Inhibition of nitric oxide synthase by cobalamins and cobinamides

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -l-[¹⁴C]arginine-to-l-[¹⁴C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN₂-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN₂-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN₂-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN₂-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with l-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the “base-off” conformation of the lower axial dimethylbenzimidazole ligand. CN₂-Cbi inhibited interferon-γ-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.     

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca²⁺ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca²⁺ mobilization study shows that l-NAME inhibited the reduction of Ca²⁺-concentration in VSM cells and reduced the increase in Ca²⁺-concentration in endothelial cells induced by SNP. This enhancement in Ca²⁺-concentration in the endothelial cells is due to a voltage-dependent Ca²⁺ channels activation. The present findings indicate that the relaxation and [Ca²⁺]_i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.     

Nitric Oxide Participates in the Brain Ischemic Tolerance Induced by Intermittent Hypobaric Hypoxia in the Hippocampal CA1 Subfield in Rats

Previous studies have shown that intermittent hypobaric hypoxia (IH) preconditioning protected neurons survival from brain ischemia. However, the mechanism remains to be elucidated. The present study explored the role of nitric oxide (NO) in the process by measuring the expression of NO synthase (NOS) and NO levels. Male Wistar rats (100) were randomly assigned into four groups: sham group, IH?+?sham group, ischemia group and IH?+?ischemia group. Rats for IH preconditioning were exposed to hypobaric hypoxia mimicking 5000 m high-altitude (P_B?=?404 mmHg, PO₂?=?84 mmHg) 6 h/day, once daily for 28 days. Global brain ischemia was established by four-vessel occlusion that has been created by Pulsinelli. Rats were sacrificed at 7th day after the ischemia for neuropathological evaluation by thionin stain. In addition, the expression of neuronal NOS (nNOS), inducible NOS (iNOS), and NO content in the hippocampal CA1 subfield were measured at 2nd day and 7th day after the ischemia. Results revealed that global brain ischemia engendered delayed neuronal death (DND), both nNOS and iNOS expression up-regulated, and NO content increased in the hippocampal CA1 subfield. IH preconditioning reduced neuronal injury induced by the ischemia, and prevented the up-regulation of NOS expression and NO production. In addition, l-NAME?+?ischemia group was designed to detect whether depressing NO production could alleviate the DND. Pre-administration of l-NAME alleviated DND induced by the ischemia. These results suggest that IH preconditioning plays a protective role by inhibiting the over expression of NOS and NO content after brain ischemia.     

Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson's disease induced by l-DOPA?

Inflammatory mechanisms are proposed to play a role in l-DOPA-induced dyskinesia. Cyclooxygenase-2 (COX2) contributes to inflammation pathways in the periphery and is constitutively expressed in the central nervous system. Considering that inhibition of nitric oxide (NO) formation attenuates l-DOPA-induced dyskinesia, this study aimed at investigating if a NO synthase (NOS) inhibitor would change COX2 brain expression in animals with l-DOPA-induced dyskinesia. To this aim, male Wistar rats received unilateral 6-hydroxydopamine microinjection into the medial forebrain bundle were treated daily with l-DOPA (21 days) combined with 7-nitroindazole or vehicle. All hemi-Parkinsonian rats receiving l-DOPA showed dyskinesia. They also presented increased neuronal COX2 immunoreactivity in the dopamine-depleted dorsal striatum that was directly correlated with dyskinesia severity. Striatal COX2 co-localized with choline-acetyltransferase, calbindin and DARPP-32 (dopamine-cAMP-regulated phosphoprotein-32), neuronal markers of GABAergic neurons. NOS inhibition prevented l-DOPA-induced dyskinesia and COX2 increased expression in the dorsal striatum. These results suggest that increased COX2 expression after l-DOPA long-term treatment in Parkinsonian-like rats could contribute to the development of dyskinesia.     

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.

Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.     

Asymmetric Dimethylarginine and Hepatic Encephalopathy: Cause,Effect or Association?

