Inclusion volume solid-phase synthesis

Solid-phase synthesis of peptides was carried out using only the volume of the solvent included in the swollen solid-phase resin beads [inclusion volume synthesis]. This approach enables (i) the use of higher concentrations of activated amino acids, resulting in increased coupling rates, (ii) drastically decreased consumption of solvents, and (iii) the construction of multiple peptide synthesizers having virtually no reaction vessels.     

Germination of Cicer arietinum seeds and thiourea-induced phytotoxicity

Thiourea, hydroxyurea, phenylthiourea, methylurea, methylthiourea, thiosemicarbazide and 2,2-dithiodipyridine affected the germination of Cicer arietinum L. cv. Castellana (chick-pea) seeds. Microscopic observations of the subapical zone of the radicle showed that thiourea induced an increase in cell volume and length when compared with control seeds germinated at 25° or 30°C in water. These results emphasize the importance of the processes controlling solute and water uptake during early germination of chick-pea seeds. In contrast to this stimulation of volume increase, the thiourea-treated seedlings were unable to synthesize chlorophyll when exposed to light. This toxic effect was reduced when thiourea was administered only during the first few hours of germination. Thiourea also caused an increase in the uptake of ³H-thymidine and ¹⁴C-leucine but it decreased their incorporation into DNA and protein, respectively. These results suggest a stimulation of plasmalemma exchange activities, but toxic or inhibitory effects on other metabolic processes necessary for normal development of seedlings.     

Automated carbohydrate synthesis as platform to address fundamental aspects of glycobiology--current status and future challenges

This award account attempts to define the status of automated carbohydrate synthesis and its applications while trying to identify areas critical for further development. In this context the work of the Seeberger laboratory over the past 10 years is reviewed. Advances and shortcomings of the first automated oligosaccharide synthesizer platform will be discussed. Using this method, access to a multitude of complex oligosaccharides has been accelerated more than 100-fold. The synthesis of usable quantities of oligosaccharides has given rise to tools that had been common-place in nucleic acid and protein biochemistry. Carbohydrate microarrays are a versatile screening platform, and affinity columns and labeled carbohydrates are beginning to aid glycobiologists. While much has been achieved, many questions remain before a generally applicable set of tools will be available to facilitate carbohydrate research much in the same way oligonucleotide and peptide biology is explored today. Application of this technology to synthetic carbohydrate antigens in synthetic vaccine candidates against parasites and bacteria is attractive and has already yielded important insights.     

Synthesis of protein,nucleosides and other organic compounds from cyanamide and potassium nitrite under possible primitive earth conditions

The mixing of cyanamide and KNO₂ produced changes from white solids to yellow liquid and then to orange solid. The gases cyanogen and ammonia were formed. No external energy was used. The reactions were carried out with a small amount of O₂. The presence of proteins in the reaction product formed 13 months after the mixing was indicated by the positive reactions of the cyanamide-KNO₂ reaction product with ninhydrin, microbiuret, and Folin reagent; the ultraviolet absorption at about 280 nm; the yield of 24% of 15 amino acids; and molecular weight measurements of more than 160 000. The presence of nucleosides, nucleic acid bases, hydrocarbons, and organic esters in the reaction product formed 2 months after the mixing was indicated by ultraviolet absorption at about 260 nm, and the results of ligand-exchange chromatography, paper chromatography, infrared analysis, mass spectral analysis, and NMR spectroscopy. Possible cyanamide-mediated dehydration reactions and mechanisms are discussed.     

光学活性叔亮氨酸合成的研究进展

由于叔丁基的特殊结构和性质 ,叔亮氨酸是重要的医药中间体和不对称合成的手性诱导模板。本文综述了近年来光学活性的叔亮氨酸的合成研究及最新进展。     

Synthesis of nucleic acids and localization of atrial natriuretic peptide in crayfish haemocytes

