Incorporation of 5-methylorcylaldehyde and methionine into the acetogenin (polyketide) gliorosein in Gliocladium roseum I.M.I. 93065

1. methyl-(14)C-labelled 1,3-dihydroxy-4,5-dimethylbenzene, 5-methylorcylaldehyde and 5-methylorsellinic acid were synthesized from orcinol and sodium [(14)C]cyanide and tested for activity as precursors of gliorosein. ring-(14)C-labelled orcylaldehyde was also prepared. 5[(14)C]-Methylorcylaldehyde was incorporated into gliorosein (36% conversion); all the radioactivity was located in the C-methyl groups. 5-Methylorsellinic acid was decarboxylated by Gliocladium roseum and the resulting phenol was secreted into the medium. 2. The formation of an enzyme-bound derivative of 5-methylorsellinic acid as the first aromatic compound in the biosynthesis of gliorosein is suggested to explain these results. 3. ring-(14)C-labelled 3,4-dihydroxy-6-methyltoluquinone was also effectively incorporated into gliorosein and related products (20% conversion). 4. Sodium [(14)C]formate and [Me-(14)C]-methionine were incorporated into gliorosein and related products (15.4 and 22.2% conversion respectively). Isolation and estimation of the radioactivity in the O-methyl and C-methyl groups in the (14)C-labelled gliorosein thus formed showed an appreciable difference in the specific activities of the two types of methyl group (14 and 15% respectively). The results in the doubly-labelled methionine experiment indicate that the C-methyl group arises in the same manner as that in ergosterol; one of the original hydrogen atoms of the methyl group is lost. This confirms that C-methylation occurs at an ethylenic group at the aliphatic level. 5. The sequence of reactions at the aromatic level leading to the formation of gliorosein is proposed as 5-methylorsellinyl-enzyme-->3-hydroxy-5-methylorsellinyl-enzyme-->3,4-dihydroxy-6-methyltoluquinol-->3,4-dimethoxy-6-methyltoluquinol-->gliorosein.     

Studies on the biosynthesis of quinones in fungi. Incorporation of 6-methylsalicylic acid into fumigatin and related compounds in Aspergillus fumigatus I.M.I. 89353

1. Orsellinic acid has been detected as a metabolite of Aspergillus fumigatus. 2. The other principal aromatic components of the medium are fumigatin and the quinol, fumigatol. Fumigatol has been shown to be dihydrofumigatin after oxidation to the quinone followed by acetylation. 3. (14)C-labelled 6-methylsalicylic acid can be hydroxylated in A. fumigatus to form orsellinic acid and decarboxylated to give m-cresol. 4. (14)C-labelled 6-methylsalicylic acid is incorporated into fumigatin and fumigatol (1.0-1.5%), but the conversion does not occur until about 2-3 days after supplementation of the medium. At this stage of growth, the organism has already synthesized approx. 20 times as much fumigatol as fumigatin and this ratio is reflected in the much lower specific activity of the quinol. 5. Supplementation of the medium with either orsellinic acid or orcinol, in addition to (14)C-labelled 6-methylsalicylic acid, greatly decreases the latter's incorporation into fumigatin. At the same time, the cultures containing these substances are stimulated to produce another quinone with relatively high specific activity. 6. 6-Methylsalicylic acid has not been detected in the medium of normal cultures. The results indicate that 6-methylsalicylic acid itself is not a direct precursor of fumigatin and fumigatol but that it is converted into a true intermediate, probably after hydroxylation to orsellinic acid. 7. Supplementation of the medium with 6-methylsalicylic acid (15-25mg./200ml.) greatly affects the metabolism of A. fumigatus. Growth is inhibited and the synthesis of fumigatol is markedly depressed in these cultures. The inhibitory effects may possibly be related in some way to the production of m-cresol.     

6-Methylsalicylic Acid Production in Solid Cultures of Penicillium patulum Occurs Only When an Aerial Mycelium Is Present

When Penicillium patulum was grown on Czapek-Dox agar, 6-methylsalicylic acid was produced as an aerial mycelium was forming. Nutrients were often plentiful in the medium when biosynthesis began. If the formation of an aerial mycelium was prevented by growing the fungus between two sheets of dialysis membrane, no 6-methylsalicylic acid was produced even when nutrients were completely consumed. If the upper sheet of dialysis membrane was stripped off cultures of the latter type, an aerial mycelium formed; concomitantly, 6-methylsalicylic acid biosynthesis was observed. We conclude that 6-methylsalicylic acid was produced only by P. patulum colonies that possessed an aerial mycelium.     

