Purified Cell Wall Fraction Research Articles

Mycobacterium bovis BCG cell wall-specific differentially expressed genes identified by differential display and cDNA subtraction in human macrophages.

We have analyzed the gene expression profile of monocytes in response to a highly purified cell wall fraction of Mycobacterium bovis BCG, a clinically approved adjuvant known as BCG cell wall skeleton (BCG-CWS). It is composed of mycolic acid, arabinogalactan, and peptidoglycan and confers Toll-like receptor 2 (TLR2)- and TLR4-dependent signaling that induces monocytes to differentiate into antigen-presenting cells (APCs). Here we report differential gene expression analysis with BCG-CWS-stimulated versus nonstimulated monocytes. BCG-CWS exerted massive induction of genes regulated by TLR signaling. Marked gene regulatory characteristics in BCG-CWS-stimulated cells compared to lipopolysaccharide (LPS)-stimulated cells follow. (i) Spliced mRNAs encoding soluble forms of TREM-1 and TREM-2 (recently discovered inflammatory-signal-amplifying receptors) were regulated by BCG-CWS, resulting in their differential expression. (ii) The genes for zinc-iron transporter protein (ZIP)-like family proteins HKE-1 and LIV-1 were induced exclusively by BCG-CWS. (iii) Interleukin-23 (IL-23), rather than IL-12p70, was induced by BCG-CWS, while interferon-inducible genes were induced only by LPS. By Northern and reverse transcription-PCR analyses, we confirmed the differential expression of more than 30 BCG-CWS regulatory genes, and their expression was compared with that of LPS and other known TLR ligands. A battery of genes responded rapidly and for a short time to LPS but for a long time to BCG-CWS. Structural analysis of the identified novel or hypothetical proteins revealed that some are potential candidates as signaling mediators or transcriptional regulators. Hence, BCG-CWS may profoundly modulate APC responses in a way distinct from that of LPS, leading to possible advantages for its adjuvant-active therapeutic potential.

Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtii y1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins.

Purification and characterization of extracellular and cell wall bound beta-glucosidases from Aspergillus kawachii.

We isolated two extracellular beta-glucosidases (EX-1: 145 kDa, EX-2: 130 kDa) and one cell wall bound beta-glucosidase (CB-1: 120 kDa) from Aspergillus kawachii and characterized their physical and kinetic properties. From the results of N-terminal amino acid sequence, enzymatic parameters and deglycosylation of the three purified enzymes, we strongly suggest that these three enzymes were products of the same gene, modified by different degree of glycosylation. All three purified beta-glucosidases adsorbed to the purified cell wall fraction of this strain. This association could dramatically improve the stability of purified enzymes. These three purified beta-glucosidases were readily inactivated even in moderate conditions but became very stable upon the addition of the purified cell wall fraction. The all purified beta-glucosidases were stable in the range of pH 2.0-9.0 and stable below 30 degrees C with 2 mg/ml of the purified cell wall fraction.

Purification and properties of cephalosporin-C deacetylase from the yeast, Rhodotorula glutinis 38B1, useful for bioconversion of 7-aminocephalosporanic acid derivatives

A 7-aminocephalosporanic acid (7-ACA) deacetylating enzyme was purified to homogeneity from Rhodotorula glutinis 38B1, whose resting cells have been previously reported as useful for the conversion of 7-ACA derivatives [Sakai et al., Appl. Environ. Microbiol., 62, 2667–2672, 1996]. The purified enzyme was a dimer comprised of identical subunits with a molecular mass of 82 kDa. The purified enzyme used cephalosporin C and several 7-ACA derivatives with low K m and high k cat values as substrates, as well as some acetyl esters with relatively long-chain alcohols. Based on this substrate specificity, the enzyme is classified as cephalosporin-C deacetylase (EC 3.1.1.41). The enzyme was most active at 35°C and pH 5.5, and was inhibited by several serine enzyme inhibitors. The purified enzyme was glycosylated on the addition of an asparagine-linked “hybrid type” oligosaccharide, and most of the enzyme activity was found in the purified cell wall fraction. The enzyme localization and kinetic properties explain the high efficiency of 7-ACA deacetylation in a resting-cell reaction.

