Isolation of a bioemulsifier from Candida lipolytica.

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Isolation of a bioemulsifier from Candida lipolytica

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH₃CN-H₂O (1:1, v/v) and MeOH-H₂O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations.     

Identification of novel α7 nicotinic receptor ligands by in silico screening against the crystal structure of a chimeric α7 receptor ligand binding domain

A hierarchical in silico screening procedure using the crystal structure of an agonist bound chimeric α7/Ls-AChBP protein was successfully applied to both proprietary and commercial databases containing drug-like molecules. An overall hit rate of 26% (pK_i ?5.0) was obtained, with an even better hit rate of 35% for the commercial compound collection. Structurally novel and diverse ligands were identified. Binding studies with [³H]epibatidine on chimeric α7/5-HT₃ receptors yielded submicromolar inhibition constants for identified hits. Compared to a previous screening procedure that utilized the wild type Ls-AChBP crystal structure, the current study shows that the recently obtained α7/Ls-AChBP chimeric protein crystal structure is a better template for the identification of novel α7 receptor ligands.     

Influence of carbon-dioxide on the growth of Spirulina sp. (MCRC-A0003) isolated from Muttukadu backwaters,South India

Growth of Spirulina sp. (MCRC-A0003), a cyanobacterium, was evaluated under different concentrations of carbon-dioxide (CO₂) (4–50 %) in a closed glass photobioreactor. Although significant CO₂ utilization by the cyanobacterial strain was observed up to 50 % concentration, complete utilization was observed only at 4, 10 and 20 % concentrations on 3rd, 6th and 8th day respectively. However, considerable reduction was witnessed in reactors containing 30–50 % CO₂ only between 6th and 9th day. A corresponding increase in the biomass and primary metabolites like chlorophyll-a, carbohydrate and protein were observed. Biomass productivity of Spirulina in reactors sparged with 4, 10 and 20 % CO₂ were 13.7, 43 and 44 % more than that in control reactor without CO₂. While CO₂ increased the levels of primary metabolites in the cyanobacterial cells, it was quite prominent in 10 % CO₂ concentration with the chlorophyll-a, carbohydrate and protein contents were 64, 183 and 626 mg g^?1 respectively. While 10 and 6.6 % increase were noticed in chlorophyll-a and protein, 17 % increase in carbohydrate levels was observed in Spirulina cells, which could be attributed to the conversion of CO₂ to carbohydrate by the cyanobacterium.     

The partial purification of a Trichogramma pretiosum pupation factor from hemolymph of Manduca sexta

Factors responsible for pupation of Trichogramma pretiosum Riley reared in vitro were partially purified. The active material was extracted with 76% ethanol from Manduca sexta (L.) hemolymph. The water soluble active fraction was chromatographed first on a Sephadex G-10® column and then on a C₁₈ cartridge (reversed phase). After removing more than 99% of the original protein, 20% of the original pupation activity remained; this fraction, however, also contained about 20% of the original hemolymph carbohydrate. The pupation factor was further separated by DEAE-HPLC into two active carbohydrate containing fractions. HPLC of the active fraction from the C₁₈ cartridge on an amino column produced some separation of the original carbohydrate, but no pupation activity was found in single fractions after this separation. However, when certain fractions were re-combined, weak pupation activity was obtained. The results indicate that when T. pretiosum were fed nutritionally rich artificial diets, at least two and possibly several polar, low molecular weight chemicals in M. sexta hemolymph were also needed and were responsible for growth and development of the parasitoids to the pupal stage. This is the first report describing the partial purification of insect growth factors affecting the growth and development of parasitoids.     

Reduced Dietary Intake of Carbohydrates by Obese Subjects Results in Decreased Concentrations of Butyrate and Butyrate-Producing Bacteria in Feces

Weight loss diets for humans that are based on a high intake of protein but low intake of fermentable carbohydrate may alter microbial activity and bacterial populations in the large intestine and thus impact on gut health. In this study, 19 healthy, obese (body mass index range, 30 to 42) volunteers were given in succession three different diets: maintenance (M) for 3 days (399 g carbohydrate/day) and then high protein/medium (164 g/day) carbohydrate (HPMC) and high protein/low (24 g/day) carbohydrate (HPLC) each for 4 weeks. Stool samples were collected at the end of each dietary regimen. Total fecal short-chain fatty acids were 114 mM, 74 mM, and 56 mM (P < 0.001) for M, HPMC, and HPLC diets, respectively, and there was a disproportionate reduction in fecal butyrate (18 mM, 9 mM, and 4 mM, respectively; P < 0.001) with decreasing carbohydrate. Major groups of fecal bacteria were monitored using nine 16S rRNA-targeted fluorescence in situ hybridization probes, relative to counts obtained with the broad probe Eub338. No significant change was seen in the relative counts of the bacteroides (Bac303) (mean, 29.6%) or the clostridial cluster XIVa (Erec482, 23.3%), cluster IX (Prop853, 9.3%), or cluster IV (Fprau645, 11.6%; Rbro730 plus Rfla729, 9.3%) groups. In contrast, the Roseburia spp. and Eubacterium rectale subgroup of cluster XIVa (11%, 8%, and 3% for M, HPMC, and HPLC, respectively; P < 0.001) and bifidobacteria (4%, 2.1%, and 1.9%, respectively; P = 0.026) decreased as carbohydrate intake decreased. The abundance of butyrate-producing bacteria related to Roseburia spp. and E. rectale correlated well with the decline in fecal butyrate.     

