Helicobacter pylori GmhB enzyme involved in ADP-heptose biosynthesis pathway is essential for lipopolysaccharide biosynthesis and bacterial virulence.

Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the ß-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

An Avocado Extract Enriched in Mannoheptulose Prevents the Negative Effects of a High-Fat Diet in Mice.

Beginning at 16 weeks of age and continuing for 44 weeks, male C57BL/6J were fed either a control (CON) diet; a high-fat (HF) diet (60% unsaturated); or the HF diet containing an extract of unripe avocados (AvX) enriched in the 7-carbon sugar mannoheptulose (MH), designed to act as a glycolytic inhibitor (HF + MH). Compared to the CON diet, mice on the HF diet exhibited higher body weights; body fat; blood lipids; and leptin with reduced adiponectin levels, insulin sensitivity, VO2max, and falls from a rotarod. Mice on the HF + MH diet were completely protected against these changes in the absence of significant diet effects on food intake. Compared to the CON diet, oxidative stress was also increased by the HF diet indicated by higher levels of total reactive oxygen species, superoxide, and peroxynitrite measured in liver samples by electron paramagnetic resonance spectroscopy, whereas the HF + MH diet attenuated these changes. Compared to the CON, the HF diet increased signaling in the mechanistic target of the rapamycin (mTOR) pathway, and the addition of the MH-enriched AvX to this diet attenuated these changes. Beyond generating further interest in the health benefits of avocados, these results draw further new attention to the effects of this rare sugar, MH, as a botanical intervention for preventing obesity.

Total synthesis of D-glycero-D-mannno-heptose 1ß, 7-bisphosphate with 3-O-amyl amine linker and its monophosphate derivative.

D-Glycero-D-mannno-heptose 1ß, 7-bisphosphate (HBPß) is an important intermediate for constructing the core structure of Gram-negative bacterial lipopolysaccharides and was reported as a pathogen-associated molecular pattern (PAMP) that regulates immune responses. HBPß with 3-O-amyl amine linker and its monophosphate derivative D-glycero-D-mannno-heptose 7-phosphate (HP) with 1α-amyl amine linker have been synthesized as candidates for immunity study of HBPß. The O3-amyl amine linker of heptose was installed by dibutyltin oxide-mediated regioselective alkylation under fine-tuned protecting condition. The stereoselective installation of 1ß-phosphate ester was achieved by NIS-mediated phosphorylation at low temperature. The strategy for installation of 3-O-amyl amine linker onto HBP derivative can be expanded to the syntheses of other conjugation-ready carbohydrates bearing anomeric phosphoester.

Nonhydrolyzable Heptose Bis- and Monophosphate Analogues Modulate Pro-inflammatory TIFA-NF-κB Signaling.

d-Glycero-d-manno-heptose-1ß,7-bisphosphate (HBP) and d-glycero-d-manno-heptose-1ß-phosphate (H1P) are bacterial metabolites that were recently shown to stimulate inflammatory responses in host cells through the activation of the TIFA-dependent NF-κB pathway. To better understand structure-based activity in relation to this process, a family of nonhydrolyzable phosphonate analogues of HBP and H1P was synthesized. The inflammation modulation by which these molecules induce the TIFA-NF-κB signal axis was evaluated in vivo at a low-nanomolar concentration (6 nM) and compared to that of the natural metabolites. Our data showed that three phosphonate analogues had similar stimulatory activity to HBP, whereas two phosphonates antagonized HBP-induced TIFA-NF-κB signaling. These results open new horizons for the design of pro-inflammatory and innate immune modulators that could be used as vaccine adjuvant.

Characterization of avocado honey (Persea americana Mill.) produced in Southern Spain.

Plant origin, physicochemical parameters and composition were analysed to characterize the avocado honey (Persea americana Mill.) from Andalusia (Southern, Spain). Ashes content, total polyphenol, and electrical conductivity corresponded to these of a typical dark honey (>80 mm scale Pfund). Regarding mineral elements, K was predominant, followed by P and Mg. Antioxidant and invertase activities presented some desirable values. In the 20 analysed samples, 48 pollen types corresponding to 33 families were identified. Avocado pollen was found in high variability (13-58%). At least a 20% was suggested to guarantee the authentic avocado honey. Perseitol, sugar-alcohol identified only in avocado honey, fundamentally contributes to distinguish this kind of honey. The content varied between 0.31 and 1.56 g/100 g. The correlation between perseitol and avocado pollen was found to be significant. A minimum concentration of 0.30 g/100 g of perseitol is suggested to characterize the proposed monofloral avocado honey.

