Purification and characterization of an enhancer-binding protein of the fibroin gene. I. Complete purification of fibroin factor 1

An enhancer-binding protein of the fibroin gene, fibroin factor 1 (FF1), has been purified to homogeneity from crude nuclear extracts of posterior silk gland cells where this gene is transcribed specifically. There is a multiplicity of FF1; the FF1 activity was eluted as at least three major fractions on column chromatographies. FF1 is able to form a stable complex with the enhancer DNA sequence in the presence of another proteinous factor named FF2, which lacks ability to bind DNA molecules by itself. One of FF1 forms, FF1a, was purified with a combination of classical purification techniques without using a sequence-specific affinity column, and identified as a protein with molecular mass 125 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To obtain homogeneous protein of FF1a, purification of more than 26,000-fold from the starting nuclear extract was necessary.     

Inhibition of 125I-human follicle-stimulating hormone binding to receptor by a low molecular weight fraction of bovine follicular fluid: inhibitor concentration is related to biochemical parameters of follicular development

Pools of follicular fluid (FF) were obtained from large or small follicles of cows which were pregnant or in the luteal phase of the estrous cycle. Cells present in each FF pool were collected by centrifugation and measured for content of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptors. Steroid levels in FF were quantitated by radioimmunoassay (RIA). Since the quantity of bovine follicular cells (mostly granulosa cells) was limited, FSH binding inhibition was studied utilizing a calf testis receptor system. Low (less than 6000) molecular weight (Mr) fractions prepared by dialysis were shown to account for most (76 to 94%) of the FSH binding inhibition (FSH-BI) present in unfractionated FF. The concentration of low Mr FSH-BI was higher in pools of FF from cows in the luteal phase of the estrous cycle than in pools of FF from pregnant cows. The concentration of low Mr FSH-BI was also higher in FF pooled from small follicles than in FF pooled from large follicles of either pregnant or luteal phase cows. Relative concentrations of receptors for gonadotropins (FSH, LH) on granulosa cells were used to rank the pools according to relative degree of follicular maturation. Other parameters of follicular maturation were concentration of estrogens and the ratio of estrogens to androgens in FF. Biochemical parameters for follicular atresia were the concentration of androgens and the ratio of estrogens to androgens in FF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay

Precontact communication between gametes is established by chemotaxis. Sperm chemotaxis toward factor(s) in follicular fluid (FF) has been demonstrated in humans and mice. In humans, the chemotactic responsiveness is restricted to capacitated spermatozoa. Here, we investigated whether sperm chemotaxis to factor(s) present in FF also occurs in rabbits and, if so, whether only capacitated spermatozoa are chemotactically responsive. Chemotaxis assays were performed by videomicroscopy in a Zigmond chamber. We measured chemotactic responsiveness as a function of FF dilution by means of a novel directionality-based method that considers the ratio between the distances traveled by the spermatozoa both parallel to the chemoattractant gradient and perpendicular to it. A peak of maximal response was observed at 10(-4) dilution of FF, resulting in a typical chemotactic concentration-dependent curve in which 23% of the spermatozoa were chemotactically responsive. In contrast, the percentage of cells exhibiting FF-dependent enhanced speed of swimming increased with the FF concentration, whereas the percentage of cells maintaining linear motility decreased with the FF concentration. The percentages of chemotactically responsive cells were very similar to those of capacitated spermatozoa. Depletion of the latter by stimulation of the acrosome reaction resulted in a total loss of the chemotactic response, whereas the reappearance of capacitated cells resulted in a recovery of chemotactic responsiveness. We conclude that rabbit spermatozoa, like human spermatozoa, are chemotactically responsive to FF factor(s) and acquire this responsiveness as part of the capacitation process.     

Extended exposure to follicular fluid is required for significant stimulation of the acrosome reaction in human spermatozoa