The methylated derivative of l-arginine, asymmetric dimethylarginine (ADMA) is synthesized in different mammalian tissues including the brain. ADMA acts as an endogenous, nonselective, competitive inhibitor of all three isoforms of nitric oxide synthase (NOS) and may limit l-arginine supply from the plasma to the enzyme via reducing its transport by cationic amino acid transporters. Hepatic encephalopathy (HE) is a relatively frequently diagnosed complex neuropsychiatric syndrome associated with acute or chronic liver failure, characterized by symptoms linked with impaired brain function leading to neurological disabilities. The l-arginine—nitric oxide (NO) pathway is crucially involved in the pathomechanism of HE via modulating important cerebral processes that are thought to contribute to the major HE symptoms. Specifically, activation of this pathway in acute HE leads to an increase in NO production and free radical formation, thus, contributing to astrocytic swelling and cerebral edema. Moreover, the NO-cGMP pathway seems to be involved in cerebral blood flow (CBF) regulation, altered in HE. For this reason, depressed NO-cGMP signaling accompanying chronic HE and ensuing cGMP deficit contributes to the cognitive and motor failure. However, it should be remembered that ADMA, a relatively little known element limiting NO synthesis in HE, may also influence the NO-cGMP pathway regulation. In this review, we will discuss the contribution of ADMA to the regulation of the NO-cGMP pathway in the brain, correlation of ADMA level with CBF and cognitive alterations observed during HE progression in patients and/or animal models of HE.     

Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands

Genome sequencing has recently shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles of these proteins remain unclear. The interactions of a series of l-arginine (l-arg) analogs and iron ligands with two recombinant NOS-like proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) have been studied by UV–visible spectroscopy. SaNOS and baNOS in their ferric native state, as well as their complexes with l-arg analogs and with various ligands, exhibit spectral characteristics highly similar to the corresponding complexes of heme-thiolate proteins such as cytochromes P450 and NOSs. However, saNOS greatly differs from baNOS at the level of three main properties: (i) native saNOS mainly exists under an hexacoordinated low-spin ferric state whereas native baNOS is mainly high-spin, (ii) the addition of tetrahydrobiopterin (H4B) or H4B analogs leads to an increase of the affinity of l-arg for saNOS but not for baNOS, and (iii) saNOS Fe^II, contrary to baNOS, binds relatively bulky ligands such as nitrosoalkanes and tert-butylisocyanide. Thus, saNOS exhibits properties very similar to those of the oxygenase domain of inducible NOS (iNOS_oxy) not containing H4B, as expected for a NOSoxy-like protein that does not contain H4B. By contrast, the properties of baNOS which look like those of H4B-containing iNOS_oxy are unexpected for a NOS-like protein not containing H4B. The origin of these surprising properties of baNOS remains to be determined.     

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopoiesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells.     

Identification and distribution of nitric oxide synthase in the brain of adult Antarctic teleosts

We investigated the presence of nitric oxide synthase (NOS) in brain of adult Antarctic teleosts by indirect immunofluorescence technique using a synthetic rat neuronal NOS (nNOS) antibody. The following species were examined: Trematomus bernacchii, Gymnodraco acuticeps, Histiodraco velifer, Cygnodraco mawsoni (haemoglobin-rich), Chionodraco hamatus and Pagetopsis macropterus (haemoglobin-free). Immunoreactive cell bodies were localized in dorsal telencephalon, in hypothalamus, in optic tectum of the mesencephalon as well as in Purkinje cells of the cerebellum. No differences were observed in the localization of the nNOS immunopositivity in the Antarctic teleosts brains examined and NOS distribution was similar to that described in other teleosts, suggesting that nitric oxide (NO) may also function as a neurotransmitter in the brain of Antarctic teleosts. A strong immunopositivity was observed in the cerebral blood vessels of the icefishes suggesting that NO may play a pivotal role in the regulation of the cerebral blood flow especially in these haemoglobin-free species.     

l-Carnitine enhances resistance to oxidative stress by reducing DNA damage in Ataxia telangiectasia cells

In this study, the modulating effect of l-carnitine on tert-butyl-hydroperoxide-induced DNA damage was compared with that of mannitol, a well known scavenger of hydroxyl radicals, both in normal and Ataxia telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines established from A. telangiectasia (A-T) patients. The alkaline version of the comet assay was employed to measure the frequency of single-strand breaks (SSBs) and alkali-labile sites induced by t-butyl-OOH immediately after treatment and at different recovery times in normal and A-T cell lines, with and without pre-treatment with l-carnitine. In addition, both the yield of induced chromosomal damage and the effect on cell proliferation were evaluated. Our results show that pre-treatment of cells with l-carnitine produced an enhancement of the rate and extent of DNA repair in A-T cell lines at early recovery time; furthermore, in samples pre-treated with l-carnitine a reduction of all types of chromosomal aberration was observed, both in A-T and in wild-type cell lines. The reducing effect of l-carnitine pre-treatment on oxidative DNA damage was more prominent than that of pre-treatment with mannitol. In conclusion, we demonstrated a protective effect of l-carnitine on oxidative stress-induced DNA damage in A-T cells, suggesting its possible role in future pharmacological applications in A-T therapy.     

