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Photosynthesis is a fundamental biosynthetic process in plants that can enhance carbon absorption and increase crop productivity. Heat stress severely inhibits photosynthetic efficiency. Melatonin is a bio-stimulator capable of regulating diverse abiotic stress tolerances. However, the underlying mechanisms of melatonin-mediated photosynthesis in plants exposed to heat stress largely remain elucidated. Our results revealed that melatonin treatment (100 μM) in tomato seedlings increased the endogenous melatonin levels and photosynthetic pigment content along with upregulated of their biosynthesis gene expression under high-temperature stress (42 °C for 24 h), whereas heat stress significantly decreased the values of gas exchange parameters. Under heat stress, melatonin boosted CO2 assimilation, i.e., Vc,max (maximum rate of ribulose-1,5-bisphosphate carboxylase, Rubisco), and Jmax (electron transport of Rubisco generation) and also enhanced the Rubisco and FBPase activities, which resulted in upregulated photosynthetic related gene expression. In addition, heat stress greatly reduced the photochemical chemistry of photosystem II (PSII) and photosystem I (PSI), particularly the maximum quantum efficiency of PSII (Fv/Fm) and PSI (Pm). Conversely, melatonin supplementation increased the chlorophyll a fluorescence parameters led to amplifying the electron transport efficiency. Moreover, heat stress decreased the actual PSII efficiency (ΦPSII), electron transport rate (ETR) and photochemical quenching coefficient (qP), while increasing nonphotochemical quenching (NPQ); however, melatonin reversed these values, which helps to fostering the dissipation of excess excitation energy. Taken together, our results provide a concrete insight into the efficacy of melatonin-mediated photosynthesis performance in a high-temperature regime.

Aging is a very complicated biological process that can change gene expressions. The Chinese rhesus macaque (Macaca mulatta lasiota; CR) is closely related to humans. We explored gene expression with increasing age and DNA methylation changes in young and old CRs. Results showed blood transcriptome and DNA methylome significantly changed from young to old CRs. The age-associated differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were associated with age-related biological features, such as immunity, blood coagulation, and biosynthetic process. The measurements of coagulation indicators confirmed old CRs had shorter coagulation time than young CRs, and the activities of coagulation factor II (FII) and factor VIII (FVIII) were enhanced in old CRs. Humans and CRs exhibited the same enhanced blood coagulation with age phenotype. Our study found aging is a critical factor affecting gene expression in CRs, and also provided new insights into the blood coagulation changes in non-human primates.

Objective Myricetin 3-O-galactoside is an active compound with pharmaceutical potential. The insufficient supply of this compound becomes a bottleneck in the druggability study of myricetin 3-O-galactoside. Thus, it is necessary to develop a biosynthetic process for myricetin 3-O-galactoside through metabolic engineering. Methods Two genes OcSUS1 and OcUGE1 encoding sucrose synthase and UDP-glucose 4-epimerase were introduced into BL21(DE3) to reconstruct a UDP-D-galactose (UDP-Gal) biosynthetic pathway in Escherichia coli. The resultant chassis strain was able to produce UDP-Gal. Subsequently, a flavonol 3-O-galactosyltransferase DkFGT gene was transformed into the chassis strain producing UDP-Gal. An artificial pathway for myricetin 3-O-galactoside biosynthesis was thus constructed in E. coli. Results The obtained engineered strain was demonstrated to be capable of producing myricetin 3-O-galactoside, reaching 29.7 mg/L. Conclusion Biosynthesis of myricetin 3-O-galactoside through engineered E. coli could be achieved. This result lays the foundation for the large-scale preparation of myricetin 3-O-galactoside.

Aluminum compounds have been observed in various brain regions, and their accumulation has been associated with many neurodegenerative disorders. Neurotoxic effects of aluminum are attributed to reactive oxygen species generation, induction of apoptosis and inflammatory reactions activation. Metalloestrogen activity of aluminum has also been linked to breast cancer progression and metastasis. In this study, taking into account the anti-apoptotic and anti-oxidant activities of estrogens in neuronal cells, which are mediated by estrogen receptors, the possible estrogenic activity of aluminum in SH-SY5Y neuroblastoma cells was studied. Our results showed that aluminum in the form of aluminum chlorohydrate (ACH) exhibited no effect on estrogen receptors transcriptional activation, and differential effect on estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) protein levels. ACH caused reduction in ERβ protein levels, and increase in its mitochondrial localization. ACH-induced reduction in ERβ protein level may be linked, at least in part, to the ACH-induced increase in ERα protein level. This statement is based on our observations showing aluminum-induced reduction in the E2-induced increase in ERα S118 phosphorylation, in MCF-7 and SH-SH5Y cells. Phosphorylation at S118 residue is known to be associated with inhibition of the ubiquitin-induced proteolytic degradation of ERα, leading to its accumulation. Since it is known that ERα negatively regulate ERβ expression, increase in ERα, may contribute to reduction in ERβ levels and subsequent weakening of its anti-apoptotic and anti-oxidant activity, justified by the observed reduction in procaspase 9, mitochondrial cytochrome c, Bcl-2, Bcl-xL and mitochondrial thioredoxin protein level, as well as by the increase in proapoptotic BAX level, in ACH treated SH-SY5Y cells. In addition, increase in mitochondrial ERβ localization may also trigger mitochondrial metabolism, suppress biosynthetic process of gluconeogenesis, as indicated by the observed reduction in the phosphoenolpyruvate carboxykinase protein level, and eventually lead to increase in reactive oxygen species (ROS) generation, known to be implicated in aluminum induced neurodegeneration. This statement was verified by the observed ACH-induced increase in ERβ mitochondrial localization, induction of the mitochondrial membrane depolarization and increase in ROS production, in neuronal-like differentiated SH-SY5Y cells.

Background Starch is a complex branched glucose polymer, mainly comprising amylose and amylopectin. The number of individual chains as a function of the number of monomer units they contain, i.e. the chain-length distributions (CLDs), are controlled by the underlying biosynthetic process occurring during plant growth. CLDs are currently commonly related to biosynthetic processes and to functional properties by dividing into arbitrarily chosen regions. However, this empiricism is not completely satisfactory: conclusions can depend on the choice of division. Biosynthesis-based models replacing this arbitrary division by model-based parameterization have been developed, but are presently rarely used, because of the complex underlying mathematics. Scope and approach These models are summarized in non-mathematical language. These give information on the biosynthetic processes producing the starch, and yield a parameterization of CLD data to give a fit that is essentially the same as experiment. Additionally, the models are sufficiently flexible that they can also fit data for modified starches. This enables the whole CLDs for both amylose and amylopectin as a small number of parameters which can be used to find statistically-valid structure-property relations. Key findings and conclusions The underlying theory and data-fitting methodology can be used both to better understand starch biosynthesis, to see what structural features control functional properties and to deduce mechanisms for observed correlations. This enables raw materials to be chosen in non-empirical ways to select and process grains and other starch sources for improved foods and other products.

