Effects of Neonatal Tactile Stimulation and Maternal Separation on the Anxiety and the Emotional Memory in Adult Female Rats

We investigated the long-lasting effects of early postnatal tactile stimulation （TS） and maternal separation （MS） on the emotional behaviors of adult female rats. A split-litter design was introduced to remove confusing factors such as maternal disturbance. Pups of the non-tactile stimulation （NTS） group did not receive any handling. Pups subjected to the TS treatment were handled and marked for approximately 30 s daily from postnatal days （PND） 2 - 9 or from PND 10 - 17. Pups subjected to the MS treatment were handled and marked in the same way as the TS pups and then individually placed in a cup with familiar nest bedding for 1 h daily. At the age of 3 months, female rats with different neonatal experiences were employed in the light/dark box test and the one-trial passive avoidance response. Both PND 2 - 9 TS and PND 10 - 17 TS groups exhibited more time spent in the illuminated chamber of the light/dark box, and longer step-through latencies in the passive avoidance response when compared to the NTS group, indicating that early life TS treatment reduced novelty-induced anxious emotion and facilitated the retention of emotional memory in adult female rats. No significant effects were found on any behavioral measures between the MS groups and the TS groups, suggesting that neonatal short-time MS treatment was not intensive enough to alter the emotional behaviors, at least in female rats. Infantile age was not an effective factor for these measures. This result supports the hypothesis that neonatal tactile stimulation and maternal separation lead to different effects on the neural development of postnatal pups.

Growth Differentiation Factor-9 Gene Expression of Mice Oocytes in Vitro and in Vivo

Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to explore the relationship between oocyte maturation and GDF-9 gene expression. Preantral follicles of lO-day old mice were isolated from the ovaries and were cultured for 12 days. Oocytes from day 2 （D2）, D4, D6, D8, DIO, D12 cultured in vitro were named the in vitro group and oocytes of day 12 （D12）, D14, D16, D18, D20, D22 grown in vivo were named the in vivo group. Follicle survival, antrum formation and maturation rate were 89.5%, 51.8% and 56.6% respectively in follicles cultured in vitro. After RT-PCR and agarose gel electrophoresis, relative mRNA abundance of GDF-9 was measured in each group of oocytes. At day 8 - 12, the GDF-9 gene expression level of oocytes in vitro was significantly lower than that in vivo （P 〈 0.05）. We conclude that M Ⅱ oocytes can be obtained from in vitro culture of preantral follicles. The GDF- 9 gene expression of oocytes varies at different growth stages in vivo. The low expression of GDF-9 in oocytes cuhured in vitro may be the cause of their low developmental capacity.

Secreted Expression of S-adenosy-L-methionine Synthetase in Pichia pastoris

[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.

Classification of Chinese Traditional Drug-"Beimu" (Bulbus Fritillariae) by Pyrolysis High Resolution Gas Chromatography-Pattern Recognition

The combination of pyrolysis high resolution gas chromatography and pat- tern recognition techniques is a powerful tool for the classification of traditional Chinese drug.A study has been completed on 55 Beimu samples of five different geographic origins: Eastern China.Central China.South-western China,North-western China and North-eastern China.Principal component analysis and SIMCA are applied to effectively classifying the samples according to the origin of the plants.The chemical information contained in the high resolution gas chromatographic data is sufficient to characterize the geographic origin of sam- pies.

Nonlinear Regression Fuzzy Analysis of Real Variable and Fuzzy Parameter

As far as the nonlinear regression method is concerned, the condition when both independent and dependent variable take the Fuzzy value, while the parameter, θ∈ΘR m the real value, have been discussed in . But for most of actual conditions, the independent variable generally takes the real value, while both parameter and dependent variable take the Fuzzy value. This paper propounded a method for the latter and its relevant Fuzzy regreession model. In addition the Fuzzy observation, matrix distribution and the rational estimation of modeling parameter have also been discussed. Furthermore, the Max min estimation of modeling parameter and its corresponding calculating sequence have also been offered to and the calculating example shows the method is feasible.

Isolation and Identification of Saccharomycetes in Wine Grape Region of Chateau Changyu Moser XV

Objective] This study was conducted to further explore the diversity of Saccharomyces cerevisiae_from Chateau Changyu Moser XV and realize better de-velopment and utilization of Saccharomyces cerevisiae resources. [Method] ln this study, the wine grape regions of Chateau Changyu Moser XV were taken as the research object. The Saccharomycetes_in the soil was isolated, screened and ob-served in the natural fermentation process of grape berry epidermis and the fruit. The 32 Saccharomycetes strains were preliminarily classified based on WL nutrient agar, and 26S rDNA D1/D2 sequence analysis was conducted. [Result] Total y, 4 kinds of Saccharomycetes were identified in this study, including Pichia kluyveri, Hanseniaspora uvarum, Saccharomyces cerevisiae_and Cryptococcus magnus. [Con-clusion] The main species of Saccharomycetes in the wine grape region of Chateau Changyu Moser XV were preliminarily determined, which provides theoretical basis and research basis for the brewing of wine with special characteristics.

