Sucrose isomerase and its mutants from Erwinia rhapontici can synthesise α-arbutin

Sucrose isomerase (SI) from Erwinia rhapontici is an intramolecular isomerase that is normally used to synthesise isomaltulose from sucrose by a mechanism of intramolecular transglycosylation. In this study, it was found that SI could synthesise α-arbutin using hydroquinone and sucrose as substrates, via an intermolecular transglycosylation reaction. Five phenylalanine residues (F185, F186, F205, F297, and F321) in the catalytic pocket of SI were chosen for sitedirected mutagenesis. Mutants F185I, F321I, and F321W, whose hydrolytic activities were enhanced after the mutation, could synthesise α-arbutin through intermolecular transglycosylation with a more than two-fold increase in the molar transfer ratio compared with wild type SI. The F297A mutant showed a strong ability to synthesise a novel α-arbutin derivative and a four-fold increase in its specific activity for intermolecular transglycosylation over the wild type. Our findings may lead to a new way to synthesise novel glucoside products such as α-arbutin derivatives by simply manipulating the Phe residues in the catalytic pocket. From the structure superposition, our strategy of manipulating these Phe residues may be applicable to other similar transglycosylating enzymes.     

The function of trehalose biosynthesis in plants

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside) occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant. Until recently, only few plant species, mainly desiccation-tolerant 'resurrection' plants, were considered to synthesise trehalose. Instead of trehalose, most other plants species accumulate sucrose as major transport sugar and during stress. The ability to synthesize sucrose has probably evolved from the cyanobacterial ancestors of plastids and may be linked to photosynthetic function. Although most plant species do not appear to accumulate easily detectable amounts of trehalose, the discovery of genes for trehalose biosynthesis in Arabidopsis and in a range of crop plants suggests that the ability to synthesise trehalose is widely distributed in the plant kingdom. The apparent lack of trehalose accumulation in these plants is probably due to the presence of trehalase activity. After inhibition of trehalase, trehalose synthesis can be detected in Arabidopsis. Since trehalose induces metabolic changes, such as an accumulation of storage carbohydrates, rapid degradation of trehalose may be required to prevent detrimental effects of trehalose on the regulation of plant metabolism. In addition, the precursor of trehalose, trehalose-6-phosphate, is probably involved in the regulation of developmental and metabolic processes in plants.     

A simple,universal, efficient PCR-based gene synthesis method: Sequential OE-PCR gene synthesis

Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience.     

Characterisation of the mob locus of Rhodobacter sphaeroides WS8: mobA is the only gene required for molybdopterin guanine dinucleotide synthesis

The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molybdopterin guanine dinucleotide. The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a molybdopterin enzyme, is unaffected. The inability of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.     

Free and membrane bound ribosomes of the cotyledons of Vicia faba (L.)

Summary Changes in the quantities of free and membrane-bound ribosomes were followed in the cotyledons of developing seeds. During the phase of storage protein synthesis, free and membrane bound ribosomes do not interchange, and as both classes synthesise protein in vivo, it is possible that they may synthesise different groups of proteins. This suggestion is discussed in relationship to the developing cotyledon.     

Sensing trehalose biosynthesis in plants

A most unexpected finding in research on plant carbohydrate metabolism is the recent discovery that angiosperms encode genes whose products are involved in trehalose metabolism. The presence and functionality of such genes has been elegantly shown by expressing Arabidopsis-derived trehalose phosphate synthase and trehalose phosphate phosphatase genes in yeast mutants lacking these enzymatic activities. Homologue sequences have now been cloned from a number of different plant species suggesting that the capacity to synthesise trehalose is ubiquitous in angiosperms. Except for Myrothamnus flabellifolius, trehalose biosynthesis has never been observed in tissues of higher plants, probably due to the presence of high levels of trehalase activity. The function of trehalose metabolism in plants is still a mystery. One of the postulated functions of trehalose metabolism in yeast is in the control of glucose repression and a similar function in sugar sensing can be proposed for plants as well.     

Rhamnolipids are conserved biosurfactants molecules: implications for their biotechnological potential

A range of isolates of Pseudomonas aeruginosa from widely different environmental sources were examined for their ability to synthesise rhamnolipid biosurfactants. No significant differences in the quantity or composition of the rhamnolipid congeners could be produced by manipulating the growth conditions. Sequences for the rhamnolipid genes indicated low levels of strain variation, and the majority of polymorphisms did lead to amino acid sequence changes that had no evident phenotypic effect. Expression of the rhlB and rhlC rhamnosyltransferase genes showed a fixed sequential expression pattern during growth, and no significant up-regulation could be induced by varying producer strains or growth media. The results indicated that rhamnolipids are highly conserved molecules and that their gene expression has a rather stringent control. This leaves little opportunity to manipulate and greatly increase the yield of rhamnolipids from strains of P. aeruginosa for biotechnological applications.     