A unique thermostable amylosucrase from Bifidobacterium thermophilum was produced as a recombinant protein with the half-life of 577 h at 50 °C. By adding 1.0 M fructose, turanose yield was improved from 22.7% to 43.3% with 1.0 M sucrose, and from 23.7% to 39.4% with 1.5 M sucrose. Sucrose consumption rate was greatest at 55 °C, but the lowest amount of turanose was produced. Thus, turanose yield from sucrose biomass was inversely proportional to reaction temperature and was highly dependent on [fructose]. Meanwhile, insoluble α-glucan yield was clearly reduced as [fructose] increased. With 1.0 M fructose + 1.0 M sucrose, glucan byproduct yield significantly decreased from 29.4% to 1.1%. Molecular weights of linear glucans were almost identical among various [sucrose]s and were homogenous with very low polydispersity. This unique dual reaction patterns of amylosucrase enzyme would be very useful for massive productions of two different biomaterials simply by changing sucrose biomass concentration.

Leaf wax n-alkanes are terrestrial plant biomarkers that have characteristics that are widely employed as proxies for climatic conditions. Understanding the relationship between different environmental factors to the amounts and types of leaf wax n-alkanes in modern plants is crucial for the application of these proxies to paleoenvironmental reconstructions. However, the effects of climate conditions on plant wax characteristics remain complicated due to the interactions among temperature, precipitation and phylogeny. To evaluate the effect of temperature with minimized interfering factors, we collected 106 Artemisia plants across a 15 °C mean annual temperature gradient along the 400 mm isohyet in China. Both total n-alkane concentration (∑alk) and carbon preference index (CPI) varied greatly but did not correlate with temperature. Average chain length (ACL) increased with temperature, especially summer temperature (TJJA, June–August), indicating that ACL could be used as proxy for temperature. The stable carbon isotope compositions of n-C27, n-C29 and n-C31 were very similar in each plant (−38.2‰ to −30.0‰, −39.0‰ to −30.2‰, −38.7‰ to −30.5‰, respectively), which reflects a similar biosynthetic process for all three n-alkane homologues of Artemisia. There was a positive relationship between δ13C of bulk leaf tissues (δ13Cbulk) and of n-alkanes (δ13Calk), and the average offset of δ13C29 relative to δ13Cbulk (eC29/bulk) was −7.1‰. Increasing trends in both δ13Cbulk and δ13C29 were found with temperature. However, correlation of δ13Cbulkwith temperature (R2 = 18%) was much weaker than that of δ13C29 with temperature (R2 = 60%). Therefore, δ13C29 appears to be a better proxy of paleotemperature than δ13Cbulk.

The green biogenic PdAu nanoparticles (bio-PdAu NPs) exhibits remarkable catalytic performance in hydrogenation, which is highly desired. However, the catalytic principles and effectiveness of bio-PdxAuy NPs in response to various catalytic systems (electrocatalysis and suspension-catalysis) are unclear. Herein, a facile synthetic strategy for bio-PdxAuy NPs synthesis with controlled size and the catalytic principles for hydrogen evolution reaction (HER) and azo dye degradation is reported. In the biosynthetic process, the size and composition of the bio-PdxAuy NPs could be precisely controlled by predesigning the precursor mass ratio of Pd/Au, and the Au proportion showed a linear relationship with the size of NPs (R2 = 0.92). The obtained bio-PdxAuy NPs exhibit variable activity in electrocatalysis (HER) and suspension-catalysis (azo dye degradation). For electrocatalysis, the formation of conductive networks that facilitates the extracellular electron transfer is crucial. It was revealed that the bio-Pd2Au8 exhibited superior electrocatalytic performance in HER/toward hydrogen evolution, with a maximum current density of 1.65 mA cm−2, which was 1.54 times higher than that commercial Pd/C (1.07 mA cm−2). The high electrocatalytic activity was attributed to its appropriate size (81.38 ± 6.14 nm) and uniform distribution on the cell surface, which promoted the extracellular electron transfer by constructing a conductive network between catalyst and electrode. However, for suspension-catalysis, the size effect and synergistic effect of bimetallic NPs have a more prominent effect on the degradation of azo dyes. As the increase of Au proportion the particle size decreases, and the catalytic activity of bio-PdxAuy improved significantly. The response principles of bio-PdxAuy proposed in this study provide a reliable reference for the rational design of bio-based bimetallic catalysts with enhanced catalytic performance.

Thraustochytrium is a marine protist producing a specific profile of nutritionally important fatty acids, including very long chain polyunsaturated fatty acids (VLCPUFAs) docosahexaenoic acid (DHA, 22:6n-3), even chain saturated fatty acids (SFAs) palmitic acid (16:0), and odd chain SFAs pentadecanoic acid (15:0). To study how these fatty acids are synthesized, a series of radiolabeled precursors were used to trace the biosynthetic process in vivo and in vitro. When Thraustochytrium was fed with long chain fatty acid intermediates such as [1–14C]-oleic acid, [1–14C]-linoleic acid and [1–14C]-α-linolenic acid, no VLCPUFAs were produced, indicating that the aerobic pathway for the biosynthesis of VLCPUFAs was not functional in Thraustochytrium. When fed with [1–14C]-acetic acid, both SFAs and VLCPUFAs were labeled, and when fed with [1–14C]-propionic acid, mainly SFAs were labeled. However, when fed with [1–14C]-acetic acid in the presence of cerulenin, a type I FAS inhibitor, only VLCPUFAs were labeled, and when fed with [1–14C]-propionic acid in the presence of cerulenin, neither SFAs nor VLCPUFAs were labeled. This result clearly indicates that the type I fatty acid synthase (FAS) in Thraustochytrium could use acetic acid and propionic acid as the primers to synthesize even chain and odd chain SFAs, respectively, and VLCPUFAs were synthesized by the PUFA synthase using acetic acid as the primer. In addition, radioactive acetic acid could label both phospholipids (PL) and triacylglycerols (TAG), and VLCPUFAs appeared first and were largely accumulated in PL, whereas TAG accumulated much more SFAs than VLCPUFAs. The in vitro assay with [1–14C]-malonyl-CoA in presence of cerulenin showed that the crude protein of Thraustochytrium produced only VLCPUFAs, not SFAs, further confirming the role of the PUFA synthase in the biosynthesis of VLCPUFAs. Collectively, these results have elucidated the biochemical mechanisms for the biosynthesis of all fatty acids in Thraustochytrium.