Three types of cells circulate in the haemolymph of the crayfish Astacus astacus, i.e., agranular haemocytes (HCs I), small-granule haemocytes (HCs II), and large-granule haemocytes (HCs III). Their proliferation, differentiation, and function remain poorly understood. Using light and electron microscopic autoradiography with [³H]-thymidine, we found that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To verify whether HCs I are proliferating progenitor cells for granular HCs, we have analyzed autographs of the HC population 1, 2, 7, and 21 days after a single [3H]-thymidine administration. Contrary to our expectations, we have failed to find labeled HCs II and HCs III. These findings have raised doubts as to the capacity of HCs I to differentiate into two other types of HCs. With the use of ³H-uridine autoradiography, it was found that RNA synthesis was the most active in HCs I and 2 and 4 times lower in HCs II and HCs III, respectively. ANP-like immunoreactivity was revealed in large granules of the HCs III by electron microscopic immunocytochemistry. We assume that the presence of ANP in secretory granules extends the possible functions of crayfish HCs and suggests their participation in the regulation of the watersalt balance and immune response.     

Thematic review series: glycerolipids. Mammalian glycerol-3-phosphate acyltransferases: new genes for an old activity

Glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15) catalyze the first step in the de novo synthesis of neutral lipids (triglycerides) and glycerophospholipids. The existence of multiple enzyme isoforms with GPAT activity was predicted many years ago when GPAT activities with distinct kinetic profiles and sensitivity to inhibitors were characterized in two subcellular compartments, mitochondria and microsomes. We now know that mammals have at least four GPAT isoforms with distinct tissue distribution and function. GPAT1 is the major mitochondrial GPAT isoform and is characterized by its resistance to sulfhydryl-modifying reagents, such as N-ethylmaleimide (NEM). GPAT2 is a minor NEM-sensitive mitochondrial isoform. The activity referred to as microsomal GPAT is encoded by two closely related genes, GPAT3 and GPAT4. GPAT isoforms are important regulators of cellular triglyceride and phospholipid content, and may channel fatty acids toward particular metabolic fates. Overexpression and knock-out studies suggest that GPAT isoforms can play important roles in the development of hepatic steatosis, insulin resistance, and obesity; GPAT isoforms are also important for lactation. This review summarizes the current state of knowledge on mammalian GPAT isoforms.     

基因合成技术研究进展

基因合成是生物学中一项最基本的、最常用的技术.对DNA调控元件、基因、途径乃至整个基因组的合成是验证生物学假设和利用生物学为人类服务的有力工具.合成生物学的快速发展对基因合成能力提出了日益迫切的需求.近年来,基于微芯片基因合成技术取得了很多令人振奋的新进展,正在向着高通量、高保真、自动化的方向发展.文中综述了DNA化学合成和基因组装及相关技术的最新研究进展和发展趋势,这些新技术正在推动着合成生物学向着更高的水平发展.     

Biocatalytic synthesis of 2′-deoxynucleotide 5′-triphosphates from bacterial genomic DNA: Proof of principle

2′-deoxynucleoside 5′-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR). These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in low- and middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solid-state bioreactor for on-demand dNTP production.     

从DNA合成技术角度阐述合成生物学

合成生物学是一个拥有巨大潜力的新兴学科,合成生物学技术的发展将会对未来生物、医药、农业、能源、材料和环保等方面产生巨大的推进作用。基因合成是合成生物学中最基本和使用最多的一种技术手段,合成生物学的快速发展对基因合成能力提出了空前需求。综述基因合成技术的发展历史、现状和未来趋势,探讨基因合成技术存合成生物学以及整个生命科学研究中的应用和重要意义。     

Inhibition by warfarin of prothrombin synthesis and of lipid-saccharide synthesis in rat liver

In vivo and in vitro studies using [³H]glucosamine incorporation into prothrombin and into glycolipids were conducted in rat liver to determine the role of lipid-saccharides in the biosynthesis of prothrombin.In vivo studies demonstrated that 10 mg warfarin/kg inhibited the incorporation of radiolabeled glucosamine into liver prothrombin and glycolipids. This inhibition was similar to the kinetics of inhibition of prothrombin synthesis in the liver.In vitro studies demonstrated a time-dependent increase in the incorporation of radiolabeled glucosamine into lipid-saccharides and prothrombin. This incorporation was inhibited 50% by 5 · 10^?4 M warfarin. Warfarin also inhibited the incorporation of radiolabeled glucosamine into glycolipids in a dose-related manner.In all studies, vitamin K-1 reversed the inhibition of glucosamine incorporation into glycolipids and into prothrombin.     