Ovarian and fat-body vitellogenin synthesis in Drosophila melanogaster

The ovary and the fat body of Drosophila melanogaster both synthesise vitellogenins in vivo. The ovary contributes nearly as much vitellogenin to the yolk of an oocyte as does the fat body. Densitometry of fluorographs and gels has been used to compare the amount of the smallest vitellogenin polypeptide, yolk protein 3, synthesised by each tissue. Cell-free translations indicate that the ovary, in contrast to the fat body, contains a much reduced level of the mRNA for yolk protein 3 compared with the mRNAs for the other vitellogenin polypeptides. However, if tissues are cultured in vitro, the underproduction of this protein by the ovary is not significant. Because young embryos have levels of this polypeptide which are expected if the ovary has a low level of its corresponding mRNA, we argue that the ovary genuinely underproduces this protein in vivo and that the relative levels synthesised by the ovary in vitro are an artefact. Egg chambers of previtellogenic stages can synthesise vitellogenins, but the maximum level of vitellogenin synthesis occurs in egg chambers of the early vitellogenic stages. We conclude that the expression of the vitellogenin genes is subject to different controls at each site of synthesis. The possible cell types responsible for ovarian vitellogenin synthesis are discussed; the follicle epithelial cells are tentatively nominated for this role. We also suggest that a specific repression mechanism for vitellogenin gene expression exists in the ovary.     

Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids

The suitability of the caco-2 cell line as a model for studying the long term impact of dietary fatty acids on intestinal lipid handling and chylomicron production was examined. Chronic supplementation of caco-2 cells with palmitic acid (PA) resulted in a lower triacylglycerol secretion than oleic acid (OA). This was coupled with a detrimental effect of PA, but not OA, on transepithelial electrical resistance (TER) measurements, suggesting a loss of structural integrity across the cell monolayer. Addition of OA reversed the adverse effects of PA and stearic acid on TER and increased the ability of cells to synthesise and accumulate lipid, but did not normalise the secretion of lipids by caco-2 cells. Increasing amounts of OA and decreasing amounts of PA in the incubation media markedly improved the ability of cells to synthesise apolipoprotein B and secrete lipids. Real time RT-PCR revealed a down regulation of genes involved in lipoprotein synthesis following PA than OA. Electron microscopy showed adverse effects of PA on cellular morphology consistent with immature enterocytes such as stunted microvilli and poor tight junction formation. In conclusion, previously reported differences in lipoprotein secretion by caco-2 cells supplemented with saturated fatty acids (SFA) and OA may partly reflect early cytotoxic effects of SFA on cellular integrity and function.     

Xmn I polymorphism in the gamma-globin gene region among Saudis

The gamma-globin chains of haemoglobin F are encoded by two non-allelic genes which synthesise two different gamma-chains referred to as G gamma and A gamma. Xmn I restricts on both sides of the genes and normally produces an 8.1-kilobase (kb) fragment containing both gamma-genes. A polymorphic site on the 5' end of the G gamma genes has been reported in some populations and it results in the production of a 7.0-kb fragment containing both gamma-genes. We investigated the Xmn I polymorphic site in normal individuals, haemoglobin S heterozygotes and sickle cell disease patients from three different regions of Saudi Arabia. In the eastern province, 10% of gamma-genes in normal individuals were linked to the 7.0-kb fragment while in the sickle cell disease patients and haemoglobin S heterozygotes the frequency of the polymorphic site was 0.932 and 0.625, respectively. In the south-western province, the frequency of the polymorphic site in normal individuals was 0.096 (similar to that in the eastern province) but in the sickle cell disease patients and sickle cell heterozygotes the frequency was 0.033 and 0.095, respectively. Finally, only 6 sickle cell disease patients from the north-western province were investigated and all had the gamma-globin gene linked to the 8.1-kb fragment. The results of this study in different regions of Saudi Arabia are presented and the possible significance of Xmn I polymorphism is suggested.     

A transfection compound series based on a versatile Tris linkage

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.     