Graphical abstract, Mycosynthesis deal with an energy-saving and eco-friendly process intended for extracellular synthesis of AgNPs, by means of cell-free filtrates of fungi Aspergillus niger and Fusarium semitectum as reducing agents. Optimization of different parameters during biosynthetic process demonstrated diverse property on production rate, the size, distribution, yield of biosynthesized AgNPs. SEM micrographs showed polydisperse spherical and ellipsoid nanoparticles (SIZE). AgNPs exhibits potential antimicrobial effect than Ag+ not in favor of E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa. These results demonstrate that mycosynthesis of AGNPs is a cost effective and eco-friendly method, resulting in particles with antibacterial properties that are efficient as an antimicrobial agent. • Characterization of Silver nano particle is widely applauded domain at present. • Mycosynthesis of AgNPs as reducing agents and exhibits potential antimicrobial effect. • Results of mycosynthesis of AgNPs is cost effective and ecofriendly.

In the present study, cadmium-based nanoparticles (NPs) were biosynthesized by incubating their precursor salts with E. coli CD-2. Transmission electron microscopy (TEM) revealed the morphology of the NPs and confirmed that the NPs were formed via an intracellular growth. Energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) determined the elemental composition of the NPs and identified the NPs as CdS. The contents of extracellular Cd2+, intracellular Cd2+ and intracellular CdS NPs were determined during the whole CdS biosynthetic process. The results demonstrated that the contents of Cd2+ and CdS NPs changed during the biosynthetic process. The colony-forming capability test showed that strain CD-2 could maintain its growth during CdS biosynthesis. Protein oxidation levels confirmed that the E. coli cells faced oxidative stress induced both by Cd2+ and CdS. Both Cd2+ and CdS NPs affected the cellular antioxidative system by upregulating related gene expression. However, different pathways might be involved to eliminate ROS induced by Cd2+ ions or CdS NPs, respectively. The expression levels of ef-tu, ftsZ, mutS and dnaK were enhanced together with CdS accumulation, indicating that the cells had to overexpress certain related genes to respond to the NPs-induced stress.

Exposure to light with certain wavelengths was demonstrated to enhance the biosynthesis of astaxanthin in Haematococcus pluvialis for industrial purposes. Photo-responsive biosynthetic process is likely to be mediated by photoreceptor proteins. Therefore, the study of photoreceptors particularly responsible for algal growth and metabolite synthesis is in urgent demand. A novel channelrhodopsin gene HpChR1 was cloned. We examined the subcellular localization and cellular functions of HpChR1 in HeLa cells by living cell imaging. HpChR1 was shown to mainly be localized to the endoplasmic reticulum and the nucleus and mediate endoplasmic reticulum calcium release in HeLa cells. HpChR1 protein comprises seven transmembrane domains at N-terminus and a putative MED15 (mediator complex subunit 15) domain at C-terminus. By using Yeast Two-Hybrid, we showed the binding partners of HpChR1 and they further assisting to interpret the functional roles of HpChR1.These results showed HpChR1 may involve in the reception and transformation of photon energy and signal as channelrhodopsin, and this may shed light on understanding photo-responsive biosynthetic mechanisms in H. pluvialis.

Functional gene fingerprinting of chemicals could be used to understand the direct gene-chemical interaction in the process of toxification from a genome-wide scale. 2, 2-bis (bromomethyl)-1, 3-propanediol (BMP) is a brominated flame retardant with widespread production but with very limited toxicological data. Here the cytotoxicity of BMP was assessed by Escherichia coli ( E. coli ) functional genome-wide knockout mutants screening and the underlying molecular mechanism was investigated. The median inhibition concentration (IC50) of BMP was 1.608 ± 0.078 mg/ml after 24 h exposure. 119 initial, including 66 sensitive and 53 resistant single gene mutants, were identified by a full library screening of BMP at the concentration of IC50. The resistant genes were significantly enriched in nucleobase-containing compound biosynthetic process (GO: 0034654) by gene ontology (GO) biological process analyses, which suggested that the pathway of DNA repair is a critical cellular process in the survival of cells exposed to BMP. Meanwhile, function annotation of all BMP responsive genes suggested the mechanism of BMP was associated with DNA damage, oxidative stress and cellular transmembrane transport process. Many genes were exclusively responsive to BMP comparing with other chemicals that has been assessed by E. coli mutant screening approach, which indicated that BMP has a distinct mode of toxic action. Overall, the functional genomic screening approach presented here provides a great tool to assess the cellular toxicological mechanism of environmental chemicals.

A simple and convenient method was proposed in this paper to develop a flow-through enzymatic micro-reactor made from polytetrafluoroethylene (PTFE). It consisted of the polydopamine layer (functioned as a primer) and layer by layer (LBL) coatings composed of polyethylenimine (PEI) and lipase. The multiple deposition of PEI and lipase was the key factor of increasing the enzyme loading on microreactor. After 8 PEI/lipase layers, enzyme loading on the inner surface of 5-m microchannel reached a maximum (350 μg to 400 μg), compared with approximately 20 μg in single layer. Microreactor with higher enzyme loading was successfully applied on transesterification of soybean oil for effective fatty acid methyl ester (FAME, biodiesel) production. A 95.2% conversion rate of biodiesel can be achieved in 53 min under optimized conditions, instead of a couple of hours in the traditional batch reaction.

The indolizidine alkaloid swainsonine (SW) is a deadly mycotoxin to livestock. The genetic mechanism of SW biosynthesis remains obscure for decades. Here we report that SW is produced via a multi-branched pathway by a hybrid nonribosomal peptide/polyketide synthase (NRPS-PKS) gene cluster in the endophytic entomopathogenic fungus Metarhizium robertsii. The precursor pipecolic acid can be redundantly converted from lysine by both clustered and unclustered genes. The hybrid enzyme that begins with a NRPS module produces three intermediates with and without domain skipping. Intriguingly, the biosynthetic process is coupled with the trans to cis non-enzymatic epimerization of C1-OH for both hydroxyl- and dihydroxyl-indolizidine intermediates. We also found that SW production is dispensable for fungal colonization of plants and the infection of insect hosts. Our finding that SW does not accumulate in planta after Metarhizium colonization provides a notable indication of safety when using this fungus to control insect pests.

Chinese black truffle (Tuber indicum) is rich in nutrition. However, commercial interests lead to the aroma components and nutrients of T. indicum being greatly affected by overexploitation without consideration of their maturity. This study investigated the proteomic and metabolomic profiles of truffle fruiting bodies at different maturities using a meta-proteomic approach. Among the 3007 identified proteins, the most up-expressed protein in the mature ascocarps was involved in the peptidyl-diphthamide biosynthetic process, while thiamine metabolism was the most differentially expressed pathway. Furthermore, a total of 54 metabolites identified upon LC-MS differed significantly, with 30 being up-expressed in the mature ascocarps, including organic acids, carnitine substances and polysaccharides. Additionally, the ash, protein, fat, crude fiber and total sugar contents were all higher in the mature ascocarps. Overall, our findings reveal that mature truffles have a higher nutritional value, providing a basis for further exploring protein functionality of T. indicum at different maturities.