Studies on the biosynthesis of phenols in fungi. Production of 4-methoxytoluquinol, epoxysuccinic acid and a diacetylenic alcohol by surface cultures of Lentinus degener I.M.I. 110525

1. 4-Methoxytoluquinol was secreted into the medium by surface cultures of the basidiomycete Lentinus degener Kalchbr. (approx. 100mg./l. of medium). In addition, epoxysuccinic acid (150–200mg.) and a long-chain diacetylenic alcohol (3mg.) were also secreted. Epoxysuccinic acid has previously been found in the culture medium of some Fungi Imperfecti. These metabolites were all synthesized during the early phase of growth but maximum production occurred some time later. 2. Supplementation of the medium with cycloheximide or 8-azaguanine inhibited the production of epoxysuccinic acid. 3. Sodium [1-¹⁴C]acetate and 6-methyl[¹⁴C]salicylic acid were not incorporated into 4-methoxytoluquinol, but [U-¹⁴C]tyrosine and [Me-¹⁴C]methionine were incorporated to the extent of 0·55 and 4·75% respectively (minimum values). Degradation studies established that the aromatic ring and C-methyl group were derived from the ring and β-carbon atom of tyrosine; the O-methyl group alone was formed from methionine.     

Use of Fluorinated Compounds To Detect Aromatic Metabolites from m-Cresol in a Methanogenic Consortium: Evidence for a Demethylation Reaction

Anaerobic sewage sludge was used to enrich a methanogenic m-cresol-degrading consortium. 6-Fluoro-3-methylphenol was synthesized and added to subcultures of the consortium with m-cresol. This caused the accumulation of 4-hydroxy-2-methylbenzoic acid. In a separate experiment, the addition of 3-fluorobenzoic acid caused the transient accumulation of 4-hydroxybenzoic acid. Inhibition with bromoethanesulfonic acid caused the accumulation of benzoic acid. Thus, the proposed degradation pathway was m-cresol → 4-hydroxy-2-methylbenzoic acid → 4-hydroxybenzoic acid → benzoic acid. The m-cresol-degrading consortium was able to convert exogenous 4-hydroxybenzoic acid and benzoic acid to methane. In addition, for each metabolite of m-cresol identified, the corresponding fluorinated metabolite was detected, giving the following sequence: 6-fluoro-3-methylphenol → 5-fluoro-4-hydroxy-2-methylbenzoic acid → 3-fluoro-4-hydroxybenzoic acid → 3-fluorobenzoic acid. The second step in each of these pathways is a novel demethylation which was rate limiting. This demethylation reaction would likely facilitate the transformation of the methyl group to methane, which is consistent with the results of a previous study that showed that the methyl carbon of m-[methyl-¹⁴C]cresol was recovered predominantly as [¹⁴C]methane (D. J. Roberts, P. M. Fedorak, and S. E. Hrudey, Can. J. Microbiol. 33:335-338, 1987). The final aromatic compound in the proposed route for m-cresol metabolism was benzoic acid, and its detection in these cultures merges the pathway for the methanogenic degradation of m-cresol with those for the anaerobic metabolism of many phenols.     

Studies on the biosynthesis of phenols in fungi. Conversion of [14C]orsellinic acid and [14C]orcinol into fumigatol by Aspergillus fumigatus I.M.I. 89353

1. Sodium [1-(14)C]acetate was incorporated into orsellinic acid and fumigatol by Aspergillus fumigatus. 2. [(14)C]Orsellinic acid was prepared biosynthetically. It was converted almost entirely into fumigatol and fumigatin within 2 days of supplementation of the medium. The apparent decrease in incorporation after a longer period of growth was due to decomposition of radioactive fumigatol and the production of relatively unlabelled material. The addition of orcinol to these cultures decreased the conversion of [(14)C]orsellinic acid into fumigatol. [(14)C]Orsellinic acid was incorporated into 3,4-dihydroxytoluquinol in both sets of cultures. 3. [(14)C]Orcinol was prepared from [(14)C]orsellinic acid after acid hydrolysis. It was also very effective as a precursor of fumigatol (60% incorporation). 4. The specific activity of fumigatin was lower than that of fumigatol at early stages of growth (4-5 days after inoculation) with all the labelled substrates that were tested. This indicated that fumigatin arose from fumigatol after oxidation in the medium. 5. The presence of orcinol in the medium greatly stimulated the incorporation of radioactivity (presumably derived from the (14)CO(2)H of orsellinic acid) into the isoprenoid compounds, ergosterol and ubiquinone, in the mycelium.     

