De novo biosynthesis of FSH like peptide by the human prostate

Human benign prostatic hyperplasia tissue is able to synthesize immunoreactive FSH (IR FSH) in vitro. The prostatic FSH is similar to pituitary FSH as evident by co-elution on Sephadex G-100. High performance gel filtration chromatography and Western blot analysis. Immunocytochemical localization studies indicate positive staining in the cytoplasm of prostatic epithelial cells. There is a two fold increase in the concentration of immunoreactive FSH in benign hyperplastic tissue as compared to normal prostate.     

De novo biosynthesis of Gastrodin in Escherichia coli

Gastrodin, a phenolic glycoside, is the key ingredient of Gastrodia elata, a notable herbal plant that has been used to treat various conditions in oriental countries for centuries. Gastrodin is extensively used clinically for its sedative, hypnotic, anticonvulsive and neuroprotective properties in China. Gastrodin is usually produced by plant extraction or chemical synthesis, which has many disadvantages. Herein, we report unprecedented microbial synthesis of gastrodin via an artificial pathway. A Nocardia carboxylic acid reductase, endogenous alcohol dehydrogenases and a Rhodiola glycosyltransferase UGT73B6 transformed 4-hydroxybenzoic acid, an intermediate of ubiquinone biosynthesis, into gastrodin in Escherichia coli. Pathway genes were overexpressed to enhance metabolic flux toward precursor 4-hydroxybenzyl alcohol. Furthermore, the catalytic properties of the UGT73B6 toward phenolic alcohols were improved through directed evolution. The finally engineered strain produced 545 mg l⁻¹ gastrodin in 48 h. This work creates a new route to produce gastrodin, instead of plant extractions and chemical synthesis.     

De novo biosynthesis of cytokinins in the biotrophic fungus Claviceps purpurea

Disease symptoms of some phytopathogenic fungi are associated with changes in cytokinin (CK) levels. Here, we show that the CK profile of ergot‐infected rye plants is also altered, although no pronounced changes occur in the expression of the host plant's CK biosynthesis genes. Instead, we demonstrate a clearly different mechanism: we report on the first fungal de novo CK biosynthesis genes, prove their functions and constitute a biosynthetic pathway. The ergot fungus Claviceps purpurea produces substantial quantities of CKs in culture and, like plants, expresses enzymes containing the isopentenyltransferase and lonely guy domains necessary for de novo isopentenyladenine production. Uniquely, two of these domains are combined in one bifunctional enzyme, CpIPT‐LOG, depicting a novel and potent mechanism for CK production. The fungus also forms trans‐zeatin, a reaction catalysed by a CK‐specific cytochrome P450 monooxygenase, which is encoded by cpp450 forming a small cluster with cpipt‐log. Deletion of cpipt‐log and cpp450 did not affect virulence of the fungus, but Δcpp450 mutants exhibit a hyper‐sporulating phenotype, implying that CKs are environmental factors influencing fungal development.     

De novo biosynthesis of docosahexaenoyl-phosphatidic acid in bovine retinal microsomes

Lysophosphatidic acid stimulated several-fold the formation of docosahexaenoyl-phosphatidic acid from 14C-labeled docosahexaenoic acid (22:6 (n-3] in the bovine retina. 1-Palmitoyl- and 1-oleoyl-sn-glycerol 3-phosphate were the preferred acceptors. Most of the activity was localized in the 105 000 X g microsomal fraction. Despite the very high content of 22:6 in the phospholipids of photoreceptor membranes, only about 1% of the microsomal activity was found in discs isolated from rod outer segments. The newly synthesized docosahexaenoyl-phosphatidic acid was further metabolized to diacylglycerols, triacylglycerols, phosphatidylcholine and phosphatidylserine. The de novo synthesis of docosahexaenoyl-phosphatidylcholine was stimulated by 1 mM CDPcholine. Lysophosphatidic acid and lysophosphatidylcholine up to 50 microM do not compete with each other for 22:6 in the formation of their respective diacylated lipids. This suggests that this fatty acid is introduced into phosphatidic acid and phosphatidylcholine via different acylation systems. We conclude that, in addition to the deacylation-acylation cycle, there is also an active pathway for the acylation of 22:6 into glycerolipids during the de novo biosynthesis of phosphatidic acid.     

De novo biosynthesis of polyunsaturated fatty acids in the cockroach Periplaneta americana

The de novo biosynthesis of 6,9,12-linolenic acid, 11,14-eicosadienoic acid, 5,11,14-eicosatrienoic acid, and arachidonic acid was demonstrated in adult female cockroaches, Periplaneta americana. These four polyunsaturated fatty acids (PUFA) were present primarily in the phospholipid (PL) fraction of both males and females. They were purified by AgNO3 thin-layer chromatography and high pressure liquid chromatography. The double bond positions of the major isomer of eicosatrienoic acid were shown to be at the delta 5,11,14 positions by gas chromatography-mass spectrometry (GC-MS) of both methoxy and epoxide derivatives and gas-liquid chromatography (GLC) and GC-MS of ozonolysis products. The other PUFAs cochromatographed with standards on both packed and capillary GLC columns. The in vivo incorporation of [1-14C]acetate into 5,11,14-eicosatrienoic acid, 11,14-eicosadienoic acid, 6,9,12-linolenic acid, and arachidonic acid was demonstrated by radio-GLC and radio-HPLC and for 5,11,14-eicosatrienoic acid by radio-GLC of ozonolysis products. The latter technique clearly demonstrated that the entire eicosatrienoic acid molecule was labeled. Thoracic tissue contained the highest amount of radiolabeled 5,11,14-eicosatrienoic acid (1.6% of total radioactivity incorporated into PL) while radiolabeled 11,14-eicosadienoic acid was found primarily in abdominal epidermal tissue (2% of total radioactivity incorporated into PL). Radiolabeled arachidonic and 6,9,12-linolenic acids comprised 0.1 and 0.02%, respectively, of the total radioactivity in the PL fraction. These data document the de novo biosynthesis of di-, tri-, and tetraunsaturated fatty acids in the American cockroach, and indicate that this animal can desaturate on both sides of the delta 9 double bond of oleic acid.     

