Purification and Chemical Analysis of Microbial Cell Flocculant Produced by Aspergillus sojae AJ7002

A bio-flocculant was isolated from the culture broth of Asp. sojae AJ 7002. It was partially purified by acetone or ethanol precipitation, by ion-exchange and gel chromatography, and by dialysis. The isolated polymer possessed chemical characteristics of a poly-hexosamine and a protein. Glucosamine and galactosamine were not acetylated. The flocculant contained 2-ketogluconic acid, but sulfur or phosphorus was not detected. This flocculant was thermo-stable and its activity varied with pH. It was suggested that the hexosamine moiety in the polymer played a major role in bio-flocculation, assisted by protein portion in enlargement of the molecular weight of the flocculant, and by 2-ketogluconic acid in endowing it with amphoteric character.     

Isolation and Biochemical Characterization of a Galactoside Binding Lectin from <Emphasis Type="Italic">Bauhinia variegata</Emphasis> Candida (BvcL) Seeds

A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized d-lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal amino acid sequence of 17 residues showed 90% sequential homology to galactose-specific legume lectins of the subfamily Caesalpinoideae.     

Mass spectrometric determination of glycosylation sites and oligosaccharide composition of insect-expressed mouse interleukin-3

The primary structure of Baculovirus-expressed mouse interleukin-3 produced in infected Bombyx mori larvae was characterized by liquid secondary ion mass spectrometry and ²⁵²Cf-plasma desorption mass spectrometry in combination with selected protein microchemical reactions. Interleukin-3 was found to consist of at least two glycoprotein species of ca. 17 000 dalton. Characterization of tryptic and S. aureus V8 protease peptides by Edman degradation combined with plasma desorption mass spectrometry showed that two N-glycosylation sites, Asn-16 and Asn-86, were present. N-Glycan residues were shown by liquid secondary ion mass spectrometry and high-performance liquid chromatography to consist of mannose, fucose, and glucosamine. The presence of galactosamine indicated that O-glycosylated residues were present, in addition to the N-glycosylated residues. Glucose was also present, which indicated incomplete processing of the insect-expressed N-linked oligosaccharides.     

Evidence for tyrosine-linked glycosaminoglycan in a bacterial surface protein.

The S-layer protein of Acetogenium kivui was subjected to proteolysis with different proteases and several high molecular mass glycosaminoglycan peptides containing glucose, galactosamine and an unidentified sugar-related component were separated by molecular sieve chromatography and reversed-phase HPLC and subjected to N-terminal sequence analysis. By methylation analysis glucose was found to be uniformly 1,6-linked, whereas galactosamine was exclusively 1,4-linked. Hydrazinolysis and subsequent amino-acid analysis as well as two-dimensional NMR spectroscopy were used to demonstrate that in these peptides carbohydrate was covalently linked to tyrosine. As all of the four Tyr-glycosylation sites were found to be preceded by valine, a new recognition sequence for glycosylation is suggested.     

Carbohydrate Composition of Vesicular Stomatitis Virus

Analysis by gas-liquid chromatography of the trimethylsilylated sugar residues of purified vesicular stomatitis virus grown in L cells or chick embryo cells revealed the presence in the whole virion of four hexoses (glucose, galactose, mannose, and fucose), two hexosamines (glucosamine and galactosamine), and 34 to 40% neuraminic acid. The isolated viral glycoprotein was devoid of galactosamine and fucose, both of which sugars were present in whole virions presumably as part of the membrane glycolipids.     

The antifouling potentiality of galactosamine characterized from Vibrio vulnificus exopolysaccharide

To gain a better insight into biofilm composition, the exopolysaccharide (EPS) of the Gram-negative bacterium Vibrio vulnificus was studied. Monosaccharide composition analysis of the wild-type and mutant V. vulnificus EPS carried out with Bio-liquid chromatography revealed the presence of d-glucosamine, d-galactose, d-glucose and d-xylose in both strains. d-Galactosamine was found only in the mutant that formed less biofilm compared to its wild-type. The influence of galactosamine on biofilm formation was then studied by adding this substance gradually to six different Gram-negative/positive bacteria associated with various autoinducers. Four bacterial species known to use the autoinducer type-2 signaling system produced less biofilm in the presence of galactosamine. No significant inhibition of biofilm formation was observed in bacteria that produce autoinducer type-1 signal molecules. Galactosamine was also immobilized on polymeric nanofibers to determine its re-usability for the study of biofilm inhibition. The immobilized galactosamine retained >65% of its initial antifouling activity after 10 repeated uses. The results of this study suggest the antifouling role of galactosamine for bacteria that produce AI-2.     

