促进剂对发酵生产辅酶Q10的影响

研究了几种促进剂对放射型根瘤菌细胞生长及其辅酶Q10发酵的影响。结果表明:在培养基中适量添加VB1、乙酸钠、花生油均可明显提高辅酶Q10的产量,使用正交试验优化后的促进剂组合可使辅酶Q10产量达到50.7 mg/L,比对照组提高33%。     

CoQ10 supplementation elevates the epidermal CoQ10 level in adult hairless mice

We have already shown that prolonged supplementation of CoQ(10) in humans reduces the wrinkle area rate and wrinkle volume per unit area in the corner of the eye. CoQ(10) supplementation is known to increase the CoQ(10) level in serum and in many organs; however, the level of CoQ(10) in skin has not yet been fully investigated yet. We examined whether CoQ(10) intake elevates the CoQ(10) and CoQ(9) levels in epidermis, dermis, serum and other organs (kidney, heart, brain, muscle and crystalline lens) in 43-week-old hairless male mice. We also established a method using a high performance liquid chromatograph equipped with an electrochemical detector (HPLC-ECD) to simultaneously quantify CoQ(9) and CoQ(10) in the tissues. CoQ(10) (0, 1, 100 mg/kg p.o.) was administered daily for 2 weeks. CoQ(10) supplementation of 100 mg/kg increased the serum and epidermal CoQ(10) levels significantly, but did not increase the CoQ(10) levels in either dermis or other organs. In conclusion, we showed that CoQ(10) intake elevates the epidermal CoQ(10) level, which may be a prerequisite to the reduction of wrinkles and other benefits related to the potent antioxidant and energizing effects of CoQ(10) in skin.     

Degradation of 4-nitrobenzoate via 4-hydroxylaminobenzoate and 3,4-dihydroxybenzoate in Comamonas acidovorans NBA-10

Comamonas acidovorans NBA-10 was previously shown to degrade 4-nitrobenzoate via 4-hydroxylaminobenzoate and 3,4-dihydroxybenzoate. Washed cells, grown on a mixture of 4-nitrobenzoate and ethanol, stoichiometrically produced ammonium and 3,4-dihydroxybenzoate from 4-nitrobenzoate under anaerobic conditions provided ethanol was present. In cell extracts 4-hydroxylaminobenzoate was degraded to ammonium and 3,4-dihydroxybenzoate, but this activity was lost upon dialysis. No requirement for a cofactor was found, but rather reduced incubation conditions were necessary to restore enzyme activity. The 4-hydroxylamino-degrading enzyme was purified and the role of this novel type of enzyme in the degradation of nitroaromatic compounds is discussed.Abbreviation 4-ABA 4-aminobenzoate - 4-NBA 4-nitrobenzoate - 4-HABA 4-hydroxylaminobenzoate - 3,4-diHBA 3,4-dihydroxybenzoate     

Treatment of CoQ10 Deficient Fibroblasts with Ubiquinone,CoQ Analogs,and Vitamin C: Time- and Compound-Dependent Effects

Background

Coenzyme Q₁₀ (CoQ₁₀) and its analogs are used therapeutically by virtue of their functions as electron carriers, antioxidant compounds, or both. However, published studies suggest that different ubiquinone analogs may produce divergent effects on oxidative phosphorylation and oxidative stress.

Methodology/Principal Findings

To test these concepts, we have evaluated the effects of CoQ₁₀, coenzyme Q₂ (CoQ₂), idebenone, and vitamin C on bioenergetics and oxidative stress in human skin fibroblasts with primary CoQ₁₀ deficiency. A final concentration of 5 µM of each compound was chosen to approximate the plasma concentration of CoQ₁₀ of patients treated with oral ubiquinone. CoQ₁₀ supplementation for one week but not for 24 hours doubled ATP levels and ATP/ADP ratio in CoQ₁₀ deficient fibroblasts therein normalizing the bioenergetics status of the cells. Other compounds did not affect cellular bioenergetics. In COQ2 mutant fibroblasts, increased superoxide anion production and oxidative stress-induced cell death were normalized by all supplements.

