Sulfated galactans from the red seaweed Nothogenia fastigiata (Nemaliales,Rhodophyta)

Fractionation of the cetrimide salts of the sulfated polysaccharides of Nothogenia fastigiata led to the isolation of a complex galactan sulfate. This product showed compositional and molecular weight heterodispersion together with composition-, temperature-, time-, and conformation-dependent molecular associations. In this sense, the behavior of the galactan sulfate is similar to that of the mannan sulfate previously isolated from the same seaweed.Formerly classified as Chaetangium fastigiatum.     

The system of galactans of the red seaweed, Kappaphycus alvarezii, with emphasis on its minor constituents

The galactans extracted with hot water from Kappaphycus alvarezii, after previous extraction at room temperature, are mainly composed of kappa-carrageenans (approximately 74%) and micro-carrageenans (approximately 3%). However, a significant percentage of these galactans (at least 14%) is composed of sulfated agarans and, possibly, agaran-type sulfated DL-hybrid galactans. These agarans are partially substituted on C-2 or C-4 or disubstituted on both positions of the beta-D-galactose units and on C-3 or C-2 and C-3 of the alpha-L-galactose residues with sulfate groups or single stubs of beta-D-xylopyranose, D-glucopyranose, and galactose or with D-glucopyranosyl-(1-->4)-D-glucopyranose side chains. Significant quantities of 2-O-methyl- and 3-O-methyl-L-galactose units are also present. A great tendency to retain Ca2+ and Mg2+, in spite of massive treatments with Na+ and K+ salts, was observed. The complexation between agarans and agarans-kappa-carrageenans through divalent cations and the possible zipper-type carbohydrate-carbohydrate interactions would be two complementary mechanisms of interactions.     

Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG’s) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor l-captopril (IC₅₀ = 3.3 μM, K_i = 1.8 μM). In vitro antimicrobial activity was demonstrated for l-captopril against Escherichia coli.     

Steroidal saponins from the rhizomes of Paris delavayi

Two new steroidal saponins, padelaosides A (1) and B (2), along with two other known steroidal saponins (3 and 4) were isolated from the rhizomes of Paris delavayi. Their structures were elucidated by 1D and 2D NMR techniques, HRFTMS, physical data and chemical methods. The two different absolute configurations of fucose, assigned as l and d that were found on compounds 1 and 2, respectively, were simultaneously reported in a natural medicine for the first time.     

The system of xylogalactans from the red seaweed Jania rubens (Corallinales, Rhodophyta)

The main acidic polysaccharides from the red seaweed Jania rubens share the general characteristics of corallinans (agar-like xylogalactans). After fractionation by ion-exchange chromatography, ten fractions were separated and characterized by sugar composition, other components, methylation, ethylation, desulfation-methylation, and NMR analyses. The main group of fractions carry the agaran disaccharidic repeating unit [-->3)-beta-d-Gal-(1-->4)-alpha-l-Gal-(1-->] substituted mainly on O-6 of the beta-d-Gal unit by beta-xylosyl side stubs, and less with sulfate or methoxyl groups, and also on O-2 of the alpha-l-Gal unit with methoxyl or sulfate, or less on O-3 of the same unit with methoxyl groups. These features are somehow common to the four members of the order already studied. However, a sugar uncommon to the order appears in moderate proportions for all the fractions: it is 3,6-anhydro-l-galactose (partly sulfated or methoxylated on O-2) replacing the l-Gal unit. Besides, several other structural features never found in the order (and uncommon in any polysaccharide) appear in some minor fractions: the presence of side stubs of 2,3-di- and 3-O-methyl-d-galactose, and also part of the 3-O-methyl-l-galactose acting as side stubs. These results show that, although the main features of the corallinean xylogalactans are common to all the species studied, each one has minor characteristics of its own.     

Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia

Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an in silico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, N^α-benzoyl-l-arginine, and N^ω-amino-l-arginine as substrates but not agmatine, l-homoarginine, N^α-benzoyl-l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424–alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k₁ = 80 s⁻¹) and hydrolysis (k₂ = 35 s⁻¹) of the intermediate were determined. The comparatively lower value of the steady-state rate constant (k_cat = 2.6 s⁻¹), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso-l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas N^ω-cyano-l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.     

