Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Oxidative phosphorylation in yeast VI. ATPase activity and protein synthesis in mitochondria isolated from nuclear mutants deficient in cytochromes

1. The oligomycin-sensitive Mg²⁺-dependent ATPase activity of mitochondria isolated from wild-type yeast Saccharomyces cerevisiae was only slightly inhibited by atractyloside at concentrations which entirely prevented oxidative phosphorylation. This indicated that most of the ATPase in these mitochondrial preparations was located outside the atractyloside-sensitive barrier and did not participate in the energy-transfer process.

2. ATPase activity of mitochondria isolated from nuclear gene mutants deficient in a single cytochrome, a, b, or c, respectively, was strongly inhibited by oligomycin. The mitochondria from these mutants, like those from the wild-type strain, were able to incorporate amino acids into protein.

3. Mitochondrial ATPase activity of single nuclear gene mutants deficient in both cytochromes a and b was only slightly inhibited by oligomycin. These mitochondria were incapable of incorporating amino acids into protein. The mitochondria from these nuclear mutants thus resembled mitochondria of cytoplasmic respiration-deficient mutants.

4. The results suggest that mitochondrial cytochromes may be coded by nuclear genes and that product(s) of mitochondrial protein synthesis may be required for integrating the cytochromes a and b and the components of the oligomycin-sensitive ATPase complex into the mitochondrial membranes.     

The mechanism of inhibition by oligomycin of oxidative phosphorylation in mitochondria

1. The concentration of specific oligomycin-binding sites in rat-liver mitochondria is 0.12 nmole/mg protein, whereas at least 10-times more oligomycin can be bound non-specifically.

2. The activity of oligomycin-inhibited processes in intact mitochondria and submitochondrial particles cannot be restored by treatment with egg lecithin or mitochondrial phospholipids.

3. Analysis of the kinetics of inhibition of State-3 respiration by oligomycin reveals that (i) after a certain lag period the inhibition by oligomycin is pseudo-first order with respect to the respiratory-control ratio, defined as the ratio of the respiratory rate at time t to that of the final inhibited site, (ii) the value of the pseudo-first-order rate constant (k₀) is dependent on the oligomycin: protein ratio, phospholipid: protein ratio, pH and temperature, (iii) the effects of various substrates and inhibitors of electron transfer on the kinetics of oligomycin inhibition can be explained by their effects on respiratory control.

4. A detailed model is proposed for the interaction of oligomycin with mitochondria. It is proposed that two conformations of the oligomycin-sensitive site are present, and that oligomycin specifically binds to the conformation that is involved in the induction of respiratory control.     

Irradiance dependency of UV-A induced phase shifts in the locomotor activity rhythm of the field mouse Mus booduga

In the nocturnal field mouse Mus booduga, the responsiveness of the circadian system to UV-A light of 2.5 W/m² and 30 minutes duration is known to be phase dependent. The results of our experiments indicate that the phase shifts evoked by UV-A at the two phases, CT14 (circadian time 14) and CT20 increases nonlinearly with irradiance. (Chronobiology International, 17(6), 777-782, 2000)     

Time-keeping system of the eel pout, Zoarces viviparus

Observations on the rhythmic activity of 71 juvenile specimens of the inter-tidal blenny Zoarces viviparus reveal an endogenous pattern of swimming at three different periodicities. Circatidal swimming, with activity peaks phased to high water or the ebb of the subjective 12.4-h tides, was found in 50 fish and was the predominant pattern seen immediately after collection, when the rhythm generally persisted for between 3 and 12 cycles. Discrete activity peaks, with a free running period of approximately 24 h were also evident in the swimming pattern of eight fish. A circadian influence was also manifest as a modulation in amplitude, phase shifts and changes in free-running period of the circa-tidal rhythm. Overall, the activity level declined with time but those fish that remained active long enough showed a semi-lunar rhythm, with maximum activity at the time of the spring tides. A comparison of the behavior of animals collected at different times of the year suggests a seasonal variation in the persistence of circatidal swimming. The results are consistent with a control system involving circatidal, circadian, and semi-lunar oscillators. (Chronobiology International, 18(1), 27-46, 2001)     