Delta 6-desaturase activity in liver microsomes of rats fed diets enriched with cholesterol and/or omega 3 fatty acids.

A glycoprotein antigen was purified from human brain by immunoaffinity chromatography using the 44D10-monoclonal IgG, and its chemical nature was investigated. The yield of antigen was estimated at 91% and a 4340-fold purification was obtained relative to the white-matter homogenate. The antigen preparation from brain was further purified by preparative SDS/polyacrylamide-gel electrophoresis (PAGE) to obtain a glycoprotein with an Mr of 80,000 consisting of a single polypeptide. Amino acid analyses revealed a composition which was high in acidic and neutral amino acids, and low in basic residues. The presence of both glucosamine and galactosamine suggested that the glycoprotein contained both N- and O-linked glycans. Neutral sugar analyses showed that fucose, galactose and mannose were present. An assay for sialic acid determined that there were approximately 20 mol of sialic acid per mol of glycoprotein. Chemical cleavage of oligosaccharides by trifluoromethanesulphonic acid followed by SDS/PAGE showed that carbohydrate accounted for 25,000 of the 80,000-Mr glycoprotein.     

Presence and cellular distribution of soybean agglutinin-binding epitopes in two strains of<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The cellular location ofN-acetylgalactosamine inBacillus subtilis strains 168 and 170 was examined by electron microscopy using gold-conjugated soybean agglutinin (SBA) as a marker. Post-embedding labeling of sectioned material showed SBA-reactive, galactosamine-containing polymers associated with the cell membrane or the cytoplasm of the two strains. This intracellular location was distinct from the concanavalin A-binding epitopes that were located over the cell wall. Labeling of whole cells (native, fixed in glutaraldehyde, or treated with proteinase K, or Tween 20) before negative staining revealed no galactosamine exposed on the surface of strain 168. On the surface of strain 168 some exposed galactosamine terminal residues were detected; their accessibility to SBA increased when Tween 20 or proteinase K was applied.     

Determination of PF-04928473 in human plasma using liquid chromatography with tandem mass spectrometry

A simple, rapid and sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) analytical method was developed for quantification of Hsp90 inhibitor PF-04928473 in human plasma, following administration of its prodrug, PF-04929113. Sample processing involved protein precipitation by addition of 0.4 mL of methanol containing internal standard (PF-04972487) to 50 μL volume of plasma sample. Chromatographic separation of PF-04928473 and PF-04972487 was achieved on a Phenomenex^® Luna C18(2) (2.0mm × 50 mm, 5 μm) column using a gradient elution method with mobile phase solvents: methanol containing 0.1% formic acid and 0.1% formic acid at a flow rate of 0.25 mL/min. Detection was performed in electrospray positive ionization mode, monitoring the ion transitions from m/z 465.1 → 350.1 (PF-04928473) and m/z 447.0 → 329.1 (PF-04972487). The retention times for PF-04928473 and PF-04972487 were 1.86 and 2.85 min, respectively. Calibration curves were generated in the range of 2–2000 ng/mL. The accuracy and precision ranged from 94.1 to 99.0% and 86.7 to 97.6%, respectively, which were calculated using quality control samples of three different concentrations analyzed in quintuplicate on four different days.     

