Evaluation of biotin responsiveness in cultured fibroblasts from patients with propionic acidemia: Absence of response by structurally altered carboxylases

We have demonstrated that, although propionyl CoA carboxylase (PCC) activity is deficient in fibroblast extracts from PCC-deficient patients belonging to the two major and two minor genetic complementation groups, the activity of another biotin-dependent carboxylase, -methylcrotonyl CoA carboxylase (MCC), is normal. Moreover, MCC activity is stimulated when the fibroblasts are cultured in high concentrations of biotin, in the same way that it is in normal fibroblasts, whereas the depressed PCC activity remains essentially unchanged. Because these results are parallel with the in vivo failure of high-dose biotin to stimulate PCC activity in peripheral blood leukocytes, we conclude that the biotin responsiveness of PCC in cultured fibroblasts from patients with PCC deficiency may be used to predict or confirm biotin responsiveness in vivo.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Increased levels of maleate cis-trans isomerase in Alcaligenes faecalis IB-14 were induced by malonate analogues such as tartronate, keto-malonate and ethyl-malonate. Similarly in the case of malonate, those analogues were not served as carbon sources for the cell growth, but they were more effective as inducer than maleate, the normal inducer of the enzyme.

The induced synthesis of the enzyme was markedly repressed in the presence of various carbon sources, e.g. oxalacetate, d- and l-malates, fumarate and succinate. More than 90 per cent of the enzyme formation was inhibited when 10^?1 m of oxalacetate or dl-malate was present in malonate medium. The repression of the enzyme synthesis by those dicarboxylic acids was always associated with remarkable promotion of the cell growth.     

Effect of Reaction Temperature on the Determination of Rat Serum IgE by the Avidin-Biotin Method

We established an avidin–biotin method for the sensitive determination of rat IgE and found that a non-specific signal was generated depending on the reaction temperature. When the sera of rats immunized or not with ovalbumin (OVA) were fractionated in a hydroxyapatite column and OVA-specific IgE was determined by the avidin–biotin method at 4°C or 37°C, OVA-specific IgE peaks were detected at 37°C, even with nonimmunized rats, but not at 4°C.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Repression of maleate cis-trans isomerase(maleate isomerase) by carbon sources and its reversal were investigated by using Alcaligenes faecalis IB-14.

The formation of maleate isomerase was induced by malonate favorably in a poor medium, whereas it was repressed in a rich medium by carbon sources such as intermediates of TCA cycle. The repression provoked by dl-malate was accompanied with remarkable promotion of the cell growth and with accumulation of a large amount of pyruvate. The enzyme levels of TCA cycle were elevated several times in the dl-malate repressed cells. It was probable to assume that the formation of maleate isomerase was subject to catabolite repression when a rapid and surplus metabolism of dl-malate via TCA cycle was conducted.

So, as an approach to reveal the chemical nature of the catabolite moiety, reversal of the catabolite repression was studied. It was demonstrated that the repression provoked by dl-malate was reversed by various cultural conditions as follows; addition of higher concentrations of malonate, divided supply of dl-malate, “anaerobic” incubation and addition of higher concentrations of ammonium ion. From physiological significances of these events, it was revealed that catabolite repression of maleate isomerase was reversed by minimizing the functioning of TCA cycle.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Maleate cis-trans isomerase in Alcaligenes faecalis IB–14 was induced by malonate and purified about 100-fold over the crude cell-free extract by treatments of ammonium sulfate fractionation, Sephadex G–100 gel filtration, DEAE-cellulose and DEAE-Sephadex A–50 column chromatography. The preparation was shown to be monodisperse on ultracentrifugal analysis and Svedberg value was found to be 3.84 S.

The enzyme was most active at pH value around 8.3 and was stable over the range of pH 5.0 to 7.0 in the presence of dithiothreitol (DTT) for a few weeks, but in the absence of it, the enzyme activity was markedly decreased, especially in the alkaline region. The enzyme activity was inhibited by various sulfhydryl reagents and oxidizing agents, whereas it was not affected by metal chelating agents. The inhibition by Hg²⁺ and PCMB was overcome by the addition of sulfhydryl compounds such as DTT, 2-mercaptoethanol, l-cysteine and glutathione. It was observed that the enzyme did not require co-factor for its function.

Kinetic studies showed that Michaelis constant for maleate was 2.8×10^?3 m and the enzyme did not catalyze the reverse reaction.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Malonate added to peptone-meat extract medium has been shown to induce maleate cis-trans isomerase in Alcaligenes faecalis IB-l4. This enzyme played an indispensable role in the enzymatic production of L-aspartic acid from maleic acid and ammonia.

