A New Method for Strain Identification of Rhizobium trifolii in Nodules

An indirect haemagglutination test has been developed for the detection of strains of Rhizobium trifolii in nodules of subterranean clover plants. Preserved sheep red blood cells, coated with isolated specific rhizobial lipopolysaccharide, were used as the indicator of agglutination; these cells were agglutinated by specific antilipopolysaccharide antibody. Detection of lipopolysaccharide antigen in a suspension of nodular tissue was carried out by reacting the suspension with antilipopolysaccharide antibody prior to the addition of coated red blood cells. The presence of antigen in the suspension was indicated by an inhibition of agglutination. The test was more sensitive than agglutination and immunodiffusion in the detection of rhizobial lipopolysaccharide antigens, and could be used for the rapid screening of large numbers of nodules.     

Purification and characterisation of a calcium-independent lectin (PjLec) from the haemolymph of the shrimp Penaeus japonicus

A natural lectin (nominated PjLec) was isolated from haemolymph of the shrimp Penaeus japonicus by affinity chromatography with fetuin-Sepharose. The result of SDS-PAGE showed that the purified PjLec protein consisted of 37kDa subunits. The native PjLec behaved as a 452kDa protein in gel filtration chromatography. Those data suggest that PjLec is composed of 12 subunits of similar molecular weight. PjLec has a broad spectrum of bacterial-agglutination activities against both Gram-positive and Gram-negative bacteria, including two Vibrio species and two other strains pathogenic for shrimp. In addition, PjLec could agglutinate all the vertebrate erythrocytes tested, and the haemagglutination was calcium-independent. The haemagglutination of PjLec was inhibited by ManNAc, Neu5A and lipopolysaccharide. Bovine submaxillary mucin, which contains mainly Neu5A, was the most potent inhibitor of PjLec (MIC of 0.0006mgml(-1)). The haemagglutination activity of PjLec was stable between pH 6 and pH 8, and was temperature-dependent. Our results suggested that PjLec may be an important humoral defence factor against bacterial infection in P. japonicus.     

Structural relationship between the polyagglutinable antigen and the core polysaccharide of Pseudomonas aeruginosa

It has been observed that each strain of the Pseudomonas aeruginosa species harbours the so-called polyagglutinable antigen (PA). Some strains may produce it in a form which is linked to the core moiety of lipopolysaccharide (LPS) and this type of PA can thus be detected by passive haemagglutination using the isolated LPS as coating antigen. Other strains synthesize PA exclusively in a free form, which is also coextractable with LPS, its presence can, however, be demonstrated by the haemagglutination inhibition test. From a polyagglutinable strain of P. aeruginosa an R-type LPS was isolated having the core-linked PA. This LPS preparation was highly immunogenic with regard to its PA moiety. The core-bound PA seems to exert an immunosuppression on the core region, hence, the polyagglutinable strains isolated from cystic fibrosis patients only engender anti-PA antibodies, whereas antibodies against both, side chain and core region of LPS, are not engendered. The mucoid exopolysaccharide also contains the PA which could possibly play an important role in the patient by protecting P. aeruginosa cells against anti-PA antibodies.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the polysaccharide chain of Ps. aeruginosa O-serogroup 7 (Lanyi) lipopolysacharide

A loosely bound lipopolysaccharide-protein complex was extracted from cells of Pseudomonas aeruginosa strain 170015 (O:7ab; Lanyi classification) by saline solution and purified from contaminant nucleic acid by Cetavlon treatment followed by precipitation in an ultracentrifuge. The saline-treated cells were re-extracted with hot aqueous phenol to give firmly bound lipopolysaccharide which was isolated from the phenol layer and purified by ultracentrifugaiton. The identity of both lipopolysaccharide preparations was proved by serological and chemical evidence. Mild acid degradation of the lipopolysaccharide resulted in the splitting off of a lipid component and led to polysaccharide which was purified by gel-filtration on a Sephadex G-50 column. The polysaccharide consisted of N-acetyl-D-fucosamine, N-acetyl-L-fucosamine and D-glucose in the ratio 1:1:1. On the basis of nuclear magnetic resonance spectra, results of methylation analysis and two sequential Smith degradations, the following structure can be assigned to the repeating unit of the polysaccharide: -3)LFucNAc(alpha 1-3)DFucNAc(beta 1-2)DGlc(beta 1-. The polysaccharide did not show serological activity whereas alkali-treated lipopolysaccharide readily sensitised sheep erythrocytes and inhibited the passive haemagglutination reaction with anti-(O:7a,b)serum. Evidence is presented that the oligosaccharide repeating units of the polysaccharide and alkali-treated lipopolysaccharide are indistinguishable. Ps. aeruginosa strain 170016 (O:7a,c) was shown to have the O-specific lipopolysaccharide identical with that from strain 170015. The presented data show that subfactors 7b and 7c in the Lanyi classification of Ps. aeruginosa O-antigens seem to relate to components of the bacterial surface other than lipopolysaccharides.     