Conclusions/Significance

These results indicate that: 1) pharmacokinetics of CoQ₁₀ in reaching the mitochondrial respiratory chain is delayed; 2) short-tail ubiquinone analogs cannot replace CoQ₁₀ in the mitochondrial respiratory chain under conditions of CoQ₁₀ deficiency; and 3) oxidative stress and cell death can be counteracted by administration of lipophilic or hydrophilic antioxidants. The results of our in vitro experiments suggest that primary CoQ₁₀ deficiencies should be treated with CoQ₁₀ supplementation but not with short-tail ubiquinone analogs, such as idebenone or CoQ₂. Complementary administration of antioxidants with high bioavailability should be considered if oxidative stress is present.     

微生物CoQ合成途径及CoQ10生产菌株的分子生物学改造

CoQ10具有呼吸链电子传递者、抗氧化性、调控基因表达等多种生理生化功能,目前不仅用作药物也用作食品添加剂。微生物发酵法是目前生产CoQ10最有效的方法。本文就有关微生物CoQ合成途径及基于CoQ合成途径的CoQ10生产菌株分子生物学改造的策略与研究进展进行了综述和展望。     

Significance of biological parameters of human blood levels of CoQ10

Ninety-one men and 143 women who were so-called normal subjects were tested for cardiac performance at rest and their blood levels of co-enzyme Q10 (CoQ10) were determined. In males, a negative relationship between progression of age and cardiac performance, and a positive relationship between progression of age and blood levels of CoQ10 were revealed. In females, a positive relationship between age and blood levels of CoQ10 was found. The mean CoQ10 blood level for both sexes was the same (0.79 +/- 0.20 micrograms/ml for males and 0.79 +/- 0.23 for females). Cardiac performance declines with age in the male population. A decreased biosynthesis and/or incorporation of CoQ10 into mitochondrial structures of muscle cells may occur with age in a normal population.     

CoQ10 deficiency diseases in adults

Deficiency of Coenzyme Q₁₀ (CoQ₁₀) in muscle has been associated with a spectrum of diseases including infantile-onset multi-systemic diseases, encephalomyopathies with recurrent myobinuria, cerebellar ataxia, and pure myopathy. CoQ₁₀ deficiency predominantly affects children, but patients have presented with adult-onset cerebellar ataxia or myopathy. Mutations in the CoQ10 biosynthetic genes, COQ2 and PDSS2, have been identified in children with the infantile form of CoQ₁₀ deficiency; however, the molecular genetic bases of adult-onset CoQ₁₀ deficiency remains undefined.     

Therapeutic effects of coenzyme Q10 (CoQ10) and reduced CoQ10 in the MPTP model of Parkinsonism

Coenzyme Q₁₀ (CoQ₁₀) is a promising agent for neuroprotection in neurodegenerative diseases. We tested the effects of various doses of two formulations of CoQ₁₀ in food and found that administration in the diet resulted in significant protection against loss of dopamine (DA), which was accompanied by a marked increase in plasma concentrations of CoQ₁₀. We further investigated the neuroprotective effects of CoQ₁₀, reduced CoQ₁₀ (ubiquinol), and CoQ₁₀ emulsions in the (MPTP) model of Parkinson's disease (PD). We found neuroprotection against MPTP induced loss of DA using both CoQ₁₀, and reduced CoQ₁₀, which produced the largest increases in plasma concentrations. Lastly, we administered CoQ₁₀ in the diet to test its effects in a chronic MPTP model induced by administration of MPTP by Alzet pump for 1 month. We found neuroprotective effects against DA depletion, loss of tyrosine hydroxylase neurons and induction of alpha-synuclein inclusions in the substantia nigra pars compacta. The finding that CoQ₁₀ is effective in a chronic dosing model of MPTP toxicity, is of particular interest, as this may be more relevant to PD. These results provide further evidence that administration of CoQ₁₀ is a promising therapeutic strategy for the treatment of PD.     