Vacuole is a prominent organelle that often occupies most of the plant cell volume. The vacuolar accumulation of secondary metabolites, also called specialized metabolites, plays important roles in environmental responses such as protecting against insect herbivores and attracting pollinators. The compartmentation of xenobiotics in the vacuole is also essential for adaptation to environmental stresses. These accumulations involve several transport systems, for which some responsible transporter proteins have been reported. Furthermore, studies on biosynthetic enzymes and transporters of secondary metabolites have revealed that vacuoles, which have been recognized for many years as a site for accumulation, also function as a site for biosynthesis of secondary metabolites and are thus actively involved in the entire biosynthetic process. In this review, we briefly summarize recent findings on vacuolar transporters involved in secondary metabolites and xenobiotics, and discuss their roles in plant adaptation to biotic and abiotic stresses, through vacuolar dynamism.

Extensive studies in various plants show that the anthocyanin biosynthetic process is affected by environmental factors and regulated by many transcription factors through sophisticated regulatory networks. However, it remains largely unclear about the roles of microRNA in this process. Here, we demonstrate that miR858a is a positive regulator of anthocyanin biosynthesis in Arabidopsis seedlings. Overexpression of miR858a enhances the accumulation of anthocyanins, whereas the reduced miR858a activity results in low levels of anthocyanins in STTM858 transgenic plants. We found that miR858a inhibits the expression of MYBL2, a key negative regulator of anthocyanin biosynthesis, by translational repression. In addition, ELONGATED HYPOCOTYL 5 (HY5) was shown to directly bind the MYBL2 promoter and represses its expression via specific histone modifications. Interestingly, we found that miR858a exhibits light-responsive expression in an HY5-dependent manner. Together, these results delineate the HY5-MIR858a-MYBL2 loop as a cellular mechanism for modulating anthocyanin biosynthesis, suggesting that integration of transcriptional and posttranscriptional regulation is critical for governing proper anthocyanin accumulation in response to light and other environmental factors.

Context Ginsenosides are regarded as the major active ingredients of ginseng, the dried taproot of Panax ginseng C. A. Mey. Our previous study has shown that several single nucleotide polymorphisms (SNPs) loci were identified in dammarenediol synthase (DS) gene, a key enzyme in the biosynthetic process of ginsenoside. Objective The current study aims to investigate the possible influence of SNPs on the content of ginsenosides in DAMAYA ginseng, a cultivar of P. ginseng . Materials and methods We selected a particular group of DAMAYA ginseng samples that were harvested in the same place of origin for SNP analysis, and then analyzed 18 ginsenosides in these samples with ultrahigh pressure liquid chromatography (UHPLC) coupled with time-of-flight mass spectrometer system (TOF/MS). Results We found that these SNPs are associated with the accumulation of active ingredients ginsenosides Rf, Rg1 and F1. Moreover, OPLS-DA analysis implied that SNP loci of 279 might play a role in influencing the content level of ginsenosides. Further bioinformatics analysis showed that Leu83aa mutant might cause structural and functional changes of DS gene, which could in turn result in the content change of dammarane ginsenosides. Discussion and conclusion Our findings suggest that specific SNPs of DS gene might be associated with the accumulation of some ginsenosides in DAMAYA ginseng and thus can be used for varietal characterization, molecular identification, qualitative evaluation of ginseng-type medicinal materials, or gene engineering and ginseng breeding.

Compared with plant-derived cellulose, developing biocellulose nanofibers (bio-CNFs) as a functional membrane material is of great interest in membrane filtration for the effective separation of emulsified oily wastewater due to its low cost, renewability, high water-holding capacity and anti-oil-fouling properties. In this work, a bio-CNF membrane was produced by cultured Gluconacetobacter xylinus through a simple biosynthetic process followed by a purification step involving alkali treatment. Herein, the bio-CNF membrane treated with 0.75 M NaOH solution formed a unique, porous nanonetwork structure with good hydrophilicity and ultralow-oil-adhesion properties, and this was successfully applied for the separation of surfactant-stabilized oil/water emulsions. The superhydrophilic and superoleophobic bio-CNF membranes exhibited superior performance for separating surfactant-stabilized oil/water emulsions, showing a separation efficiency of ~99% and a high permeate flux recovery ratio (above 94%) for long-term separation processes.

Waste biomass of two major milk weeds was separately given combination of chemical-mechanical treatments in order to expose natural cellulosic micro and nano-fibrillated structures (MFCs and NFCs). Compositional and FTIR analysis were initially used to confirm the removal of impurities. WA-XRD and FESEM analysis of MFCs further revealed presence of highly crystalline, fine-shaped materials that resemble carbonaceous structures. HR-TEM analysis further exposed morphology of the two nanofibrils possessing a well entangled network (diameter ≤80 nm) comprising coil shaped fine spots of size up to 80 nm–220 nm. Morphology even got clearer when STEM and EDX mapping of the NFCs assured that observed fine-spots are integral and possibly due to certain biosynthetic process. Presence of calcium elements in two types of NFCs provided important evidence of exposure of nanocellulose-xyloglucan-pectin configuration responsible for coil-shaped fine-spots resembling “biomechanical hot spots”.

Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24 h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain.

The analyses of endogenous substances as biomarkers presents challenges that are distinctly different from the analyses of drugs or other xenobiotic substances. This is particularly true for estrogens. When no matrix is available which does not contain some level of the biomarker of interest, specificity cannot be demonstrated. Therefore it cannot be known whether the analyte signal includes a response from another substance. This uncertainty is increased by the fact that biomarkers are often created as part of a complex biosynthetic process that also creates a large number of substances with very similar structures and sometimes the same mass. Because of this, the two most powerful selectivity tools in the analysis of drugs, mass selective detection and MS/MS, are often rendered ineffective. The only remaining selectivity tool is chromatography and as will be demonstrated these separations can be very challenging. Failure to achieve specificity is perhaps the leading cause for inaccuracy of biomarker data and inter-laboratory variability.

Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [(35)S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-(35)S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the (35)S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia.

ω-Hydroxyundec-9-enoic acid and n -heptanoic acid are valuable building blocks for the production of flavors and antifungal agents as well as bioplastics such as polyamides and polyesters. However, a biosynthetic process to allow high productivity and product yield has not been reported. In the present study, we engineered an Escherichia coli -based biocatalytic process to efficiently produce ω-hydroxyundec-9-enoic acid and n -heptanoic acid from a renewable fatty acid (i.e., ricinoleic acid). Expression systems for catalytic enzymes (i.e., an alcohol dehydrogenase of Micrococcus luteus , a Baeyer–Villiger monooxygenase of Pseudomonas putida KT2440, an esterase of Pseudomonas fluorescens SIK WI) and biotransformation conditions were investigated. Biotransformation during stationary growth phase of recombinant E. coli in a bioreactor allowed to produce ω-hydroxyundec-9-enoic acid and n -heptanoic acid at a rate of 3.2 mM/h resulting in a final product concentration of ca. 20 mM. The total amount of ω-hydroxyundec-9-enoic acid and n -heptanoic acid produced reached 6.5 g/L (4.0 g/L of ω-hydroxyundec-9-enoic acid and 2.5 g/L of n -heptanoic acid). These results indicate that the high value carboxylic acids ω-hydroxyundec-9-enoic acid and n -heptanoic acid can be produced from a renewable fatty acid via whole-cell biotransformation.

