Pro-apoptotic activity of lipidic α-amino acids isolated from Protopalythoa variabilis

Lipidic α-amino acids (LAAs) have been described as non-natural amino acids with long saturated or unsaturated aliphatic chains. In the continuing prospect to discover anticancer agents from marine sources, we have obtained a mixture of two cytotoxic LAAs (1a and 1b) from the zoanthid Protopalythoa variabilis. The anti-proliferative potential of 14 synthetic LAAs and 1a/1b were evaluated on four tumor cell lines (HCT-8, SF-295, MDA-MB-435, and HL-60). Five of the synthetic LAAs showed high percentage of tumor cell inhibition, while 1a/1b completely inhibited tumor cell growth. Additionally, apoptotic effects of 1a/1b were studied on HL-60 cell line. 1a/1b-treated cells showed apoptosis morphology, loss of mitochondrial potential, and DNA fragmentation.     

Structurally Diverse Sesquiterpenoids with NO Production and α-Glucosidase Inhibitory Activities from the Fruits of Schisandra chinensis

Chemical fractionation of the AcOEt partition, generated from the EtOH extract of the fruits of Schisandra chinensis, afforded a series of sesquiterpenyl constituents including two new cadinanes, a new eudesmane, two new widdranes (a handling artefact and a new natural product), a new bisabolane and two new natural cuparane enantiomers, along with 15 known structurally related analogs. Structures of the new compounds were unambiguously characterized by interpretation of detailed spectroscopic data including ESI-MS and 1D/2D NMR, with their absolute configurations being established by electronic circular dichroism (ECD) calculation and induced ECD experiment. The inhibitory effects of all the isolates against α-glucosidase and lipopolysaccharide (LPS) induced nitric oxide (NO) production in murine RAW264.7 macrophages, as well as their antibacterial and cytotoxic potential, were evaluated, with selective compounds showing moderate α-glucosidase and NO inhibitory activity. Notably, canangaterpene III exhibited the most significant NO inhibitory effect with an IC₅₀ value of 31.50±1.49 μM.     

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the ¹⁴C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg⁻¹ protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.     

Crystal Structure of Monomeric Photosystem II from Thermosynechococcus elongatus at 3.6-? Resolution

The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form of PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C222₁, contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 Å. The x-ray diffraction pattern of the PSIIcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSIIcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone Q_A, two heme groups, 11 β-carotenes, 22 lipids, seven detergent molecules, and the Mn₄Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of non-crystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular (∼87°) to the crystallographic b-axis, further information about the structure of the Mn₄Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form.     

An α-glucan isolated from root of Isatis Indigotica,its structure and adjuvant activity

The root of Isatis indigotica is a traditional Chinese herbal medicine. An α-glucan (IIP-A-1) was firstly isolated from the roots. In this study we elucidated the chemical structure of IIP-A-1 and determined its adjuvant activity by co-immunizing mice with H1N1 influenza virus split and recombinant hepatitis B surface antigen (HBsAg), respectively. The polysaccharide was pretreated with periodate oxidation, Smith degradation and methylation in order to analyze its structure using GC, HPGPC, FT-IR, NMR and GC-MS. The adjuvant effect was evaluated by determining the antibody titers of serum against H1N1 influenza and HBsAg using ELISA. The proliferation and TNF-α secretion of macrophages administrated with different dose of IIP-A-1 were measured in vitro. The results of this study revealed that IIP-A-1 was an α-glucan with the molecular weight of 3,600 Da. The backbone was α-(1?→?4)-D-glucan with (1?→?6) branch chain. The α-glucan could significantly enhance the immune response of mice immunized with H1N1 influenza or HBsAg in vivo and exert good dose-dependent effects on the proliferation and the TNF-α secretion of macrophages in vitro. These results supported that IIP-A-1 was expected to be an efficacious adjuvant candidate for prophylactic and therapeutic vaccines.     