A comparison of the kinetics of aluminum (Al) uptake and distribution in roots of wheat (Triticum aestivum) using different aluminum sources. A revision of the operational definition of symplastic Al

The lack of information about the movement of aluminum (Al) across the plasma membrane presents a significant barrier to the elucidation of resistance mechanisms which may involve exclusion of Al from the symplast. An understanding of mechanistic aspects of exclusion requires the estimation of symplastic Al levels. Such measurements may be achievable through the use of a kinetic approach. A kinetic protocol was developed to characterize the accumulation and distribution of Al in various cellular compartments in roots of wheat (Triticum aestivum L.). The kinetics of uptake and desorption were similar when Al was supplied as AIK(SO4)2 or as AlCl3. When both salts were supplied at low concentration (50 μM), Al bound to a purified cell wall fraction could be reduced to less than 10–20% of non‐exchangeable Al, if roots were washed for 30 min in citric acid following exposure. In contrast, when AlK(SO4)2 was supplied at a high concentration (200 μM), a strong linear phase of uptake into cell wall material was observed, which accounted for approximately 48% of non‐exchangeable Al in roots. These results suggest that the use of low concentrations of Al in simple salt solutions is required to minimize accumulation of non‐exchangeable Al in the apoplasm. A series of multiple‐desorption experiments confirmed that citric acid was effective in removing Al from the cell wall compartment of roots exposed to Al for short periods (3 h). However, long exposures (48 h) appeared to create conditions conducive to the accumulation of non‐exchangeable Al in the cell wall. In experiments where uptake from solutions containing 50 μM AlCl3 was followed by desorption in citric acid, non‐exchangeable Al in microsomal membrane fractions represented less than 4% of total non‐exchangeable Al. Thus, we can exclude the plasma membrane and cell wall as major sites for accumulation of non‐exchangeable Al in short exposure studies. Although we cannot provide unequivocal evidence for the localization of Al within the symplast, use of simple salt solutions followed by desorption in citric acid provides the best kinetic technique currently available for the quantitation of Al in the symplasm.

Effect of fluchloralin on protein synthesis, free amino acids and hydroxyproline content in groundnut (Arachis hypogaea L.)

SUMMARYCultivar TMV‐2 of groundnut plant {Arachis hypogaea L.) was grown in a nutrient solution containing fluchloralin at the rate of either 2 mg litre‐1 or 4 mg litre“1. Protein synthesis and hydroxyproline content in the cell walls of roots, stem and leaves were determined. Free amino acids content and total ammonia in leaves and roots were also analysed. Presence of fluchloralin did not adversely affect protein synthesis. No significant effect of herbicide was observed on hydroxyproline content of a purified cell wall fraction of groundnut roots, stem and leaves. The total amount of ammonia increased in roots and leaves of plants which received the higher concentration of fluchloralin. With the exception of aspartic acid, asparagine, glutamic acid and glutamine, free amino acids content decreased considerably with herbicide treatment. Alanine and glycine were strongly reduced. It is suggested that transamination reactions could be affected and the process of senescence may be enhanced.

Kinetics of Aluminum Uptake in Triticum aestivum L

The identity of a linear phase of aluminum (Al) uptake in Triticum aestivum was investigated by analysis of the kinetics of Al uptake by excised roots and purified cell wall fractions. Classical interpretation of kinetic data suggests that a linear phase of uptake with time reflects uptake across the plasma membrane; however, in studies with Al the possibility that the linear phase of uptake includes accumulation of Al in both the symplasm and the apoplasm has not been discounted. In our experiments, we observed a linear phase of Al uptake at both ambient and low temperatures, although the rate of uptake at 0 degrees C was 53 to 72% less than at 23 degrees C, depending on cultivars. This nonsaturable phase of uptake at low temperature suggests that a portion of the linear phase of Al uptake is nonmetabolic. Furthermore, analysis of Al in cell wall fractions isolated from excised roots pretreated with Al suggests that the linear phase of uptake includes a cell wall component. When excised roots were pretreated with Al, accumulation of Al in purified cell wall material included a linear phase that could not be desorbed with a 30 minute wash in citrate. The rates of linear-phase accumulation of Al by cell wall material and cell contents were similar. In contrast, the linear phase of in vitro uptake of Al by purified cell wall material was completely desorbed by a 30 minute wash with citrate. These results suggest that the linear phase of Al uptake observed in excised roots of T. aestivum included metabolism-dependent binding of Al in apoplasm.

Filamentous fungi as metal biosorbents: A review

Common filamentous fungi (e.g. Penicillium spp, Rhizopus spp) serve as sources of highly effective heavy metal biosorbents. Derivatives of fungal biomass range from purified cell wall fractions to whole, heat killed mycelium otherwise unmodified. Limited trials suggest a moderate degree of regenerability. When compared with commercial ion-exchange resins, carbons and metal oxides, fungal derivatives generally perform well. Fungal systems offer considerable versatility with respect to metals taken up and bound including the cations Fe, Ni, Cu, Zn, Ag, Cd, La, Pb, Th, and U as well as Cr and Mo anions. Versatility also extends to physico-chemical conditions such as pH and heat tolerance. A deterrent to the application of these materials to waste water management has been a lack of standardized conditions along with the inability to make interspecies comparisons. However, potentials for use of filamentous fungi as biosorbents and their tailoring by genetic techniques to meet specific needs, recommend them for further research and development.

