Dietary Supplementation of β-Carotene Reveals miRNAs Involved in the Regulation of Carotenoid Metabolism in Crassostrea gigas

Carotenoids play crucial physiological roles in animals.A comprehensive investigation into the mechanism of carotenoid metabolism in oysters will establish a theoretical foundation for further development of its carotenoid-rich traits.However,the information on the function of miRNA in β-carotene metabolism in oysters is limited.To elucidate the mechanisms underlying miRNA regulation of carotenoid metabolism in oysters,we compared the expressions of miRNA in digestive gland tissues of Pacific oyster(Crassostrea gigas)fed with aβ-carotene supplemented diet and a normal diet,respectively.A total of 690 candidate miRNAs in the Pacific oyster digestive gland tissues were identified,including 590 known miRNAs and 111 unknown miRNAs.Three differentially expressed miRNAs were obtained in the carotenoid-fed and normal groups,associated to 137 differentially expressed target genes.Moreover,the GO enrichment analysis revealed that the differentially expressed target genes were mainly involved in transmembrane transport activity.KEGG enrichment showed that the differentially expressed target genes were involved in ABC transport.Analysis of the mRNA-miRNA network revealed that novel0025 played a central role in carotenoid metabolism,and it was negatively correlated with the expression of 46 mRNAs.In addition,down-regulated expression of novel0025 upregulated the expression of the lipoprotein gene LOC105342186,suggesting a potential regulatory role in carotenoid metabolism.Our results provide useful information for elucidating the miRNA regulation mechanism during carotenoids metabolism in the Pacific oyster.

Aβ-Carotene Ketolase Gene NfcrtO from Subaerial Cyanobacteria Confers Drought Tolerance in Rice

Nostoc flagelliforme is a terrestrial cyanobacterium that can resist many types of stressors,including drought,ultraviolet radiation,and extreme temperatures.In this study,we identified the drought tolerance gene NfcrtO,which encodes aβ-carotene ketolase,through screening the transcriptome of N.flagelliforme under water loss stress.Prokaryotic expression of NfcrtO under 0.6 mol/L sorbitol or under 0.3 mol/L NaCl stress significantly increased the growth rate of Escherichia coli.When NfcrtO was heterologously expressed in rice,the seedling height and root length of NfcrtO-overexpressing rice plants were significantly higher than those of the wild type(WT)plants grown on½Murashige and Skoog solid medium with 120 mmol/L mannitol at the seedling stage.Transcriptome analysis revealed that NfcrtO was involved in osmotic stress,antioxidant,and other stress-related pathways.Additionally,the survival rate of the NfcrtO-overexpression lines was significantly higher than that of the WT line under both hydroponic stress(24%PEG and 100 mmol/L H_(2)O_(2))and soil drought treatment at the seedling stage.Physiological traits,including the activity levels of superoxide dismutase,peroxidase,catalase,total antioxidant capacity,and the contents of proline,trehalose,and soluble sugar,were significantly improved in the NfcrtO-overexpression lines relative to those in the WT line under 20%PEG treatment.Furthermore,when water was withheld at the booting stage,the grain yield per plant of NfcrtO-overexpression lines was significantly higher than that of the WT line.Yeast two-hybrid analysis identified interactions between NfcrtO and Dna J protein,E3 ubiquitin-protein ligase,and pyrophosphate-energized vacuolar membrane proton pump.Thus,heterologous expression of NfcrtO in rice could significantly improve the tolerance of rice to osmotic stress,potentially facilitating the development of new rice varieties.