Proteins of the Thermus thermophilus ribosome Purification of several individual proteins and crystallization of protein TL7

The procedure of selective removal of eight proteins from the 50 S ribosomal subunit of the extreme thermophilic bacterium Thermus thermophilus has been developed based on extraction at 60°C in the presence of 0.5 M or 1 M NH₄Cl and 50% ethanol. CM-Sepharose CL column chromatography of the protein mixture under non-denaturing conditions yielded five proteins with a purity of 95% or higher. Crystals of one of these proteins, namely TL7 (probably an analog of L6 protein from the Escherichia coli ribosome) have been obtained using the ‘hanging drop’ method with ammonium sulphate as a precipitant.     

Isolation and analysis of cell wall material from beeswing wheat bran (Triticum aestivum)

Cell wall material (CWM) isolated from beeswing wheat bran contains 66% carbohydrate, 12% Klason lignin, 6% protein and 4% ash. The relative proportions of sugars in the CWM are arabinose 34%, xylose 26%, galactose 2%, glucose 32% and uronic acid 6%. The uronic acid was shown to consist of glucuronic acid and its 4-O-Me analogue in the ratio 1.8:1. Partial acid hydrolysis of the CWM yielded neutral sugars and a uronic acid fraction. The latter was shown to contain Glc p A-(1→2)-Xyl p and Glc p A-(1→2)-O-Xyl p-(1→4)-Xyl p and their 4-O-Me substituted uronic acid analogues. Methylation analysis of the whole CWM and partially degraded methylated CWM revealed the nature of the constituent glycosidic linkages. From the combined evidence we infer that the major structural features of the non-cellulosic polysaccharides are a linear chain of xylopyranose units joined by (1→4)-linkages, and arabinofuranose, xylose, galactose (and uronic acid) end groups, which in at least some of the polysaccharides, are attached directly by (1→2)- and/or (1→3)-linkages to the xylan chain. The CWM has been fractionated by successive extractions with water at 80°, 0.2 M (NH₄)₂C₂O₄ at 80°, Na chlorite/HOAc at 70°, 0.2 M (NH₄)₂C₂O₄ at 80°, 1 M and 4 M KOH, and the neutral sugar composition of the fractions determined. It is concluded from these and other experiments that the CWM contains two main types of polysaccharides, the arabinoxylans and cellulosic polymers, and that phenolic ester linkages play a role in holding them together.     

Hydroxyproline-rich bacterial agglutinin from potato : extraction, purification, and characterization

A protein, extracted from Katahdin potato (Solanum tuberosum L. cv `Katahdin') tubers and purified by ion exchange chromatography and gel filtration, agglutinates avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum, but only weakly agglutinates virulent strains. The agglutinin has very low hemagglutinating activity (in contrast to potato lectin) and is a glycoprotein containing about 61% carbohydrate. The carbohydrate moiety contains 91% (weight%) arabinose, 5% galactose, 3% glucose, and 1% glucosamine. The protein portion is rich in hydroxyproline (42%), lysine (16%), serine (9%), and proline (9%). The entire agglutinin has a molecular weight of 91,000 ± 5,000 and is very basic (pI > 11). Shape estimations based on the concentration dependence of the sedimentation coefficient, the high viscosity ([η] = 92.7), the frictional coefficient (f/f_o = 2.15), and axial ratio (a/b = 25) indicate that the agglutinin is a prolate ellipsoid.     

Characterization and inhibition of invertases in sugar cane juice

(NH₄)₂SO₄ fractionation followed by Sephadex G-200 chromatography of sugar cane juice gave an acid invertase with MW of 380 000 and 23.5% carbohydrate and a neutral invertase with MW of 66 000 and 22% carbohydrate. For acid invertase, K_m is 2.8 mM and V_max is 2.7 μmol sucrose hydrolysed/hr/mg protein. For neutral invertase, K_m and V_max are 0.32 mM and 2.8 μmol hydrolysed/hr/mg protein, respectively. Inhibition of both invertases by either lauryl sulfate or metasilicate is not competitive.     