Unraveling the B. pseudomallei Heptokinase WcbL: From Structure to Drug Discovery.

Gram-negative bacteria utilize heptoses as part of their repertoire of extracellular polysaccharide virulence determinants. Disruption of heptose biosynthesis offers an attractive target for novel antimicrobials. A critical step in the synthesis of heptoses is their 1-O phosphorylation, mediated by kinases such as HldE or WcbL. Here, we present the structure of WcbL from Burkholderia pseudomallei. We report that WcbL operates through a sequential ordered Bi-Bi mechanism, loading the heptose first and then ATP. We show that dimeric WcbL binds ATP anti-cooperatively in the absence of heptose, and cooperatively in its presence. Modeling of WcbL suggests that heptose binding causes an elegant switch in the hydrogen-bonding network, facilitating the binding of a second ATP molecule. Finally, we screened a library of drug-like fragments, identifying hits that potently inhibit WcbL. Our results provide a novel mechanism for control of substrate binding and emphasize WcbL as an attractive anti-microbial target for Gram-negative bacteria.

Evaluation of 7-O-galloyl-D-sedoheptulose, isolated from Corni Fructus, in the adipose tissue of type 2 diabetic db/db mice.

The aim of the present study was to evaluate the beneficial effects of 7-O-galloyl-D-sedoheptulose (GS), isolated from Corni Fructus, using type 2 diabetic mice. GS was orally administered to db/db mice at doses of 20 and 100 mg/kg body weight per day for 6 weeks, and the effects of GS on biochemical factors in serum and adipose tissue were investigated. To define the underlying mechanism of these effects, protein expressions related to lipid metabolism, inflammation, fibrosis, and apoptosis, were measured. The results showed that levels of glucose, leptin, insulin, C-peptide, resistin, tumor necrosis factor-α, interleukin-6, triglycerides, total cholesterol, non-esterified fatty acids, high-density lipoprotein cholesterol, very low-density lipoprotein cholesterol/low-density lipoprotein cholesterol, reactive oxygen species (ROS), and thiobarbituric acid-reactive substance (TBARS) in serum were down-regulated, while adiponectin was augmented by GS treatment. In addition, the elevated lipid, ROS, and TBARS contents in adipose tissue as well as serum levels in db/db mice were significantly decreased by the oral administration of GS. From protein analysis, the decreased expressions of peroxisome proliferator activated receptor (PPAR)α, PPARγ, and B-cell lymphoma 2 were up-regulated in the adipose tissue of db/db mice. The administration of GS significantly decreased sterol regulatory element binding protein-1, nuclear factor-kappa ?>Bp65, cyclooxygenase-2, inducible nitric oxide synthase, monocyte chemotactic protein-1, intracellular adhesion molecule-1, phosphor c-Jun N-terminal kinase, activator protein-1, transforming growth factor-ß1, Bax, cytochrome c, and caspase-3 expressions. These results suggest that GS acts as a regulator of oxidative stress, inflammation, fibrosis, and apoptosis in the adipose tissue of db/db mice.

7-O-galloyl-D-sedoheptulose ameliorates renal damage triggered by reactive oxygen species-sensitive pathway of inflammation and apoptosis.

OBJECTIVES: This study was carried out to verify the preventive effects of 7-O-galloyl-d-sedoheptulose (GS), a phenolic compound isolated from Corni Fructus, underlying diabetic renal damage in type 2 diabetes. METHODS: GS was orally administered to db/db mice at doses of 20 and 100 mg/kg body weight per day for six weeks, and its effects were compared with those of the vehicle in db/db and m/m mice. KEY FINDINGS: In the serum and kidney, biochemical factors and expression of protein related to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, apoptosis and inflammation were examined. GS treatment attenuated serum and renal oxidative stress through reduction of reactive oxygen species and lipid peroxidation and increase in the ratio of glutathione and its oxidised form. Importantly, GS reduced renal protein expression of Nox-4 and p22(phox) (one of the subunits of NADPH oxidase), pro-apoptotic factors (such as Bax and cytochrome c) and nuclear factor-kappa B-targeting pro-inflammatory inducible nitric oxide synthase and cyclooxygenase-2. CONCLUSIONS: These renoprotective effects of GS were achieved through attenuation of diabetes-induced oxidative stress and its sensitive protein expression associated with inflammation and apoptosis in db/db mice.