The pectic enzymes of Aspergillus niger. A mercury-activated exopolygalacturonase

1. An exopolygalacturonase was separated from a mycelial extract of Aspergillus niger with a 290-fold purification and a recovery of 8·6%. 2. The enzyme displayed its full activity only in the presence of Hg²⁺ ions; K_A for mercuric chloride was about 6×10⁻⁸m. 3. The mercury-activated enzyme progressively removed the terminal galacturonic acid residues from α-(1→4)-linked galacturonide chains and converted digalacturonic acid, trigalacturonic acid, tetragalacturonic acid and pectic acid into galacturonic acid.     

Organization of Electron Transport in Photosystem II of Spinach Chloroplasts According to Chelator Inhibition Sites

The organization of electron transport in photosystem II of spinach (Spinacia oleracea) chloroplasts was studied by means of various chelators and uncouplers. The partial reactions used included H₂O→methyl viologen, H₂O→silicomolybdic acid H₂O→ferricyanide, and H₂O→dimethylbenzoquinone. Three types of chelator inhibition were found (a) inhibition common to all pathways and presumably affecting the Mn or water oxidation site in photosystem II (salicylaldoxime, dithizone, acridine, 4,4,4-trifluoro-1-(2-thienyl)-1,1-butanedione, 4,4,4-trifluoro-0-(2-furyl)-1,3-butanedione; (b) strong inhibition of the H₂O→silicomolybdic acid pathway in presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea by lipophilic chelators (bathocuproine, tertoctylcatechol) but stimulation by orthophenanthroline; and (c) 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-insensitive dimethylbenzoquinone reduction inhibited by all phenanthrolines while ferricyanide reduction was remarkably stimulated by bathophenanthroline but inhibited by orthophenanthroline and bathocuproine. The action of lipophilic chelators on silicomolybdic acid reduction presumes the presence of a metallo protein in photosystem II. The differential action of bathophenanthroline on dimethylbenzoquinone and ferricyanide reduction indicated the possible existence of a metalloprotein in this pathway which is different from the site of orthophenanthroline inhibition.     

Cell Walls of Germinating Uredospores: II. Carbohydrate Polymers

Polymeric carbohydrates in ¹⁴C-labeled germ tube and uredospore walls of Uromyces phaseoli var. typica were studied by permethylation and by enzymatic hydrolysis. The native structure of the uredospore wall limited the effectiveness of both techniques with this wall, but evidence for two distinct polysaccharides was obtained. A linear (1→3) glucan, containing minor quantities of (1→6) linkages, may account for most of the glucose in the uredospore wall. A second uredospore polymer was a glucomannan similar to one reported for other rust fungi in that it consisted of approximately equal numbers of β(1→3) and β(1→4) mannosidic linkages with glucose as a minor component at the nonreducing end. Branching, most likely by (1→6) mannose links, was low. In contrast to uredospore wall, considerably more germ tube polysaccharide was accessible to enzymes and to methylation. Methylation studies indicate that (1→3) glucose and mannose bonds occur predominantly. Evidence from hydrolysis with exo- (β)-(1→3) glucanase suggests distinct wall regions of β(1→3) glycan, highly branched by (1→6) bonds, as well as wall regions of a glucomannan with alternating (1→3) glucose and (1→3) mannose residues. Polymer heterogeneity was indicated by differences in the proportions of mannose, glucose, and galactose as reducing end groups in different solubility fractions. In germ tube walls, but not in uredospore walls, glucosamine apparently existed as part of chitin polymer as evidenced by the isolation of N,N-diacetylchitobiose from chitinase digestion.     