蝙蝠蛾鳞翅虫草Samsoniella hepiali模式菌株线粒体基因组系统发育分析

蝙蝠蛾鳞翅虫草Samsoniella hepiali是重要的药用虫生真菌,根据现代分类系统,隶属于虫草科Cordycipitaceae鳞翅虫草属Samsoniella。本研究对蝙蝠蛾鳞翅虫草模式菌株(ICMM 82-2)线粒体基因组进行测序、组装和注释,发现其线粒体基因组成闭合环状,大小为24 246 bp。基因区域占比为85.10%,共编码42个基因,包括15个蛋白质编码基因(PCGs)、2个rRNA基因和25个tRNA基因。15个PCGs均以ATG为起始密码子,以TAA为终止密码子,偏好密码子为UUA。各氨基酸使用频率具有较大差异,其中亮氨酸(Leu)出现频率最高。25个tRNA基因可转运全部20种氨基酸,其中19个tRNA基因二级结构为三叶草结构。本研究基于线粒体基因组中14个PCGs基因串联数据集,构建肉座菌目6科50个物种的系统发育树,进一步证实蝙蝠蛾鳞翅虫草模式菌株系统发育位置隶属于肉座菌目Hypocreales虫草科Cordycipitaceae鳞翅虫草属Samsoniella。通过虫草科线粒体基因组共线性分析,发现其中12个物种有5个同源区,3个物种存在6个同源区,同源区域A和B长度变异较大。与其他虫草科物种相比,蝙蝠蛾鳞翅虫草线粒体基因组较小,同源区域相对较短。蝙蝠蛾鳞翅虫草模式菌株线粒体基因组信息有助于鉴别其物种特异性,确定其系统发育关系,将为虫草系统发育研究提供新思路,为继续研究鳞翅虫草属物种药用价值提供系统发育依据。     

Failure of extraocular light to facilitate circadian rhythm reentrainment in humans

Although extraocular light can entrain the circadian rhythms of invertebrates and nonmammalian vertebrates, almost all studies show that the mammalian circadian system can only be affected by light to the eyes. The exception is a recent study by Campbell and Murphy that reported phase shifts in humans to bright light applied with fiber-optic pads behind the knees (popliteal region). We tested whether this extraocular light stimulus could accelerate the entrainment of circadian rhythms to a shift of the sleep schedule, as occurs in shift work or jet lag. In experiment 1, the sleep/dark episodes were delayed 8h from baseline for 2 days, and 3h light exposures were timed to occur before the temperature minimum to help delay circadian rhythms. There were three groups: (1) bright (about 13,000 lux) extraocular light from fiber-optic pads, (2) control (dim light, 10-20 lux), and (3) medium-intensity (about 1000 lux) ocular light from light boxes. In experiment 2, the sleep/dark episodes were inverted, and extraocular light was applied either before the temperature minimum to help delay circadian rhythms or after the temperature minimum to help advance rhythms. Circadian phase markers were the salivary dim light melatonin onset (DLMO) and the rectal temperature minimum. There was no evidence that the popliteal extraocular light had a phase-shifting effect in either experiment. Possible reasons for phase shifts in the Campbell and Murphy study and not the current study include the many differences between the protocols. In the current study, there was substantial sleep deprivation before the extraocular light was applied. There was a large shift in the sleep/dark schedule, rather than allowing subjects to sleep each day from midnight to noon, as in the Campbell and Murphy study. Also, when extraocular light was applied in the current protocol, subjects did not experience a change from sleeping to awake, a change in posture (from lying in bed to sitting in a chair), or a change in ocular light (from dark to dim light). Further research is necessary to determine the conditions under which extraocular light might produce phase shifts in human circadian rhythms. (Chronobiology International, 17(6), 807-826, 2000).     

Biogenesis of mitochondria 36

Summary The isolation and characterisation of a mutant affecting the assembly of mitochondrial ATPase is reported. The mutation confers resistance to oligomycin and venturicidin and sensitivity of growth on nonfermentable substrates to low temperature (19°). Genetic analysis indicates that the phenotype is due to a single mutation located on the mitochondrial DNA which is probably allelic with the independently isolated oligomycin resistance mutation [oli1-r].Growth of the mutant at the non-restrictive temperature (28°) yields mitochondria in which the ATPase appears more sensitive to oligomycin than that of the sensitive parental strain. However, when the enzyme is isolated free from the influence of the membrane strong resistance to oligomycin is evident. These data suggest that the component responsible for the oligomycin resistance of the ATPase is part of or subject to interaction with the mitochondrial inner membrane.Measurements of the ATPase content of mitochondria indicate that ATPase production is impaired during growth at 19° C. In addition, studies of the maximum inhibition of mitochondrial ATPase activity by high concentrations of oligomycin suggest a selective lesion in ATPase assembly at low temperature. The nett result is that during growth at 19° only about 10% of the normal level of ATPase is produced of which less than half is membrane integrated and thus capable of oxidative energy production.We propose that the mutation affects a mitochondrially synthesised membrane sector peptide of the ATPase which defines the interaction of F₁ ATPase with specific environments on the mitochondrial inner membrane.     