Though malonate in the medium inhibited the growth of A. faecalis IB-14 to some extent, cells grown in the medium containing 10^-1 M of malonate showed the highest level of the enzyme activity. Specific activity of the enzyme of malonate-grown cells was approximately ten times as strong as that of maleate-grown cells, while, in basal mediumgrown cells, cis-trans isomerization of maleate did not occur at all. Maleate was utilized not only as carbon source of the cell growth but also as inducer for the formation of maleate cis-trans isomerase. On the other hand, malonate was not utilized as carbon source and the metabolism of it within the cell was rather restricted within narrow limits. Thus it was concluded that malonate was a gratuitous inducer for the formation of the enzyme, while maleate, which was considerably metabolized, was normal inducer.

It was demonstrated that most of radioactivity of l-C¹⁴-malonate taken up by cells was localized in particle fraction which sedimented at 105,000×g for 120 minutes, whereas radioactivity of 1-4-C¹⁴-maleate was uniformly distributed in both particulate and soluble fractions. Maleate cis-trans isomerase activity, in turn, was detected exclusively in soluble fraction in both malonate and maleate induced cells.     

Reorganization of cytoskeletal proteins of mouse oocytes mediated by integrins

To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for superovulation. The zona-free oocytes were incubated separately with specific ligand of integrins,an active RGD peptide, in vitro for certain period of time. RGE peptide and mouse capacitated sperm were used as controls. Freshly ovulated oocytes and those treated with different factors were immunostained with FITC-labeled anti-actin antibody, then detected with confocal microscope. The results demonstrated that freshly ovulated mouse oocytes, oocytes incubated for 2 h in vitro and those treated with control RGE peptide for 15 min showed hardly visible fluorescene or only thin fluorescence in plasma membrane region. Oocytes coincubated with sperms for 15 min and those treated with active RGD peptide for 10 min, 30 min and 2 hours respectively had strong and thick fluorescence in the plasma membrane and cortical region of oocytes, and some of them showed asymmetrically fluorescent distribution. It is proved that integrins on membrane are ligated directly with cytoskeletal protein. Integrins binding with their ligands regulate reorganization of cytoskelal protein, which may be involved in transmembrane signaling in egg activation.     

Reorganization of cytoskeletal proteins of mouse oocytes mediated by integrins

Integrins, as transmembrane signalling receptors,initiate a series of events of intracellular signal trans-duction by ligating with their ligands. In the process ofintegrin-mediated transmembrane signal transduction,the roles of intracellular cytoskeletal proteins havebeen described in many types of cells[1—4]. On thebasis of the well-documented investigations, Clark andhis colleagues raised the functional pattern of integrinsmediating transmembrane signal transduction. It issuggested that th…     

酶法拆分生物素中间体及其反应条件的优化

探讨多种具有水解酯键的商品化酶作用于生物素中间体1（1H-呋喃[3,4-d]并咪唑-6-氢-1,3-二苄基-2,4-二酮）的两种异构体,在水-有机相中进行选择性水解结果并进行了活性比较,从而找到一种活性较高的中性脂肪酶。最后对该酶最佳反应条件（水/有机相体积比、有机溶剂的选择、温度、pH值）作了研究,并建立快速鉴定两种异构体的方法。该酶的最佳反应条件为：在50ml苯或甲苯为介质,加水6ml,35℃ pH7,反应6h,产物的EE值为99%。     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.     

Induction of meiotic maturation in mouse oocytes by adenosine analogs

In this study we have examined the meiosis-inducing influence of adenosine analogs in mouse oocytes. When a varied group of nucleosides and nucleotides were tested on overnight cultures of hypoxanthine-arrested, cumulus cell-enclosed oocytes (CEO), halogenated adenosine nucleosides, but not native adenosine, exhibited a significant meiosis-inducing capability. When tested under a variety of conditions, meiotic induction by 8-bromo-adenosine (8-Br-Ado) and a second adenosine analog, methylmercaptopurine riboside (MMPR), was especially potent in denuded oocytes (DO) compared to CEO and was not dependent on the type of inhibitor chosen to maintain meiotic arrest. Germinal vesicle breakdown (GVB) was stimulated with rapid kinetics and was preceded by an increase in AMP-activated protein kinase (AMPK) activity. Moreover, compound C, an inhibitor of AMPK, blocked the meiosis-inducing activities of both adenosine analogs. When tested for an effect on meiotic progression to metaphase II (MII) in spontaneously maturing CEO, 8-Br-Ado and the AMPK activator, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), increased the percentage of MII-stage oocytes, but MMPR decreased this number. Adenosine and inhibitors of de novo purine synthesis had no effect on the completion of maturation, while compound C suppressed this process. These results support the proposition that oocyte AMPK mediates the positive influence of AICAR and 8-Br-Ado on both the initiation and completion of meiotic maturation. The role of AMPK in MMPR action is less clear.     

