Platelet-derived Growth Factor Differentially Regulates the Expression and Post-translational Modification of Versican by Arterial Smooth Muscle Cells through Distinct Protein Kinase C and Extracellular Signal-regulated Kinase Pathways

The synthesis of proteoglycans involves steps that regulate both protein and glycosaminoglycan (GAG) synthesis, but it is unclear whether these two pathways are regulated by the same or different signaling pathways. We therefore investigated signaling pathways involved in platelet-derived growth factor (PDGF)-mediated increases in versican core protein and GAG chain synthesis in arterial smooth muscle cells (ASMCs). PDGF treatment of ASMCs resulted in increased versican core protein synthesis and elongation of GAG chains attached to the versican core protein. The effects of PDGF on versican mRNA were blocked by inhibiting either protein kinase C (PKC) or the ERK pathways, whereas the GAG elongation effect of PDGF was blocked by PKC inhibition but not by ERK inhibition. Interestingly, blocking protein synthesis in the presence of cycloheximide abolished the PDGF effect, but not in the presence of xyloside, indicating that GAG synthesis that results from PKC activation is independent from de novo protein synthesis. PDGF also stimulated an increase in the chondroitin-6-sulfate to chondroitin-4-sulfate ratio of GAG chains on versican, and this effect was blocked by PKC inhibitors. These data show that PKC activation is sufficient to cause GAG chain elongation, but both PKC and ERK activation are required for versican mRNA core protein expression. These results indicate that different signaling pathways control different aspects of PDGF-stimulated versican biosynthesis by ASMCs. These data will be useful in designing strategies to interfere with the synthesis of this proteoglycan in various disease states.     

浓香型白酒发酵过程微生物合成正丙醇途径解析

【目的】揭示浓香型白酒窖内发酵过程与正丙醇合成相关的微生物和代谢途径。【方法】通过对浓香型白酒窖内发酵过程酒醅中微生物的宏转录组进行分析,解析与正丙醇合成相关的微生物和代谢途径,并验证相关微生物合成正丙醇的能力。【结果】浓香型白酒窖内发酵过程中有3条可能的酒醅微生物合成正丙醇的途径。真菌主要通过2-甲基苹果酸代谢途径和苏氨酸代谢途径合成正丙醇,细菌则主要通过丙酸代谢途径合成并参与苏氨酸代谢途径。宏转录组测序分析表明,这3条途径对白酒窖内发酵过程正丙醇的合成与积累均有贡献,并且微生物通过这3条途径合成正丙醇的时期和能力存在较大差异。此外,对分离自酒醅的酵母和乳酸菌合成正丙醇能力分析发现,它们均与浓香型白酒窖内发酵过程正丙醇的合成有关。【结论】本研究揭示了浓香型白酒窖内发酵过程中正丙醇合成相关的微生物和代谢途径,为阐明白酒发酵过程中正丙醇的形成机制奠定了理论基础。     

细菌不饱和脂肪酸合成研究进展

脂肪酸不仅是细菌细胞膜组分，还是许多生物活性物质的合成原料。不饱和脂肪酸(unsaturated fatty acid, UFA)具有更低的相变温度，是细菌调节细胞膜流动性的重要分子，因此UFA合成途径是重要的抗菌药物筛选靶点。细菌可利用厌氧途径合成UFA，其中模式生物大肠杆菌利用经典的FabA-FabB途径合成UFA，但不同细菌中UFA合成的厌氧途径具有多样性，相关催化酶类也不尽相同；细菌还可以利用需氧途径合成UFA，利用脂肪酸脱饱和酶直接将饱和脂肪酸(saturated fatty acid, SFA)转化为不饱和脂肪酸，而不同脱饱和酶会生成不同结构的UFA，在逆境耐受、致病力等多方面发挥重要作用；细菌还可以利用单加氧酶，将脂肪酸合成途径中癸酰酰基载体蛋白(acyl carrier protein, ACP)转化为顺-3-癸烯酰ACP，并最终合成UFA。细菌脂肪酸合成相关的其他酶类在UFA合成或不同种类UFA调节中也发挥着重要作用。本文系统地总结了细菌UFA合成途径与相关酶类的多样性研究进展，旨在为进一步了解细菌UFA合成机制，并以此为靶点开发抗菌药物等方面提供理论支撑。     

Fat synthesis in adipose tissue. An examination of stoichiometric constraints.