Role of Zearalenone Lactonase in Protection of Gliocladium roseum from Fungitoxic Effects of the Mycotoxin Zearalenone

Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of ≤10 μg/ml. The fungitoxic effect declined in the order zearalenone > α-zearalenol > β-zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.     

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57.

A 65-kb region of DNA from Streptomyces viridochromogenes Tü57, containing genes encoding proteins involved in the biosynthesis of avilamycins, was isolated. The DNA sequence of a 6.4-kb fragment from this region revealed four open reading frames (ORF1 to ORF4), three of which are fully contained within the sequenced fragment. The deduced amino acid sequence of AviM, encoded by ORF2, shows 37% identity to a 6-methylsalicylic acid synthase from Penicillium patulum. Cultures of S. lividans TK24 and S. coelicolor CH999 containing plasmids with ORF2 on a 5.5-kb PstI fragment were able to produce orsellinic acid, an unreduced version of 6-methylsalicylic acid. The amino acid sequence encoded by ORF3 (AviD) is 62% identical to that of StrD, a dTDP-glucose synthase from S. griseus. The deduced amino acid sequence of AviE, encoded by ORF4, shows 55% identity to a dTDP-glucose dehydratase (StrE) from S. griseus. Gene insertional inactivation experiments of aviE abolished avilamycin production, indicating the involvement of aviE in the biosynthesis of avilamycins.     

Identification of an O-Acyltransferase Gene (oacB) That Mediates 3- and 4-O-Acetylation of Rhamnose III in Shigella flexneri O Antigens

O antigen (O polysaccharide) is an important and highly variable cell component present on the surface of cells which defines the serospecificity of Gram-negative bacteria. Most O antigens of Shigella flexneri, a cause of shigellosis, share a backbone composed of →2)-α-l-Rhap^III-(1→2)-α-l-Rhap^II-(1→3)-α-l-Rhap^I-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various substituents, giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha^I, and phosphorylation with phosphoethanolamine on Rha^II or/and Rha^III. Recently, two new O-antigen modifications, namely, O-acetylation at position 3 or 4 of Rha^III and position 6 of GlcNAc, have been identified in several S. flexneri serotypes. In this work, the genetic basis for the 3/4-O-acetylation on Rha^III was elucidated. Bioinformatic analysis of the genome of S. flexneri serotype 2a strain Sf301, which carries 3/4-O-acetylation on Rha^III, revealed an O-acyltransferase gene designated oacB. Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not serotype 6, which has a different O-antigen backbone structure. The oacB gene is carried by a transposon-like structure located in the proA-adrA region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in S. flexneri. These findings enhance our knowledge of S. flexneri O-antigen modifications and shed light on the origin of new O-antigen variants.     

Characterization of the oligosaccharides from lipid-linked oligosaccharides of mung bean seedlings

Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseolus aureus) seedlings in the presence of GDP-[¹⁴C] mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P-4 columns. Five different oligosaccharides were purified in this way. Based on their relative elution constants (K_d) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man₇GlcNAc₂, Man₅GlcNAc₂, Man₃GlcNAc₂, Man₂GlcNAc₂, and ManGlcNAc₂. These oligosaccharides were treated with endoglucosaminidase H and α- and β-mannosidase, and the products were examined on Biogel P-4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB³H₄ reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as follows: E, Manβ-GlcNAc-GlcNAc; D, Manα1→3Manβ-GlcNAc-GlcNAc; C, Manα1→2Manα1→3Manβ-GlcNAc-GlcNAc; B, Manα1→2Manα1→2Manα1→ 3(Manα1→6)Manβ-GlcNAc-GlcNAc; and A, Manα1→2Manα1→ 2Manα1→3(Manα1→ [Manα1→6]Manα1→6) Manβ-GlcNAc-GlcNAc. The synthesis of the Man₇GlcNAc₂ was greatly diminished when tunicamycin (10 μg/ml) was added to the incubation mixtures.     

Herbicide Transformation: I. Studies with Whole Cells of Fusarium solani

A strain of Fusarium solani isolated from soil by enrichment techniques used propanil (3′, 4′-dichloropropionanilide) as a sole source of organic carbon and energy for growth in pure culture. The primary product of the transformation of propanil by F. solani was isolated and identified as 3,4-dichloroaniline (DCA). This compound accumulated in the medium to a level (80 μg/ml) which stopped further herbicide utilization. Herbicide utilization by F. solani was influenced by various environmental and nutritional factors. It was more sensitive to acid than alkaline pH. Added glucose and yeast extract increased the rate of propanil decomposition, and the reduced aeration retarded growth of the fungus and herbicide utilization. The growth of F. solani on propionate was inhibited by added DCA.     