Circadian period lengths of lipid synthesis mutants (cel,chol-1) of Neurospora show defective temperature,but intact pH-compensation

The influence of extracellular pH on the circadian sporulation rhythm of Neurospora crassa has been investigated for the mutants chol-1 and cel. Both mutants have a defect in the lipid synthesis pathway and require either choline or palmitate, respectively, as supplements for normal growth. The chol-1 and cel mutants also show an impaired temperature-compensation when growing on minimal medium. We investigated the possible correlation between loss of temperature- and pH-compensation in cel and chol-1 similar to the correlation found earlier for the frq⁷ mutant. Our results show that the cel and the chol-1 mutants, although defective in temperature-compensation have an intact pH-compensation of their circadian rhythms. At present, the products of the frq-locus are the only components of the clock that affect the sporulation rhythm of Neurospora both through pH- and temperature-compensation.     

Affinities of ATP for the dinitrophenol-induced ATPase

1. 1. The effect of Mg²⁺ on ATP-dependent processes catalysed by intact rat-liver mitochondria can be explained quantitatively by the formation of Mg-ATP complexes that cannot act as a substrate for the adenine nucleotide translocator.

2. 2. The dinitrophenol-induced ATPase is characterized by two affinities of ATP: K_m(1) = 6.7 μM and K_m(2) = 63 μM, which contribute to the extent of 70% and 30%, respectively, to the total ATPase activity under the standard conditions employed.

3. 3. K_m(1) of ATP is competitively increased by atractyloside, and is insensitive to changes in cation concentration or to oligomycin or aurovertin.

4. 4. K_m(2) is as sensitive to atractyloside as the K_m(1) and is also insensitive to oligomycin. However, it is increased by decreasing the cation concentration, and disappears in the presence of aurovertin.

5. 5. It is proposed that two conformations of the adenine nucleotide translocator exist, characterized by their different affinities for ATP. The distribution of the enzyme over these two conformations appears to be a function of the energy state of the mitochondria (coupled or uncoupled).

On the Role of Energy Metabolism in Neurospora Circadian Clock Function

Neurospora crassa (bdA) mycelia were kept in liquid culture. Without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. Maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at CT 0-6 hr. Pulses of metabolic inhibitors such as vanadate (Na₃Vo₄), molybdate (Na₂MoO₄: 2 H₂O), N-ethylmaleimide (NEM), azide (NaN₃), cyanide (NaCN) and oligomycin phase shift the circadian conidiation rhythm of Neurospora crassa. Maximal advance phase shifts are observed at about CT 6 with all inhibitors.

Pulses of N,N'dicyclohexylcarbodiimide (DCCD) and light phase shift the conidiation rhythm following a phase response curve different from those of the other agents (maximal advance at about CT 18-24). The phase shifts with DCCD and light are significantly larger in the wild type compared to the mitochrondrial mutant poky. Such differences are not found in PRCs of the protein synthesis inhibitor cycloheximide.

[³¹P] NMR spectra of wild type Neurospora crassa and the clock mutants frq 1 and frq 7 which differ in their circadian period lengths did not reveal differences in the concentrations of adenine nucleotides, pyridine nucleotides or sugar phosphates. Starvation causes drastic changes of the levels of adenine nucleotides, phosphate and mobile polyphosphate without effecting phase or period length of the circadian rhythm.     

Temporal organization of feeding in Syrian hamsters with a genetically altered circadian period

The variation in spontaneous meal patterning was studied in three genotypes (tau +/+, tau +/-and tau -/-) of the Syrian hamster with an altered circadian period. Feeding activity was monitored continuously in 13 individuals from each genotype in constant dim light conditions. All three genotypes had on average six feeding episodes during the circadian cycle (about 20h in homozygous tau mutants and 22h in heterozygotes compared with 24h in wild-type hamsters). Thus, homozygous tau mutant hamsters had significantly more feeding episodes per 24h than wild types, and heterozygotes were intermediate. The average duration of feeding bouts (FBs) was indistinguishable (around 30 minutes) among the three genotypes, whereas the intermeal (IM) intervals were significantly shorter for homozygote tau mutant hamsters (99 minutes), intermediate for heterozygotes (116 minutes), and the longest for wild-type hamsters (148 minutes). Thus, the meal-to-meal duration was on average 25% shorter in homozygous tau mutants (16% in heterozygous) than in wild-type hamsters. The reduction of the circadian period has a pronounced effect on short-term feeding rhythms and meal frequency in hamsters carrying the tau mutation. (Chronobiology International, 18(4), 657-664, 2001)     