Properties of colony-stimulating factors produced by macrophage cell lines and hybrid cells

Colony-stimulating factors (CSFs) produced by two simian virus 40(SV40) transformed macrophage cell lines (BAM1 and BAM3), and three hybrids (HM3-11, HM3-12, and HM3-14) derived from fusion between BAM3 and a Chinese hamster cell line (hs222-16) were examined. HM3-11 and HM3-14 produce two molecular species of CSF, which are not found in the conditioned media from cultures of BAM1 and BAM3 or lipopolysaccharide (LPS), phorbolmyristate-acetate (PMA), and zymosan-stimulated BAM3. HM3-12, which is classified into another group in terms of CSF secretion, does not produce these two CSFs. On the basis of various criteria, one of these CSF species (peak 1-CSF) was characterized as a macrophage-colony-stimulating factor (M-CSF). The other CSF (peak 2-CSF) induced a group of bone marrow cells in granulocytes and macrophages as well as growth of a mast cell line, IC2. This CSF has an apparent molecular weight of 18,000, estimated by SDS-polyacrylamide gel electrophoresis. Unlike interleukin 3 (IL3) from WEHI-3 cells, the growth factor activity of peak 2-CSF binds to DEAE-Sephacel. Thus, peak 2-CSF is similar to a granulocyte-macrophage colony-stimulating factor (GM-CSF) rather than to IL3. The anti L cell CSF serum does not inhibit the CSF activity in Chinese hamster fibroblast conditioned medium, and the IC2 cells do not respond to Chinese hamster lung conditioned medium (CHLCM), suggesting that peak 1- and peak 2-CSF are of mouse origin.     

Evaluation of hepatic-metastasis risk of colorectal cancer upon the protein signature of PI3K/AKT pathway

Liver is the most common organ of colorectal cancer (CRC) metastasis, and hepatic metastasis (HM) is regulated by complex protein network. Hence, we initiated a proteomic survey to seek interrelated multiplex markers related with HM. A total of 34 unique differential proteins were identified in the primary tumor tissues from 14 CRC patients with/without HM. A differential protein cluster, consisting of 17 proteins throughout PI3K/AKT pathway, was deduced and validated by Western blot. A three-protein signature elicited from the protein cluster, phosphorylated IkappaBalpha, TNFalpha and MFAP3L, was detected by immunohistochemistry on 105 pairs of CRC and normal samples. The positive protein signature was specifically correlated with HM (P < 0.001), and classified the HM risk of CRC patients with high sensitivity (92.85 +/- 4.87%) and specificity (94.94 +/- 2.5%). The high-risk group had significantly decreased overall survival (P < 0.001). Furthermore, RKO and HT29, two colon cancer cells with different expression status of the protein signature, were used to construct the nude mouse model of HM. And the HM occurrence of RKO cell (4/5) was dramatically higher than that of HT29 cell (1/5). Therefore, the protein signature derived from PI3K/AKT pathway is likely a promising multiplex biomarker for HM of CRC.     