A modified determination of coenzyme Q10 in human blood and CoQ10 blood levels in diverse patients with allergies

Two situations required a modified determination of coenzyme Q10 (CoQ10) in human blood and organ tissue. Blood from patients with AIDS and cancer raised apprehensions about safety to an analyst, and the number of specimens for analysis is increasing enormously. A modified determination replaces silica gel-TLC with disposable Florisil columns, and steps were simplified to allow more analyses per unit time. Data from the modified determination are quantitatively compatible with data from older and tedious procedures. This determination was used for blood from 36 diverse patients with allergies. The mean CoQ10 blood level of these patients is not different from the mean level of so-called normal individuals, but approximately 40% (14/36) of these allergic patients had levels up to 0.65 micrograms/ml, which is the level of dying class IV cardiac patients. The biosynthesis of CoQ10 in human tissues is a complex process that requires several vitamins and micronutrients, so that countless vitamin-unsupplemented Americans may be deficient in CoQ10. The relationship of allergies to autoimmune mechanisms and immunity, and the established relationship of CoQ10 to immune states, may be a rationale for therapeutic trials of administering CoQ10 to patients with allergies who have low CoQ10 blood levels and are very likely deficient.     

COQ4 Mutations Cause a Broad Spectrum of Mitochondrial Disorders Associated with CoQ10 Deficiency

Primary coenzyme Q10 (CoQ₁₀) deficiencies are rare, clinically heterogeneous disorders caused by mutations in several genes encoding proteins involved in CoQ₁₀ biosynthesis. CoQ₁₀ is an essential component of the electron transport chain (ETC), where it shuttles electrons from complex I or II to complex III. By whole-exome sequencing, we identified five individuals carrying biallelic mutations in COQ4. The precise function of human COQ4 is not known, but it seems to play a structural role in stabilizing a multiheteromeric complex that contains most of the CoQ₁₀ biosynthetic enzymes. The clinical phenotypes of the five subjects varied widely, but four had a prenatal or perinatal onset with early fatal outcome. Two unrelated individuals presented with severe hypotonia, bradycardia, respiratory insufficiency, and heart failure; two sisters showed antenatal cerebellar hypoplasia, neonatal respiratory-distress syndrome, and epileptic encephalopathy. The fifth subject had an early-onset but slowly progressive clinical course dominated by neurological deterioration with hardly any involvement of other organs. All available specimens from affected subjects showed reduced amounts of CoQ₁₀ and often displayed a decrease in CoQ₁₀-dependent ETC complex activities. The pathogenic role of all identified mutations was experimentally validated in a recombinant yeast model; oxidative growth, strongly impaired in strains lacking COQ4, was corrected by expression of human wild-type COQ4 cDNA but failed to be corrected by expression of COQ4 cDNAs with any of the mutations identified in affected subjects. COQ4 mutations are responsible for early-onset mitochondrial diseases with heterogeneous clinical presentations and associated with CoQ10 deficiency.     

Ubiquinol-10 ameliorates mitochondrial encephalopathy associated with CoQ deficiency