Hydroxyproline and proline content of cell walls of sunflower, peanut and cotton grown under salt stress

Proline and hydroxyproline content of the cell walls of peanut ( Arachis hypogea L. cv. Shulamit), cotton ( Gossypium hirsutum L. cv. sj) and sunflower ( Helianthus annuus L. cv. Saffola SO222) were determined. Proline concentration in the leaves of all plants species tested was approximately 110 nmol · mg −1 dry matter and higher than in the root and stem. Hydroxyproline concentration was highest in the root of all species tested and was between 30 and 60 nmol·mg −1 dry matter. This was 5–10 times higher than the concentration in the stem and leaf. There was no significant effect of salt stress on proline and hydroxyproline concentration in “purified cell wall fraction” of sunflower. Salt stress (100 mmol·l −1NaCl) reduced substantially plant growth of 53-day-old sunflowers, stem volume decreased from 21.3 ± 5.2 ml to 3.8 ± 1.6 ml, dry matter content of stem decreased from 719 ± 186 mg to 88±33 mg and the number of leaves per plant decreased from 18 to 13. These results suggest that inhibition of plant growth, by salt stress, was accompanied by inhibition of cell wall proteins (extensins) synthesis. Therefore, changes in the physicochemical properties of cell wall accompanying the osmotic adjustment should be sought in other posttranslational modifications of extensin(s), either glycosylation or inter-and/or intramolecular cross-linking in the cell wall.

Isolation and Characterization of Cell Wall Components of the Unicellular Cyanobacterium Synechococcus sp. PCC 6307

SUMMARY: Cell walls and outer membranes, free of thylakoids and cytoplasmic membranes, were isolated from the unicellular cyanobacterium Synechococcus sp. PCC 6307. Electron microscopy revealed C-shaped cell wall fragments, which were partially converted to outer membrane vesicles after removal of the peptidoglycan by lysozyme digestion. The major constituents of the outer membrane were proteins and lipopolysaccharide, while lipids and carotenoids were minor components. The polypeptide patterns of the outer membranes were dominated by two major proteins (M r 52000 and 54000). Five strongly polar lipids (unidentified), free fatty acids and small amounts of sulpholipid were detected in extracts of partially purified cell wall fractions, but monogalactosyldiglyceride and digalactosyldiglyceride were not found. The peptidoglycan layer (10 nm thick) was also isolated. Its chemical composition indicated an Alγ-type structure. The degree of cross-linkage was 57%. A polysaccharide, consisting of fucose, mannose, galactose and glucose, was bound to the peptidoglycan, most likely via muramic acid 6-phosphate.

Subject index

Proline and hydroxyproline content of the cell walls of peanut (Arachis hypogea L. cv. Shulamit), cotton (Gossypium hirsutum L. cv. sj) and sunflower (Helianthus annuus L. cv. Saffola SO222) were determined. Proline concentration in the leaves of all plants species tested was approximately 110 nmol · mg−1 dry matter and higher than in the root and stem. Hydroxyproline concentration was highest in the root of all species tested and was between 30 and 60 nmol·mg−1 dry matter. This was 5–10 times higher than the concentration in the stem and leaf. There was no significant effect of salt stress on proline and hydroxyproline concentration in “purified cell wall fraction” of sunflower. Salt stress (100 mmol·l−1NaCl) reduced substantially plant growth of 53-day-old sunflowers, stem volume decreased from 21.3 ± 5.2 ml to 3.8 ± 1.6 ml, dry matter content of stem decreased from 719 ± 186 mg to 88±33 mg and the number of leaves per plant decreased from 18 to 13. These results suggest that inhibition of plant growth, by salt stress, was accompanied by inhibition of cell wall proteins (extensins) synthesis. Therefore, changes in the physicochemical properties of cell wall accompanying the osmotic adjustment should be sought in other posttranslational modifications of extensin(s), either glycosylation or inter-and/or intramolecular cross-linking in the cell wall.

Lymphoid cell responses to bacterial cell wall components: murine B-cell responses to a purified cell wall moiety of Actinomyces.

A mitogenic component, designated fraction C (Fr C), has been purified from a mutanolysin enzyme digest of Actinomyces cell walls by CM Sephadex C-25 ion-exchange and G-100 gel filtration chromatography. Good mitogenic responses were obtained with Fr C over a broad dose range with peak mitogenesis seen with 500 micrograms/culture. Fraction C (mol. wt. = 35,000-40,000) consists of 75% carbohydrate and 23% protein, is non-dialysable, resistant to heat, lysozyme or protease treatment, and partially sensitive to base, and all mitogenic activity is destroyed by either periodate or acid treatment. Fraction C is a B-cell mitogen since it induced responses in nude (nu/nu) and nu/+ BALB/c spleen cell cultures and purified splenic B-cell cultures, but did not stimulate purified splenic T-cell cultures. Similar mitogenic fractions for B cells have been obtained from cell walls of A. naeslundii and from a human isolate of A. viscosus. Good polyclonal IgM synthesis and plaque-forming cell responses to hapten or erythrocytes were obtained in vitro with the purified cell wall fractions derived from all three Actinomyces strains studied. These results indicate that the Actinomyces cell wall possesses a carbohydrate-rich component which activates B cells and may represent a common determinant of this genus.