Agarans from the red seaweed Polysiphonia nigrescens (Rhodomelaceae, Ceramiales)

Polysiphonia nigrescens was sequentially extracted with water at room temperature, 70 and 90 degrees C. The extracts were analyzed and the major one, isolated at 70 degrees C, was fractionated by ion-exchange chromatography, eluting with water and solutions of increasing sodium chloride concentration; five main fractions were separated. Structural analysis, carried out by methylation analysis and NMR spectroscopy, showed that four of these were partially cyclized agarans that were highly substituted on C-6 mainly with sulfate, although methyl ether and single stubs of beta-D-xylose were found in minor proportions. A fifth fraction comprising 6-sulfated agarose was also isolated. The use of 2D NMR techniques allowed us to assign the 1H and 13C NMR resonances of the G6S-->L6S diad for the first time.     

Using native hydantoinase promoter to induce d-carbamoylase soluble expression in Escherichia coli

By use of PCR, the genes encoding d-carbamoylase from A. radiobacter TH572 were cloned in plasmid pET30a and transformed into Escherichia coli BL21 (DE3) to overexpress d-carbamoylase. However, almost all of the protein remained trapped in inclusion bodies. To improve the expression of the properly folded active enzyme, a constitutive plasmid of pGEMT-DCB was constructed using the native hydantoinase promoter (P_Hase) whose optimal length was confirmed to 209 bp. Furthermore, the RBS region in the downstream of P_Hase was optimized to increase the expression level, so the plasmid pGEMT-R-DCB was constructed and transformed into E. coli strain Top10F′. The enzyme activity of Top10F′/pGEMT-R-DCB grown at 37 °C was found to be 0.603 U/mg (dry cell weight, DCW) and increase 58-fold over cells of BL21 (DE3) harboring the plasmid pET-DCB grown at 28 °C.     

Immunostimulatory activity of sulfated galactans isolated from the red seaweed Gracilaria fisheri and development of resistance against white spot syndrome virus (WSSV) in shrimp

Sulfated galactans (SG) were isolated from the red seaweed Gracilaria fisheri (G. fisheri). Chemical analysis revealed SG contains sulfate (12.7%) and total carbohydrate (42.2%) with an estimated molecular mass of 100 kDa. Structure analysis by NMR and FT-IR spectroscopy revealed that SG is a complex structure with a linear backbone of alternating 3-linked β-d-galactopyranose and 4-linked 3,6-anhydrogalactose units with partial 6-O-methylate-β-d-galactopyranose and with sulfation occurring on C4 of d-galactopyranose and C6 of l-galactopyranose units. SG treatment enhanced immune parameters including total haemocytes, phenoloxidase activity, superoxide anions and superoxide dismutase in shrimp Penaeus monodon. Shrimp fed with Artemia salina enriched with SG (100 and 200 μg ml⁻¹) and inoculated with white spot syndrome virus (WSSV) showed a significantly lower mortality rate and lower viral VP 28 amplification and expression than control. The results suggest that SG from G. fisheri exhibits immune stimulatory and antiviral activities that could protect P. monodon from WSSV infection.     

Development of an interference-free biosensor for l-glutamate using a bienzyme salicylate hydroxylase/l-glutamate dehydrogenase system

An amperometric biosensor was developed for the interference-free determination of l-glutamate with a bienzyme-based Clark electrode. This sensor is based on the specific dehydrogenation by l-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with salicylate hydroxylase (SHL, EC 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation of l-glutamate consuming NAD⁺. The product, NADH, initiates the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement of l-glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (1 s) and a short recovery (1 min). It shows a linear detection range between 10 μM and 1.5 mM l-glutamate with a detection limit of 3.0 μM. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from other amino acids and electroactive substances.     

Unusual structures in the polysaccharides from the red seaweed Pterocladiella capillacea (Gelidiaceae,Gelidiales)

Sequential extraction of tetrasporic Pterocladiella capillacea with water at room temperature and then at 50 degrees C led to the isolation of two products that were each fractionated with cetrimide to give a soluble fraction and a precipitate. The precipitates were then subjected to fractional solubilization in solutions of increasing sodium chloride concentration. The whole treatment yielded two major fractions in each case, one soluble in the cetrimide medium and the other soluble in 0.5 M NaCl, which were further fractionated by anion-exchange chromatography. Structural analysis, carried out by methylation, desulfation-methylation, 13C NMR spectroscopy and determination of the absolute configuration of the 2,6-di-O-methylgalactose units in the permethylated products, indicated the presence of xylogalactans, with low content of 3,6-anhydrogalactose and low molecular weight. These polysaccharides varied in the level of xylopyranosyl and sulfate substitution, primarily on the 6-position of the 3-linked beta-D-galactopyranosyl and on the 3-position of the 4-linked alpha-galactopyranosyl units. Moreover, herein we report, for the first time, the presence of 3-substituted, 4-linked D-galactopyranosyl residues in an alga belonging to the Gelidiales.     