The requirement for net balance of synthesis, degradation and transport for all intermediates in the pathways from glucose to fat imposes constraints on the balance of fluxes between different pathways. Linear programming has been used to examine the interactions between these constraints on metabolism in adipocytes and the requirement for efficiency in the conversion of glucose into fat. The circumstances under which excessive ATP synthesis would accompany this conversion have been investigated.     

Arabidopsis loss-of-function mutant in the lysine pathway points out complex regulation mechanisms

In plants, the amino acids lysine, threonine, methionine and isoleucine have L-aspartate-beta-semialdehyde (ASA) as a common precursor in their biosynthesis pathways. How this ASA precursor is dispersed among the different pathways remains vague knowledge. The proportional balances of free and/or protein-bound lysine, threonine, isoleucine and methionine are a function of protein synthesis, secondary metabolism and plant physiology. Some control points determining the flux through the distinct pathways are known, but an adequate explanation of how the competing pathways share ASA in a fine-tuned amino acid biosynthesis network is yet not available. In this article we discuss the influence of lysine biosynthesis on the adjacent pathways of threonine and methionine. We report the finding of an Arabidopsis thaliana dihydrodipicolinate synthase T-DNA insertion mutant displaying lower lysine synthesis, and, as a result of this, a strongly enhanced synthesis of threonine. Consequences of these cross-pathway regulations are discussed.     

De novo synthesis and recycling pathways of sphingomyelin in rat Sertoli cells

Sertoli cells from 19-day-old rats have two molecular species of sphingomyelin (SM1 and SM2) with different kinetic characteristics and fatty acid composition. Here, we have studied the incorporation of [14C]-choline and [14C]-palmitic acid into SM in presence or absence of fumonisin B1, an inhibitor of ceramide synthesis, and beta-chloroalanine, an inhibitor of sphinganine synthesis. The contributions of de novo synthesis and recycling pathways were estimated by analysis of the inhibition caused by these drugs. SM1 was synthesized more by sphingosine recycling, and SM2 was synthesized principally by ceramide recycling than SM1. De novo synthesis seems to be important for the two SM types, but our results showed that this pathway is more extensively utilized by SM2. In conclusion, using Sertoli cell cultures, we have shown for the first time that in the same cell different molecular species of SM are synthesized by different pathways.     

On the Origin of Metabolic Pathways

The heterotrophic theory of the origin of life is the only proposal available with experimental support. This comes from the ease of prebiotic synthesis under strongly reducing conditions. The prebiotic synthesis of organic compounds by reduction of CO₂ to monomers used by the first organisms would also be considered an heterotrophic origin. Autotrophy means that the first organisms biosynthesized their cell constituents as well as assembling them. Prebiotic synthetic pathways are all different from the biosynthetic pathways of the last common ancestor (LCA). The steps leading to the origin of the metabolic pathways are closer to prebiotic chemistry than to those in the LCA. There may have been different biosynthetic routes between the prebiotic and the LCAs that played an early role in metabolism but have disappeared from extant organisms. The semienzymatic theory of the origin of metabolism proposed here is similar to the Horowitz hypothesis but includes the use of compounds leaking from preexisting pathways as well as prebiotic compounds from the environment.     

Strange organelles--Plasmodium mitochondria lack a pyruvate dehydrogenase complex

Our understanding of the Plasmodium mitochondrion and apicoplast has been greatly assisted by the genome sequence project. Sequence data have seeded recent research showing that the apicoplast is the site of several anabolic pathways including fatty acid synthesis. The discovery of an active apicoplast pyruvate dehydrogenase complex implies this enzyme generates the acetyl-CoA needed for fatty acid synthesis. However, the absence of a corresponding mitochondrial complex suggests that energy generation in Plasmodium is considerably different from pathways described in other eukaryotes.     

Probabilistic pathway construction

Expression of novel synthesis pathways in host organisms amenable to genetic manipulations has emerged as an attractive metabolic engineering strategy to overproduce natural products, biofuels, biopolymers and other commercially useful metabolites. We present a pathway construction algorithm for identifying viable synthesis pathways compatible with balanced cell growth. Rather than exhaustive exploration, we investigate probabilistic selection of reactions to construct the pathways. Three different selection schemes are investigated for the selection of reactions: high metabolite connectivity, low connectivity and uniformly random. For all case studies, which involved a diverse set of target metabolites, the uniformly random selection scheme resulted in the highest average maximum yield. When compared to an exhaustive search enumerating all possible reaction routes, our probabilistic algorithm returned nearly identical distributions of yields, while requiring far less computing time (minutes vs. years). The pathways identified by our algorithm have previously been confirmed in the literature as viable, high-yield synthesis routes. Prospectively, our algorithm could facilitate the design of novel, non-native synthesis routes by efficiently exploring the diversity of biochemical transformations in nature.     

Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway.

In addition to the CDP-choline pathway for phosphatidylcholine (PC) synthesis, the liver has a unique phosphatidylethanolamine (PE) methyltransferase activity for PC synthesis via three methylations of the ethanolamine moiety of PE. Previous studies indicate that the two pathways are functionally different and not interchangeable even though PC is the common product of both pathways. This study was designed to test the hypothesis that these two pathways produce different profiles of PC species. The PC species from these two pathways were labeled with specific stable isotope precursors, D9-choline and D4-ethanolamine, and analyzed by electrospray tandem mass spectrometry. Our studies revealed a profound distinction in PC profiles between the CDP-choline pathway and the PE methylation pathway. PC molecules produced from the CDP-choline pathway were mainly comprised of medium chain, saturated (e.g. 16:0/18:0) species. On the other hand, PC molecules from the PE methylation pathway were much more diverse and were comprised of significantly more long chain, polyunsaturated (e.g. 18:0/20:4) species. PC species from the methylation pathway contained a higher percentage of arachidonate and were more diverse than those from the CDP-choline pathway. This profound distinction of PC profiles may contribute to the different functions of these two pathways in the liver.     

Glycerophospholipid acquisition in Plasmodium - A puzzling assembly of biosynthetic pathways

Throughout the Plasmodium life cycle, malaria parasites repeatedly undergo rapid cellular growth and prolific divisions, necessitating intense membrane neogenesis and, in particular, the acquisition of high amounts of phospholipids. At the intraerythrocytic stage, glycerophospholipids are the main parasite membrane constituents, which mostly originate from the Plasmodium-encoded enzymatic machinery. Several proteins and entire pathways have been characterized and their features reported, thereby generating a global view of glycerophospholipid synthesis across Plasmodium spp. The malaria parasite displays a panoply of pathways that are seldom found together in a single organism. The major glycerophospholipids are synthesized via ancestral prokaryotic CDP-diacylglycerol-dependent pathways and eukaryotic-type de novo pathways. The parasite exhibits additional reactions that bridge some of these routes and are otherwise restricted to some organisms, such as plants, while base-exchange mechanisms are largely unexplored in Plasmodium. Marked differences between Plasmodium spp. have also been reported in phosphatidylcholine and phosphatidylethanolamine synthesis. Little is currently known about glycerophospholipid acquisition at non-erythrocytic stages, but recent data reveal that intrahepatocytic parasites, oocysts and sporozoites import various host lipids, and that de novo fatty acid synthesis is only crucial at the late liver stage. More studies on the different Plasmodium developmental stages are needed, to further assemble the different pieces of this glycerophospholipid synthesis puzzle, which contains highly promising therapeutic targets.     

Synthetic biology of secondary metabolite biosynthesis in actinomycetes: Engineering precursor supply as a way to optimize antibiotic production

Actinomycetes are a rich source for the synthesis of medically and technically useful natural products. The genes encoding the enzymes for their biosynthesis are normally organized in gene clusters, which include also the information for resistance (in the case of antibacterial compounds), regulation, and transport. This facilitates the manipulation of such pathways by molecular genetic techniques. Recent advances in DNA sequencing and analytical chemistry revealed that not only new strains isolated from yet unexplored habitats, but also already known strains possess a large potential for the synthesis of novel compounds. Synthetic Biology now offers a new perspective to exploit this potential further by generating novel pathways, and thereby novel products, by combining different biosynthetic steps originating from different bacteria. The supply of precursors, which are subsequently incorporated into the final product, is often already organized in a modular manner in nature and may directly be exploited for Synthetic Biology. Here we report examples for the synthesis of building blocks and possibilities to modify and optimize antibiotic biosynthesis, exemplary for the synthesis of the manipulation of the synthesis of the glycopeptide antibiotic balhimycin.     