Bacterial cellulose (BC), produced by aerobic bacteria received ample of attention due to its unique physiochemical properties compared to plant cellulose. Intense researches on BC mainly focus on biosynthetic process to achieve low-cost preparation and high cellulose production. BC has been used as biomaterial for medical field, electrical instrument and food ingredient. However, BC alone has limited capabilities to fulfil current demand on high-performance biomaterials. Hence, BC composite has been introduced to enhance BC properties through addition of reinforcement materials. This review discusses current knowledge in fermentation process and potential application of BC including its application in food industry.

Phylloquinone (vitamin K1) is an essential nutrient for both humans and animals; it plays a key role in blood coagulation amongst other processes. In this work we describe the scale-up of a biosynthetic process for phylloquinone production using the cyanobacterium Anabaena cylindrica. A 50 L bubble column photo-bioreactor was used to cultivate A. cylindrica and the effects of a wide range of operating conditions including superficial velocities (0.67–6.7 cm s−1), day lengths (12–24 h), sparger designs and medium compositions were examined. The column design and superficial velocity had minimal impact on phylloquinone production, while changes to the medium composition and day length had large impacts. By varying these factors we were able to achieve final phylloquinone titres of the order 280 μg L−1 and productivities of 40 μg L−1 day−1, which are approximately double the values previously obtained. The biomass produced has clear applications in human and animal nutrition as its phylloquinone concentration was approximately 330 μg g−1 (dry basis); this being ten times higher than rich dietary sources. Conclusions regarding the effect of photo-bioreactor design and operating conditions can be applied to the production of phylloquinone, as well as the scale-up of cyanobacterial cultures more broadly.

In this work, Au Pd core-shell nanoparticles (NPs) biosynthesized through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (Au-Pd/AtPCS1-E. coli) with catalytic enhanced chemiluminescence (CL) and benzyl alcohol oxidation (BAO) was investigated. Such biosynthesis of Au Pd core-shell NPs was obviously enhanced due to insertion of the gene sequence of Arabidopsis thaliana phytochelatin synthase (AtPCS1) to a plasmid vector (pET-28b) of Escherichia coli (E. coli). The obtained Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (AtPCS1-E. coli) could generate phytochelatins (PCs, (γ-Glu-Cys)n-Gly, n > 1) for efficient capture and enrichment of Au3+. The component and morphology of Au Pd core-shell NPs were checked through X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscopy (TEM) and energy dispersive spectrometer (EDS). Catalytic CL (in H2O2-luminol system) and BAO (in H2O2-benzyl alcohol system) effect with different experimental conditions were examined, respectively. These results revealed that multifunctional PCs could effectively facilitate biosynthetic process of Au Pd core-shell NPs with better distribution, higher yield and lower cost while stronger CL intensity and higher conversion could be obtained for further quantitative analysis and application.

A pot experiment was conducted to investigate into effects of molybdenum (Mo) on the secondary metabolic process of glycyrrhizic acid (GA). One-year-old seedlings were grown in pots with washed vermiculite and sand. Hoagland nutrition solution was irrigated with four concentrations: 0, 0.52, 5.2 and 10.4 mg L −1 . The accumulations of GA and its biosynthetic precursors ( β -amyrin and squalene) and then expression of the key synthase ( β -amyrin synthase, β -AS) were studied on 35, 70 and 105 d. In the early stage, that was on the 35 and 70 d, the contents of squalene and GA, and the expression of β - AS gene under 0.52 and 5.2 mg L −1 Mo treatments were significantly higher than that under 0 and 10.4 mg L −1 Mo. There was a contrary result of β -amyrin. However, the content of squalene under 0 mg L −1 Mo was the highest on 105 d. Thus, it suggested an appropriate concentration of Mo could promote the accumulation of GA, by affecting the biosynthetic process of GA at a certain time. Practically, the time and amount of application of Mo on Glycyrrhiza uralensis should be the noted.

A simple and ecofriendly biosynthetic process has been developed for silver nanoparticles using the aqueous extract of gum olibanum (Boswellia serrata), a renewable natural plant biopolymer. The water soluble compounds in the gum serve as dual functional reducing and stabilizing agents. The effect of concentration of gum and silver nitrate; and reaction time on nanoparticle synthesis was studied. The UV–visible spectroscopy, transmission electron microscopy and X-ray diffraction techniques were used to characterize the synthesized nanoparticles. By tuning the reaction conditions, size controlled spherical nanoparticles of around 7.5 ± 3.8 nm was achieved. Using Fourier transform infrared spectroscopy and Raman spectroscopy, a probable mechanism involved in reduction and stabilization of nanoparticles has been explained. The produced silver nanoparticles exhibited substantial antibacterial activity on both the Gram classes of bacteria. By virtue of being biogenic and encapsulated with proteins, these surface functionalized nanoparticles can be easily integrated for various biological applications.

Hundred and twenty microbial isolates could be isolated from different soil samples collected from different localities in Egypt. One of the bacterial cultures AZ-SH-B8 was found to produce a wide spectrum antimicrobial agent when cultivated on plastic wastes. The bacterial culture AZ-SH-B8 could be isolated from a soil sample collected from Sharkia governorate, Egypt. From the taxonomic features, the bacterial isolate AZ-SH-B8 matches with Pseudomonas aeurginosa in the morphological, physiological and biochemical characters and confirmation by using API20E system. Thus, it was given the suggested name P. aeurginosa , AZ-SH-B8. The parameters controlling the biosynthetic process of antimicrobial agent formation includes: innoculum size, different pH values, different temperatures, different incubation period, different carbon and nitrogen sources and different mineral salts concentrations (KNO 3 , K 2 HPO 4 , MgSO 4 ·7H 2 O and KCl) were fully investigated. The active metabolite was extracted using n-butanol (1:1, v/v) at pH 7.0. The separation of the active ingredient and its purification was performed using both thin layer chromatography (TLC) and column chromatography (CC) techniques. The physico-chemical characteristics of the purified antibiotic viz. color, melting point, solubility, elemental analysis, spectroscopic characteristics and chemical reactions have been investigated. This analysis indicates a suggested empirical formula of C 13 H 21 N 3 O 6 S 2 . The minimum inhibition concentrations “MICs” of the purified antimicrobial agent were also determined. The purified antimicrobial agent was suggestive of belonging to Sparsomycin antibiotic produced by P. aeurginosa , AZ-SH-B8.