Engineering of β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces cerevisiae

The conversion of r-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps ofketolation are catalyzed by β-carotene hydroxylase （CrtZ） and β-carotene ketolase （CrtW） respectively. Here, astaxanthin biosynthesis path- way was constructed in Saccharomyces cerevisiae by introducing heterologous CrtZ and CrtW into an existing high r-carotene producing strain. Both genes crtZ and crtW were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE Scl 18060 with CrtW from Brevundimonas vesicu- laris DC263 and CrtZ from Alcaligenes sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of crtZ in strain SyBE_Scl 18060 was replaced from FBAlp to TEFlp, the astaxanthin yield was increased by 30.4% （from 3.4 to 4.5 mg/g DCW）. In the meanwhile, 33.5-fold increase on crtZ transcription level and 39.1-fold enhancement on the transcriptional ratio of crtZ to crtWwere observed at early exponential phase in medium with 4% （w/v） glucose. Otherwise, although the ratio of crtZ to crtW were increased at mid-, late-exponential phases in medium with 2% （w/v） glucose, the transcription level of both crtZ and crtW were actually decreased during the whole timecourse, consequently leading to no significant improve- ment on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance lation by employing the key desired compounds accumu- enzyme（s） in microbes.

Study on the interaction between β-carotene and gut microflora using an in vitro fermentation model

β-Carotene,a typical non-oxygenated carotenoid,is the most efficient source of retinol(VA).The low bio-availability ofβ-carotene lead to large accumulation in colon;however,the relationship betweenβ-carotene and gut microflora remains unclear.This study intends to explore the interaction betweenβ-carotene and gut microflora using an in vitro fermentation model.After 24 h fermentation,the degradation rate ofβ-carotene was(64.28±6.23)%,which was 1.46 times that of the group without gut microflora.Meanwhile,the production of VA was nearly 2 times that of the group without gut microflora,indicating that the gut microflora can metabolizeβ-carotene into VA.β-Carotene also influences the production of short-chain fatty acids(SCFAs),the production of total SCFAs in 0.5 mg/mLβ-carotene(BCM)group was(44.00±1.16)mmol/L,which was 2.26 times that of the blank control(BLK)group.Among them,the production of acetic acid in BCM group was(19.06±0.82)mmol/L,which was 2.64 time that of the BLK group.Furthermore,β-carotene significantly affected the structure and composition of gut microflora,increasing the abundance of Roseburia,Parasutterella and Lachnospiraceae,and decreasing the abundance of Dialister,Collinsella and Enterobacter(P<0.05).This study provides a new way to understand howβ-carotene works in human body with gut microflora.

Flavanone and flavonoid hydroxylase genes regulate fiber color formation in naturally colored cotton

Using naturally colored cotton(NCC)can eliminate dyeing,printing and industrial processing,and reduce sewage discharge and energy consumption.Proanthocyanidins(PAs),the primary coloration components in brown fibers,are polyphenols formed by oligomers or polymers of flavan-3-ol units derived from anthocyanidins.Three essential structural genes for flavanone and flavonoid hydroxylation encoding flavanone-3-hydroxylase(F3H),flavonoid 3’-hydroxylase(F3’H)and flavonoid 3’5’-hydroxylase(F3’5’H)are initially committed in the flavonoid biosynthesis pathway to produce common precursors.The three genes were all expressed predominantly in developing fibers of NCCs,and their expression patterns varied temporally and spatially among NCC varieties.In GhF3Hi,GhF3’Hi and GhF3’5’Hi silenced lines of NCC varieties XC20 and ZX1,the expression level of the three genes decreased in developing cotton fiber,negatively correlated with anthocyanidin content and fiber color depth.Fiber color depth and type in RNAi lines changed with endogenous gene silencing efficiency and expression pattern,the three hydroxylase genes functioned in fiber color formation.GhF3H showed functional differentiation among NCC varieties and GhF3’H acted in the accumulation of anthocyanin in fiber.Compared with GhF3’H,GhF3’5’H was expressed more highly in brown fiber with a longer duration of expression and caused lighter color of fibers in GhF3’5’H silenced lines.These three genes regulating fiber color depth and type could be used to improve these traits by genetic manipulation.