Studies on the Host-finding Mechanisms of Neotylenchus linfordi

The plant-parasitic nematode, Neotylenchus linlordi, congregated around colonies or filtrates from mycelia of Gliocladium roseum, Rhizoctonia solani, Pyrenochaeta terrestris and Chaetomium indicum. The average time required for the nematodes to reach the fungal colonies ranged from less than 4 hr for G. roseum to 20 hr for R. solani. Nematodes first circled near the point of introduction, then moved toward the fungus or filtrate. Several methods of measuring the response of N. linfordi to G. roseum culture filtrate were evaluated. The response was strongest when the test materials were assayed on an agar disk submerged in water agar and the introduced nematodes suspended in agar in a center well midway between the test materials. Filtrates obtained from cultures of G. roseum incubated between 12 and 21 days in potato dextrose broth, were most active. The attractants were small thermostable molecules, soluble in methyl alcohol and unaffected by pH. A yellow pigment with properties similar to a mixture of aurantiogliocladin, rubrogliocladin, and gliorosein was shown to be one of the active materials. The response of N. linfordi to the G. roseum filtrate was not associated with any nutritive factors which would result in reproduction.     

Changing Emergence of Shigella Sero-Groups in Bangladesh: Observation from Four Different Diarrheal Disease Hospitals

Background

Shigellosis continues to be a public health challenge for developing countries, including Bangladesh. The aim of the study is to demonstrate recent changes in Shigella sero-groups and their geographical diversity.

Methods

Data were extracted from data archive of four diarrheal disease surveillance systems. A 2% sub sample from urban Dhaka Hospital (2008–2011; n = 10,650), and 10% from urban Mirpur Treatment Centre (2009–2011; n = 3,585), were enrolled systematically; whereas, all patients coming from the Health and Demographic Surveillance System area in rural Matlab (2008–2011; n = 6,399) and rural Mirzapur (2010–2011; n = 2,812) were included irrespective of age, sex, and disease severity. A fresh stool specimen was collected for identification of Shigella spp. Of them, 315 (3%) were positive for Shigella in Dhaka, 490 (8%) from Matlab, 109 (3%) from Mirpur and 369 (13%) from Mirzapur and considered as analyzable sample size.

Results

Among all Shigella isolates regardless of age, significant decreases in percentage of S. flexneri over time was observed in Mirpur (55→29%; p value of χ²-for trend = 0.019) and Mirzapur (59→47%; p = 0.025). A non-significant decrease was also seen in Dhaka (58→48%), while in Matlab there was a non-significant increase (73→81%). Similar patterns were observed among under-5 children at all sites. Emergence of S. sonnei was found in Dhaka (8→25%; p<0.001) and Mirpur (10→33%; p = 0.015), whereas it decreased in Mirzapur (32→23%; p = 0.056). The emergence of S. boydii was seen in all ages in Mirzapur [(3→28%; p<0.001); (3→27%; p<0.001)]. On the other hand, we saw non-significant percent reductions in S. boydii in Dhaka [overall (25→16%); under-5 (16→9%)]. Decreasing rates of Shigella dysenteriae were observed in Matlab, Mirpur and Mirzapur; whereas, in Dhaka it remained unchanged.

Conclusion and Significance

Emergence of S. sonnei and S. boydii as important infectious diarrhea etiologies and variations in geographical diversity underscore the need for monitoring, with possible implications for vaccine development.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

Inhibition of glycosidases by aldonolactones of corresponding configuration: Preparation of (1-->5)-lactones by catalytic oxidation of pyranoses and study of their inhibitory properties

