethylornithine·HCl·H₂O) treatment was evaluated on macrophage and natural killer (NK) cell tumoricidal activity. DFMO administered alone, 1% or 2% in drinking water, inhibited 49.4% or 88.0% of B16 melanoma growth in vivo, respectively. Administration of C. parvum alone, three doses of 300 g each, inhibited tumor growth 57.4%. When administered together, DFMO and C. parvum treatment resulted in 89.8% (1% DFMO) or 97.4% (2% DFMO) inhibition of melanoma growth depending upon the dose of DFMO. C. parvum-treated animals had increased levels of macrophage-mediated tumoricidal activity directed against B16 melanoma cells in vitro, however, NK cell activity was reduced. DFMO treatment alone had no effect on macrophage or NK cell tumoricidal activity. In animals receiving both C. parvum and DFMO treatments macrophage-mediated tumoricidal activity was augmented. These results demonstrate that C. parvum can augment the antitumor activity of DFMO in vivo, possibly through macrophage activation. Furthermore, in contrast to many other cancer chemotherapeutic drugs, DFMO is apparently not immunosuppressive regarding tumoricidal effector cells.     

Close linkage between mouse genes determining the two forms of complement component C6 and component C7, and cis action of a C6 Regulatory Gene

Two forms of mouse complement component C6, with molecular weights (M _rs) of 90 and 100 kilodaltons (kd), are present in the sera from certain inbred strains such as the CBA strain; other strains, such as the BALB/c and DBA/2 strains, have only the 90 kd C6A form. The present work was undertaken to determine whether the two M _r forms were the products of genes coding at separate loci. We screened sera from mice from a number of inbred strains by isoelectric focusing and found one strain, AKR, exhibiting allotypic structural variations of C6 forms. To distinguish the various types, we designated the 90 kd types from CBA and AKR mice C6A1 and C6A2, respectively, and the corresponding 100 kd types C6B 1 and C6B2, respectively. Mice possessing only one M _r form were all typed as C6A1. Results of breeding experiments strongly suggested that the two M _r forms of C6 are coded for at two closely linked loci. Sera from a number of inbred strains were also screened for a complement C7 polymorphism by means of isoelectric focusing and functional overlay. C7 from all strains, excepting the AKR strain, produced identical C7 band patterns. AKR C7 produced a unique band pattern, and results of breeding experiments with AKR and BALB/c mice showed the C6 and C7 loci to be closely linked. In addition, we identified a regulatory gene for C6 production. The gene apparently requires androgen to facilitate C6 production in the majority of strains. In these strains C6 activity is virtually absent from female sera. However, we observed moderate levels of C6 activity in sera from IS/Cam females, indicating that, in this strain, male physiological androgen levels are not necessary for C6 production. IS/Cam possess one form of circulating C6 which appears identical with BALB/c C6A1, and therefore IS/Cam mice differ from AKR mice at both the C6 structural and regulatory loci. These two strains were thus suitable for use in breeding experiments to determine the manner of action of the regulatory gene. Results showed that it acted in a cis manner.Abbreviations used in this paper M _r molecular weight - kd kilodaltons - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - IEF isoelectric focusing - Slp sex-limited protein - MHC major histocompatibility complex