Jacob’s Syndrome and Deficiency of 11-Beta-Hydroxylase Enzyme Association Revealed by a Statural Advance: A Case Report

47XYY syndrome is a rare sex chromosome variant with an extra Y chromosome. Most patients with a 47XYY karyotype have a normal phenotype. This disorder seems to be associated with a higher risk of developing behavior and cognitive problems, tall stature and infertility in adulthood. We report here a rare case of 47, XYY syndrome associated with an 11-beta-hydroxylase deficiency revealed by a stature advance along with precocious puberty after obtaining informed consent from parents. To our knowledge, this is the first case reported in the literature.

Dynamic Accumulation of β-carotene and Dry Matter in Orange-fleshed Sweetpotato [Ipomoea batatas(L.) Lam] and Their Correlation Analysis

Using three orange-fleshed sweetpotato varieties as experimental materials,16 economic traits at 60 d,75 d,90 d,105 d,120 d and 135 d after planting were measured,so as to revealing the dynamic variations of β-carotene and dry matter accumulation in roots and their relationships with economic traits in orangefleshed sweetpotato.The results showed that the dynamic variations of β-carotene accumulation in tubers varied hugely among different varieties.Interesting,the βcarotene content of all three varieties showed a significant decrease after 120 d,while the dry matter content of them performed a similar ＂fluctuation-type＂.Correlation analysis indicated that β-carotene content of three orange-fleshed sweetpotato varieties had no significant correlation with dry matter content and photosynthetic parameters,but the correlation with other economic traits also varied among varieties.

Observation of Fundamental and High Order Raman Modes of β-Carotenes in Pd OECC Thin Film by SERS

Raman spectra of purified oxygen evolution core complexes (Pd OECC) thin films on silver mirror substrates have been taken over the frequency range of 250-3100 cm -1 by surface enhanced Raman scattering (SERS). Besides the fundamental frequency modes of β_carotene in Pd OECC, many weak peaks are observed. According to the selection rules of overtone and combination bands, most of them are attributed to the second_order Raman spectra of β_carotene. Compared with the SERS of normal Pd OECC, the SERS of Pd OECC after strong illumination shows a decrease in scattering intensity and an increase in line widths, indicating changes of conformation and micro_environment of β_carotene. The results of SERS are consistent with the changes of absorption spectrum of Pd OECC induced by strong illumination. There are no changes that can be ascribed to new vibration bands, so it is deduced that Pd OECC on the silver mirror is identical to that in the solution. In summary, SERS proved a good method to study the photodamage mechanism of photosynthesis.

Effect of α-synuclein on the promoter activity of tyrosine hydroxylase gene

Objective To approach the associated mechanism by which α-synuclein （α-Syn） might regulate the metabolism of dopamine. Methods A DNA fragment, located at --495 to ＋25 of the human tyrosine hydroxylase （TH） gene, was amplified by PCR and inserted into the pGL3-Basic luciferase reporter vector. The recombinant plasmid pGL3-THprom was transfected into a dopammergic cell line MES23.5 or a α-Syn over-expressed MES23.5 （named MES23.5/hα-Syn^＋）. The promoter activity was detected by the Dual Luciferase Assay System. Results The luciferase activities in the MES23.5 cells transfected with pGl.,3-Basic, pGL3-THprom, and pGL3-Control vectors were 5.60±0.67, 26.80±4.11, and 32.90±4.75, respectively. On the other hand, the luciferase activity of pGL3-THprom in the MES23.5 （26.80±4.11） was significantly higher than that in the MES23.5/hα-Syn^＋（14.40±0.61） （P〈0.01）. Conclusion These results indicate that the -495 to ＋25 region in the TH gene possesses promoter activity for controlling the gene expression, and that α-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.