Production of raw-starch-hydrolysing α-amylase from the newly isolated Geobacillus thermodenitrificans HRO10

An extracellular raw-starch-digesting α-amylase was isolated from Geobacillus thermodenitrificans HRO10. The culture conditions for the production of α-amylase by G. thermodenitrificans HRO10 was optimized in 1.2–l bioreactor using full 2⁴ and 3² factorial designs. From the optimal reaction conditions, a model (Y = − 594.206 − 0.178T² − 8.448pH² + 6.020TpH − 0.005T²pH²) was predicted, which was then used for α-amylase production. In the bioreactor studies, the enzyme yield under optimized conditions (pH 7.1, 49°C) was 30.20 U/ml, a 51% improvement over the results (19.97 U/ml) obtained when the traditional one-factor-at-a-time method was employed. This α-amylase does not require extraneous calcium ions for activity, which may be a commercially important observation.     

Segregation analysis of α-L-fucosidase activity

Summary Complex segregation analysis of plasma -L-fucosidase in 45 British families provides evidence for an additive major gene causing low activities of fucosidase. There was no significant evidence of polygenic heritability or common family environment.     

Recombinogenic activity of Pantoprazole? in somatic cells of Drosophila melanogaster

Pantoprazole^® is one of the leading proton pump inhibitors (PPIs) used inthe treatment of a variety of diseases related to the upper gastrointestinal tract.However, studies have shown an increased risk of developing gastric cancer,intestinal metaplasia and hyperplasia of endocrine cells with prolonged use. In thepresent study, the somatic mutation and recombination test (SMART) was employed todetermine the mutagenic effects of Pantoprazole on Drosophilamelanogaster. Repeated treatments with Pantoprazole were performed on72-hour larvae of the standard (ST) and high bioactivation (HB) crosses atconcentrations of 2.5, 5.0, and 10.0 μM. In addition, doxorubicin (DXR) wasadministered at 0.4 mM, as a positive control. When administered to ST descendants,total number of spots were statistically significant at 2.5 and 5.0 μMconcentrations. For HB descendants, a significant increase in the total number ofspots was observed among the marked transheterozygous (MH) flies. Through analysis ofbalancer heterozygous (BH) descendants, recombinogenic effects were observed at allconcentrations in descendants of the HB cross. In view of these experimentalconditions and results, it was concluded that Pantoprazole is associated withrecombinogenic effects in Drosophila melanogaster.     

The α-amylase of the beetle Callosobruchus chinensis properties

C. chinensis larval amylase is activated by Ca(2+) and inhibited by Cl(-) and EDTA (K(i) 6.7x10(-3)m). GSH and 2-mercaptoethanol activate, presumably at different sites, as 2-mercaptoethanol interferes with Ca(2+) activation, whereas GSH enhances it. The inhibition by iodoacetic acid and N-ethylmaleimide (K(i) 1.55x10(-2)m) suggest that free thiol groups are essential for activity. The pH optimum of 5.2-5.4 is moved to 5.6-5.8 by Ca(2+) and 2-mercaptoethanol. The activation energy is 7270 cal/mol, and is not affected by Ca(2+) and 2-mercaptoethanol. K(m) for soluble starch is 2.3mg/ml.     

Origin of α-mannosidase activity in CSF

The α-mannosidase activity in human frontal gyrus, cerebrospinal fluid and plasma has been analyzed by DEAE-cellulose chromatography to investigate the origin of the α-mannosidase activity in cerebrospinal fluid (CSF). The profile of α-mannosidase isoenzymes obtained in CSF was similar to that in the frontal gyrus but different from that in human plasma. In particular the two characteristic peaks of lysosomal α-mannosidase, A and B, which have a pH-optimum of 4.5 and are found in human tissues, were present in both the frontal gyrus and CSF. In contrast the majority of α-mannosidase activity in human plasma was due to the so called intermediate form, which has a pH-optimum of 5.5. The results suggest that the intermediate form of α-mannosidase in plasma does not cross the blood–brain barrier and that the α-mannosidase activity present in the cerebrospinal fluid is of lysosomal type and of brain origin. Thus the α-mannosidase activity in cerebrospinal fluid might mirror the brain pathological changes linked to neurodegenerative disorders such as Parkinson's disease.     

Plasmodium vivax malaria elimination: should innovative ideas from the past be revisited?