The effect of human follicular fluid (FF) on the incidence of spontaneous acrosome reactions (AR) in human spermatozoa was examined over a 24-25 h period using electron microscopy. Suspensions of motile spermatozoa were prepared by a swim-up method in Earle's medium, known to support in-vitro fertilization. After adjusting the concentration to 10 x 10(6) cells/ml, suspensions were diluted 1:1 with medium (control) or FF, the latter giving a final concentration of 50% FF. In addition, at 5 h and 24 h an aliquant of the control suspension was removed, diluted 1:1 with FF and incubated for 1 h; the three suspensions were examined at 6 h and 25 h. Continuous exposure to 50% FF stimulated the AR, the effect being significant (P less than 0.001) at 25 h. However, the 1-h short exposure of spermatozoa to FF did not produce an increase in AR, even after 24 h preincubation. In a separate series of experiments, the effect of continuous incubation for 24 h in increasing concentrations of FF was investigated. A significant linear dose-dependent effect on the AR was observed with all concentrations assessed (P less than 0.01 for 12.5% FF and P less than 0.001 for 25, 50, 75 and 100% FF, compared with FF-free control). Therefore, human FF can stimulate the AR, but only after a continuous exposure to FF. A short exposure to FF, even after 24 h preincubation, does not trigger an increased AR response.     

Simultaneous determination of thiamphenicol,florfenicol and florfenicol amine in swine muscle by liquid chromatography–tandem mass spectrometry with immunoaffinity chromatography clean-up

A rapid and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512 ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4–50 ng/g, the average recoveries were 85.2–98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0 μg/kg.     

Correlation between follicular fluid content and the results of in vitro fertilization and embryo transfer. III. Proteoglycans

Using a specific proteoglycan (PG) radioimmunoassay (RIA) in which human cartilage antiserum was directed against the PG protein core, the PG content of follicular fluid (FF) obtained from 42 women undergoing in vitro fertilization (IVF) and embryo transfer (ET) was studied as a function of IVF-ET outcome. Inhibition curves of purified PG cartilage preparations were parallel to those of large and small nonstimulated follicles and follicles that had been stimulated by a luteinizing hormone-releasing hormone (LHRH) agonist, d-tryptophan-6 (Decapeptyl: D-Trp6 analogue, Beaufour Laboratories, IPSEN Biotech, Paris, France), and human menopausal gonadotropin (hMG). While FF levels of immunoreactive PG-like material (Ir-PG) did not differ according to IVF-ET outcome, highly significant negative correlations were obtained between FF 17 beta-estradiol levels and FF Ir-PG levels in oocyte groups where pregnancy was obtained, i.e., oocytes were fertilized and cleaved, and pregnancy followed either for each ET or for one of two embryos reimplanted. The correlation persisted but weakened when all groups were pooled together. No correlation was observed between FF Ir-PG and progesterone. RIA or bioassay showed a positive correlation between FF inhibin and Ir-PG for the group in which each ET led to a pregnancy. Ir-PG concentrations were significantly greater in smaller than in larger follicles collected from untreated women. Upon induction of ovulation with either pure follicle-stimulating hormone (FSH), FSH + human chorionic gonadotropin (hCG), or D-Trp6/hMG + hCG, this difference no longer appeared. These results indicate that the reduction of Ir-PG concentrations constitutes an index of follicular maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of follicular fluid collected from various diameter follicles on the progression of nuclear maturation and developmental competence of pig oocytes

Supplementing in vitro maturation medium with porcine follicular fluid (FF) improves maturation rate, male pronucleus formation, and monospermic fertilization of pig oocytes. This study examined, (1) if there are differences in FF derived from large follicles (LF, 5–6 mm in diameter) and small follicles (SF, 3–4 mm in diameter) on the effect of supplementing the maturation medium with FF on the progression of nuclear maturation, fertilization rate, and developmental competence of porcine oocytes; (2) whether the FF source influences the effect of the FF on the maturation medium on the survival rate and proliferation rate of cumulus cells (CCs) and the expansion of cumulus-oocyte-complexes (COCs); (3) whether the oocyte source (oocytes collected from LFs or SFs) influences the effect of FF on the progression of the nuclear maturation of oocytes; (4) whether the factors in the FF that affect the kinetics of nuclear maturation are proteins, and the range of the molecular weight of the FF factors.

In experiment 1, adding FF from LFs (LFF) significantly accelerated nuclear maturation and improved the fertilization rate; the developmental ratio was comparable with those of adding FF from SFs (SFF). In experiment 2, adding LFF, but not SFF, improved the CC survival rate, although the FF source did not affect the proliferation rate. Expansion of COCs was greater with SFF than LFF. In experiment 3, LFF promoted nuclear maturation of oocytes collected from only LFs. There was a significant interaction between the FF source and the oocyte source in the effect on nuclear maturation stages at 36 h of maturation. In experiment 4, treatment of FF with heat or trypsin diminished the difference between the effect of LFF and SFF on the progression of nuclear maturation. In addition, the predominant effect of LFF compared to that of SFF on nuclear maturation was not affected by ultrafiltration of the FF with a 30-kDa filter, but was diminished by ultrafiltration with a 100-kDa filter. The present study suggests that some proteins present in LFF that range in molecular weight from 30 to 100 kDa improve the developmental competence of oocytes probably via progression of nuclear maturation and cumulus cells viability.     