Production and biological function of volatile esters in Saccharomyces cerevisiae

The need to understand and control ester synthesis is driven by the fact that esters play a key role in the sensorial quality of fermented alcoholic beverages like beer, wine and sake. As esters are synthesized in yeast via several complex metabolic pathways, there is a need to gain a clear understanding of ester metabolism and its regulation. The individual genes involved, their functions and regulatory mechanisms have to be identified. In alcoholic beverages, there are two important groups of esters: the acetate esters and the medium-chain fatty acid (MCFA) ethyl esters. For acetate ester synthesis, the genes involved have already been cloned and characterized. Also the biochemical pathways and the regulation of acetate ester synthesis are well defined. With respect to the molecular basis of MCFA ethyl ester synthesis, however, significant progress has only recently been made. Next to the characterization of the biochemical pathways and regulation of ester synthesis, a new and more important question arises: what is the advantage for yeast to produce these esters? Several hypotheses have been proposed in the past, but none was satisfactorily. This paper reviews the current hypotheses of ester synthesis in yeast in relation to the complex regulation of the alcohol acetyl transferases and the different factors that allow ester formation to be controlled during fermentation.     

Similar sensitivity of human lymphoblast cells proficient or deficient in nucleotide biosynthesis to the cytotoxic and cell cycle effects of 3-aminobenzamide

3-Aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, induces cytotoxicity and cell cycle delays in exposed cells. 3AB has been reported to inhibit de novo nucleotide synthesis in human lymphoblasts. To determine if the 3AB-mediated effects are related to this inhibition of DNA synthesis, cytotoxicity and cell cycle progression in human lymphoblastoid cell lines, proficient or deficient in salvage nucleotide synthesis pathways, were determined after exposures to 3AB. In addition, changes in cell cycle progression were followed after treatment with 3-aminobenzoic acid (ABA), a nonactive analog of 3AB, and 5-methylnicotinamide (5MN), a less potent inhibitor of poly(ADP-ribose) synthesis. All three lines responded similarly to the different treatments. Cells deficient in salvage nucleotide synthesis pathways were no more sensitive to the cytotoxic or cell cycle effects of 3AB, ABA or 5MN. Thus, effects on nucleotide synthesis are not responsible for either cytotoxicity or the retarded cell growth found in human lymphoblastoid cells exposed to inhibitors of poly(ADP-ribose) synthesis.     

Concentration matters!! ppGpp, from a whispering to a strident alarmone

Bacteria have developed sophisticated signal transduction pathways to sense and respond to environmental stresses. These pathways include intracellular regulators that elicit adaptive changes in the physiology of the cell. Extensive work, mostly performed in Escherichia coli, showed that the modified nucleotide ppGpp plays a key regulatory role by co-ordinating the cellular responses to adverse environmental conditions. In this issue of Molecular Microbiology, Traxler et al. define two sets of ppGpp-dependent genes that are expressed at different times after induction of ppGpp synthesis. Their results suggest that quantitative differences in the ppGpp intracellular concentration determine the precise pattern of gene expression during adaptation process: low levels of ppGpp suffice to activate the Lrp regulon, which, by activating the synthesis of some amino acid pathways, can generate a negative feedback loop while high levels activate RpoS and a feed-forward amplification of the general stress response. These dose-dependent effects on gene expression open new perspectives on the complex regulatory pathways mediated by ppGpp during environmental adaptation.     

Enzymatic synthesis of nucleotide sugars

The present review gives a survey on the biosynthetic pathways of nucleotide sugars which are important for the in vitro synthesis of mammalian glycoconjugates. With respect to the use of these enzymes in glycotechnology the availability as recombinant enzymes from different sources, the large-scale synthesis of nucleotide sugars and their in situ regeneration in combination with glycosyltransferases are summarized and evaluated.     

Effect of the nitrogen source on glutamine and alanine biosynthesis in Neurospora crassa. An in vivo 15N nuclear magnetic resonance study

The influences of different nitrogen sources on the relative rates of biosynthesis of glutamine and alanine have been studied by 15N nuclear magnetic resonance spectroscopy of intact Neurospora crassa mycelia suspensions. The rate of glutamine synthesis was fastest after growth in media deficient in free ammonium ion, whereas it was slowest following growth in media containing both glutamic acid and glutamine. The reverse trend was observed for the biosynthesis of alanine. A competition between the two biosynthetic pathways for the same substrate, glutamic acid, was found to limit the rate of alanine synthesis when glutamine synthesis was rapid. The observed in vivo rates of these reactions are compared to the reported specific activities of the enzymes catalyzing the reactions, and implications of these results for nitrogen regulation of these pathways under various physiological conditions are discussed.