Carbohydrate Responsive Proteins in the Roots of Pennisetum americanum

The effect of changes in carbohydrate status on the synthesis of specific proteins was investigated in millet (Pennisetum americanum L., Leeke, Tift 23B₁E₁) seedlings grown in sterile solution culture. Carbohydrate status was altered by extended darkness and sucrose feeding. Root proteins from intact seedlings were labeled with [³⁵S]methionine, phenol-extracted, separated by two-dimensional gel electrophoresis, and visualized by autoradiography. In four separate experiments, two proteins showed a consistent change in labeling when root carbohydrate levels were varied between 200 and 1000 micromole hexose per gram residual dry weight. Labeling of the first protein (P₄₇, M_r 47 kD) increased as the carbohydrate levels rose above 500 micromole hexose per gram residual dry weight. Labeling of the second protein (P₃₄, M_r 34 kD) increased as carbohydrate levels declined from 500 to 200 micromole hexose per gram residual dry weight. Under extreme conditions, when carbohydrate levels fell below 100 micromole hexose per gram residual dry weight, the labeling pattern of most proteins was drastically altered. It is suggested that P₄₇ and P₃₄ are `carbohydrate responsive proteins,' i.e. proteins whose concentrations are controlled either directly or indirectly by tissue carbohydrate status. In contrast, the changes in protein labeling that occur once carbohydrate pools are depleted may be involved in adaptation to periods of prolonged starvation.     

Purification of rat chondrosarcoma xylosyltransferase

The chondroitin sulfate chain-initiating enzyme, UDP-d-xylose:core protein β-d-xylosyltransferase has been purified over 600-fold from the high-speed supernatant fraction of a rat chondrosarcoma. The purification procedure involved differential centrifugation, gel chromatography on Sephadex G-200, and affinity chromatography on a matrix consisting of core protein bound to Sepharose. The purified enzyme was homogenous by electrophoretic and immunological criteria, had a molecular weight between 95,000 and 100,000 and contained approximately 10% carbohydrate. The K_m value for UDP-xylose was 1 × 10^?5, m and for the core-protein acceptor was 330 mg/liter.     

Purification and characterization of a phosphonic acid-containing glycoprotein from the cell membranes of Tetrahymena

A protein which contains 2-aminoethylphosphonic acid (AEP) has been isolated from the ciliate protozoan Tetrahymena thermophila. The protein contains about 30% carbohydrate with both N- and O-glycosidic linkages to the polypeptide and 8% AEP which is attached only to the O-linked glycoside. The amino group of AEP is unreactive to dansyl chloride as is the amino terminus of the protein. The polypeptide portion of the molecule, M_r 22,500, contains 22% glycine, 5.5% hydroxyproline, and is quite acidic. The phosphoprotein is found in the cell membranes. Its synthesis is inhibited by tunicamycin to the same extent which the antibiotic inhibits cell division.     

Purification and characterization of alkane solubilizing factor produced by Pseudomonas PG-1

The normal hexadecane emulsifying and solubilizing factor (PG-1 ESF C₁₆) produced by Pseudomonas PG-1 during growth on n-hexadecane was isolated and purified. The factor was composed of protein, carbohydrate and lipid, which were largely undialyzable. Ca²⁺ was necessary for activation and heat stability of the factor. Particle size of the factor was less than 10 nm. All the protein along with 68–74% of the carbohydrate in the factor was obtained in a single protein peak by gel filtration chromatography using Biogel P-30. The isolated protein fraction showed a 1–5 fold increase in n-hexadecane solubilizing activity. The isolated protein was shown to be a homogeneous, monomeric protein with a molecular weight of approximately 11,000 daltons by SDS-PAGE. The protein and carbohydrate moieties in the isolate were separated by DEAE-cellulose chromatography. Neither purified protein nor carbohydrate showed n-hexadecane solubilizing activity separately, but when these were mixed full activity was restored. Hydrocarbon emulsifying activity was confined to the lipid fraction, which was isolated to the extent of 85% from the Biogel P-30 column by ethyl ether extraction.     