Variation of visual detection over the 24-hour period in humans

A circadian rhythm for visual sensitivity has been intensively assessed in animals. This rhythm may be due to the existence of a circadian clock in the mammalian eye, which could account for fluctuating sensitivity to light over the day in certain species. However, very few studies have been devoted to the human visual system. The present experiment was designed to assess a possible rhythm of visual sensitivity using a psychophysical method over the whole 24h period. Twelve subjects underwent visual detection threshold measures in a protocol that allowed one point every 2h. The results show that the visual detection threshold changes over the 24h period, with high thresholds in the morning, a progressive decrease over the day and the early night, and an increase during the last part of the night. These data suggest that a circadian rhythm influences visual sensitivity to mesopic luminance in humans. (Chronobiology International, 17(6), 795-805, 2000)     

两种不同来源的绿色木霉降解木质纤维素研究

利用纤维素酶高产菌绿色木霉Trichoderma viride降解木质纤维素是实现废料资源化的重要手段。本研究选取来自不同生境的两株T. viride,分别以玉米秸秆和甘草药渣为基质,测定两者滤纸纤维素酶（filter paper cellulase,FPase）活性和还原糖产量。从时间、温度、水分、pH 4个方面比较两株T. viride的环境适应性和不同基质的差异性。结果表明,以玉米秸秆为基质,T. viride XJ最适初始料液比为1:4-1:5.5,T. viride AG最适初始料液比为1:5-1:5.5。初始料液比1:5.5时,T. viride AG产FPase活性显著高于T. viride XJ。两株T. viride最适发酵温度均为28℃,各温度处理下不同菌株间无显著差异。两株T. viride均表现为还原糖消耗。以甘草药渣为基质,T. viride XJ最适初始料液比为1:2-1:2.5,T. viride AG最适初始料液比为1:3-1:3.5。料液比高于1:3,T. viride AG产FPase活性显著高于T. viride XJ。T. viride AG最适发酵温度为28℃,T. viride XJ最适发酵温度为23-28℃。温度低于28℃,T. viride XJ产FPase活性显著高于T. viride AG。两株T. viride均表现为还原糖积累。两株T. viride最适初始pH均为6-7,最适发酵时间均为3d。最优发酵条件下FPase活性：T. viride AG>T. viride XJ。对T. viride产FPase诱导能力：甘草药渣>玉米秸秆。变差分解表明两株T. viride产FPase活性差异主要源于菌株对生境的生态适应。比较分析菌种来源、基质类型、环境条件对T. viride发酵效果的影响,将有助于该菌大规模应用性研究。     

高效木质素降解菌的筛选及其对玉米秸秆的降解效果

本研究利用愈创木酚和苯胺蓝固体培养基对菌株进行初筛,利用形态学和分子生物学对筛选出的菌株进行鉴定,以黄孢原毛平革菌Phanerochaete chrysosporium CGMCC 5.0776为对照,利用其对玉米秸秆进行预处理并测定木质素和纤维素的降解率,测定筛选菌株在预处理玉米秸秆过程中漆酶、锰过氧化物酶（manganese peroxidase,MnP）和木质素过氧化物酶（lignin peroxidase,LiP）活性。结果表明:利用愈创木酚和苯胺蓝固体培养基,从16株白腐真菌菌株中筛选出2株具有较高漆酶或MnP活性的菌株,鉴定其为桦栓孔菌Trametes betulina (L.) Pilát（ZT-153）和亚黑管孔菌Bjerkandera fumosa (Pers.) P. Karst.（ZT-307）,测定T. betulina ZT-153和B. fumosa ZT-307对玉米秸秆酸不溶木质素降解效率分别为13.60%和21.87%,较对照P. chrysosporium CGMCC 5.0776高1.58%和9.85%,对纤维素的降解率较低,分别为4.10%和4.50%。2株菌株在预处理玉米秸秆过程中,T. betulina ZT-153表现出漆酶和MnP活性,B. fumosa ZT-307只表现出LiP活性。其中B. fumosa ZT-307对玉米秸秆酸不溶木质素的降解效率最高,在秸秆资源的综合利用方面具有较好的潜力和应用前景。     

Staining Mitochondria in Saccharomyces cerevisiae

After testing various procedures (amidoblack 10B, acid fuchsin-methyl blue, Luxol fast blue MBS-phloxine, toluidine blue O, Jams green B and pinacyanol), three stains can be recommended for staining both types of mitochondria (globose and threadlike) in the cells of Saccharomyces cerevisiae: (1) 0.1% solution of amidoblack 10B in citrate buffer (pH 3.0) for 10 min; (2) 0.01% solution of toluidine blue O in phosphate buffer (pH 6.0) for 30 min; (3) 0.01% solution of Janus green B in distilled water (pH 5.6) for 30 min. The latter stain is most specific because its staining reaction depends upon the action of the mitochondrial enzyme cytochrome c oxidase. Yet, low concentrations and short incubation periods must be applied to avoid poisoning of the cell metabolism.     