Synthesis of acryloyl guar gum and its hydrogel materials for use in the slow release of l-DOPA and l-tyrosine

Acryloyl guar gum (AGG) and its hydrogel materials were synthesized for use as carriers and slow release devices of two pro-drugs, l-tyrosine and 3,4-dihydroxy phenylalanine (l-DOPA). To evaluate their structure-properties relationship, these were characterized by scanning electron micrography (SEM), FTIR spectroscopy and swelling studies. The hydrogel materials responded to the change of pH of the swelling medium, and exhibited reversible transitions in 0.9% saline solution. These were loaded with two pro-drugs, and their cumulative release behavior was studied at pH 2.2 and pH 7.4. The hydrogel materials exhibited structure-property relationship in the release of these pro-drugs. The % cumulative release of l-tyrosine was the maximum from the AGG-g-poly(methacrylic acid), while the maximum release of l-DOPA was observed from AGG-g-poly(AAc) in both the media. On the other hand, the AGG-g-poly(2-hydroxyethyl methacrylate) and AGG-g-poly(2-hydroxypropyl methacrylate) retained 42.33% and 49.05% of the drug even after 12 h.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Galactans from Cryptonemia species. Part II: Studies on the system of galactans of Cryptonemia seminervis (Halymeniales) and on the structure of major fractions

Cryptonemia seminervis biosynthesizes a family of d,l-hybrid galactans based on the classical 3-linked β-d-galactopyranosyl→4-linked α-d- and α-l-galactopyranosyl alternating sequence (A-units→B-units) with major amounts of α-d- and α-l-galactose and 3,6-anhydro-d- and l-galactose and lesser percentages of 3,6-anhydro-2-O-methyl-l-galactose, 2-O-methyl-, 4-O-methyl- and 6-O-methylgalactoses. The dispersion of structures in this family is based on five structural factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and glycosyl side-chain (4-O-methyl galactopyranosyl and/or xylosyl); (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio d,l-galactoses and d,l-3,6-anhydrogalactoses also in the B-units, (d) the formation of diads and (e) the sequence of the diads in the linear backbone. Considering these variables it is not unexpected to find in the fractions studied at least 18 structural units producing highly complex structures. Structural studies carried out in two major fractions (S2S-3 and S2S-4) showed that these galactans were formed mainly by β-d-galactopyranosyl 2-sulfate (20 and 11.9 mol %), β-d-galactopyranosyl 2-sulfate 4,6-O-(1′-carboxyethylidene) (8.9 and 6.0 mol %) and β-d-galactopyranosyl 2,6-sulfate (5.4 and 18.6 mol %), together with 3,6-anhydro-α-l-galactopyranosyl (11.4 and 7.3 mol %) and 3,6-anhydro-α-l-galactopyranosyl 2-sulfate (4.9 and 15.4 mol %) and minor quantities of 12-15 other structural units.Preparative alkaline treatment carried out on fraction (S2S-3) produced a quantitative formation of 3,6-anhydro α-l-galactopyranosyl units from precursor units (α-l-galactose 6-sulfate and α-l-galactose 2,6-sulfate). Kinetic studies on this 3,6-anhydro cyclization show a rate constant of 5.2 × 10⁴ s⁻¹ indicating diads of the type G→L6S/2,6S. Data from chemical, spectroscopic and kinetic studies suggest that, in S2S-3, the agaran block in the d,l-hybrid galactan is composed of the following diads: G(6R)→L6S/2,6S and G2S(P)(2,6S)→LA(2S)(2R)(2M) and the carrageenan block of G2S(P)→D(2S)(2,3S)(3S)(3,6S) in a molar ratio of agaran to carrageenan structures of ∼2:1.     

Antioxidative defense organization in retroperitoneal white adipose tissue during acclimation to cold—The involvement of l-arginine/NO pathway

1. Retroperitoneal white adipose tissue (RpWAT) antioxidative defense was investigated in untreated, l-arginine-treated and N^ω-nitro-l-arginine methyl ester (l-NAME)-treated rats kept at 4±1 °C (1, 3, 7, 12, 21 and 45 days) and compared to control rats at 22±1 °C.