A comparative review of the structure and biosynthesis of thyroglobulin

Thyroglobulin, the major iodoglycoprotein of the thyroid (M_r 669 kDa) has a sedimentation coefficient of 19 S and an isoelectric point (pI) of 4.4–4.7. The protein has been isolated and purified from saline extracts of the gland of several animal species, by methods such as ammonium sulfate fractionation, DEAE-cellulose chromatography and Sepharose 4B/6B gel-filtration. DEAE-cellulose chromatography of thyroglobulin from many species, by linear gradient, yielded a complex elution pattern, while camel thyroglobulin showed only a major and minor peak. As an iodoprotein, the protein has 0.1–2.0% iodine. The amino acid and iodoamino acid composition of thyroglobulins, in general, is similar. However, a high thyroxine content (15 mol/mol protein) has been noted for buffalo species. Asparagine or aspartic acid has been reported as the major N-terminal amino acid for thyroglobulins of several animal species whereas glutamic acid is the sole N-terminal amino acid for buffalo thyroglobulin. As a glycoprotein, thyroglobulin contains 8–10% total carbohydrate with galactose, mannose, fucose, N-acetyl glucosamine and sialic acid residues. The carbohydrate in the protein is distributed as two distinct units, A and B. In addition, human thyroglobulin has carbohydrate unit C. The occurrence of sulfate and phosphate as Gal-3-SO₄ and Man-6-PO₄, respectively, has been reported in few species. The quaternary structure of native thyroglobulin is comprised of two equal sized subunits of 330 kDa. However, the protein appears to contain 4–8 non-identical units in few species. The synthesis of thyroid hormones occurs in the matrix of the protein and is regulated by pituitary thyrotropin. The role of tyrosine residues 5 and 130 in thyroxine synthesis has been well documented.     

α2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes

Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor α₂-macroglobulin (α₂M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α₂M binding site to the C terminal end of HB3VAR06, and demonstrate that α₂M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to α₂M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with α₂M and markedly lowers the concentration of α₂M required for rosetting. Finally, HB3VAR06⁺ IEs share the capacity to bind α₂M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit α₂M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the α₂M—(and IgM-) binding domains of PfEMP1, which would be independent of the host receptor specificity of clinically important PfEMP1 antigens.     

The isolation and partial characterization of α2-macroglobulin from the serum of the ostrich (Struthio camelus)

• 1.1. α₂-Macroglobulin (α₂M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α₂M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α₂M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
• 2.2. α₂M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn²⁺-affinity chromatography.
• 3.3. Ostrich α₂M migrated as a single band (M_r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α₂ M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
• 4.4. Isoelectric focusing revealed a pI of 5.3.
• 5.5. The amino acid composition of ostrich α₂M is typical of an α₂M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
• 6.6. α₂M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α₂M.
• 7.7. Ostrich α₂M seems to show many physical, chemical and kinetic properties similar to those of other known α₂Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.

     

Studies of nicotinic acetylcholine receptor protein from rat brain: II. Partial purification

The pharmacological specificity of the binding of ¹²⁵I-labeled α-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the α-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12 600-fold has been obtained. Binding of ¹²⁵I-labeled α-bungarotoxin to this purified acetylcholine receptor protein is saturable with a K_d of 1·10^?8 M. Nicotine and acetylcholine iodide at concentrations of 10^?5 M inhibit ¹²⁵I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10^?4 M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10^?4 M. These data support the isolation of partially purified nicotinic acetylcholine receptor protein.     

The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review

Pseudomonas aeruginosa (Pa) produces several surface-associated adherence factors or adhesins which promote attachment to epithelial cells and contribute to the virulence of this pathogen. Among them, the type-4 pilus accounts for about 90% of the adherence capability of Pa to human lung pneumocyte A549 cells. Furthermore, it is responsible for more than 90% of the virulence in AB.Y/SnJ mice. Pa type-4 pili display a tip-base differentiation with the adherence function located at the tip of the pilus. All Pa pili prototypes characterized so far contain an intrachain disulfide loop (DSL) of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin. In Pa, this DSL comprises the epithelial cell-binding domain. Despite little sequence homology, DSL-containing peptides of different pilin prototypes seemingly reveal striking structural similarities. Two β-turns within the loop and the disulfide bridge impose significant structural rigidity on the DSL pilin peptide, suggesting a conformationally conserved binding domain. Insertions of C-terminal pilin peptides with disrupted DSL displayed on the surface of bacterial S-layer mediate the same receptor binding characteristics as pili, indicating that a DSL is not essential in maintaining the functionality of the binding domain. Pa pili bind specifically to the carbohydrate moiety of the glycosphingolipids (GSL) asialo-G_M1 and asialo-G_M2 and, to a much weaker extent, to lactosyl ceramide and ceramide trihexoside. The disaccharide sequence GalNAcβ(1-4)Gal, common in both asialo-G_M1 and asialo-G_M2, likely represents the minimal structural receptor motif recognized by the pili. Pa pili also bind to surface-localized proteins of human epithelial cells and other cell types, suggesting that non-sialylated GSL and (glyco)proteins function as receptors of pili. In addition to the major pilus adhesin, exoenzyme S and, as recent studies indicate, flagella, are further protein adhesins of Pa with GSL receptor binding specificities similar to those of pili.