Characterization of cross-reacting serotypes of Campylobacter jejuni

Some strains of Campylobacter jejuni react with more than one reference antiserum from the serotyping scheme based on heat-stable lipopolysaccharide antigens. To investigate the molecular basis of these cross-reactions, lipopolysaccharides from the reference strains for serotypes 4, 13, 16, 43, and 50 and isolates recovered during two different outbreaks of C. jejuni enteritis were analyzed by passive haemagglutination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis coupled with silver staining or immunoblotting. The results showed that lipopolysaccharides from the reference strains and the isolates reacted with antisera prepared against heterologous strains in various combinations and that both silver-stainable, low Mr and non-silver-stainable, high Mr lipopolysaccharide components provided the antigenic determinants associated with the cross-reactions. There were strain-to-strain differences in the structural and antigenic properties of these macromolecules and shared antigenic determinants were not always provided by a common structure. Analysis of the silver-stained lipopolysaccharide profiles, outer membrane protein patterns, and chromosomal DNA restriction patterns indicated that strains with the same lipopolysaccharide profile could have the same outer membrane protein pattern and the same DNA restriction pattern. These results provided evidence for the presence of clones within this antigenic complex and implicated antigenic variation in some strains as the phenomenon responsible for the multiplicity of cross-reactions.     

Immunological study of antisera to to artificial O-antigen of Pseudomonas aeruginosa

O-sera were obtained by immunizing rabbits with artificial antigens: polysaccharide extracted from the lipopolysaccharide of P. aeruginosa (strain No. 868; 03a, 3d, 3e), or the high-molecular-weight or low-molecular-weight fraction of this polysaccharide, complexed with a natural protein (human IgG or rabbit globulin). The antisera to these antigenic complexes were highly O-specific. Antisera to the complexes of polysaccharide-protein and high-molecular-weight fraction-protein were more active in the passive haemagglutination reaction, slide agglutination test and immunodiffusion test in agar gel than was antiserum to the low-molecular-weight fraction-protein complex. The artificial antigens prepared and employed in the study are apt to be used for the preparation of monospecific immune sera.     

Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain

Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase. HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases. HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation. The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272. Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method. The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272.     

Immunochemical investigations of antigens isolated fromBacteroides ovatus strain ATCC 8483

A saline extract (SE) and a phenol/water extract (WL) were prepared fromBacteroides ovatus strain ATCC 8483. A fraction CS was isolated from the culture supernatant. WL was further split by ultracentrifugation into lipopolysaccharide (LPS) and supernatant (L₁). Fractions SE, WL, LPS and L₁ reacted serologically with homologous antiserum but did not cross-react with antisera against heterologousBacteroides serotypes. Fraction CS was inactive in haemagglutination, haemagglutination inhibition and immunoelectrophoresis tests. SE, WL, LPS and L₁ proved to be serologically heterogeneous. A distinct serological specificity for SE was demonstrated. The serological reactivity in SE and WL was not altered after treatment with proteolytic enzymes yet completely destroyed in WL and partially in SE by sodium metaperiodate. SE, WL, LPS and L₁ contained the sugar components rhamnose, fucose, ribose, mannose, galactose, glucose and glucosamine in different molar ratios for each fraction. Galactosamine was found in WL and LPS, uronic acid in WL and L₁. Two unidentified aminohexoses were detected in WL, one of which was also detectable in L₁ and SE. 2-Keto-3-deoxyaldonic acid was demonstrated in LPS and L₁ after strong acid hydrolysis.     

Atmospheric heavy metal input to forest soils in rural areas of Denmark

Atmospheric bulk deposition of heavy metals (HM) was measured from 1972/73 to the present time at five to ten forest sites in rural areas of Denmark. From 1979, HM in aerosols were measured at one to four forest sites. On the basis of these long-term continuous measurements, the atmospheric inputs to the forest floor have been calculated. Yearly HM emission estimates to the European atmosphere seems to correlate well with yearly average values of HM deposition, as well as with HM concentrations in the ambient atmosphere. HM emissions have been estimated since the 1950s. Using the correlation between emission and deposition, HM deposition values maybe extrapolated in reverse chronological order. The accumulated atmospheric HM deposition has been estimated in this way over a period of 50 years.     