Coenzyme Q10 (CoQ₁₀) deficiency (MIM 607426) causes a mitochondrial syndrome with variability in the clinical presentations. Patients with CoQ₁₀ deficiency show inconsistent responses to oral ubiquinone-10 supplementation, with the highest percentage of unsuccessful results in patients with neurological symptoms (encephalopathy, cerebellar ataxia or multisystemic disease). Failure in the ubiquinone-10 treatment may be the result of its poor absorption and bioavailability, which may be improved by using different pharmacological formulations. In a mouse model (Coq9^X/X) of mitochondrial encephalopathy due to CoQ deficiency, we have evaluated oral supplementation with water-soluble formulations of reduced (ubiquinol-10) and oxidized (ubiquinone-10) forms of CoQ₁₀. Our results show that CoQ₁₀ was increased in all tissues after supplementation with ubiquinone-10 or ubiquinol-10, with the tissue levels of CoQ₁₀ with ubiquinol-10 being higher than with ubiquinone-10. Moreover, only ubiquinol-10 was able to increase the levels of CoQ₁₀ in mitochondria from cerebrum of Coq9^X/X mice. Consequently, ubiquinol-10 was more efficient than ubiquinone-10 in increasing the animal body weight and CoQ-dependent respiratory chain complex activities, and reducing the vacuolization, astrogliosis and oxidative damage in diencephalon, septum–striatum and, to a lesser extent, in brainstem. These results suggest that water-soluble formulations of ubiquinol-10 may improve the efficacy of CoQ₁₀ therapy in primary and secondary CoQ₁₀ deficiencies, other mitochondrial diseases and neurodegenerative diseases.     

Effects of dissolved oxygen concentration and DO-stat feeding strategy on CoQ10 production with Rhizobium radiobacter

The cell growth and CoQ₁₀ (coenzyme Q₁₀) formation of Rhizobium radiobacter WSH2601 were investigated in a 7-1 bioreactor under different dissolved oxygen (DO) concentrations. A maximal CoQ₁₀ content (C/B) of 1.91 mg/g dry cell weight (DCW) and CoQ₁₀ concentration of 32.1 mg/l were obtained at the appropriate DO concentration of 40% (of air saturation). High DO concentration was favourable to the cell growth of Rhizobium radiobacter WSH2601. In order to achieve the maximal yield of CoQ₁₀ production, a new DO-stat feeding strategy was proposed, which significantly improved cell growth and CoQ₁₀ formation. With this strategy, the maximal CoQ₁₀ concentration and DCW reached 51.1 mg/l and 23.9 g/l, respectively, which were 67 and 44.8% higher than those obtained in the batch culture with DO concentration controlled.     

Cellular redox activity of coenzyme Q10: effect of CoQ10 supplementation on human skeletal muscle

In this paper, we report results obtained from a continuing clinical trial on the effect of coenzyme Q 10 (CoQ 10 ) administration on human vastus lateralis (quadriceps) skeletal muscle. Muscle samples, obtained from aged individuals receiving placebo or CoQ 10 supplementation (300 mg per day for four weeks prior to hip replacement surgery) were analysed for changes in gene and protein expression and in muscle fibre type composition. Microarray analysis (Affymetrix U95A human oligonucleotide array) using a change in gene expression of 1.8-fold or greater as a cutoff point, demonstrated that a total of 115 genes were differentially expressed in six subject comparisons. In the CoQ 10 -treated subjects, 47 genes were up-regulated and 68 down-regulated in comparison with placebo-treated subjects. Restriction fragment differential display analysis showed that over 600 fragments were differentially expressed using a 2.0-fold or greater change in expression as a cutoff point. Proteome analysis revealed that, of the high abundance muscle proteins detected (2086 ±115), the expression of 174 proteins was induced by CoQ 10 while 77 proteins were repressed by CoQ 10 supplementation. Muscle fibre types were also affected by CoQ 10 treatment; CoQ 10 -treated individuals showed a lower proportion of type I (slow twitch) fibres and a higher proportion of type IIb (fast twitch) fibres, compared to age-matched placebo-treated subjects. The data suggests that CoQ 10 treatment can act to influence the fibre type composition towards the fibre type profile generally found in younger individuals. Our results led us to the conclusion that coenzyme Q 10 is a gene regulator and consequently has wide-ranging effects on over-all tissue metabolism. We develop a comprehensive hypothesis that CoQ 10 plays a major role in the determination of membrane potential of many, if not all, sub-cellular membrane systems and that H 2 O 2 arising from the activities of CoQ 10 acts as a second messenger for the modulation of gene expression and cellular metabolism.     