Repeated assessment of the endogenous 24-hour profile of blood pressure under constant routine

The impact of environmental and behavioral factors on the 24-h profile of blood pressure (BP) has been well established. Various attempts have been made to control these exogenous factors, in order to investigate a possible endogenous circadian variation of BP. Recently, we reported the results of the first environmentally and behaviorally controlled laboratory study with 24-h recordings of BP and heart rate (HR) during maintained wakefulness. In this constant-routine study, a pronounced endogenous circadian rhythm of HR was found, but circadian variation of BP was absent. This result suggested that the circadian rhythm of BP observed in earlier controlled studies, with sleep allowed, was evoked by the sleep-wake cycle as opposed to the endogenous circadian pacemaker. In order to verify our previous finding during maintained wakefulness, we repeated the experiment five times with six normotensive, healthy young subjects. Statistical analyses of the hourly measurements of BP and HR confirmed the replicable presence of an endogenous circadian rhythm of HR, as well as the consistent absence of an endogenous circadian variation of BP. Thus, this study provided additional evidence that the 24-h profile of BP—as observed under normal circumstances—is the sole result of environmental and behavioral factors such as the occurrence of sleep, and has no endogenous circadian component. (Chronobiology International, 18(1), 85-98, 2001)     

A Circadian Clock of Drosophila: Effects of Deuterium Oxide and Mutations at the period Locus

Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (per^L), shorten (per⁵), or abolish (per°) overt circadian rhythmi-city. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (per⁵ per^L, and per°) and wild-type Drosophila melanogaster (per⁺) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages ofdeuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).     

RETINAL CIRCADIAN RHYTHMS IN HUMANS *

Circadian rhythms in the retina may reflect intrinsic rhythms in the eye. Previous reports on circadian variability in electrophysiological human retinal measures have been scanty, and the results have been somewhat inconsistent. We studied the circadian variation of the electrooculography (EOG), electroretinography (ERG), and visual threshold (VTH) in subjects undergoing a 36h testing period. We used an ultrashort sleep-wake cycle to balance effects of sleep and light-dark across circadian cycles. Twelve healthy volunteers (10 males, 2 females; mean age 26.3 years, standard deviation [SD] 8.0 years, range 19-40 years) participated in the study. The retinal functions and oral temperature were measured every 90 min. The EOG was measured in the light, whereas the ERG and the VTH were measured in the dark. Sleep was inferred from activity detected by an Actillume monitor. The EOG peak-to-peak responses followed a circadian rhythm, with the peak occurring late in the morning (acrophase 12:22). The ERG b-wave implicit time peaked in the early morning (acrophase 06:46). No statistically significant circadian rhythms could be demonstrated in the ERG a-wave implicit time or peak-to-peak amplitude. The VTH rhythm peaked in the early morning (acrophases 07:59 for blue and 07:32 for red stimuli). All retinal rhythms showed less-consistent acrophases than the temperature and sleep rhythms. This study demonstrated several different circadian rhythms in retinal electrophysiological and psychophysical measures of healthy subjects. As the retinal rhythms had much poorer signal-to-noise ratios than the temperature rhythm, these measures cannot be recommended as circadian markers. (Chronobiology International, 18(6), 957-971, 2001)     

不同银杏内酯对黑曲霉线粒体结构及功能的影响

真菌感染作为威胁人类健康的传染性疾病之一,是普遍的全球性问题。本研究通过在黑曲霉的孢子悬浮液中添加不同浓度的银杏内酯,收集黑曲霉菌丝体,评估黑曲霉线粒体的结构和功能,考察不同银杏内酯B和C对黑曲霉的抗真菌效果。透射电子显微镜扫描发现经银杏内酯B处理后的黑曲霉线粒体表现出明显的空泡化,基质结构被破坏;线粒体三羧酸循环中关键酶琥珀酸脱氢酶和苹果酸脱氢酶活性下降,线粒体膜电位降低,丙二醛和活性氧水平显著升高。研究结果初步揭示了银杏内酯以剂量依赖的方式损坏线粒体膜的完整性,破坏线粒体结构,进一步损害线粒体功能;银杏内酯B的抗黑曲霉效果要显著高于银杏内酯C。