CTRP3/cartducin promotes proliferation and migration of endothelial cells

Nicotinic acid (niacin) has been used clinically to manage dyslipidemia for many years. The molecular target of nicotinic acid was unknown until the recent revelation of human G-coupled receptor HM74a as the high affinity receptor for nicotinic acid. In searching for a cell line expressing endogenous human HM74a receptor, we have identified that the A431 cell line, a human epidermoid cell line, expresses a high level of HM74a receptor. An HM74a-specific real time PCR probe set was designed and the mRNA levels of HM74a in A431 and 32 other cultured cell lines were measured quantitatively. When the mRNA expression of HM74a in A431 cells was compared to that in human primary preadipocytes, adipocytes and adipose tissue, we found that the level in A431 was about 10- fold higher than that in adipocytes and adipose tissue. The ratio of HM74a:HM74 mRNA was measured quantitatively and it was determined to be 3:2 in A431 cells. The function of the HM74a receptor in A431 cells was evaluated for its ability to inhibit forskolin-induced cAMP production. Pertussis toxin treatment abolished the inhibition. Our data suggest that the A431 cell line may serve as a cellular model for further investigation of niacin/HM74a-mediated signal transduction in modulating metabolism. A431 cell line may also provide a valuable cell model to study prostaglandin production upon HM74a activation to improve our understanding of niacin/HM74a-mediated skin flushing. The first two author contributed equally.     

Comparative study of alleviating effects of GSH, Se and Zn under combined contamination of cadmium and chromium in rice (Oryza sativa)

A hydroponic experiment was conducted to study the ameliorative effects of separate or combined application of exogenous glutathione (GSH), selenium (Se) and zinc (Zn) upon 20 μM cadmium (Cd) plus 20 μM chromium (Cr) heavy metal stress (HM) in rice seedlings. The results showed that HM caused a marked reduction in seedling height, chlorophyll content (SPAD) and biomass, and activities of catalase (CAT) and ascorbate peroxidase (APX) in leaves and H⁺-ATPase in roots/leaves, but elevated superoxide dismutase (SOD) and guaiacol peroxidase (POD) activities in leaves with elevated malondialdehyde (MDA) accumulation both in leaves and roots over the control. The best mitigation effect was recorded in HM+GSH+Zn and HM+GSH (addition of GSH+Zn and GSH to HM solution), which greatly alleviated HM-induced growth inhibition and oxidative stress. Compared with HM alone, HM+GSH and HM+GSH+Zn markedly reduced Cr uptake and translocation but not affected Cd concentration; improved H⁺-ATPase activity and Fe, Zn, Mn uptake and translocation, and repressed MDA accumulation. Meanwhile exogenous GSH and GSH+Zn counteracted HM-induced response of antioxidant enzymes, via suppressing HM-induced dramatic increase of root/leaf SOD and leaf POD activities, and elevating stress-depressed leaf APX and leaf/root CAT activities.     

Characterization of strain specific typing antisera for genetic monitoring of inbred strains of rats

Strain-specific typing antisera (SSTA) were prepared for six inbred strains of rats by using a pooled immunization protocol. The SSTA were used in both a haemagglutination assay and a complement dependent microcytotoxicity assay to compare the usefulness of the two test systems. Both assays were simple, reliable and repeatable, and each system had distinct advantages and disadvantages. The haemagglutination assay was faster and required less specialized equipment than the microcytotoxicity assay. On the other hand, interpretation of results in the microcytotoxicity assay was easier and more objective. It was concluded that SSTA could be used with the microcytotoxicity assay and/or the haemagglutination assay to provide a simple and effective genetic monitoring method for inbred strain of rats.     

Recurrence of the T666M calcium channel CACNA1A gene mutation in familial hemiplegic migraine with progressive cerebellar ataxia.