1. A method was devised for the preparation of (1→5)-lactones from pyranose sugars and uronic acids by platinum-catalysed oxidation with gaseous oxygen in aqueous solution at acid pH. It was applied to mannose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, galacturonic acid, galactose, l-arabinose and d-fucose. 2. Only the first three yielded products that could be obtained in the solid state without decomposition. In every case, however, the oxidation product in aqueous solution behaved as the aldono-(1→5)-lactone, and was more inhibitory towards the appropriate glycosidases than any aldonolactone preparation hitherto examined. 3. The stabilities of the oxidation products were studied, and their interconversion with the (1→4)-lactones was demonstrated. Ring-opening does not appear to be mandatory for this isomeric change, which in some instances is very rapid. 4. To explain all the inhibitory effects observed with aldonolactones on glycosidases of corresponding configuration, it is tentatively postulated that inhibition may be due entirely to the (1→5)-lactone, and that any inhibitory effect seen with the (1→4)-lactone is a measure of the extent and speed of its conversion into the (1→5)-lactone in aqueous solution.     

Structural studies on heparan sulphate from human lung fibroblasts. Characterization of oligosaccharides obtained by selective periodate oxidation of d-glucuronic acid residues followed by scission in alkali

1. ³H- and ³⁵S-labelled heparan sulphate was isolated from monolayers of human lung fibroblasts and subjected to degradations by (a) deaminative cleavage and (b) periodate oxidation/alkaline elimination. Fragments were resolved by gel- and ion-exchange-chromatography. 2. Deaminative cleavage of the radioactive glycan afforded mainly disaccharides with a low content of ester-sulphate and free sulphate, indicating that a large part (approx. 80%) of the repeating units consisted of uronosyl-glucosamine-N-sulphate. Blocks of non-sulphated [glucuronosyl-N-acetyl glucosamine] repeats (3–4 consecutive units) accounted for the remainder of the chains. 3. By selective oxidation of glucuronic acid residues associated with N-acetylglucosamine, followed by scission in alkali, the radioactive glycan was degraded into a series of fragments. The glucuronosyl-N-acetylglucosamine-containing block regions yielded a compound N-acetylglucosamine–R, where R is the remnant of an oxidized and degraded glucuronic acid. Periodate-insensitive uronic acid residues were recovered in saccharides of the general structure glucosamine–(uronic acid–glucosamine)_n–R. 4. Further degradations of these saccharides via deaminative cleavage and re-oxidations with periodate revealed that iduronic acid may be located in sequences such as glucosamine-N-sulphate→iduronic acid→N-acetylglucosamine. Occasionally the iduronic acid was sulphated. Blocks of iduronic acid-containing repeats may contain up to five consecutive units. Alternating arrangements of iduronic acid- and glucuronic acid-containing repeats were also observed. 5. ³H- and ³⁵S-labelled heparan sulphates from sequential extracts of fibroblasts (medium, EDTA, trypsin digest, dithiothreitol extract, cell-soluble and cell-insoluble material) afforded similar profiles after both periodate oxidation/alkaline elimination and deaminative cleavage.     

Biosynthesis and Secretion of Cryptogein, a Protein Elicitor Secreted by Phytophthora cryptogea

The phytopathogenic fungi Phytophthora subspecies elicit hypersensitive-like necroses on their nonhost tobacco (Nicotiana tabacum), with the exception of the tobacco pathogen Phytophthora nicotianæ. In culture, these fungi—except P. nicotianæ—secrete proteins, called elicitins, that cause these remote leaf necroses and are responsible for the incompatible reaction. These proteins protect tobacco against invasion by the agent of the tobacco black shank, P. nicotianæ, which is unable to produce such an elicitor. Cryptogein, secreted by Phytophthora cryptogea, has been purified, sequenced, and characterized as an elicitin, a novel family of 10 kilodalton holoproteins. In the present paper, we examined the secretion and biosynthesis of this protein elicitor from P. cryptogea culture. Results showed that the secretion of cryptogein began later than its synthesis and stopped earlier, simultaneously with mycelium growth, when the nitrogen source in the culture medium was nearly exhausted. Electrophoretic patterns of total protein from mycelium extracts and N-terminal sequence analysis showed that cryptogein accumulated in the mycelium in its mature form. The comparison of the immunoselected in vitro translation products with ³⁵S in vivo-labeled cryptogein showed that cryptogein was synthesized as a preprotein with a signal peptide removed cotranslationally before the secretion into the culture medium. Immunoselected in vitro-synthesized products were subjected to radiosequencing to clearly determine the N-terminal position and the size (20 amino acids) of the signal peptide. Cryptogein did not undergo any other posttranslational modification.