Changes of 5-hydroxytryptamine and tryptophan hydroxylase expression in the ventral horn of spinal cord

Objective To investigate changes of 5-hydroxytryptamine （5-HT） and its synthesis rate-limiting enzyme tryp-tophan hydroxylase （TPH） in the ventral horn of spinal cord after exercise-induced fatigue, and to further discuss the mecha- nism of exercise-induced central fatigue at spinal level. Methods Sixteen healthy adult Wistar rats were randomly divided into 2 groups： exercise-induced fatigue group and control group. Immunohistochemical staining for 5-HT and TPH in the ventral horn were performed and analysized quantitatively. The mean optic densities of 5-HT and TPH positive fibers or terminals were measured by computerized image analyzer. Results Both 5-HT and TPH positive fibers/terminals decreased in the exercise-induced fatigue group. The immunohistochemical staining was weaker and the mean optic densities decreased obviously in the fatigue group compared with those in the control group （P 〈 0.05）. Conclusion 5-HT and TPH in the ventral horn of spinal cord might be involved in exercise-induced fatigue.

Measurement and Influence Factors of the Flowability of Microcapsules with High-content β-Carotene

The flowability of five kinds of microencapsulation powders,with differentβ-carotene contents and by two alternative particle-forming technologies i.e.spray-drying and starch-catching beadlet technology,was meas- ured.The actual flow properties of the five powders were compared based on bin-flow test,and three flow indexes (Hausner ratio,repose angle and flow index)were measured.It was found that the repose angle is the most suitable index to reflect the flowability of these powders for the particle properties would not be altered due to compaction or tapping during the measuring process.Particle size and particle size distribution play most important roles in the flowability of these granular materials,which was also influenced by other factors like shape,surface texture,sur- face roughness,etc.Microcapsules with wall material of gelatin and a layer of modified starch absorbed on the sur- face showed excellent flowabilities and good mechanical properties,and they are favorable for tabletting to supply β-carotene.

Application of derivative ratio spectrophotometry for determination of β-carotene and astaxanthin from Phaffia rhodozyma extract

A derivative ratio spectrophotometric method was used for the simultaneous determination of β-carotene and astaxanthin produced from Phaffia rhodozyma. Absorbencies of a series of the standard carotenoids in the range of 441 nm to 490 nm demonstrated that their absorptive spectra accorded with Beer’s law and that the additivity when the concentrations of β-carotene and astaxanthin and their mixture were within the range of 0 to 5 μg/ml, 0 to 6 μg/ml, and 0 to 6 μg/ml, respectively. When the wavelength interval (?λ) at 2 nm was selected to calculate the first derivative ratio spectra values, the first derivative amplitudes at 461 nm and 466 nm were suitable for quantitatively determining β-carotene and astaxanthin, respectively. Effect of divisor on derivative ratio spectra could be neglected; any concentration used as divisor in range of 1.0 to 4.0 μg/ml is ideal for calculating the derivative ratio spectra values of the two carotenoids. Calibration graphs were established for β-carotene within 0?6.0 μg/ml and for astaxanthin within 0?5.0 μg/ml with their corresponding regressive equations in: y=?0.0082x?0.0002 and y=0.0146x?0.0006, respectively. R-square values in excess of 0.999 indicated the good linearity of the calibration graphs. Sample recovery rates were found satisfactory (>99%) with relative standard deviations (RSD) of less than 5%. This method was suc- cessfully applied to simultaneous determination of β-carotene and astaxanthin in the laboratory-prepared mixtures and the extract from the Phaffia rhodozyma culture.

Biochemical changes in phenols,flavonoids,tannins,vitamin E,β-carotene and antioxidant activity during soaking of three white sorghum varieties