Synthesis of Novel AZA-Analogues of Tiazofurin with 2-[5,5-bis(Hydroxymethyl)Pyrrolidin-2-yl] Framework as Sugar Mimic

The novel aza-analogues of tiazofurin (TZF) with 2-[5,5-bis(hydroxymethyl)pyrrolidin-2-yl] moiety, as sugar mimic, were synthesized from O,O-cyclohexylidene derivative of 4,4-bis(hydroxymethyl)-4-nitrobutanal in multi-gram scale. The synthetic route consisted of three stages: (i) the synthesis of corresponding derivative of 5,5-bis(hydroxymethyl)pyrrolidine-2-carbonitrile, (ii) the construction of ethyl thiazole-2-carboxylate part by the conversion of the pyrrolidine-2-carbonitrile into the N-trifluoroacetyl derivative followed by cyclocondensation with L-cysteine ethyl ester and then by dehydrogenation, and (iii) the final transformation of the ethyl thiazole-4-carboxylate into the aza-analogues of TZF. The TZF aza-analogues were evaluated for their antiviral activities in cell-culture-based assays.     

Syntheses of oligomannosides in solution and on a soluble polymer support: a comparison

The alpha-(1-->6)-linked and the alpha-(1-->2)-linked linear mannotetraose glycosides and, respectively, and the branched mannopentaoside [R=CH2(CH2)2CH2Cl] were synthesised by conventional methods in solution, using trichloroacetimidate donors, and the products were obtained in 39%, 42% and 40% overall yield, respectively. For comparative purposes, the same two linear tetrasaccharides were prepared by use of MPEG as a soluble polymer support, the yields being 34% and 14%, respectively. An attempted MPEG-supported synthesis of the branched pentasaccharide was unsuccessful. The merits and shortcomings of oligosaccharide syntheses on MPEG are discussed.     

The Distribution of Enzymes Which Synthesize Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate in Monkey, Rabbit, and Ground Squirrel Retinas

The activity of adenosinetriphosphate:nicotinamide adenylyltransferase (EC 2.7.7.1) was measured in all the layers of monkey, rabbit, and ground squirrel retinas. Nicotinamide adenine dinucleotide (NAD) kinase (EC 2.7.1.23) distribution was measured in monkey and rabbit retinas. An attempt was made to measure NAD synthetase (EC 6.3.5.1), but the activities in the retinal layers were too low to produce a reliable increment in the levels of endogenous NAD. In monkey retina the adenylyl transferase was highest by far in the outer and inner nuclear layers, lower and variable in ganglion cell and fiber layers, and almost absent elsewhere. Rabbit retina differed in that activity was nearly absent in the outer nuclear layer, whereas in the ground squirrel outer nuclear layer activity was double that of the inner nuclear layer. The species differences suggest that adenylyl transferase is almost absent from cone cell nuclei and high in rod cell nuclei. NAD kinase distribution in monkey retina was almost the mirror image of that of adenylyl transferase.     

Synthesis of RNA by Rhodomicrobium vannielii swarmer cells

Abstract Protein synthesis in Rhodomicrobium vannielii swarmer cells, incubated anaerobically in the dark, is dependent upon a rifampicin-sensitive step, indicating a dependence upon de novo RNA synthesis. In addition, toluene treatment has shown that the motile, non-differentiating swarmer cells have the capacity to initiate and sustain RNA synthesis. The major form of the DNA-dependent RNA polymerase responsible for this RNA synthesis has been identified.     

DCM和DMF对DCC-HOBt系统催化酯化及酰化反应的影响

DCC-HOBt系统在DCM和DMF溶剂中,催化Fmoc-Leu-OH与王氏树脂的酯化反应,其结合率分别为18％和10％．该系统催化Fmoc-Phe-OH与H2N-Leu-resin的酰化反应中,溶剂为DCM时,反应30min,其结合率为100％;当DCM：DMF体积比为1：3．5时,反应130min,其结合率为81％,表明DCC-HOBt系统宜在DCM非极性溶剂中催化肽键的形成．     

Suppression of Programmed Cell Death by Intracellular cAMP Is not Mediated by Expression of Genes Encoding an Inhibitor of Apoptosis