Enzymatic Synthesis of Acarviosyl-maltooligosaccharides Using Disproportionating Enzyme 1

The soluble and insoluble fractions obtained after sonication and centrifugation of Bifidobacterium adolescentis M101–4 cells were examined, and both of these fractions exhibited mitogenic activity in art assay of murine splenocytes and Peyer’s patch cells in vitro. The soluble fraction was further treated by a 6-step procedure involving proteinase K-treatment, ultrafiltration with a 50-kDa cut-off molecular-sieving membrane, anion-exchange chromatography, dialysis, ultrafiltration through a 6-kDa cut-off membrane filter, and gel-filtration to yield a soluble high molecular weight fraction (SHF) which was effective for stimulating the proliferation of murine splenocytes. Almost three quarters of this fraction by weight was found to consist of carbohydrates containing glucose and galactose as major constituents, and the average molecular weight was estimated to be between 60,000 and 2,460,000, with the main peak at 1,550,000 Da, by the retention time of gel permeation chromatography. A structural analysis by ¹H- and ¹³C-nuclear magnetic resonance and methylation indicated that SHF contained polysaccharides consisting of -4Galp1-, -4Glcp1-, and -6Glcp1- as the major residues, and Galf1- and -6Galf1- as the minor residues. Immunopotentiating SHF was found to contain galactofuranosyl residues as characteristic constituents which had not been previously detected in other soluble fractions from Gram-positive bacteria.     

Isolation and Chemical Composition of the Sheath of Sphaerotilus natans

A sheathed bacterium, Sphaerotilus natans, was cultured with vigorous shaking in a medium containing peptone. Then the biomass was harvested and treated with lysozyme, sodium dodecyl sulfate, and protease. With treatment, 1.6 mg of sheaths was obtained from 15 mg of biomass. For the preparation of sheaths of high purity, cultivation must be in the absence of glucose with sufficient aeration to prevent poly(3-hydroxybutyrate) accumulation. Carbohydrate (54.1%), protein (12.2%), and lipid (1-3%) were detected in the sheaths by colorimetric reactions and solvent extraction. Gas-liquid chromatography showed glucose and galactosamine to be present in the molar ratio of 1:4. The most abundant amino acids in the sheath protein were glycine (49.2 mol%) and cysteine (24.6 mol%). The sheaths were resistant to agents that reduce disulfide bonds (dithiothreitol and 2-mercaptoethanol) and to protease. However, sheathes were degraded completely by hydrazine, and a heteropolysaccharide composed of glucose and galactosamine (1:4) was released. The weight-average molecular weight of the polysaccharide was estimated to be 1.2×10⁵ by gel filtration chromatography with a low-angle laser-light scattering photometer and a rotation index detector. A ladder of 1.5-kDa peptides separable by sodium dodecyl sulfate gel electrophoresis was obtained by partial hydrolysis of sheaths, suggesting the sheath protein has repeating units of 1.5 kDa.     

Kaposi’s sarcoma-associated herpesvirus processivity factor-8 dimerizes in cytoplasm before being translocated to nucleus

The processivity factor-8 (PF-8) of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays an essential role in viral lytic replication. PF-8 forms homodimers in solution and is observed as a dimer on the DNA. Here, we show that PF-8 dimerizes in cells and that amino acid residues 1-21 and residues 277-304 of PF-8 (396R) are required for dimerization in vivo. Importantly, we demonstrate that PF-8 dimerizes in the cytoplasm before being translocated to the nucleus. The significance of PF-8 cytoplasmic dimerization as a possible first step in the formation of a prereplication complex is discussed.     

Management of sorghum grain mold through induced resistance mechanism using foliar spray of Pseudomonas fluorescens strains and non-conventional chemicals: a field study

Abstract

The study involved in-vitro and field studies using Pseudomonas fluorescens strains and some non-conventional chemicals for sorghum grain mold management. The culture filtrate of P. fluorescens strains PF-1 and PF-2 inhibited spore germination (p?<?0.001) and mycelial growth of grain mold fungi. Also, tested chemicals and commercial fungicides showed significant mycelial growth inhibition (p?<?0.001). PF-1, PF-2, ZnSO₄·7H₂O, salicylic acid and propiconazole were potentially effective and hence were tested in field (by foliar spraying). Two set of experiments were conducted simultaneously using naturally and artificially (Fusarium challenged) induced infected sorghum panicles. The lowest infection (score 4.2) was recorded with propiconazole. Disease severity was well controlled by PF-1 and PF-2, compared to ZnSO₄·7H₂O and salicylic acid. Furthermore, disease control was validated by determining total phenolic, flavonoid content and differentially induced phenolic acids (HPLC analysis) in harvested grains. Additionally, results of biochemical-traits, compatibility studies and antibiotic sensitivity of strains were found to be favourable.     