Seventy-five actinomycete isolates were isolated from different soil samples collected from different localities in Egypt. One of the actinomycete culture AZ-SH-29 from three cultures was found to produce a wide spectrum antimicrobial agent. The actinomycete isolate AZ-SH-29 matches with Streptomyces ramulosus in the morphological, physiological, and biochemical characters. Thus, it was given the suggested name Streptomyces ramulosus -AZ-SH-29. The parameters controlling the biosynthetic process of the antimicrobial agent formation including; different pH values, different temperatures, different incubation period, and different carbon and nitrogen sources, were fully investigated. The active metabolite was extracted using ethyl acetate (1:1, v/v) at pH 7.0. The separation of the active ingredient and its purification were performed using column chromatography (CC) technique. The physico-chemical characteristics of the purified antimicrobial agent have been investigated. This analysis indicates a suggested imperical formula of C 20 H 25 N 2 O 5 . The minimum inhibition concentrations “MICs” of the purified antibiotic and antiviral test were also determined. The purified antimicrobial agent was suggestive of being belonging to Depsipeptide (Mikamycin) group (Vernamycin-A antibiotic) produced by Streptomyces ramulosus , AZ-SH-29.

Lycopene β-cyclase (LCYb) and lycopene ɛ-cyclase (LCYe) are believed to be crucial genes to lycopene cyclization and downstream carotenoid accumulation. To illuminate the gene expression profiles and their regulation on downstream carotenoids, expression profiles of these two genes and contents of several corresponding carotenoids were determined by real-time PCR and HPLC, respectively, in relative shorter intervals during breaker and maturation stage of Guoqing No. 1 satsuma mandarin (Citrus unshiu Marcow) and Cara Cara navel orange (Citrus sinensis Osbeck). Data showed that the expression profiles of these two cyclase genes and their relative expression levels between the peels and the pulps at the same stage all varied with cultivars. But in general, accumulation of lutein and β-carotene experienced a similar tendency in both cultivars and were up-regulated by expression of LCYb and LCYe in spite of poor correlation in the pulps. Meanwhile, lycopene abundance was negatively correlated with the expression of LCYe as expected, but hardly correlated with LCYb in the pulps of Cara Cara navel orange, indicating that LCYe might exert a more prominent influence on lycopene cyclization and downstream carotenoids formation. Results of carotenoids accumulation suggest that the biosynthetic process of carotenoids would shift from upstream β, ɛ- or β, β-carotenoids such as lutein and β-carotene to downstream β, β-carotenoids during fruit development.

In Saccharomyces cerevisiae, the mitochondrial inner membrane readily allows transport of cytosolic NAD(+), but not NADPH, to the matrix. Pos5p is the only known NADH kinase in the mitochondrial matrix. The enzyme phosphorylates NADH to NADPH and is the major source of NADPH in the matrix. The importance of mitochondrial NADPH for cellular physiology is underscored by the phenotypes of the Δpos5 mutant, characterized by oxidative stress sensitivity and iron-sulfur (Fe-S) cluster deficiency. Fe-S clusters are essential cofactors of proteins such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S] in mitochondria. Intact mitochondria isolated from wild-type yeast can synthesize these clusters and insert them into the corresponding apoproteins. Here, we show that this process of Fe-S cluster biogenesis in wild-type mitochondria is greatly stimulated and kinetically favored by the addition of NAD(+) or NADH in a dose-dependent manner, probably via transport into mitochondria and subsequent conversion into NADPH. Unlike wild-type mitochondria, Δpos5 mitochondria cannot efficiently synthesize Fe-S clusters on endogenous aconitase or imported ferredoxin, although cluster biogenesis in isolated Δpos5 mitochondria is restored to a significant extent by a small amount of imported Pos5p. Interestingly, Fe-S cluster biogenesis in wild-type mitochondria is further enhanced by overexpression of Pos5p. The effects of Pos5p on Fe-S cluster generation in mitochondria indicate that one or more steps in the biosynthetic process require NADPH. The role of mitochondrial NADPH in Fe-S cluster biogenesis appears to be distinct from its function in anti-oxidant defense.

Streptococcus suis is an important pathogen in swine, and it also represents an emerging zoonotic agent. Zebrafish as a model for the evaluation of virulence of S. suis has been demonstrated before. Here, an Affymetrix Zebrafish GeneChip was used to identify alterations in gene expression of zebrafish injected with S. suis serotype 2 strain HA9801. The results showed that 189 genes were differentially expressed, of which 125 genes were upregulated and 64 genes were downregulated. Gene Ontology category and KEGG pathway were analyzed for differentially expressed genes. Upregulated genes were involved in response to bacterium, immune response, inflammatory response, complement activation, defense response. Three genes (encoding serum amyloid protein A, matrix metalloproteinase 9 and apoptosis-related cysteine protease) and genes involved in the regulation of IL-6 biosynthetic process, which have previously been implicated in the response to S. suis infection in other organisms, were also upregulated. Downregulated genes played roles in glycolysis, carbohydrate metabolic process, amino acids metabolism, behavior and muscle. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR on 12 representative genes. The data may provide further validation of this model, which will contribute to understanding of S. suis pathogenic mechanisms.

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation.

Bulbs of cv. Colorado garlic were irradiated at dormancy with a dose of 60 Gy of gamma rays and stored for 8 months, during which period the content and fatty acid composition of phospholipids (PL), glycolipids (GL) and neutral lipids were analyzed on three occasions. No significant changes were observed a few hours after irradiation, but the treatment resulted in a considerable reduction in lipid and fatty acid content 150 and 240 days post-harvest, with a concomitant reduction in the process of sprout growth. In total lipid, all fatty acids including the major linoleic acid (18:2) decreased, the largest decrease being in linolenic acid (18:3). The latter was a relatively minor component of PL (phosphatidylcholine and -ethanolamine) and a major acyl group of GL (monogalactosyl- and digalactosylglycerol). Radioinhibition had the opposite effect on polyunsaturated fatty acids of PL and GL, the 18:3/18:2 ratio decreasing in the former and increasing in the latter. Accretion of lipids and fatty acids is a normal biosynthetic process accompanying sprout growth, and the long-term effects of irradiation are interpreted to reflect a delay or slowing down of such process. Fil: Perez, Monica Beatriz. Universidad Nacional del Sur; Argentina Fil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur; Argentina Fil: Croci, Clara Ana. Universidad Nacional del Sur; Argentina