The elevation of intracellular cAMP content is accompanied by expression of genes whose promoter contains a Ca²⁺-cAMP responsive element. In vascular smooth muscle cells (VSMC), activation of cAMP signaling blocks apoptosis triggered by serum deprivation. In the present study we investigated the role of gene expression in the inhibition of apoptosis by cAMP. In VSMC transfected with E1A adenovirus, incubation in the absence of serum for 6 h led to 20-fold elevation of chromatin fragmentation and 10-fold activation of caspase-3 activity, these being employed as markers of apoptosis. Forskolin induced activation of cAMP signaling was accompanied by 50% elevation of RNA synthesis and completely abolished the development of apoptosis during the initial 6 h incubation in growth factor-free medium. In 12 h apoptosis in forskolin-treated VSMC was slowly developed and after 24 h the content of chromatin fragments was 2-fold less than in control cells. Addition of actinomycin D and cycloheximide completely blocked RNA synthesis and decreased protein synthesis by 80%, respectively. Neither compound affected baseline apoptosis or its inhibition by forskolin. More than 70 newly phosphorylated proteins were observed by 2D-electrophoresis of VSMC after incubation with forskolin for 3 h; in 24 h the number of phosphoproteins triggered by forskolin was decreased by 2-3-fold. These results show that suppression of VSMC apoptosis under activation of cAMP signaling is mediated via posttranslational modification of pre-existing intermediates of the apoptotic machinery rather than by de novo synthesis of inhibitors of programmed cell death.     

Synthèses ďacides nucléiques et de protéines au cours de ľinitiation de bourgeons adventifs sur des explantats de Cichorium intybus cultivés in vitro

Synthesis of nucleic acids and proteins during initiation of vegetative buds from explants of Cichorium intybus cultivated in vitro .
Explants of 6 mm diameter and 2 mm thickness from roots of Cichorium intybus L. (var. WitIoof cv. "Zoom"), grown in vitro in the presence of kinetin (l0_–6 M ), produced only buds. By comparing the histological events and the biochemical modifications which occurred during the neoformation of buds, it is possible to distinguish two distinct phases. The first phase starts immediately after the excision of the explants and continues for 30 h, when the mitotic index is at its highest. This corresponds to a phase of cellular activation, which is characterized by early synthesis of RNA, permitting the synthesis of proteins necessary for duplication of DNA and cell divisions. The second period starts after 30 h and ends after approximately 72 h of culture, at which time the first bud meristematic nodules were detected. This is a preparatory phase for organogenesis and above all related to synthesis of RNA and proteins.     

The levels of ecdysteroids in uninjured and leg-autotomized nymphs of Blattella germanica (L.)

The levels of ecdysteroids in control and leg-autotomized first-instar nymphs of Blattella germanica were determined by radioimmunoassay from hatching to the time of the first ecdysis. Uninjured nymphs showed a distinct release of ecdysteroids half-way through the stadium, and this resulted in the commencement of the moult cycle which formed the cuticle of the second instar. Cockroaches which had legs autotomized at 48 h after hatching (i.e. before the control ecydsteroid release) had their instar duration increased by that time period. Releases of ecdysteroids and events of the moulting cycle were also postponed by the 48 h period. The titre of ecdysteroids in injured animals was double that of controls. Nymphs were also autotomized at 96 h (i.e. after the normal release of ecdysteroids) but no changes in instar duration, ecdysteroid releases, or events of the moult cycle were recorded. The effects of injury, prothoracicotropic hormone activity and ecdysteroid release are discussed.     

Chemical and enzymatic synthesis of glycopolymers

The development of efficient, fast, flexible and general synthetic routes to glycopolymers is an ongoing challenge and much progress has been made in recent years. Chemical coupling methods have become increasingly sophisticated to fine-tune reactivity of reagents by fortuitous choices of anomeric activating group and protecting groups. As a result, oligosaccharide synthesis has become more predictable and reliable even to the extent that first examples of saccharide library syntheses in solution and on the solid phase have been published. In biology, the repertoire of biocatalysts that can be used for glycoside synthesis is ever-increasing, and enzyme-catalysed glycosylation steps have been successfully incorporated into synthetic strategies.