Human Kaposi's sarcoma herpesvirus processivity factor-8 functions as a dimer in DNA synthesis

In the absence of other proteins, the DNA polymerase (Pol-8) of Kaposi's sarcoma herpesvirus incorporates only several nucleotides from a primer template. However, association with the Kaposi's sarcoma herpesvirus processivity factor (PF-8) enables Pol-8 to incorporate thousands of nucleotides. Unlike the well described sliding clamp processivity factors, eukaryotic proliferating cell nuclear antigen and Escherichia coli beta-subunit, PF-8 and other herpesvirus processivity factors do not require a clamp loader or ATP to bind to template DNA. To begin to understand the mechanism used by PF-8 to achieve processivity, we have now purified PF-8 and demonstrated that it is a dimer both in solution and on the DNA. Mutational analysis of the PF-8 protein (396R) indicates that residues between 277 and 304 as well as the N-terminal 21 amino acids are required for dimerization. The results further correlate PF-8 dimerization with binding to Pol-8 and stabilizing Pol-8 on primer template. Notably, although removal of only 26 residues from the C terminus of PF-8 does not affect its ability to form dimers on DNA or to bind Pol-8, only short DNA chains (<100 nucleotides) are synthesized. This indicates that full-length PF-8 is necessary to enable Pol-8 to incorporate thousands of nucleotides. Interestingly, cross-linking of the processivity factor UL44 of cytomegalovirus reveals that it is a dimer in solution also.     

Purification of the Early Sporulation Gene spo0F Product of Bacillus subtilis

The RsaI fragment (750 base pairs) containing the entire early sporulation gene spo0F of Bacillus subtilis was inserted in the downstream region of the P_R promoter of the expression vector, pEBR-151. The recombinant plasmid thus obtained was introduced into Escherichia coli HB101 and the synthesis of the spo0F gene product was induced by a temperature shift up. After induction for 7 hr, a protein of molecular weight 14,000 (14 K protein) was overproduced to about 9% of the total cellular protein. The 14 K protein was purified to 94% purity by four steps of column chromatography. Deletion analysis and the sequence determination of the NH₂-terminal amino acid residues of the purified 14 K protein confirmed that the 14 K protein is the spo0F gene product.     

Characterization of an endo-alpha-N-acetyl galactosaminidase from Diplococcus pneumoniae.

Evidence is presented for the presence in filtrates of Diplococcus pneumoniae of an endo-glycosidase capable of acting on the O-glycosidic linkage between N-acetyl galactosamine and serine or threonine residues. The glycosidase was partially purified by chromatography on Affi-Gel 202. The resulting preparation acted on glycopeptides from mouse melanoma, fetuin and pig submaxillary mucin to release a disaccharide characterized as galactosyl-N-acetyl galactosamine. The enzyme had no action on phenyl α-N-acetyl-D-galactosaminide, asialo ovine submaxillary mucin or monosialoganglioside. A similar activity was detected in a commercial preparation of Clostridium perfringens neuraminidase.     

Cys92, Cys101, Cys197, and Cys203 Are Crucial Residues for Coordinating the Iron–Sulfur Cluster of RhdA from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

By proteomic analysis, we found a rhodanese-like protein(RhdA) from Acidithiobacillus ferrooxidans ATCC 23270 whose C-terminal contained a cysteine motif (Cys-XX-Trp-XX-Cys), known to bind iron–sulfur clusters. But so far, there were no articles to confirm the existence of iron–sulfur cluster in RhdA. In this study, RhdA gene from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The UV–Vis scanning and EPR spectra results indicated that the wild-type proteins contained an iron–sulfur cluster. Site-directed mutagenesis results revealed that the four cysteines Cys92, Cys101, Cys197, and Cys203 were crucial residues for iron–sulfur cluster binding.     