2. Cold-acclimation-induced RpWAT weight decrease was accompanied by a decline in glutathione level and increased activity of manganese superoxide dismutase (MnSOD), glutathione S-transferase (GST), catalase, glutathione peroxidase and glutathione reductase at different time-points.

3. l-arginine accelerated RpWAT weight decrease, the increase in MnSOD and GST activities and the prolonged increase of catalase, MnSOD and GST activities. l-NAME delayed cold-induced catalase activity increase and tissue weight decrease. Prolonged l-NAME-treatment had a similar effect on RpWAT as l-arginine.

4. Results suggest the involvement of l-arginine/NO pathway in RpWAT oxidative metabolic augmentation induced by cold-acclimation.

alpha-D-Glucuronosyl-(1-->3)-L-galactose,an unusual disaccharide from polysaccharides of the hornwort Anthoceros caucasicus

Acid hydrolysis of cell wall-rich material from thalli of the hornwort Anthoceros caucasicus yielded substantial amounts of an unusual disaccharide (1). Hydrolysis of 1 yielded only GlcA, Gal and unhydrolysed 1. Compound 1 was identified as alpha-D-GlcpA-(1-->3)-L-Gal by 1H and 13C NMR spectroscopic analysis and by the susceptibility of its monosaccharide units to phosphorylation by enantiomer-specific kinases. Compound 1 was not detected in acid hydrolysates of other land plants including mosses, leafy and thalloid liverworts, lycopodiophytes and euphyllophytes; it was also absent from charophytes. The Anthoceros polysaccharide that yields 1 was partially extractable in cold aqueous buffer (pH 4.7) and Na(2)CO(3), but not in EDTA or NaOH, suggesting that it was not a typical pectin or hemicellulose. The yield of 1 from various polysaccharide fractions correlated with the yields of Xyl, suggesting a previously unreported polymer containing D-GlcA, L-Gal and Xyl. The existence of a unique polysaccharide in an evolutionarily isolated plant (Anthoceros) supports the view that major steps in plant phylogeny were accompanied by significant changes in cell wall composition.     

Construction and co-expression of polycistronic plasmid encoding d-hydantoinase and d-carbamoylase for the production of d-amino acids

d-Hydantoinase and d-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the d-carbamoylase and avoid intermediate accumulation, the d-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble d-hydantoinase and d-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, d-p-hydroxyphenylglycine (d-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield.     

Single-step bioconversion for the preparation of l-gulose and l-galactose

Both carbohydrate monomers l-gulose and l-galactose are rarely found in nature, but are of great importance in pharmacy R&D and manufacturing. A method for the production of l-gulose and l-galactose is described that utilizes recombinant Escherichia coli harboring a unique mannitol dehydrogenase. The recombinant E. coli system was optimized by genetic manipulation and directed evolution of the recombinant protein to improve conversion. The resulting production process requires a single step, represents the first readily scalable system for the production of these sugars, is environmentally friendly, and utilizes inexpensive reagents, while producing l-galactose at 4.6 g L⁻¹ d⁻¹ and l-gulose at 0.90 g L⁻¹ d⁻¹.     

Sulfated mannans from the red seaweed Nemalion helminthoides of the South Atlantic

Nemalion helminthoides, collected in the Argentine South Atlantic coast, was extracted with hot water and the crude product fractionated using cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration and seven fractions were separated and analyzed. Structural analysis of the main fractions, those soluble in 3.0 and 4.0 M NaCl (yields 21.0% and 13.8%, respectively) and those insoluble in 4.0 M NaCl (yield 20.0%), indicated that this seaweed biosynthesizes (1 → 3)-linked α-d-mannans that are sulfated at positions 4 and 6. Three mannan fractions comprising considerable amounts of xylose were also isolated in very low total yield (2.0%). The fractions that were soluble in 3.0 and 4.0 M NaCl showed low antiherpetic activity whereas this activity was considerable for the fraction solubilized in 2.0 M NaCl (yield 0.5%) which contained single stubs of β-d-xylose. A xylan, soluble in cetrimide solution, containing (1 → 3, 1 → 4)-linked β-d-xylose residues, was also isolated in minor amount.