De novo biosynthesis and localization of immunoreactive inhibin (10.7 kDa) in normal human stomach.

We have previously reported the occurrence of inhibin-like peptide in gastric juice of normal men. In the present investigation, normal gastric mucosa was shown to synthesize inhibin, in vitro, as measured by 3H-leucine incorporation (Maximum at 18 h). Furthermore, the immunohistochemical localization studies demonstrated its presence in the acid secreting parietal cells and basal region of foveolar epithelium of gastric mucosa. Surprisingly, the protein secreting zymogen cells remained unstained.     

De novo mutations in human genetic disease

New mutations have long been known to cause genetic disease, but their true contribution to the disease burden can only now be determined using family-based whole-genome or whole-exome sequencing approaches. In this Review we discuss recent findings suggesting that de novo mutations play a prominent part in rare and common forms of neurodevelopmental diseases, including intellectual disability, autism and schizophrenia. De novo mutations provide a mechanism by which early-onset reproductively lethal diseases remain frequent in the population. These mutations, although individually rare, may capture a significant part of the heritability for complex genetic diseases that is not detectable by genome-wide association studies.     

De novo purine and pyrimidine biosynthesis during thymocyte concanavalin A stimulation

Measurement of the incorporation of [¹⁴C]formate and [¹⁴C]HCO₃⁻ enable us to characterize the activity of the synthetic pathways of purine and pyrimidine nucleotides in vitro throughout the process of the conA stimulation of rat thymocyte populations enriched for immunocompetent cells by isopycnic centrifugation. Our results show that de novo synthesis compensates for the total absence of exogenous nitrogenous bases and nucleosides in the culture medium. The magnitude of the proliferative response in media supplemented with dialysed fetal calf serum (FCS) was found to be the same as that observed when complete FCS was used in the culture medium. The induction of de novo synthesis (1) contributes to the expansion of the free nucleotide pool (the quantity of ATP measured in the perchloric acid (PCA)-soluble material of a same number of cells is increased by a factor of 10); (2) supplies the nucleotides necessary for nucleic acid synthesis (the total number of cells is increased by a factor of 3 after 4 days culture period). The activity of the metabolic pathways involved appears to be solicited by the dynamic requirements for thymocyte stimulation. For each step in the cellular activation a steady state of adenylic nucleotide synthesis and condensation into nucleic acids is established.     

De novo synthesis of DNA in human platelets

Platelets, incubated with radiolabeled thymidine and purified free of contaminating nucleated cells, were analyzed for their ability to synthesize DNA. The only DNA species isolated from these purified platelets was mitochondrial DNA. The CsCl gradient-purified platelet DNA was treated with the restriction endonucleases EcoRI, HindIII and HpaI yielding the expected pattern for human mitochondrial DNA. Nitrocellulose blots of the electrophoresed, restriction endonuclease-treated DNA were fluorographed. All of the DNA fragments generated by the restriction enzymes were labeled, indicating de novo synthesis. This was further substantiated by inhibition of DNA synthesis by ethidium bromide and 2',3'-dideoxythymidine. Platelet DNA appeared to become greatly fragmented after 4 to 7 days storage while all of the thymidine incorporated was observed in intact mitochondrial DNA. These results indicate a continuous degradation of platelet mitochondrial DNA with no apparent repair mechanism. The ability of platelets to synthesize DNA may be associated with the protein synthetic capacity of platelets previously described.     

De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation

Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1) FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.     

De novo purine biosynthesis in the crustacean Artemia: Influence of salinity and geographical origin

Summary In vivo studies of the incoporation of [U-¹⁴C]glycine into purine nucleotides have established the de novo pathway for purine biosynthesis in Artemia sp. during the early period of larval development. This pathway can be modified by the salt concentration of the incubation media. In addition, Artemia of different geographical origins may differ with respect to the detection, functionality and variability of this metabolical pathway.Abbreviations ADP adenosine, diphosphate - ASN acid soluble nucleotides - ATP adenosine triphosphate - DNA desoxyribonucleic acid - GDP guanosine diphosphate - GP₄G pl, p4-diguanosine 5-tetraphosphate - HPLC high performance liquid chromatography - PCA perchloric acid - RNA ribonucleic acid     

De novo biosynthesis of prostaglandins by the Australian field cricket, Teleogryllus commodus

The accumulation and metabolism of certain polyunsaturated fatty acids by testes from the Australian field cricket, Teleogryllus commodus, are described. Testes accumulated a substantial proportion (about 16%) of label from radioactive C20:3n6 that was injected into the haemocoel. Fifty percent of the label accumulated by testes was associated with the phospholipid fraction, whereas in the remainder of the body 30% was incorporated into the phospholipid fraction. Prostaglandins (PG) E1, E2 and F2 alpha were quantified in extracts of the testes of adult insects by radioimmunoassay. Label from injected radioactive C18:2n6, C20:3n6 and C20:4n6 was recovered as prostaglandins PGE and PGF. The radioactivity from C18:2n6 that was recovered as PGE1 and PGF1 alpha indicated elongation/desaturation to C20:3n6 followed by conversion to PG. Since C18:2n6 is readily formed from acetate in T. commodus, these findings indicate the de novo biosynthesis of C20 polyunsaturated fatty acids and prostaglandins by this species.