Gonadotropin-induced murine oocyte maturation in vivo is not associated with decreased cyclic adenosine monophosphate in the oocyte-cumulus cell complex

The cyclic adenosine monophosphate (cAMP) content of intact oocyte-cumulus cell complexes at various times after the induction of oocyte maturation in mice in vivo was correlated with the time of commitment by the oocytes to undergo germinal vesicle breakdown (GVB) and metabolic coupling between the oocyte and cumulus cells. Seventy-nine percent of the oocytes either underwent GVB or were committed to do so by 2 h after injection of human chorionic gonadotropin (hCG). This occurred without a decrease in the coupling between cumulus cells and the oocyte and with increasing cAMP levels in the oocyte-cumulus cell complex. Maintenance of threshold levels of cAMP within mammalian oocytes appears essential for the maintenance of meiotic arrest, but data presented here suggest that oocyte maturation in mice is induced by gonadotropins in nonatretic follicles in vivo by some mechanism other than one which decreases the cAMP content of the intact oocyte-cumulus cell complex.     

Progressive defects observed in mouse sperm during the course of three generations of selenium deficiency

Three successive generations of mice were fed a Torula yeast based Se-deficient diet with or without 0.1 ppm Se in the drinking water. The Se-deficient mice, in the course of three generations, showed a decrease in body weight, testis weight, epididymal weight, and sperm production. The percentage of morphologically abnormal sperm increased in successive generations. The majority of sperm defects were found in the midpiece region of the tail. Many of these aberrant sperm were motile. A progressive decrease in fertility was noted during the first two generations of Se deficiency. This system confirms the essential role of Se in spermatogenesis and provides a model for the evaluation of the primary effect of Se deprivation on the structural development of sperm.     

A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate-dependent meiotic arrest in murine oocytes

The effects of the putative maturation inhibitor in porcine follicular fluid on gonadotropinstimulated reversal of cyclic adenosine monophosphate (cAMP)-maintained meiotic arrest in mouse oocytes in vitro were assessed in this study. When cumulus cell-enclosed oocytes were cultured in a suboptimal inhibitory concentration of dibutyryl cAMP (dbcAMP), the effect of follicle-stimulating hormone (FSH) on oocyte maturation was initially inhibitory at 3 hr, but stimulatory at 6 hr. Supplementation of the medium with an ultrafiltrate of porcine follicuiar fluid (PM10-filtrate) completely suppressed FSH-promoted reversal of inhibition at 6 hr. Charcoal extraction eliminated this effect of the PM10-filtrate. FSH reversed the inhibition of maturation of cumulus cell-enclosed oocytes maintained by a high concentration of dbcAMP and suboptimal concentrations of the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX), during a 21–22-hr culture period. However, the effect of a completely inhibitory concentration of IBMX was not reversed by gonadotropin. A component of serum was also found to inhibit FSH reversal of dbcAMP-maintained meiotic arrest, and this activity was removed by charcoal extraction. In addition, when oocytes were cultured in medium containing a suboptimal concentration of dbcAMP plus a low molecular weight fraction (< 1,000) of porcine follicular fluid, porcine serum, or fetal bovine serum, a synergistic inhibition of maturation was observed. Experiments with highly purified gonadotropins revealed that reversal of dbcAMP-maintained meiotic arrest occurred only in response to FSH; neither highly purified luteinizing hormone nor human chorionic gonadotropin could mimic this action of FSH. Also, this effect was mediated by the cumulus cells, since FSH could not reverse dbcAMP-maintained meiotic arrest in denuded oocytes. Furthermore, elevating cAMP levels in denuded oocytes augmented, rather than reversed, the inhibitory action of dbcAMP on oocyte maturation. These data therefore suggest that dbcAMP- or IBMX-maintained meiotic arrest in vitro is reversed by an FSH-stimulated, cAMP-dependent process mediated by the cumulus cells and demonstrate that a factor present both in follicular fluid and serum prevents this action of the gonadotropin.     