Pseudomonas aeruginosa bacteriophage phi PLS27-lipopolysaccharide interactions

We investigated the phi PLS27 receptor in Pseudomonas aeruginosa strain PAO lipopolysaccharide (LPS) by analyzing a resistant mutant. This mutant, which was designated AK1282, had the most defective LPS yet reported for a P. aeruginosa rough mutant; this LPS contained only lipid A, 2-keto-3-deoxyoctonate, heptose, and alanine as major components. In addition, this LPS lacked galactosamine, which is present in the inner core of the LPS of other rough mutants. The loss of galactosamine but only a small decrease in the alanine content indicated that the core of strain PAO LPS differed from the core structure which has been suggested for the LPS of other well-characterized P. aeruginosa strains. Our analysis also indicated that galactosamine residues may be crucial for phi PLS27 receptor activity of the LPS. Electrodialysis of LPS and conversion to salt forms (sodium or triethylamine) influenced the phage-inactivating capacity of the LPS, as did the medium in which the inactivation occurred; experiments performed in 1/10-strength broth resulted in much lower PhI50 (concentration of LPS causing a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) values than experiments performed in regular-strength broth. Sonication of the LPS also increased the phage-inactivating capacities of the LPS preparations.     

Molecular cloning,nucleotide sequence and expression of the structural gene for a thermostable alkaline protease from Bacillus sp. no. AH-101

Summary Alkaliphilic Bacillus sp. no. AH-101 produces an extremely thermostable alkaline serine protease that has a high optimum pH (pH 12–13) and shows keratinolytic activity. The gene encoding this protease was cloned in Escherichia coli and expressed in B. subtilis. The cloned protease was identical to the AH-101 protease in its optimum pH and thermostability at high alkaline pH. An open reading frame of 1083 bases, identified as the protease gene, was preceded by a putative Shine-Dalgarno sequence (AAAGGAGG) with a spacing of 11 bases. The deduced amino acid sequence revealed a pre-pro-peptide of 93 residues followed by the mature protease comprising 268 residues. AH-101 protease showed slightly higher homology to alkaline proteases from alkaliphilic bacilli (61.2% and 65.3%) than to those from neutrophilic bacilli (54.9–56.7%). Also AH-101 protease and other proteases from alkaliphilic bacilli shared common amino acid changes and a four amino acid deletion when compared to the proteases from neutrophilic bacilli. AH-101 protease, however, was distinct among the proteases from alkaliphilic bacilli in showing the lowest homology to the others.Correspondence to: H. Takami     

80 Purification and characterization of male and female gamete recognition molecules of a marine red alga aglaothamnion oosumiense

In red algae, fertilization begins with gamete‐gamete contact between the trichogyne cell wall of the female carpogonium and spermatial coverings. During the fertilization in Aglaothamnion oosumiense, reproductive cells interact with each other through sex specific adhesion molecules on the surface of spermatia and trichogyne. The gamete binding is highly selective suggesting the presence of recognition factors along their surfaces. In the previous studies, we have reported that spermatial binding to trichogynes of a red alga, Aglaothamnion oosumiense is mediated by a lectin‐carbohydrate complementary system. Spermatial binding to trichogynes was inhibited by pre‐incubation of trichogynes with N‐acetyl‐D‐galactosamine and D‐glucose and hence lectins specific to these sugars were expected to present on the surfaces of trichogyne cell wall. We have isolated a new lectin from Aglaothamnion oosumiense by the use of agarose bound N‐acetyl‐D‐galactosamine affinity chromatography and named it as rhodobindin. Rhodobindin agglutinated human erythrocytes as well as spermatia of Aglaothmanion oosumiense. The agglutinating activity of this lectin was inhibited by N‐acetyl‐D‐galactosamine and N‐acetyl‐D‐glucosamine. SDS‐PAGE results showed that this lectin may be monomeric. The molecular weight was determined as 21,876 dalton by matrix‐assisted laser desorption ionization (MALDI) mass‐spectrometry. N‐terminal amino acid sequence of the lectin was analyzed and revealed to have no identity with those of known proteins. The complementary male glycoprotein was also isolated and purified by the use of SBA‐agarose affinity chromatography. The subtractive cloning was carried out to characterize the recognition molecules.