gamma-Secretase is a multimeric membrane protein complex comprised of presenilin (PS), nicastrin (Nct), Aph-1, and Pen-2. It is a member of an atypical class of aspartic proteases that hydrolyzes peptide bonds within the membrane. During the biosynthetic process of the gamma-secretase complex, Nct and Aph-1 form a heterodimeric intermediate complex and bind to the C-terminal region of PS, serving as a stabilizing scaffold for the complex. Pen-2 is then recruited into this trimeric complex and triggers endoproteolysis of PS, conferring gamma-secretase activity. Although the Pen-2 accumulation depends on PS, the binding partner of Pen-2 within the gamma-secretase complex remains unknown. We reconstituted PS1 in Psen1/Psen2 deficient cells by expressing a series of PS1 mutants in which one of the N-terminal six transmembrane domains (TMDs) was swapped with those of CD4 (a type I transmembrane protein) or CLAC-P (a type II transmembrane protein). We report that the proximal two-thirds of TMD4 of PS1, including the conserved Trp-Asn-Phe sequence, are required for its interaction with Pen-2. Using a chimeric CD4 molecule harboring PS1 TMD4, we further demonstrate that the PS1 TMD4 bears a direct binding motif to Pen-2. Pen-2 may contribute to the activation of the gamma-secretase complex by directly binding to the TMD4 of PS1.

Carboxylesterases represent a large class of hydrolytic enzymes that are involved in lipid metabolism, pharmacological determination, and detoxication of organophosphorus pesticides. These enzymes have several notable structural features including two intramolecular disulfide bonds. This study was undertaken to test the hypothesis that the disulfide bonds are required during catalysis by stabilizing the catalytically active conformation. Hydrolase B, a rat liver microsomal carboxylesterase, was reduced by dithiothreitol, electrophoretically separated and assayed for hydrolysis. Contrary to the hypothesis, reduced hydrolase B was as active as the native enzyme on the hydrolysis of 1-naphthylacetate, and sulfhydryl alkylation following reduction caused no changes in the hydrolytic activity. Interestingly, substitution of a disulfide bond-forming cysteine with an alanine caused marked reduction or complete loss of the catalytic activity, suggesting that disulfide bond formation plays a role in the biosynthetic process of hydrolase B. In support of this notion, refolding experiments restored a significant amount of hydrolytic activity when hydrolase B was unfolded with urea alone. In contrast, little activity was restored when unfolding was performed in the presence of reducing agent dithiothreitol. These results suggest that formation of the disulfide bonds plays a critical role in folding hydrolase B into the catalytically active conformation, and that the disulfide bonds play little role or function redundantly in maintaining this conformation during catalysis.

Prostaglandins are locally acting hormones that have remarkable variety of physiological functions. They are rapidly synthesized in several types of vertebrate cells as oxygenated metabolites of arachidonic acid in response to various stimuli. In many insect species they are biosynthesized in fat body and hemocytes mainly in response to bacterial infections. In the present study, we administered synthetic analog of prostaglandin F2alpha, the most prominent of the prostaglandins to the 48 h old fifth instar silkworm, Bombyx mori L. at a single dose of 4 microg per larva to study its effects on the larval growth pattern and silk synthesis. The possible role of PGF2alpha at altering the quantum of silk synthesis by controlling the silk gene expression was also studied. The genomic DNA was isolated from the posterior silk gland on Days 5 and 7 of the fifth instar from the prostaglandin treated and the control larvae and were random amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of PGF2alpha in the silk biosynthesis controlling the silk gene expression. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of PGF2alpha analog in the silk biosynthetic process with its physiological and molecular implications are discussed.

Carrier proteins are considered “lynchpin” enzymes of biosynthetic pathways. The E. coli acyl carrier protein (ACP) is composed of multiple alpha helices that form a hydrophobic, solvent-protected pocket that sometimes provides a cavernous hiding spot to protect substrates bound to the ACP's phosphopantetheine (Ppant) arm. The action of “chain sequestration” is thought to be important for driving the biosynthetic process. In this study, selected amino acid residues inside the ACP sequestration channel were targeted as possible incorporation sites for para-substituted aromatic nitrile or alkyne vibrational spectroscopic probes. Using stop codon suppression, non-canonical amino acids were incorporated into the ACP via a co-transformed aminoacyl tRNA synthetase. In addition, artificial substrates containing unique vibrational labeling groups like nitriles were covalently ligated to the ACP's Ppant arm. The vibrational frequencies of these labeling groups, measured via IR absorption or Raman scattering, report on mainly the solvent exposure of the CN group, and the lineshape reports the distribution of environments around the label on either the protein or the growing substrate. IR and Raman results that report on the sequestration of substrates of different length, as well as methodological advances that enable this spectroscopic approach, will be discussed.

The cellular organelles translating the genetic code into proteins, the ribosomes, are large, asymmetric, flexible, and unstable ribonucleoprotein assemblies, hence they are difficult to crystallize. Despite two decades of intensive effort and thorough searches for suitable sources, so far only three crystal types have yielded high-resolution structures: two large subunits (from an archaean and from a mesophilic eubacterium) and one thermophilic small subunit. These structures have added to our understanding of decoding, have revealed dynamic aspects of the biosynthetic process, and have indicated the strategies adopted by ribosomes for interacting between themselves as well as with inhibitors, factors and substrates.

This work examines the lipid composition and metabolism of bovine red blood cells infected by apicomplexan Babesia parasites, organisms closely related to Plasmodium sp. We found that erythrocytes infected with Babesia bovis (i-RBC) accumulate lipids and show striking increases in phosphatidylcholine, phosphatidic acid, diacylglycerol and cholesteryl esters as compared to uninfected erythrocytes cultured under the same conditions (n-RBC). A similar pattern was observed in cultures of erythrocytes infected with Babesia bigemina. The lipid profile of purified B. bovis merozoites showed that phosphatidylcholine is the most abundant phospholipid in this parasite (31.8% +/- 2.8 of total phospholipid), markedly differing from bovine n-RBC, in which it is only a minor component (4.8% +/- 0.6). B. bovis cultures incorporate radiolabeled choline into complex lipids, especially phosphatidylcholine, with minor amounts recovered in sphingomyelin and lysophosphatidylcholine. When [14C] stearate was used as precursor, the labeling pattern again gave the highest incorporation into phosphatidylcholine, with lesser incorporation in sphingomyelin, phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. Diacylglycerol and small amounts of cholesteryl esters were the only labeled neutral lipids found. B. bovis also incorporates [3H] myo-inositol into phosphatidylinositol. Parallel incubations with n-RBC as a control yielded no incorporation into either polar or neutral lipids with any precursor. These results indicate that the lipid changes observed in i-RBC can be explained on the basis of the lipid biosynthetic activities of the babesial parasite. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid methyl esters from phospholipids of i-RBC and n-RBC showed the same qualitative composition in both. However, i-RBC had higher ratios of saturated to unsaturated fatty acids and B. bovis cultures did not desaturate [14C] stearate. Cholesterol was the only sterol detected by GC-MS. Phospholipase A2 treatment of i-RBC and n-RBC revealed no enhanced hemolytic effects in i-RBC, suggesting that the erythrocyte membrane phospholipid composition is essentially unaltered by the parasite. Labeling of i-RBC or n-RBC with [125I] Bolton-Hunter resulted in an enhanced phosphatidylserine labeling in i-RBC. This study provides the first data on B. bovis lipid constitution and biosynthesis. They show that phosphatidylcholine formation is the main biosynthetic process in these cells. The striking differences in the contents of phosphatidylcholine between host erythrocytes and the parasite suggests that it may be a useful target for both chemotherapy and immunoprophylaxis against bovine babesiosis.