Familial hemiplegic migraine (HM) is an autosomal dominant migraine with aura. In 20% of HM families, HM is associated with a mild permanent cerebellar ataxia (PCA). The CACNA1A gene encoding the alpha1A subunit of P/Q-type voltage-gated calcium channels is involved in 50% of unselected HM families and in all families with HM/PCA. Four CACNA1A missense mutations have been identified in HM: two in pure HM and two in HM/PCA. Different CACNA1A mutations have been identified in other autosomal dominant conditions: mutations leading to a truncated protein in episodic ataxia type 2 (EA2), small expansions of a CAG trinucleotide in spinocerebellar ataxia type 6 and also in three families with EA2 features, and, finally, a missense mutation in a single family suffering from episodic ataxia and severe progressive PCA. We screened 16 families and 3 nonfamilial case patients affected by HM/PCA for specific CACNA1A mutations and found nine families and one nonfamilial case with the same T666M mutation, one new mutation (D715E) in one family, and no CAG repeat expansion. Both T666M and D715E substitutions were absent in 12 probands belonging to pure HM families whose disease appears to be linked to CACNA1A. Finally, haplotyping with neighboring markers suggested that T666M arose through recurrent mutational events. These data could indicate that the PCA observed in 20% of HM families results from specific pathophysiologic mechanisms.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

Antiphage sera were produced in rabbits against the HM-phages of Clostridium saccharoperbutylacetonicum; on the basis of cross-neutralization experiments with homologous and heterologous antisera, the twelve HM-phages were classified into three serological groups, termed I, II and III. Group I contained seven phages, i.e., HM 1, HM 2, HM 8, HM 9, HM 10, HM 11 and HM 12. Group II contained four phages, i.e., HM 3, HM 4, HM 5 and HM 6, and group III one phage, i.e., HM 7. This classification was in accord with morphological one that was reported in the preceding paper. By using the K value of antisera, the degree of serological relatedness among the phages within groups I and II was demonstrated. On the bases of serological similarities and of dissimilarities in host-rang specificity, the phages of groups I and II are considered as host range mutants derived from an identical ancestor, HM 1 and HM 3, respectively.     

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd⁻/b⁺ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de- O -acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de- O -acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy- d - manno -octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.     

Rheology of Mixed Pectin Solutions

The effects of sucrose (S) and pectin (P) concentrations and the ratio between two distinct pectins (R) on the rheological behavior of diluted pectin systems were evaluated simultaneously using the surface response methodology. The systems were composed of a mixture of two high methoxy pectins with different degree of methyl esterification values (HM1/HM2) and of a mixture of a high-methoxy with an amidated low-methoxy pectin (HM1/LMA). For the HM1/HM2 systems, the multivariate analysis showed that the sucrose and pectin concentrations exerted statistically significant (p < 0.05) linear effects on the consistency index k and viscosity, the influence of pectin being about five times higher than that of sucrose. The pectin concentration and the ratio between the different pectins were shown to be significant with respect to the rheological parameters of the HM1/LMA systems. Evaluating the influence of the ratio between the different pectins, a synergistic effect on the structure reinforcement was observed when mixing HM1 and LMA in similar proportions, indicating the importance of the presence of hydrophobic interactions between methyl ester groups in addition to the stronger hydrogen bonding in junction zone stabilization. In general, the conditions in which hydrogen bonds were favored in relation to hydrophobic interactions led to systems with higher pseudoplasticity.     

Diversity and heavy metal tolerance of endophytic fungi from Dysphania ambrosioides,a hyperaccumulator from Pb–Zn contaminated soils

The diversity and heavy metal (HM) tolerance of endophytic fungi (EF) associated with Dysphania ambrosioides, a hyperaccumulator from two Pb–Zn contaminated sites were investigated. A total of 237 culturable EF were isolated and identified to 43 taxa based on morphological characteristics and rDNA internal transcribed spacer analysis, of which 13 occurred as endophytes of both sites, while other taxa were only found in either site. The colonization rate, dominant genera, community structure of EF as well as the HM content in the plant from two sites were significantly different. We suggest that these differences may result from the difference in the soil HM content: lower HM content in the soil, more EF in the plant, which may enhance the plant HM accumulation and thus result higher HM in it. HM tolerance tests indicated that 50% of the isolates exhibited HM tolerance. Among them, two isolates exhibited better HM tolerance, of which FT2G59 could tolerate Pb, Zn, and Cd, and the minimum inhibitory concentration (MIC) of them were 30–50, >?680, 20–30?mmol/l, respectively. While, the isolate FT2G7 could tolerate Cd, and the MIC was 30–50?mmol/l. These isolates may have potential application in phytoremediation.