L-Amino acid oxidase from Naja naja oxiana venom

A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (− 20 °C, − 70 °C) and loses its activity by heating at 70 °C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC₅₀ of 0.094 μM and 0.036 μM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.     

Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H₂S) donor Na₂S with IC₅₀ values of ∼2.4·10⁻⁵ and ∼7.9·10⁻⁵ m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H₂S does not interfere with substrate or cofactor binding. The IC₅₀ decreased to ∼1.5·10⁻⁵ m at pH 6.0 and increased to ∼8.3·10⁻⁵ m at pH 8.0. Preincubation of concentrated nNOS with H₂S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H₂S and NO. Likewise, NADPH oxidation was inhibited with an IC₅₀ of ∼1.9·10⁻⁵ m in the presence of Arg and BH4 but exhibited much higher IC₅₀ values (∼1.0–6.1·10⁻⁴ m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na₂S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC₅₀ ∼ 1.3–2.0·10⁻⁵ m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H₂S/NO at modest concentrations of H₂S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H₂S formation.     

Clonidine induces calcitonin gene-related peptide expression via nitric oxide pathway in endothelial cells

The present study was to determine whether clonidine could induce calcitonin gene-related peptide (CGRP) production and the underlying mechanisms. Human umbilical vein endothelial cells were treated with clonidine and the dose–effect or time–effect relationship of clonidine on CGRP production was examined. Youhimbine (a α₂-adrenoceptor blocker) and l-NAME (an antagonist of nitric oxide synthase, NOS) were chosen to explore the role of α₂-adrenoceptor and nitric oxide pathway in the effect of clonidine on endothelial cell-derived CGRP production. The level of CGRP mRNA or protein was detected by Real Time-PCR or radioimmunoassay. Nitric oxide content was measured by nitroreduction assay. The study showed that clonidine was able to induce CGRP mRNA (α- and β-isoforms) expression in a dose-dependent manner in endothelial cells. The effect of clonidine on endothelial cell-derived CGRP synthesis and secretion was attenuated in the presence of youhimbine. l-NAME treatment could also inhibit clonidine-induced CGRP synthesis and secretion concomitantly with the decreased NO content in culture medium. These results suggest that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of α₂-adrenoceptor, which is related to the NO pathway.     

Glycosylated nervogenic acid derivatives from Liparis condylobulbon (Reichb.f.) leaves

Three new nervogenic acid glycosides, 1-O-α-l-rhamnopyranosyl 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoate, 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoic acid, and bis{3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoyl} 1,2-O-β-d-glucopyranose, which we named condobulbosides A–C, were isolated from a methanol extract of the leaves of Liparis condylobulbon together with an apigenin C-glycoside, schaftoside. Their structures were established on the basis of spectral techniques, namely, UV, IR, HR-MS spectroscopy, both 1D and 2D NMR experiments, and chemical reactions.     

Tetrahydrobiopterin, L-arginine and vitamin C actsynergistically to decrease oxidative stress, increase nitricoxide and improve blood flow after induction of hindlimbischemia in the rat

Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is a potent vasodilator and signaling molecule that plays an essential role in vascular remodeling of collateral arteries and perfusion recovery in response to hindlimb ischemia. In ischemic conditions, decreased NO bioavailability was observed because of increased oxidative stress, decreased l-arginine and tetrahy-drobiopterin. This study tested the hypothesis that dietary cosupplementation with tetrahydrobiopterin (BH4), l-arginine, and vitamin C acts synergistically to decrease oxidative stress, increase nitric oxide and improve blood flow in response to acute hindlimb ischemia. Rats were fed normal chow, chow supplemented with BH4 or l-arginine (alone or in combination) or chow supplemented with BH4 + l-arginine + vitamin C for 1 wk before induction of unilateral hindlimb ischemia. Cosupplementation with BH4 + l-arginine resulted in greater eNOS expression, Ca²⁺-dependent NOS activity and NO concentration in gastrocnemius from the is-chemic hindlimb, as well as greater recovery of foot perfusion and more collateral artery enlargement than did rats receiving either agent separately. The addition of vitamin C to the BH4 + l-arginine regimen did further increase these dependent variables, although only the increase in eNOS expression reached statistical significances. In addition, rats given all three supplements demonstrated significantly less Ca²⁺-independent activity, less nitrotyrosine accumulation, greater glutathione:glutathione disulfide (GSH:GSSG) ratio and less gastrocnemius muscle necrosis, on both macroscopic and microscopic levels. In conclusion, cosupplementation with BH4 + l-arginine + vitamin C significantly increased vascular perfusion after hindlimb ischemia by increasing eNOS activity and reducing oxidative stress and tissue necrosis. Oral cosupplementation of l-arginine, BH4 and vitamin C holds promise as a biological therapy to induce collateral artery enlargement.     