Triacylglycerol (TG) biosynthetic enzymes were characterized in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora. When the membrane or lipid body fraction of this fungus was incubated with [14C]oleoyl-CoA without adding exogenous acyl acceptors, radioactivity was incorporated predominantly into TG, indicating that diacylglycerol acyltransferase (DGAT) used endogenous diacylglycerol to incorporate [14C]oleoyl-CoA into TG. Adding glycerol 3-phosphate or lysophosphatidic acid increased radiolabeled phosphatidic acid (PA) in the membrane fraction, which reflected the presence of glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT). Label accumulation did not occur in lysophosphatidic acid when glycerol 3-phosphate was added, suggesting that GPAT was rate-limiting in sequential acylation. In the lipid body fraction, adding lysophosphatidic acid similarly increased radiolabeled PA, whereas adding glycerol 3-phosphate caused much lower increase in radiolabeled PA. Quantitative assays for GPAT, LPAAT, phosphatidic acid phosphatase (PAP), and DGAT essentially confirmed the results obtained from [1-14C]oleoyl-CoA incorporation; LPAAT had the highest activity in the membrane and lipid body fractions, GPAT was significantly lower in the lipid body fraction, and DGAT was much higher in the lipid body fraction. GPAT and LPAAT in the membrane fraction had a strong preference toward oleoyl-CoA as a substrate over palmitoyl-CoA. Results indicate that TG biosynthetic enzymes had different subcellular distribution with the sequence of enrichment in the lipid body fraction, i.e., GPATLPAAT approximately PAPDGAT. This may reflect a TG biosynthetic process from endoplasmic reticulum membranes to lipid bodies in the fungus.

Suitable conditions for the fluorescent labeling of the reducing residue of amylose with 2-aminopyridine were examined. Amylose of up to 38.5 nmol was labeled with a constant labeling efficiency. The same efficiencies were obtained for amyloses having a number-average degree of polymerization (dpn) of 521-4400. The analysis of labeled amylose on size-exclusion HPLC with refractive index and fluorescence detection enabled the determination of dpn and dp distribution on a molar basis. The analysis of eight amylose specimens from seven botanical sources (potato, sweet potato, barley, wheat, indica rice, japonica rice, and maize) gave dpn values in good agreement with those determined by a conventional colorimetric method. The molar-based distributions of these amyloses were characteristic of botanical source and revealed the presence of several molecular species with different dp not detectable in the distribution on a weight basis. Small amyloses with a dp less than 10(3) were predominant in the cereals while amyloses with a dp over 10(3) were predominant in the tubers, suggesting a difference in the biosynthetic process determining the dp distribution of amylose between cereals and tubers.

Treatment of 2-hydroxy-isoflav-3-ene with acid afforded 2-arylbenzofuran in good yield, and a mechanism for this chemical conversion involving loss of one carbon unit was described. This reaction scheme was suggested as a plausible chemical analogy for the corresponding biosynthetic process of 2-arylbenzofuran of isoflavonoid origin, and a new biosynthetic scheme depicting 2-hydroxy-isoflav-3-ene as the possible common intermediate for both 2-arylbenzofuran and 3-arylcoumarin was proposed. Synthesis of a potent antimicrobial 2-arylbenzofuran phytoalexin, vignafuran, was also achieved by use of this chemical scheme.

5-Aminolevulinic acid (ALA) is the key precursor for chlorophyll biosynthesis. It is made in the chloroplast in a process that is dependent on transfer RNA (tRNA), and that involves two main steps. In the first pivotal step, tRNA charged with glutamate (glutamyl-tRNA) (Glu-tRNA) is reduced to glutamate-1-semialdehyde (GSA) in a reaction catalyzed by Glu-tRNA reductase (GluTR). The Glu-tRNA is common to the biosyntheses of both proteins and porphyrins. Glutamate-1-semialdehyde-2,1-amino-mutase (GSA-AM) then catalyzes the conversion of GSA to ALA. The genes that encode these enzymes and glutamate-specific tRNA have now been characterized from a variety of plants, and it has therefore become possible to understand the components and regulation of this essential biosynthetic process in higher plants in considerable detail.

Cyclosporin A is synthesized by cyclosporin synthetase, a multienzyme polypeptide. This enzyme catalyzes at least 40 reaction steps in an assembly belt-like mechanism. It activates all constituent amino acids of cyclosporin A to thioesters via amino acyladenylates and carries out specific N-methylation reactions. During elongation, the activated amino acids are linked by peptide bonds leading to enzyme-bound nascent peptide chains. Some of the linear peptides of the growing cyclosporin A chain were isolated and their N-terminal amino acid was determined. D-Alanine at position 8 of the cyclosporin A molecule was found to be a starting amino acid in the biosynthetic process of cyclosporin A formation. Four intermediate peptides of the growing peptide chain of cyclosporin A could be isolated and identified. All of them represent partial sequences of cyclosporin A starting with D-alanine. That these intermediate peptides were bound by thioester linkage to cyclosporin synthetase could be demonstrated by liberation of the peptides with performic acid. The peptides strongly suggest the stepwise synthesis of a single linear peptide precursor of cyclosporin A.

In order to elucidate the biosynthetic process of cellulose and curdlan, 13C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing d -(1-13C)glucose, d -(2-13C)glucose, d -(4-13C)glucose, or d -(6-13C)glucose as the carbon source, and their structures were determined by 13C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.

Chimeric genes composed of the class I patatin promoter, A1aB1b preproglycinin cDNA and its modified versions (IV+4Met and V+4Met) containing four contiguous methionine codons and nopaline synthase gene terminator were inserted into the genome of potato by Agrobacterium-mediated transformation. Transcripts of the chimeric genes were detected specifically in tubers. Translational products were observed in parenchyma cells of the tubers of potato plants transformed with the normal and modified glycinin cDNAs. This distribution is consistent with the expression specificity of the patatin promoter. The expression levels of the normal and the modified glycinins were 0.2–1.0% of the total soluble tuber proteins. Both the normal and the modified glycinins expressed in the tubers were present as proglycinin trimers. These results indicate that the modifications in IV+4Met and V+4Met did not disrupt any stage in the biosynthetic process of glycinin in potato tubers.