A novel two-gene requirement for the octanoyltransfer reaction of Bacillus subtilis lipoic acid biosynthesis

The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM and ywfL, which we have renamed lplJ, lipM and lipL respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to Escherichia coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulphur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilisΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coliΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion article provide evidence that LipM and LipL catalyse sequential reactions in a novel pathway for lipoic acid biosynthesis.     

A novel signal transduction system for development of uric acid biosensors

Applied Microbiology and Biotechnology - Uric acid (UA) is an important biomarker for clinical diagnosis. Here, we present a novel signal transduction system for the development of UA biosensors...     

A novel chemiluminescence paper microfluidic biosensor based on enzymatic reaction for uric acid determination

In this work, chemiluminescence (CL) method was combined with microfluidic paper-based analytical device (μPAD) to establish a novel CL μPAD biosensor for the first time. This novel CL μPAD biosensor was based on enzyme reaction which produced H(2)O(2) while decomposing the substrate and the CL reaction between rhodanine derivative and generated H(2)O(2) in acid medium. Microchannels in μPAD were fabricated by cutting method. And the possible CL assay principle of this CL μPAD biosensor was explained. Rhodanine derivative system was used to reach the purpose of high sensitivity and well-defined signal for this CL μPAD biosensor. And the optimum reaction conditions were investigated. The quantitative determination of uric acid could be achieved by this CL μPAD biosensor with accurate and satisfactory result. And this biosensor could provide good reproducible results upon storage at 4°C for at least 10 weeks. The successful integration of μPAD and CL reaction made the final biosensor inexpensive, easy-to-use, low-volume, and portable for uric acid determination, which also greatly reduces the cost and increases the efficiency required for an analysis. We believe this simple, practical CL μPAD biosensor will be of interest for use in areas such as disease diagnosis.     

A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acid

Cytochrome P450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)═O]+? termed Compound I (Cpd I). The proximal cysteine thiolate ligand is a critical determinant of the spectral and catalytic properties of P450 enzymes. To explore the effect of an increased level of donation of electrons by the proximal ligand in the P450 catalytic cycle, we recently reported successful incorporation of SeCys into the active site of CYP119, a thermophilic cytochrome P450. Here we report relevant physical properties of SeCYP119 and a detailed analysis of the reaction of SeCYP119 with m-chloroperbenzoic acid. Our results indicate that the selenolate anion reduces rather than stabilizes Cpd I and also protects the heme from oxidative destruction, leading to the generation of a new stable species with an absorbance maximum at 406 nm. This stable intermediate can be returned to the normal ferric state by reducing agents and thiols, in agreement with oxidative modification of the selenolate ligand itself. Thus, in the seleno protein, the oxidative damage shifts from the heme to the proximal ligand, presumably because (a) an increased level of donation of electrons more efficiently quenches reactive species such as Cpd I and (b) the protection of the thiolate ligand provided by the protein active site structure is insufficient to shield the more oxidizable selenolate ligand.     

A novel system A isoform mediating Na+/neutral amino acid cotransport

A cDNA clone encoding a plasma membrane alanine-preferring transporter (SAT2) has been isolated from glutamatergic neurons in culture and represents the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and is expressed in most tissues, including heart, adrenal gland, skeletal muscle, stomach, fat, brain, spinal cord, colon, and lung, with lower levels detected in spleen. No signal is detected in liver or testis. In the central nervous system, SAT2 is expressed in neurons. SAT2 is significantly up-regulated during differentiation of cerebellar granule cells and is absent from astrocytes in primary culture. The functional properties of SAT2, examined using transfected fibroblasts and in cRNA-injected voltage-clamped Xenopus oocytes, show that small aliphatic neutral amino acids are preferred substrates and that transport is voltage- and Na(+)-dependent (1:1 stoichiometry), pH-sensitive, and inhibited by alpha-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of system A. Kinetic analyses of alanine and MeAIB uptake by SAT2 are saturable, with Michaelis constants (K(m)) of 200-500 microm. In addition to its ubiquitous role as a substrate for oxidative metabolism and a major vehicle of nitrogen transport, SAT2 may provide alanine to function as the amino group donor to alpha-ketoglutarate to provide an alternative source for neurotransmitter synthesis in glutamatergic neurons.     