Bacillus subtilis and Enterococcus faecium co-fermented feed regulates lactating sow's performance,immune status and gut microbiota

Fermented feed (FF) is widely applied to improve swine performance. However, the understandings of the effects of FF on the immune status and gut microbiota of lactating sows and whether probiotics are the effective composition of FF are still limited. The present study aimed to investigate the performance, immune status and gut microbiota of lactating sows fed with a basal diet supplemented with Bacillus subtilis and Enterococcus faecium co-fermented feed (FF), with the probiotic combination (PRO) of B. subtilis and E. faecium and control diet (CON) as controls. Compared with the CON group, FF group remarkably improved the average daily feed intake of sows and the weight gain of piglets, while significantly decreased the backfat loss, constipation rate of sows and diarrhoea incidence of piglets. The yield and quality of milk of sows in FF group were improved. Besides, faecal acetate and butyrate were promoted in FF group. Additionally, FF increased the level of IgG, IgM and IL-10 and decreased the concentration of TNF-α in serum. Furthermore, FF reduced the abundance of Enterobacteriaceae and increased the level of Lactobacillus and Succiniclasticum, which were remarkably associated with growth performance and serum immune parameters. Accordingly, microbial metabolic functions including DNA repair and recombination proteins, glycolysis and gluconeogenesis, mismatch repair and d -alanine metabolism were significantly upregulated, while amino acid metabolism was downregulated in FF group. Overall, the beneficial effects of FF were superior to PRO treatment. Altogether, administration of FF during lactation improved the performance and immune status, and modulated gut microbiota of sows. Probiotics are not the only one effective compound of FF.     

Inhibition and subsequent rebound of FSH secretion following treatment with bovine follicular fluid in the ewe

Plasma concentrations of gonadotropins were examined after treatment of ewes with bovine follicular fluid (FF) in four experiments. Mean concentrations of FSH were significantly decreased by FF treatment. The FSH depression appeared to continue throughout the length of treatment when the duration of treatment was 2-4 days. However, in an experiment in which the treatment period was 8 days, mean concentrations of FSH initially declined and then returned to control levels during the last 4 days of treatment. In all experiments, a rebound in FSH concentrations was found 24-36 h after cessation of FF treatment. The magnitude of this rebound appeared to be proportional to the degree of FSH suppression during FF treatment.     

The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains

The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.     

油茶肉质果和肉质叶营养成分分析与评价

采用常规生化分析方法,测定了油茶（Camellia oleifera Abel.）肉质果、肉质叶基本营养成分,用原子吸收光谱法测定微量元素含量,用高效液相色谱仪测定维生素C的含量,用氨基酸全自动分析仪测定氨基酸的种类,应用模糊识别法和氨基酸比值系数法对油茶肉质果、肉质叶蛋白质营养价值进行了全面评价,并与12种常见热带、亚热带水果进行对照比较。结果表明油茶肉质果、肉质叶富含糖、酸、蛋白质、氨基酸、脂肪。其微量元素和维生素C含量高于一些常见水果。油茶肉质果、肉质叶的必需氨基酸（EAA）占氨基酸总量（TAA）的37%和35%,其蛋白营养价值要优于12种水果。因此,油茶肉质果、肉质叶的营养价值优于一些常见水果,是一种值得开发利用的优质水果。     

Effect of follicular fluid and oestradiol on the luteinization of rat granulosa cells in vitro.

The effect of pig follicular fluid (FF), total or oestrogen-free, and of oestradiol-17 beta on the luteinization and progesterone secretion of rat granulosa cells was investigated during 4 days in culture. Both FF and oestrogen-free FF modified the differentiation of the granulosa cells, particularly their size and the appearance of their nucleoli. Addition of total FF or oestradiol-17 beta (50, 100 or 500 ng/ml) to the control medium markedly increased the progesterone secretion of the granulosa cells, but oestrogen-free FF and dialysed fetal calf serum had no effect. It was concluded that (1) FF could modify the morphological changes of the rat granulosa cells in vitro, but could not inhibit their secretion of progesterone, (2) the granulosa cells were able to synthesize progesterone regardless of their stage of differentiation, (3) oestradiol 17 beta had a direct stimulatory effect on progesterone secretion by granulosa cells in vitro. The possible mode of action of FF upon luteinization is discussed.     