Relationship between structure and inhibitory effect of arginine analogues on neuronal nitric oxide synthase activity

Since nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) froml-arginine (Arg) which has an amidino group in its molecule, we, examined the effect of 29 kinds of Arg analogues on neuronal NOS (nNOS) activity in the rat brain. None of the Arg analogues acted as a substrate for nNOS. Diamidinocystamine, hirudonine, and guanethidine inhibited nNOS activity to 67.3%, 64.2% and 74.1%, respectively, but their inhibitory efficiency was lower than N^G-monomethyl-l-arginine (to 36.5%) which is a well known NOS inhibitor. Dimethylguanidine and N-benzoylguanidine also significantly inhibited nNOS activity to 88.0% and 90.7%, respectively. Whereas almost all of the NOS inhibitors previously reported were synthesizdd by substituting the amidino nitrogen of Arg, none of these new inhibitors were substituted at this position. Furthermore, hirudonine, which is a naturally occurring compound, was thought to act as an agonist at polyamine binding site of the N-methyl-d-aspartate type of glutamate receptor complex. It is also interesting that guanethidine, an antihypertensive agent, inhibit nNOS activity. These new drugs are useful for the investigation not only of the chemical nature of nNOS but also of the physiologic function of NO.     

Initial characterization of a recombinant kynureninase from Trypanosoma cruzi identified from an EST database

Kynureninase has been described in bacteria, fungi and animals as an enzyme involved in the catabolic degradation pathway of l-tryptophan. This pyridoxal 5′-phosphate (PLP)-dependent enzyme catalyzes the hydrolytic cleavage of l-kynurenine and 3-hydroxy-l-kynurenine to yield l-alanine and either anthranilic or 3-hydroxyanthranilic acid, respectively. We identified a putative kynureninase gene from a Trypanosoma cruzi project aiming at the structural and functional characterization of more than 100 proteins differentially expressed during metacyclogenesis. This gene encodes a protein similar in size and sequence to kynureninases from other sources. This open reading frame was cloned and the recombinant enzyme was overexpressed. Recombinant T. cruzi kynureninase was purified to homogeneity and its identity was confirmed by mass spectrometry. The apparent molecular mass of the native T. cruzi kynureninase was estimated by gel filtration, suggesting that the protein is a homodimer. Circular dichroism spectrum indicated a mixture of α-helix and β-sheet structure, expected for an aminotransferase fold. l-kynurenine, preferentially hydrolyzed by prokaryotic inducible kynureninases, and 3-hydroxy-l-kynurenine, the preferred substrate in fungi and vertebrates, are both catabolized equally well by T. cruzi kynureninase. Further experimental assays will be performed to fully understand the importance of this enzyme for T. cruzi metabolism.     

Structure of the oligosaccharides isolated from Dalbergia sissoo Roxb. leaf polysaccharide

A water-soluble polysaccharide isolated from Dalbergia sissoo Roxb. leaves was purified and major homogeneous fraction obtained by GPC. Complete hydrolysis of the polysaccharide followed by paper chromatography and GLC analysis indicated the presence of l-rhamnose, d-glucuronic acid, d-galactose and d-glucose in molar ratio of 1:1:2:2.33, respectively. Partial hydrolysis of the polysaccharide furnished one tri-[I], one hepta-[II] and one nona-[III] saccharides. Hydrolysis of the oligosaccharide I, II and III followed by GLC analysis furnished d-glucose and l-rhamnose (2:1); l-rhamnose, d-galactose and d-glucuronic acid (1:3:3); and l-rhamnose, d-galactose and d-glucose (1:3:5), respectively. Methylation analysis and periodate oxidation of the oligosaccharide I indicated the presence of two non reducing glucose units linked to rhamnose by 1→2 and 1→4 linkages, respectively. Oligosaccharide II is a branched molecule with a main chain consisting of 1,3-linked β-d-galactopyranosyl (2 mol), 1,3,4 linked α-l-rhamnopyranosyl (1 mol) and 1,4,6 linked β-d-galactopyranosyl unit (1 mol) and non reducing β-d-glucuronic acid at the end along with side chains of β-d-glucouronopyranosyl units (2 mol). Oligosaccharide III is also a branched molecule with a main chain consisting of 1,3,4 linked α-l-rhamnopyranosyl (1 mol), 1,2,4 linked β-d-glucopyranosyl (1 mol), 1,3 and 1,4 linked β-d-galactopyranosyl (2 and 1 mol, respectively) having β-d-glucopyranosyl as a non reducing end.