A novel system for mild hypothermia

Mild hypothermia has been shown to provide protective effects in patients with ischemia (e.g. acute stroke and heart attack), but traditional methods for inducing, maintaining, and reversing hypothermia are slow, difficult to administer and control, and uncomfortable for patients. An innovative method produces mild, wholebody hypothermia (32 degrees C to 34 degrees C) by use of an endovascular heat exchanger placed into the inferior vena cava. A closed-loop system accurately changes core body temperature with average cool-down rates of 4.8 degrees C per hour, tight-target temperature control of +/- 0.1 degree C, and average rewarm rates of 1.9 degrees C per hour. By enhancing the mixing of blood in the vicinity of the heat exchanger, the disposable, small-diameter catheter efficiently exchanges heat between the closed-loop circulating fluid and the blood stream in response to body temperature. A control algorithm adapts to body physiology and thermal mass to mitigate temperature excursions. Flexible, bellow-shaped segments along the length of the catheter allow precise maneuvering within blood vessels. Heparin, covalently bonded to the catheter, helps control thrombogenicity. This novel design has potential clinical applications in cerebrovascular surgery, acute stroke, acute myocardial infarction, cardiac arrest, and fever control.     

A novel enzymatic reaction for converting DNA to CO-DNA

We have found previously that DNA from both the chick cerebrum and cardiac muscle has a modified structure. We named this novel DNA, CO-DNA. CO-DNA is a form of DNA in which a carbonyl group is attached to C-1 of the 2-deoxyribose and to the nitrogenous base. Therefore, 3-deoxyglucosone is the sugar constituent for CO-DNA. We found previously that the modification of the sugar moiety in DNA occurs around embryonic day 12 in the chick embryo. In this study, we isolated enzymes for the conversion of DNA to CO-DNA from chick cerebra. In our reaction system, uniformly labeled 14C-glucose was used as substrate. During incubation, the radioactivity was incorporated into DNA. From the analysis of 14C-labeled deoxynucleoside, the radioactive sugar was confirmed to be 3-deoxyglucosone. We propose a series of reactions involved in the conversion of DNA to CO-DNA: (1) DNA-enzyme complex is formed during preincubation, (2) 14C-glucose is transformed to 14C-3-deoxyhexonic acid, (3) 14C-3-deoxyhexonic acid is subsequently transformed into the sugar-phosphate, which is a mixture of phosphorylated 14C-3-deoxyhexonic acid and phosphorylated 14C-3-deoxyglucosone, (4) 2-deoxyribose in DNA is replaced with 14C-3-deoxyglucosone through its intermediate phosphorylated form, and (5) DNA is finally converted to CO-DNA.     

A digestion bomb for wet ashing biological materials

The design and construction of a wet digestion chamber resistant to acids and bases for hard and soft tissues is described. Loss of volatile elements is prevented. Reproducible complete recoveries for a variety of inorganic metal ions are routinely achieved.     

A novel riboregulator switch system of gene expression for enhanced microbial production of succinic acid

In this paper, a novel riboregulator Switch System of Gene Expression including an OFF-TO-ON switch and an ON-TO-OFF switch was designed to regulate the expression state of target genes between “ON” and “OFF” by switching the identifiability of ribosome recognition site (RBS) based on the thermodynamic stability of different RNA–RNA hybridizations between RBS and small noncoding RNAs. The proposed riboregulator switch system was employed for the fermentative production of succinic acid using an engineered strain of E. coli JW1021, during which the expression of mgtC gene was controlled at “ON” state and that of pepc and ecaA genes were controlled at the “OFF” state in the lag phase and switched to the “OFF” and “ON” state once the strain enters the logarithmic phase. The results showed that using the strain of JW1021, the yield and productivity of succinic acid can reach 0.91 g g^?1 and 3.25 g L^?1 h^?1, respectively, much higher than those using the strains without harboring the riboregulator switch system.     

Nitro-fatty acid reaction with glutathione and cysteine. Kinetic analysis of thiol alkylation by a Michael addition reaction

Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M(-1)S(-1) and 355 M(-1)S(-1), respectively, at pH 7.4 and 37 degrees C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A2 and 15-deoxy-Delta(12,14)-prostaglandin J2. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pKa of target thiols increased. Increasing concentrations of the detergent octyl-beta-d-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.     