Glycerol-induced renal damage improved by 7-O-galloyl-D-sedoheptulose treatment through attenuating oxidative stress.

The protective effect of 7-O-galloyl-D-sedoheptulose (GS), isolated from Corni Fructus as an active component, against acute renal failure (ARF) induced by glycerol was investigated. The administration of GS led to a decline in the levels of blood urea nitrogen and creatinine; on the other hand, it did not have a significant effect on creatinine clearance. Furthermore, GS also significantly decreased the urine volume and fractional excretion of sodium, but it increased the urine osmolarity, suggesting the protective role of GS against renal dysfunction. Oxidative stress under ARF was attenuated by GS through the inhibition of lipid peroxidation, scavenging of reactive oxygen species (ROS), and elevation of the antioxidative status. Renal oxidative stress is related to the overproduction of ROS by nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase; therefore, in the present study, the protein expression of p22(phox) and NAD(P)H oxidase-4 (Nox-4) was investigated. GS down-regulated the protein expression of p22(phox); on the other hand, it did not significantly affect the expression of Nox-4. This indicates that GS inhibits the production of superoxide by regulating a component of NAD(P)H oxidase, p22(phox). Furthermore, GS down-regulated the expressions of nuclear factor-κB (NF-κΒ) and inducible nitric oxide (NO) synthase (iNOS), suggesting that GS protects against NO-induced inflammatory pathological conditions under ARF through the regulation of NF-κB and iNOS expressions. The present study indicates that GS exerts a protective effect against ARF through the recovery of renal dysfunction and attenuation of renal oxidative stress by regulating related protein expression.

Novel action of 7-O-galloyl-D-sedoheptulose isolated from Corni Fructus as a hypertriglyceridaemic agent.

OBJECTIVES: We investigated the lipid-lowering activity of 7-O-galloyl-D-sedoheptulose, an active component of Corni Fructus, and related mechanisms. METHODS: Rats were fed a high-fructose diet for 6 days, followed by treatment with 7-O-galloyl-D-sedoheptulose, 5, 10 or 20 mg/kg per day, or fenofibrate (positive control). KEY FINDINGS: The high-fructose diet induced an increase in body weight, hypertriglyceridaemia, hyperglycaemia and hypertension. Administration of 7-O-galloyl-D-sedoheptulose significantly reduced the levels of triglyceride in the serum and liver (being more effective than fenofibrate) but did not lead to changes in liver weight or hepatic function, whereas fenofibrate increased the liver weight markedly. The preventive effect of 7-O-galloyl-D-sedoheptulose against the accumulation of triglyceride and cholesterol was related to the up-regulation of peroxisome proliferator-activated receptor alpha expression. CONCLUSIONS: The present study supports the role of 7-O-galloyl-D-sedoheptulose as a promising agent against hypertriglyceridaemia without hepatic side-effects.

Indonesian medicinal plants. XXIV. Stereochemical structure of perseitol x K+ complex isolated from the leaves of Scurrula fusca (Loranthaceae).

A complex of perseitol (D-glycero-D-galacto-heptitol) and K+ ions in a molar ratio of 20:1 was isolated from the leaves of Scurrula fusca (Loranthaceae), which has been traditionally used for the treatment of cancer in Sulawesi Island, Indonesia. The stereochemical structure of the complex in H2O solution has been elucidated by use of several kinds of NMR techniques. Furthermore, it has been found that the complex exhibits a potent inhibitory effect on [3H]-leucine incorporation for protein synthesis in Ehrlich ascites tumor cells in mice.

Characterisation of the abnormal pancreatic D and A cell function in streptozotocin diabetic dogs: studies with D-glyceraldehyde, dihydroxyacetone, D-mannoheptulose, D-glucose, and L-arginine.