Objective:To investigate the changes in total phenols,flavonoids,tannins,vitamin E, β-carotene and antioxidant activity during soaking of three white sorghum varieties.Methods: The changes in total phenols,total ilavonoids,tannins,phenolic acids compounds,flavonoid components,vitamin E,P-carotene and antioxidant activity during soaking of sorghum grains were determined.Results:Total phenols,total flavonoids,tannins,vitamin E,P-carotene and antioxidant activity in raw sorghum were ranged from 109.21 to 116.70,45.91 to 54.69,1.39 to 21.79 mg/100 g,1.74 to 5.25,0.54 to 1.19 mg/kg and 21.72%to 27.69%and 25.29%to 31.97%,respectively. The above measured compounds were significantly decreased after soaking.p-Hydroxybenzoic acid,vanillic acid,syringic acid and cinnamic acid represent the major phenolic acids in Dorado variety.While ferulic acid,p-coumaric acid,gallic acid and caffeic acid represent the major phenolic acids in Shandaweel-6.On the other hand,protocatechuic acid represents the major phenolic acids in Giza-15.Regarding flavonoids components,Dorado was the highest variety in kampferol and naringenin while Shandaweel-6 was the highest variety in luteolin, apigenin,hypersoid,quercelin and christen.Finally,Giza-15 was the highest variety in catechin. Phenolic acids,flavonoid compounds and antioxidant activities were decreased after soaking. Conclusions:Sorghum varieties have moderate quantities from total phenols,total flavonoids, tannins,phenolic acids compounds,flavonoid components,vitamin E,P-carotene and antioxidant activity which decreased after soaking.

In vitro Effect of Dithiocarbamate Pesticides and of CaNa_2EDTA on Human Serum Dopamine-β-hydroxylase

Serum dopamine-β-hydroxylase (DBH) inhibition has been reported in lead workers treated with CaNa2EDTA and in alcoholic patients repeatedly treated with the alcohol aversive drug Disulfiram. The mechanism of inhibition involves Cu-chelation at the active site of DBH. The effect of CaNa2EDTA and Disulfiram on serum DBH has been compared to the effect of dithiocarbamate pesticides in vitro for the possible use of serum DBH determination for the biological monitoring of workers exposed to these pesticides.Most dithiocarbamates inhibit human serum DBH at micromoIar concentrations (range of I50, 0.027-1.6 μmol/L). The inhibitory potency increased from methyl- and dimethyl dithiocarbamates to diethyl dithiocarbamates up to the most potent ethylene bisdithiocarbamates. The I50 of CaNa.EDTA was 3.8 mol/L, higher than those of dithiocarbamates. Copper addition to the test system rcactivated at stoichiometric concentrations dithiocarbamaie-inhibited DBH indicating that both base line values and percent of inhibition can be calculated in a single blood sample. Results suggest that serum DBH determination couId be useful in case of acute poisoning involving high doses of dithiocarbamate pesticides

Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra

Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson＇s disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson＇s disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson＇s disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson＇s disease were established and verbascoside（60 mg/kg） was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase m RNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were significantly higher than the Parkinson＇s model group. These findings suggest that the mechanism by which verbascoside treats Parkinson＇s disease is related to the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra.

Cloning and Expression Profile of Deoxyhypusine Snyhtase Gene and Deoxyhypusine Hydroxylase Gene in Silkworm, Bombyx mori

Deoxyhypusine snyhtase （DHS） and deoxyhypusine hydroxylase （DOHH） are the two enzymes that catalyze the synthesis of hypusine within eukaryotic initiation factor 5A （eIF5A）. Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we described the cloning and expression of two full-length cDNAs, encoding respectively DHS-like protein and DOHH-like protein from Bombyx mori by using the methods of bioinformatics, RACE, and RT-PCR technology, named as BmDHS and BmDOHH. Sequencing results indicate that they are 1 311 and 1 874 bp in length including complete open reading frame （ORF） 1 116 and 915 bp, which encode 371 amino acids （molecular weight is about 41.11 kD and isoelectric point is 5.84） and 304 amino acids （molecular weight is about 34.30 kD and isoelectric point is 4.86）, respectively. BmDHS contains only 1 exon, and BmDOHH contains 4 exons and 3 introns. The deduced amino acid sequence of BmDHS contains a deoxyhypusine synthase domain from 47 to 361 amino acid residues, and the deduced amino acid sequence of BmDOHH contains 6 E-Z type HEAT repeat domains （23-52, 54-83, 87-116, 177-206, 208-237, and 241- 270）. Compared to DHS and DOHH amino acid sequences from other species, such as Homo sapiens and Drosophila melanogaster, both silkworm DHS protein and DOHH protein have more than 55% identity. The conservative regions are very similar with each other. The phylogenetic tree analysis indicated that not only DHS but also DOHH from different species has genus-specific features. The expressions of BmDHS and BmDOHH are no tissue and stage specific in our tested samples.