The biosynthesis and nutritional uses of carotenoids

Carotenoids are isoprenoid molecules that are widespread in nature and are typically seen as pigments in fruits, flowers, birds and crustacea. Animals are unable to synthesise carotenoids de novo, and rely upon the diet as a source of these compounds. Over recent years there has been considerable interest in dietary carotenoids with respect to their potential in alleviating age-related diseases in humans. This attention has been mirrored by significant advances in cloning most of the carotenoid genes and in the genetic manipulation of crop plants with the intention of increasing levels in the diet. The aim of this article is to review our current understanding of carotenoid formation, to explain the perceived benefits of carotenoids in the diet and review the efforts that have been made to increase carotenoids in certain crop plants.     

Discovery of novel fragments inhibiting O-acetylserine sulphhydrylase by combining scaffold hopping and ligand–based drug design

Several bacteria rely on the reductive sulphur assimilation pathway, absent in mammals, to synthesise cysteine. Reduction of virulence and decrease in antibiotic resistance have already been associated with mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, inhibition of cysteine biosynthesis has emerged as a promising strategy to find new potential agents for the treatment of bacterial infection. Following our previous efforts to explore OASS inhibition and to expand and diversify our library, a scaffold hopping approach was carried out, with the aim of identifying a novel fragment for further development. This novel chemical tool, endowed with favourable pharmacological characteristics, was successfully developed, and a preliminary Structure–Activity Relationship investigation was carried out.     

Phosphomannose isomerase and phosphomannomutase gene disruptions in Streptomyces nodosus: impact on amphotericin biosynthesis and implications for glycosylation engineering

Streptomycetes synthesise several bioactive natural products that are modified with sugar residues derived from GDP-mannose. These include the antifungal polyenes, the antibacterial antibiotics hygromycin A and mannopeptimycins, and the anticancer agent bleomycin. Three enzymes function in biosynthesis of GDP-mannose from the glycolytic intermediate fructose 6-phosphate: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMPP). Synthesis of GDP-mannose from exogenous mannose requires hexokinase or phosphotransferase enzymes together with PMM and GMPP. In this study, a region containing genes for PMI, PMM and GMPP was cloned from Streptomyces nodosus, producer of the polyenes amphotericins A and B. Inactivation of the manA gene for PMI resulted in production of amphotericins and their aglycones, 8-deoxyamphoteronolides. A double mutant lacking the PMI and PMM genes produced 8-deoxyamphoteronolides in good yields along with trace levels of glycosylated amphotericins. With further genetic engineering these mutants may activate alternative hexoses as GDP-sugars for transfer to aglycones in vivo.     

Histamine in the Insect Nervous System: Distribution, Synthesis and Metabolism

Abstract: The distribution of histamine in the nervous systems of the locust, the cockroach, and the sphinx moth was mapped and the capacity of locust nervous tissue to synthesise and metabolise histamine was assessed. In all three species the highest levels of histamine were present in the retina and in the lamina neuropil of the optic lobe. Lower levels of histamine were detectable throughout the nervous system. In the locust the retina was shown to synthesise considerable quantities of histamine. The optic lobe and metathoracic ganglion synthesised smaller, though significant, amounts of histamine. Metabolic in activation of histamine in locust nervous tissue was shown to occur primarily via oxidation to imidazole-4-acetic acid and via N-acetylation to N -acetyl histamine. Whereas the retina and the optic lobe formed the two metabolic products in approximately equal proportions, the metathoracic ganglion produced almost three times as much N- acetyl histamine as imidazole-4-acetic acid.     

Synthesis of novel analogues of the delta opioid ligand SNC-80 using REM resin.

Focused libraries of delta opioid ligands were synthesised using REM resin methodology. Several high affinity compounds were identified with good selectivity over the mu opioid receptor. Automated REM resin recycling was used to synthesise larger amounts of ligand for in vivo studies.     

Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense

Disruption of ipdC, a gene involved in indole-3-acetic acid (IAA) production by the indole pyruvate pathway in Azospirillum brasilense Sp7, resulted in a mutant strain that was not impaired in IAA production with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to tryptophan produced IAA if tryptophan was present but did not synthesise IAA from indole. Similar results were obtained for a mutant strain with additional mutations in the genes ipdC and trpD. This suggests the existence of an alternative Trp-dependent route for IAA synthesis. On gluconate as a carbon source, IAA production by the ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA, both in the wild-type and in the ipdC mutant. IAA production from tryptamine was strongly decreased when gluconate was the carbon source.