Inductions of Adjuvant Arthritis in Rats and Footpad Swelling Reaction in Mice by Injection with the Purified Cell Wall Fraction from in vitro Grown Mycobacterium lepraemurium Hawaii

Mycobacterium lepraernuriurn Hawaii grown on one per cent Ogawa's egg yolk mediums1) was collected, washed and delipidated. Purified cell wall fraction (CWF) was prepared from the delipidated cells by sonicating and by digesting with trypsin and chymotrypsin. CWF was suspended in Freund's incomplete adjuvant (FIA) or in saline, or sometimes in oil-in-water solution, and then injected into the footpads of rats or mice. Arthritis induced by injecting with 100μg or 300μg of CWF into rat footpad was seen in the joints of both sides of the hind and front legs and in the joints of the tail. These symptoms were similar to those induced with Freund's complete adjuvant6).When the same CWF was injected into the mouse footpad, I could find the localized footpad swelling but not find the same kind of arthritis as seen in rats. Mouse footpad swelling reaction which was not depended on the mouse strains, showed a dose response to CWF (Fig. 2). Injection with CWF in FIA induced severer and longer reaction than with CWF in saline (Figs. 2, 3 and 6). This f ootpad reaction in mouse seems to be not related to delayed-type hypersensitivity, but due to inflammatory reaction with tissue damages, because reinjection of CWF into another footpad on the 30 th day after primary injection with CWF caused no specific reaction (Figs. 4 and 5). When CWF was injected in oil-in-water solution, comparatively high rate of footpad swelling reaction was observed for longer time (Fig. 6). It seemes to be due to making a little damage to the tissue and to giving long-term stimulation to host.It is well known that whole cells of various of Mycobacterium, Wax D from M. tuberculosis and cell wall fractions from various strains of mycobacteria have adjuvancy5, 6, 7). These cells or cellular fractions consist of mycolic acid, arabinogalactan and mucopeptide as a standard cell wall skeleton. M. lepraemurium Hawaii grown in vivo and in vitro also have the mycolic acid-arabinogalactan-mucopeptide complex in its cell wall fraction2, 3). Therefore, it is reasonable that CWF from M. lepraemurium showed the similar adjuvancy to other mycobacteria. It is reported that cell walls of M. bovis BCG act as an adjuvant and cannot nonspecifically enhance cell-mediated immunity21). Injection of CWF into mouse footpad might not induce delayed-type hypersensitivity without any antigen.

Cell wall differences of patient and soil isolates of Cryptococcus neoformans.

Two isolates of Cryptococcus neoformans were studied to determine whether or not differences in chemical composition of the cell walls existed. Aerated cultures of both isolates were grown at 37°C. in a synthetic liquid medium. A purified cell wall fraction was obtained by repeated passage of cells through a French pressure cell followed by differential centrifugation. Chemical analyses of cell wall fractions of each of these strains showed quantitative differences in 3 major groups of compounds. The isolate from the patient contained a higher percentage of hexose and a lower percentage of hexosamines and lipids than did the soil isolate.

Antigenic properties of the cell wall and other fractions of the yeast form of Histoplasma capsulatum.

Pine, Leo (Communicable Disease Center, Atlanta, Ga.), Clarence J. Boone, and Dave McLaughlin. Antigenic properties of the cell wall and other fractions of the yeast form of Histoplasma capsulatum. J. Bacteriol. 91:2158-2168. 1966.-Yeast-form cells of Histoplasma capsulatum were fractionated in an attempt to obtain complement-fixing antigens with greater specificity than those of the currently used whole yeast form. No specific fraction was isolated. Rupture of the whole cell produced soluble and insoluble highly antigenic fractions. The soluble fractions rapidly lost antigenicity. Marked reduction of activity of some particulate fractions was also observed. The purified cell wall, stripped of its protein components, was antigenic, relatively stable, and demonstrated low cross-reactivity with heterologous sera. All fractions cross-reacted with sera from cases of North American blastomycosis to a greater degree than did the whole yeast-form antigen of H. capsulatum. The production of certain cellular fractions varied with the strains used. In complement-fixation tests, insignificant differences in specificity and antigenicity were found with whole-cell antigens from 13 H. capsulatum strains as well as with purified cell wall fractions prepared from 2 of these strains.