Dark rearing maintains tyrosine hydroxylase expression in retinal amacrine cells following optic nerve transection

The present study examined changes in retinal tyrosine hydroxylase （TH） expression in rats having undergone optic nerve transection and housed under a normal day/night cycle or in the dark. The aim was to investigate the effects of amacrine cells on axonal regeneration in retinal ganglion cells and on the synapses that transmit visual signals. The results revealed that retinal TH expression gradually decreased following optic nerve transection in rats housed under a normal day/night cycle reaching a minimum at 5 days. In contrast, retinal TH expression decreased to a minimum at 1 day following optic nerve transection in dark reared rats, gradually increasing afterward and reaching a normal level at 5 7 days. The number of TH-positive synaptic particles correlated with the TH levels indicating that dark rearing can help maintain TH expression during the synaptic degeneration stage （5 7 days after optic nerve injury） in retinal amacrine cells.

Isomerization and degradation of all-trans-β-carotene during in-vitro digestion

To investigate the behavior of all-trans-β-carotene during digestion,in-vitro digestion coupled with HPLC-DAD,Raman spectroscopy and Fourier transform-infrared spectroscopy were used to monitor it.All-trans-β-carotene reduced by 75%during the in-vitro digestion and had a highest degradation during intestinal digestion compared with oral and gastro digestion.All-trans-β-carotene occurred isomerization and degradation during oral digestion and occurred degradation during gastro and intestinal digestion.Isomers were identifi ed as 15-cis-β-carotene and 9-cis-β-carotene,degradation products were compounds with function group of C—O,C—O—C or C=C—C=C.The biological fate ofβ-carotene during digestion was clarifi ed,and one of the reasons for low bioavailability ofβ-carotene was explained by high degradation rate during digestion.

Mutation Breeding of β-carotene Producing Strain B. trispora by Low Energy Ion Implantation

Ion beam bioengineering technology as a new mutation approach has been widely used in the biological breeding field. In this paper the application of low energy nitrogen ion implantation in the β-carotene producing strain, Blakeslea trispora（-） was investigated. The effects of different fermentation conditions on β-carotene production by a high yield strain were examined. Results showed that two β-carotene high yielding strains B.trispora（-） BH3-701 and BH3-728 were screened out and the averaged production of β-carotene was raised by 178.7% and 164.6% respectively after five passages in the shaking flasks. Compared with the original strain, the highest yield strain BH3-701 was potent in accumulating β-carotene, especially in the later stage, and greatly increased production efficiency.

Experimental measurement and correlation of solubility of β-carotene in pure and ethanol-modified subcritical water

For the first time,the solubility ofβ-carotene in pure and ethanol-modified subcritical water(SW)using the static method was determined.The experimental runs were performed at a temperature ranging from 298.15 to 403.15 K and 0–10%(w/w)of ethanol as a modifier at a constant pressure of 5 MPa.Samples were analyzed by UV–vis spectrophotometer.The solubility ofβ-carotene was found to range from 1.084×10^-8 to 227.1×10^-8 mol fractions in the subcritical water in above mentioned conditions.The obtainedβ-carotene solubility data were correlated using the linear model and modified Apelblat model.The obtained results showed the modified Apelblat model was better for estimating the solubility ofβ-carotene in SW.The values of the rootmean-square deviation(RMSD)between experimental and correlated data were calculated and used as the index of validity and accuracy for the model.Also,thermodynamic properties of the solution such as the Gibbs free energy of solution,enthalpy,and entropy of solution were estimated.