A novel role for N-glycans in the ERAD system

The endoplasmic reticulum (ER) provides a quality-control system for newly synthesized secretory and membrane proteins. Any improperly folded or incompletely assembled oligomers are retained in the ER, and they are retro-translocated into the cytosol when misfolding persists, where they are destroyed by the proteasome through ubiquitylation. This disposal process is called ER-associated degradation (ERAD). Although much is known about the fate of ERAD substrates near the point of degradation, little information is available about how these proteins are recognized, retained, and targeted for translocation and ubiquitylation machinery. Recent studies indicate that N-linked oligosaccharides attached to nascent proteins function as tags for several processes of a quality-control system, such as individual steps of ER-retention, selection for ERAD substrates, and ubiquitylation. In this review, I describe recent advances in the molecular basis of the ERAD system, particularly those mediated by N-glycan recognition molecules.     

A novel method for nucleic acid sequence determination

We describe a novel sequencing methodology which should be readily and completely automated. The method relies on fragmentation of a nucleotide or deoxynucleotide sequence into short fragments, and subsequent quantitation of the fragments by hybridization to oligo-deoxynucleotides on a solid support. The original sequence may be reconstructed from the resulting table of fragment frequencies. We present a specific protocol which would allow practical implementation of this approach.     

A high-performance system for automation of the polymerase chain reaction

A high-performance PCR system has been developed which reduces the time required for PCR, increases the throughput, reduces reagent consumption and ensures reproducibility of amplification. Integration of sophisticated temperature control with optimally designed vessels has resulted in an amplification system which produces unique benefits. These include rapid amplification, the elimination of the need for oil, even for small volumes, and a microplate format which provides liquid handling automation benefits.     

A novel Ganoderma lucidum G0119 fermentation strategy for enhanced triterpenes production by statistical process optimization and addition of oleic acid

A novel enhanced triterpenes fermentation production process by Ganoderma lucidum G0119 with the addition of oleic acid in the medium has been developed and optimized. All of the six exogenous additives tested were found to exhibit stimulatory effect on mycelial growth and triterpenes biosynthesis by G. lucidum. The results show that oleic acid addition had significant role in promoting triterpenes production. The optimal concentration and time of oleic acid addition were determined to be 30 mL/L and 0 h, respectively. Furthermore, three significant factors influencing triterpenes production were identified as glucose, magnesium sulfate and temperature using the Plackett–Burman design. The optimized conditions by central composite design were 27.83 g/L glucose, 1.32 g/L magnesium sulfate, 26.2°C temperature. The triterpenes fermentation yield with the optimized medium based on actual confirmatory experimental data in 6 L fermentor was 1.076 g/L versus the statistical model predicted value of 1.080 g/L. Our innovatively developed triterpenes fermentation production technology and process has been proven to produce high triterpenes productivity and yield conceivably useful for industrial production.     

A novel hybrid seed system for plants

A two-component hybrid seed system has been developed that is broadly applicable and provides for effective generation and maintenance of the male-sterile parent, hybrid seed production and full restoration of fertility in the hybrid seed. The technology is based on the functional interaction of two loci that are inserted in the same position on two homologous chromosomes, and thus are 'linked in repulsion', and that jointly code for male sterility and herbicide resistance, both traits being expressed in heterozygous plants only. The localization to the same locus on a chromosome is achieved by the genetic transformation of plants with a construct containing both genetic elements (loci), and subsequent derivatization from the primary pro-locus of the two precursor lines using site-specific deletions. The functional interaction of the two loci is achieved through intein-based trans -splicing of two pairs of complementary protein fragments that provide for male sterility and herbicide resistance. Unlike the hybrid seed systems that are currently in use, the technology relies on the genetic modification of just one parent, and is therefore much simpler to develop and use. Arabidopsis has been used for the proof of principle presented here, but the essential elements of the technology are generic and have been shown to work in many crop species.     

A novel framework for the cell-free enzymatic production of glucaric acid

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ± 0.9 mM (3.0 ± 0.2 g l⁻¹) and a molar yield of 35.2 ± 2.3% using non-immobilised enzymes, and a titre of 8.1 ± 0.2 mM (1.70 ± 0.04 g l⁻¹) and a molar yield of 20.2 ± 0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ± 0.02 g/h/l for free enzymes and 0.170 ± 0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.