In the 1950s, the strategy of adding chloroquine to food salt as a prophylaxis against malaria was considered to be a successful tool. However, with the development of Plasmodium resistance in the Brazilian Amazon, this control strategy was abandoned. More than 50 years later, asexual stage resistance can be avoided by screening for antimalarial drugs that have a selective action against gametocytes, thus old prophylactic measures can be revisited. The efficacy of the old methods should be tested as complementary tools for the elimination of malaria.     

Syntheses of α-D-glucosyl-D-fructoses by use of a reversed hydrolysis activity of α-glucosidase

Summary -D-Glucosyl-D-fructoses were synthesized by use of a reversed hydrolysis activity of -glucosidase fromSaccharomyces sp. Although -D-glucosyl-(1–1)-D-fructose was synthesized predominantly by the incubation of D-glucose solution in the presence of -glucosidase (batch method reaction), -(1–4)-linked disaccharide was a major product in a procedure by use of an immobilized -glucosidase column and an activated carbon column (column method reaction).     

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rond?nia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.     

Three new iridoid glycosides isolated from the traditional herb Siphonostegia chinensis with NF-κB inhibitory activity

Siphonoids A–C (1–3), among which siphonoids A-B (1–2) are rare (E)-p-coumaroyl iridoids, were isolated from Siphonostegia chinensis along with ten known iridoids (4–13) and four lignans (14–17). The structures of the compounds were established by 1D- and 2D-NMR spectroscopy and mass spectrometry. Most of the iridoids isolated from S. chinensis were found to possess (E)-p-coumaroyl iridoid subtype skeletons. Hence, this type of iridoid could be regarded as a chemotaxonomic marker of S. chinensis. The inhibition activities for the NF-κB pathway of iridoids (1–6) were detected. The present results showed that compounds 1–2 and 4–6 processed moderate activity towards the inhibition of NF-κB.     

Identification of a Novel Isoform of the Chloroplast-Coupling Factor α-Subunit

Studies were conducted to identify a 64-kD thylakoid membrane protein of unknown function. The protein was extracted from chloroplast thylakoids under low ionic strength conditions and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four peptides generated from the proteolytic cleavage of the wheat 64-kD protein were sequenced and found to be identical to internal sequences of the chloroplast-coupling factor (CF₁) α-subunit. Antibodies for the 64-kD protein also recognized the α-subunit of CF₁. Both the 64-kD protein and the 61-kD CF₁ α-subunit were present in the monocots barley (Hordeum vulgare), maize (Zea mays), oat (Avena sativa), and wheat (Triticum aestivum); but the dicots pea (Pisum sativum), soybean (Glycine max Merr.), and spinach (Spinacia oleracea) contained only a single polypeptide corresponding to the CF₁ α-subunit. The 64-kD protein accumulated in response to high irradiance (1000 μmol photons m⁻² s⁻¹) and declined in response to low irradiance (80 μmol photons m⁻² s⁻¹) treatments. Thus, the 64-kD protein was identified as an irradiance-dependent isoform of the CF₁ α-subunit found only in monocots. Analysis of purified CF₁ complexes showed that the 64-kD protein represented up to 15% of the total CF₁ α-subunit.     