Pancreatic D and A cell function is deranged in streptozotocin diabetes. To investigate this, the effect of D-glyceraldehyde, dihydroxyacetone, D-mannoheptulose and glucose variations during arginine stimulation on the release of somatostatin and glucagon from the isolated pancreas of normal and streptozotocin diabetic dogs was studied. Concentrations of the trioses, D-glyceraldehyde (1.25 and 2.5 mmol/l) and dihydroxyacetone (11 mmol/l), which normally stimulate D cells, did not influence the release of somatostatin in the diabetic dog. However, the higher concentration of D-glyceraldehyde (5 mmol/l) and dihydroxyacetone (11 mmol/l), which normally stimulate D cells, did not influence the release of somatostatin in the diabetic dog. However, the higher concentration of D-glyceraldehyde (5 mmol/l) suppressed D cell ssecretion in the diabetic animals at 0 and 8.3 mmol/l glucose. A cell secretion was significantly suppressed at the higher glucose level in response to both 2.5 and 5 mmol/l of te triose. This inhibition may be explained by a non-specific effect induced by the high dose of this triose. The addition of 5 mmol/l mannoheptulose, which normally reduces glucose-induced somatostatin secretion and stimulates glucagon release, did not affect hormone secretion. In both the diabetic and the normal animals, arginine (5 mmol/l) stimulated somatostatin and glucagon secretion. Although arginine was able to stimulate D and A cell secretion in the diabetic dogs, it was however unable to restore the response to changes in glucose concentration between 1.4 and 8.3 mmol/l to normal. These results demonstrate that the abnormal pancreatic D and A cell function in streptozotocin diabetes is characterised by an impaired response to glucose and certain glucose metabolites and probably results from a specific defect in glucose recognition.

[Action of soluble carbohydrates from avocado (Persea gratissima Gaertner) fruit on utilization of calcium in the rat]. / Action des glucides solubles du fruit de l'avocatier (Persea gratissima Gaertner) sur l'utilisation du calcium chez le rat.

Ethanolic extract containing soluble sugars was prepared from avocados (variety americana) coming from Madagascar. In this extract, thin layer and paper chromatography showed the presence of a ketoheptose, the mannoheptulose, in amount of 0.1 g/ml extract or 1.1 g/100 g fresh pulp. Physiological experiments were performed with avocado extract and comparatively with a sample of commercial mannoheptulose. Six-month-old rats were given by stomach tube 10 mM CaCl2 solution (+ 45Ca) containing a carbohydrate. Depending on the group, the carbohydrate was 20 and 100 mM glucose (control), D-mannoheptulose 200, 100, 50 and 25 mM, avocado extract concentrated or diluted 1/2, 1/4, 1/8, 1/16. Blood samples were taken at different times after the administration and plasma radioactivity was measured. Rats were sacrificed at 24 hours; femur and axis radioactivity was used as a measure of calcium absorption. Both mannoheptulose and avocado extract were potent on calcium absorption. This absorption increased progressively with sugar concentration, in mannoheptulose and avocado groups. The interpretation of these results was discussed and also their application to the Nutrition in tropical and subtropical countries.

[Chemotypes of "Shigella flexneri" R mutants and related phage receptors. I. -- Chemical study of the lipopolysaccharides (author's transl)]. / Chimiotypes de mutants R de Shigella flexneri et récepteurs de phages. I. -- Etude chimique des lipopolyosides

The F6R rough mutants isolated from Shigella flexneri F6S, serotype 5b, and the FH rough mutants, derived from other serotypes of S. flexneri, were chemotyped according to the chemical analysis of their lipopolysaccharides. Further, the following stages of lipopolysaccharide core biosynthesis in S. flexneri have been established: --(KDO)3--heptose--heptose--glucose--galactose; the last three stages are: either --glucose--glucosamine--glucose, or --glucosamine--glucose--glucose. The results of the chemical study of the R lipopolysaccharides are compatible with the assumption of the existence of a similar core in all considered S. flexneri serotypes.

Comparison of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium.

The cell envelope structure of Salmonella typhimurium LT2, which has a heptose-deficient lipopolysaccharide (LPS), is significantly different from that of an isogenic strain with a normal LPS. The rough strain, when examined by freeze-etching, lacks most surface structures that are routinely present in the smooth strain (surface particles and flagella) and has few transmemberane studs in the cytoplasmic membrane (those present are generally found in aggregates), and the outer membrane cleavage is substantially stronger than that of the smooth strain. These envelope differences were independent of both growth temperature and culture age. Examination of ultrathin sections indicated that the rough strain has an outer membrane which forms a much more defined double-track artifact than the smooth strain. The addition of MgCl2 to the growth medium of the rough strain decreased the extent of outer membrane cleavage, and flagella became evident in freeze-etched preparations. The presence of supplemental MgCl2 in the growth medium, which resulted in these morphological changes in the rough strain, also produced growth at a previously restrictive temperature and a decrease in the leakage of periplasmic enzymes. The smooth strain was unaltered morphologically or physiologically by MgCl2 under identical conditions. It is suggested that the outer membrane of the rough strain is more planar.