Cell density-dependent changes of glycosphingolipid biosynthesis in cultured human skin fibroblasts

In this study, the glycosphingolipid biosynthesis was investigated in the sparse and the confluent cell populations of cultured human skin fibroblasts.The human skin fibroblast cell populations were metabolically pulse labeled with ¹⁴C-galactose (48 h). The amounts of ¹⁴C-radioactivity (cpm) incorporated into extracted and purified total cellular glycosphingolipid fractions were counted by -scintillation and the individual glycosphingolipid species were separated by high performance thin layer chromatography and visualized by autoradiography. The relative labeling (%) of individual newly synthesized glycosphingolipid species was detected by densitometric scanning of autoradiographic glycosphingolipid patterns.The incorporation of ¹⁴C-label into total glycosphingolipids per cell increased significantly as the cell-density increased, referring to five fold higher rate of glycosphingolipid biosynthesis de novo in cells at confluency vs. sparse populations. The total newly synthesized glycosphingolipid pattern (100%) of sparse cell populations showed a significant predominance of the gangliosides (70%) over the neutral glycosphingolipids (30%), with ganglioside GM2 as the major species followed by monohexosyl-ceramide. Oppositely, the newly synthesized neutral glycosphingolipids (67%) predominated over the gangliosides (33%) in cells at confluency (contact inhibition). Cells reaching confluency were characterized by: (a) a dramatic increase of absolute amount of all newly synthesized neutral glycosphingolipid species, particularly the most abundant monohexosyl-ceramide and trihexosyl-ceramide, but also of the ganglioside GM3; (b) a drastic decrease of absolute amount of newly synthesized ganglioside GM2. The specific shift in newly synthesized glycosphingolipid pattern in cells reaching confluency suggests a down-regulation of biosynthetic pathway primarily at the level of N-acetylgalactosaminyl-transferase. A possible involvement of glycosphingolipids in cell density-dependent regulation of cell growth through establishment of the direct intermolecular intermembrane interactions is discussed.     

Regulatory mechanisms of UDP-glucuronic acid biosynthesis in cultured human skin fibroblasts

Kinetic parameters and regulatory properties of UDPGDH extracted from cultured human skin fibroblasts were determined and compared with those of UDPGDH from cornea and epiphysial-plate cartilage. Fibroblast enzyme showed an affinity for UDPG 7 times higher than cartilage enzyme and 42 times higher than cornea enzyme. UDP-xylose acted as a co-operative allosteric inhibitor, but under the same experimental conditions fibroblast enzyme was significantly less inhibited. These results were in agreement with the different GAG production of the cells we studied. Fibroblast UDPGDH activity was regulated by the NAD/NADH ratio and it was also affected by modifications of extracellular matrix composition. A significant increase of UDPGDH affinity for UDPG was observed after the treatment of the monolayers with Chase ABC.     

Thromboxane A2 biosynthesis in human lung fibroblasts WI-38.

A fibroblast of human lung origin (WI-38) synthesizes thromboxane A₂ from the prostaglandin endoperoxide PGH₂. Thromboxane A₂ synthesis was demonstrated by radio thin layer chromatography, gas chromatography/mass spectrometry, and by bioassay. This is the first demonstration of thromboxane A₂ biosynthesis in a homogeneous cell population other than the human platelet.     

代谢工程在微生物法产辅酶Q_(10)中的应用

辅酶Q10作为细胞呼吸链上的重要组成部分在电子传递过程中发挥着重要的作用。辅酶Q10的抗氧化、抗衰老功能使其广泛应用于医药、食品和化妆品等行业。CoQ10市场需求不断增加,这使得大规模提高CoQ10工业化生产的产量显得十分必要。目前,主要依靠从自然界中筛选到的各种微生物作为生产菌种发酵生产CoQ10,但这些原始生产菌种由于产量低、营养要求高等各种原因很难实现大规模发酵生产。随着对CoQ10生物合成途径以及代谢调控机制的了解清楚,通过对易于商业化生产的优良宿主细胞(如大肠杆菌)进行代谢工程的改造,有助于促进代谢工程菌的CoQ10工业化生产发展。