SKAP2 regulates Arp2/3 complex for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes

SKAP2 (Src kinase-associated phosphoprotein 2), a substrate of Src family kinases, has been suggested to be involved in actin-mediated cellular processes. However, little is known about its role in mouse oocyte maturation. In this study, we thus investigated the expression, localization, and functions of SKAP2 during mouse oocyte asymmetric division. SKAP2 protein expression was detected at all developmental stages in mouse oocytes. Immunofluorescent staining showed that SKAP2 was mainly distributed at the cortex of the oocytes during maturation. Treatment with cytochalasin B in oocytes confirmed that SKAP2 was co-localized with actin. Depletion of SKAP2 by injection with specific short interfering RNA caused failure of spindle migration, polar body extrusion, and cytokinesis defects. Meanwhile, the staining of actin filaments at the oocyte membrane and in the cytoplasm was significantly reduced after these treatments. SKAP2 depletion also disrupted actin cap and cortical granule-free domain formation, and arrested a large proportion of oocytes at the telophase stage. Moreover, Arp2/3 complex and WAVE2 expression was decreased after the depletion of SKAP2 activity. Our results indicate that SKAP2 regulates the Arp2/3 complex and is essential for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes.     

Melatonin alleviates the deterioration of oocytes from mice subjected to repeated superovulation

Induction of repeated superovulation with exogenous hormones is widely used in assisted reproductive technology (ART). Though it is generally safe, emerging evidence has indicated that repeated superovulation may compromise oocyte quality. However, few studies have explored how to ameliorate such impairment. Because melatonin has beneficial influences on oocytes in various detrimental environments, we aimed to explore whether melatonin could protect mouse oocytes after repeated superovulation. We found that repeated superovulation markedly reduced meiotic maturation and disrupted spindle organization and chromosome alignment. Furthermore, we observed reduced mitochondrial content and enhanced early apoptosis in oocytes from mice subjected to repeated superovulation. In addition, 5-methylcytosine (5mc) fluorescence intensity was lower in oocytes from experimental mice than in those from control mice, indicating that repeated superovulation disrupts genomic DNA methylation, and elevations in reactive oxygen species levels indicated that repeated superovulation also induces oxidative stress. Conversely, melatonin administration improved oocyte maturation and attenuated the observed defects. Interestingly, supplementation with melatonin during in vitro maturation had the same protective effects on oocytes as in vivo melatonin administration. In summary, our results show that melatonin can improve oocyte quality after repeated superovulation and thus provide a potential strategy to improve ART efficiency.     

High-fat-diet impaired mitochondrial function of cumulus cells but improved the efficiency of parthenogenetic embryonic quality in mice

Global human health has been compromised by high-fat diets. This study aimed to investigate the relationship between a high-fat diet and parthenogenetic embryo quality. Mice fed a high-fat or a normal diet was used as treated or control groups, respectively. Estradiol (E₂), total cholesterol (TC) and total triglyceride (TG) were detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Cumulus-oocyte complexes (COCs) were collected from the mice in the treated and control groups. The ultrastructure of COCs, the expression level of genes involved in mitochondrial and nuclear functions in cumulus cells and oocytes quality were evaluated with transmission electron microscopy, real-time quantitative polymerase chain reaction (RT-PCR) and artificial parthenogenesis, respectively. The results showed that the efficiency of parthenogenetic embryonic development in vitro was significantly higher in the treated group than in the control group (p?p?P450 arom were higher in the treated group than the control group (p?p?相似文献   

Comparison of the properties of histidine ammonia-lyase in normal and histidinemic mutant mice

The histidinemic (his/his) mutant mouse shows greatly reduced skin and liver histidine:ammonia-lyase (HAL; EC 4.3.1.3) activity compared with normal mice. Liver HAL activity in the mutant is heat and salt labile and is inhibited at high substrate concentrations. Two HAL components have been identified in the normal mouse liver, a minor component with properties similar to those of HAL of the mutant mouse and a major component which is heat and salt stable and insensitive to substrate inhibition. Immunotitration with anti-HAL antibody shows that the livers of mutant mice contain no detectable antigenically cross-reacting HAL protein. It is concluded, therefore, that the his allele is a null allele at a structural or regulatory locus for the major HAL enzyme and maps close to the HAL-regulatory locus Hsd and that the low residual HAL activity in the mutant is due to another enzyme.This work was supported by an MRC Project Grant to H.K. and USPHS Grant GH21002 to S.M.A.