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional ��1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc₃Man₉ GlcNAc₂-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the α1,3- and the α1,6-mannose-linked residue from GDP-mannose to Man₁GlcNAc₂-PP-Dol forming the Man₃GlcNAc₂-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX₇E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys²³⁰, as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.Asparagine-linked glycosylation is an essential protein modification highly conserved in eukaryotes (1–4), and several features of this pathway even occur in prokaryotes (5–7). In eukaryotes, biosynthesis of N-glycans starts with the assembly of the common core oligosaccharide precursor Glc₃Man₉ GlcNAc₂-PP-Dol, the glycan moiety of which is subsequently transferred onto selected Asn-Xaa-(Ser/Thr) acceptor sites of the nascent polypeptide chain by the oligosaccharyl-transferase complex (8–10). The initial steps of the dolichol pathway up to Man₅GlcNAc₂-PP-Dol take place on the cytosolic site of the endoplasmic reticulum (ER),² using sugar nucleotides as glycosyl donors. Upon translocation of the heptasaccharide to the luminal site, which is facilitated by Rft1 (11) and another not yet identified protein (12), it is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol. It has been demonstrated that the pathway operates sequentially in an ordered fashion based on differences in the substrate specificity of the various glycosyltransferases (13). In the yeast Saccharomyces cerevisiae, alg mutants (for asparagine-linked glycosylation) have been isolated, defective in lipid-linked oligosaccharide (LLO) assembly (14–17), and shown to be invaluable to define the pathway as well as to isolate the genes encoding the respective glycosyltransferases by complementing a particular phenotype characteristic of the respective mutant. Likewise various mutant cell lines from mammalian origin have been described that produce truncated lipid-linked oligosaccharides (18–20).One of the temperature-sensitive alg mutants, alg2, was shown to accumulate lipid-linked Man₂GlcNAc₂ at the restrictive temperature (15), indicating that alg2 might have a defect in the glycosyltransferase catalyzing the transfer of the third, α1,6-linked mannose, i.e. in the formation of the branched pentasaccharide Man₃GlcNAc₂-PP-Dol (see Fig. 8). On the other hand, biochemical studies in human fibroblasts from a patient with a defect in the human ALG2 ortholog, causing congenital disorder of glycosylation type CDG1i, pointed to a role in the transfer of the second, α1,3-linked mannose residue, because no elongation of Man(1,6)ManGlcNAc₂-PP-Dol occurred (21). In contrast, control fibroblasts were able to do so, albeit with reduced efficiency when compared with Man(1,3)ManGlcNAc₂-PP-Dol as glycosyl acceptor. Because a bioinformatic approach of the yeast data base did not reveal an unknown open reading frame that might encode an additional putative mannosyltransferase being involved in LLO synthesis, we reasoned that ALG2 may have a dual function, i.e. synthesizing both Man₂GlcNAc₂-PP-Dol and Man₃GlcNAc₂-PP-Dol. While the current study was in progress, evidence was presented that a membrane fraction from Escherichia coli, expressing ALG2 from yeast, is able to carry out an α1,3- and α1,6-mannosylation to form the branched pentasaccharide intermediate (22). However, the contribution of native E. coli enzymes could not entirely be ruled out. So far Alg2 has not been studied biochemically in yeast. Here, we confirm and extent this finding by investigating Alg2 in yeast. We first established a radioactive in vitro assay and demonstrate that Alg2, immunoprecipitated from detergent extracts of yeast microsomal membranes, is indeed sufficient to catalyze both elongation of Man₁GlcNAc₂-PP-Dol to Man₂GlcNAc₂-PP-Dol and subsequently to Man₃GlcNAc₂-PP-Dol. Furthermore we investigated the membrane topology of Alg2 mannosyltransferase. Evidence will be presented that Alg2 is composed only of the two N-terminal of four predicted transmembrane domains (TMDs), whereas the C-terminal hydrophobic sequences contribute to ER localization merely in a nontransmembrane manner. Surprisingly, none of the four domains is essential for Alg2 activity because deletion of either the two N-terminal or C-terminal domains gives rise to an active transferase. Finally, we perform a mutational analysis of Alg2 and identify amino acids required for its activity.Open in a separate windowFIGURE 8.Early steps of lipid-linked oligosaccharide formation on the cytosolic side of the ER membrane. Biosynthesis starts with the transfer of a GlcNAc-phosphate to dolichol phosphate with formation of the pyrophosphate bond, catalyzed by Alg7. The second step is catalyzed be the dimeric Alg14/Alg13 complex, whereby membrane-bound Alg14 recruits cytosolic Alg13 to the membrane with formation of the active GlcNAc transferase. Following the addition of the β1,4-linked mannose by Alg1, Alg2 catalyzes, as demonstrated here, both the transfer of the α1,3- and α1,6-linked mannose. The two final α1,2-mannose residues are transferred by Alg11, before the Man₅GlcNAc₂-PP heptasaccharide is translocated across the ER membrane to the lumen, where further elongation takes place to the full-length core saccharide. All of the sugar residues are donated by sugar nucleotides.