Constitution of the cell envelope of Haemophilus influenzae in relation to competence for genetic transformation.

Cell envelopes of Haemophilus influenzae have been prepared by breakage in a French pressure cell followed by differential centrifugation. The envelope fraction may be resolved into an inner-membrane (light) and an outer-membrane (heavy) fraction on density gradients. Envelopes from competent cells possess elevated levels of lipopolysaccharide with a composition different from that of log-phase cell envelopes. Three apparently new polypeptides have been observed in envelopes from competent cells by gel electrophoresis in sodium dodecyl sulfate; additional quantitative alterations in the profiles of membrane polypeptides also company the development of the capacity to transport deoxyribonucleic acid. Most of the polypeptide changes are confined to the outer membrane; one new polypeptide is associated with the inner cytoplasmic membrane of competent cells. Protein synthesis during competence developement is rquired for the change in lipopolysaccharides and in the envelope polypeptides to occur.

Studies on lipopolysaccharides isolated from strains of Neisseria gonorrhoeae.

Lipopolysaccharides, extracted by phenol-water from five strains fo Neisseria gonorrhoeae, were purified by treatment with ribonuclease followed by multiple washes. These preparations were fatal to mice when administered in submicrogram amounts with actinomycin D, the LD50 values varying from 4 to 16 mug/kg. Analyses showed that all preparations contained glucose, galactose, glucosamine, heptose, 2-keto-3-deoxyoctonic acid and phosphate. All the lipopolysaccharides contained the same fatty acids, namely beta-OH-10:0, beta-OH-12:0, beta-OH-14:0, 12:0, 14:0,16:0, 16:1, 18:0 and 18:1. We were unable to detect significant differences between the lipopolysaccharides of virulent and avirulent gonococci or between penicillin-sensitive and resistant strains. Gonococcal lipopolysaccharides appeared to lack O-antigen side chains.

[A comparative immunochemical study of the subtypes of serologic type I of the agent of pseudotuberculosis]. / Sravnitel'noe immunokhimicheskoe issledovanie podtipov serologicheskogo tipa I vozbuditelia psevdotuberkuleza

It was shown that Y. pseudotuberculosis strains causing the Far-Eastern scarlatina-like fever in the Primorsk region belonged to subtype IB-ipopolysaccharides of the standard strain of subtype IB and of the local strain were closely affiliated by the analytic data and monosaccharide composition, but differed from the lipopolysaccharide of strains belonging to subtype IA. Living vaccines should be used to obtain the sera against the subtypes IA and IB of the causative agent.

Characterization of endotoxin from Fusobacterium necrophorun.

Endotoxic lipopolysachharide (LPS) was obtained from phenol-water extraction of cell walls prepared from mass-cultivated Fusobacterium necrophorum. The LPS was relatively free of nucleic acids and low in protein, and constituted about 4% of the cell walls. Upon acid hydrolysis, some of the components detected were hexosamines (7.0%), neutral and reducing sugars (50.5%), heptose (6.4%), 2-keto-3-deoxyoctonate (0.8%), lipid A (21.0%), and phosphorus (1.7%). Under electron microscopy the LPS appeared mainly as ribbon-like trilaminar structures, and upon chemical treatment it displayed a behavior resembling that reported in certain enterobacterial LPS. The LPS was lethal to mice, 11-day-old chicken embryos, and rabbits. Endotoxicity in mice was enhanced at least 1,380-fold by the addition of 12.5 mug of actinomycin D. Induced tolerance to lethal effect of the endotoxin and rapidly acquired resistance to infection by F. necrophrum viable cells were also demonstrated in mice. The endotoxin produced both localized and generalized Shwartzman reactions as well as biphasic pyrogenic responses in rabbits. These results firmly establish the presence of a classical endotoxin in F. necrophorum, thus providing strong support to our recent suggestion that cell wall-associated components may contribute significantly to the pathogenicity of F. necrophorum.