Maintenance mechanism of polymorphism at the α-Gpdh locus in Drosophila melanogaster

Polymorphism at the alpha-Gpdh locus was studied in Drosophila melanogaster. Using two different lines, one marked by the F allele (FF line) another by the S allele (SS line), four populations were initiated, two in which the initial frequency of F was 0.1 and two in which it was 0.9. They have been observed for 34 generations. From the fifth generation on, the equilibrium frequency in the four cages was about 0.60. Viability has been measured during the evolution of te populations while F frequencies changed and recombinations between the FF and SS lines occurred. It has been evaluated in synthetic populations built with different frequencies: (1) from the original FF and SS lines and (2) from FF and SS lines extracted after 34 generations of joint evolution. In all three cases, the FF viability depended on the frequency of the F allele. The similarity of the three linear regressions implies that alpha-Gpdh locus or other closely linked loci is the target of the selection in the populations analyzed here.     

Molecular Mechanisms of Fenofibrate-Induced Metabolic Catastrophe and Glioblastoma Cell Death

Fenofibrate (FF) is a common lipid-lowering drug and a potent agonist of the peroxisome proliferator-activated receptor alpha (PPARα). FF and several other agonists of PPARα have interesting anticancer properties, and our recent studies demonstrate that FF is very effective against tumor cells of neuroectodermal origin. In spite of these promising anticancer effects, the molecular mechanism(s) of FF-induced tumor cell toxicity remains to be elucidated. Here we report a novel PPARα-independent mechanism explaining FF''s cytotoxicity in vitro and in an intracranial mouse model of glioblastoma. The mechanism involves accumulation of FF in the mitochondrial fraction, followed by immediate impairment of mitochondrial respiration at the level of complex I of the electron transport chain. This mitochondrial action sensitizes tested glioblastoma cells to the PPARα-dependent metabolic switch from glycolysis to fatty acid β-oxidation. As a consequence, prolonged exposure to FF depletes intracellular ATP, activates the AMP-activated protein kinase–mammalian target of rapamycin–autophagy pathway, and results in extensive tumor cell death. Interestingly, autophagy activators attenuate and autophagy inhibitors enhance FF-induced glioblastoma cytotoxicity. Our results explain the molecular basis of FF-induced glioblastoma cytotoxicity and reveal a new supplemental therapeutic approach in which intracranial infusion of FF could selectively trigger metabolic catastrophe in glioblastoma cells.     

In vitro maturation and early developmental capacity of bovine oocytes cultured in pure follicular fluid and supplementation with follicular wall

Mammalian oocytes mature in follicular fluid (FF), surrounded by follicular cells. In the present study, in vitro maturation of bovine oocytes cultured in FF from dominant follicles 15-17mm in diameter (with various forms of heat pretreatment) and supplementation with follicular wall from follicles 3-5mm in diameter (FW1) were examined. Heat pretreatment of FF was as follows: (1) no treatment (FF1); (2) 56 degrees C for 30min (FF2); and (3) 100 degrees C for 20s (FF3). After IVM in FF1, oocytes underwent IVF and IVC and embryo development was assessed (up to the morula stage). The rate of oocyte maturation was decreased in pure FF1 versus control (44.5% versus 62.8%, P<0.001). In the control medium, FW1 did not significantly affect nuclear maturation. By contrast, the addition of FW1 to FF1 increased the rate of matured oocytes approximately two-fold (85.9% versus 45.6%, P<0.001). Furthermore, the maturation rate in the FF+FW1 system declined (from 85.9 to 71.0%, P<0.001), whereas that in the FF system increased (from 45.6 to 71.6%, P<0.001) with increased temperature of the FF treatment. Supplementation of the control medium with FW1 increased the yield of morulae (42.6% versus 13.7%, P<0.001). However, the stimulatory effect of FW1 on the morula rate was much higher in pure FF1 (72.5% versus 31.7%, P<0.001). These findings indicated, for the first time, the stimulatory impact of FW1 on in vitro maturation and early developmental capacity of bovine oocytes cultured in pure FF from dominant follicles. We also inferred that bovine FF constituents affecting bovine oocyte maturation and the meiosis-promoting ability of the FW were heat-labile.