Pollen Mother Cell Miosis and Male Gametophyte Development of Pumpkin

[Objective] Pollen mother cell miosis and male gametophyte development of pumpkin were observed in this study, to provide some cytological basis for pumpkin anther or microspore culture. [Method] Ehrlich＇s hematoxylin staining-methyl salicylate clearing technique was used for observation and research of the variation of cell structure and chromosomal behavior during pollen mother cell miosis and male gametophyte development of ‘Tianhong＇ pumpkin. [Result] The meiosis in pollen moth- er cells of pumpkin was simultaneous cytokinesis. In the process of nuclear division, nuclear membrane and nucleolus of pumpkin pollen mother cells gradually disappeared in the metaphase I and reappeared in telophase I , phragmoplast formed between the two generated crescent-shaped nuclei without cell wall, the phragmoplast gradually disappeared in the metaphase II and reappeared in telophase II. Phragmoplast spread outward from the center of spindle during the second division was connected with that formed on the central interface of two nuclei during the first division, cell wall of microspores generated from periphery to center. Most of the tetrads contained four sub-cells while a few contained extra small cells. During the period of uniuclete microspore at periphery, the single nucleolus split into 2-3 or more small nucleoli, mature pollen grain was two-celled. Mononucleate pollen cells were mostly appeared in the flower buds with length of 1.0-2.0 cm, which could be used as an important indicator to collect materials for anther or microspore culture. [Conclusion] This study laid the foundation for research of the cytogenetics of pumpkin.

Molecular Identification of Gallibacterium from Hen and Analysis of Its 16S rRNA Gene

Two strains of Gallibacterium, isolated from one laying hen flock in Zhengzhou City of Henan Province, were identified by the morphological observation, genus -specific PCR, and analysis of 16S rRNA gene, which was used to generate the phylogenetic tree, with the 21 members of the 12 genera belonging to Pasteurellaceae to analyze the homology. Two strains were named Yu-ZZ-HL-I-SLG and Yu-ZZ-HL-II-GZ. The comparative result of the 16S rDNA sequence shows that the 2 isolated strains are identical in sequence; the highest identity （99.9%） was observed between the isolated strain and one of the strains of Gallibacterium anatis （AF22.8002）, the homologies between the isolated strain and 3 strains of gallibacterium accessed in NCBI （AF22.8016, Gallibacterium genomosp. 1, AF228017, Gallibacterium genomosp. 2, AF22.8018, Gal- libacterium genomosp. 1 ） were above 97.1%, higher than that of the isolated strain and the other strains of the other 11 genera which were between 90.7% -93.2%. It can be seen from the phylogenetic tree that the 2 isolated strains and the other 4 strain of gallibacterium fell into the same branch, furthermore the 2 isolated strains and the strain of Gallibacterium anatis locate in an internal branch, indicating that the 2 isolated strains belong to Gallibacterium anatis.

Comparative Studies on the Changes of Microtubule Distribution and Reorganization During the Meiotic Stages of Development in Normal (IR36) and a Temperature/photoperiod Sensitive Male Sterile Line (Peiai 64S) of Rice ( Oryza sativa )

Changes in the pattern of organization of microtubules in the meiotic stages of development of pollen (i.e. from pre-meiotic interphase to more or less metaphase I) of a normal (IR36) and a temperature/photoperiod sensitive male sterile line (Peiai 64S) of rice were studied using immunofluorescence confocal microscopy. In IR36, from pre-meiotic interphase to metaphase I, the pattern of microtubule distribution in the meiocytes underwent a series of changes. Some new organizational patterns of microtubules (that have not been described before) were observed during microsporogenesis, including the existence of a broad band of perinuclear microtubules at the diakinesis stage of development. The pattern of microtubule distribution in the meiocytes of the male sterile line, Peiai 64S, was quite different front that seen in IR36. In Peiai 64S, the microtubules showed abnormal patterns of distribution from pre-meiotic interphase to metaphase I. For example the broad band of perinuclear microtubules seen at diakinesis in IR36 was much disorganized and loosened in Peiai 64S. The spindles formed were also very abnormal and different from the normal spindle. The appearance of abnormal microtubule distribution in the early stages of microsporogenesis may contribute to the malformation and ultimate abortion of pollen in Peiai 64S.

Effects of MEK inhibitor U0126 on meiotic progression in mouse oocytes: microtuble organization, asymmetric division and metaphase Ⅱ arrest

In this study we used U0126, a potent and specific inhibitor of MEK, to study the roles of MEK/ERK/p90rsk signaling pathway in the meiotic cell cycle of mouse oocytes. The phosphorylation of MAP kinase and p90rsk in the oocytes treated with 1.5 μMU0126 was the same as that in oocytes cultured in drug-free medium. With 1.5 μM U0126 treatment, the spindles appeared normal as they formed in oocytes, but failed to maintain its structure.Instead, the spindle lost one pole or elongated extraordinarily. After further culture, some oocytes extruded gigantic polar bodies (＞30 μm) that later divided into two small ones. Some oocytes underwent symmetric division and produced two equal-size daughter cells in which normal spindles formed. In oocytes with different division patterns,MAP kinase was normally phosphorylated. When the concentration of U0126 was increased to 15 mM, the phosphorylation of both MAPK and p90rsk were inhibited, while symmetric division was decreased. When incubating in medium containing 15 μM U0126 for 14 h, oocytes were activated, but part of them failed to emit polar bodies. MII oocytes were also activated by 15 μM U0126, at the same time the dephosphorylation of MAP kinase and p90rsk was observed. Our results indicate that 1) MEK plays important but not indispensable roles in microtubule organization;2) MEK keeps normal meiotic spindle morphology, targets peripheral spindle positioning and regulates asymmetric division by activating some unknown substrates other than MAP kinase/p90rsk; and 3) activation of MEK/ERK/p90rsk cascade maintains MII arrest in mouse oocytes.

Mechanism of separating muscovite and quartz by flotation

Flotation experiments were performed to investigate the separation of muscovite and quartz in the presence of dodecylamine(DDA), tallow amine(TTA) and dodecyltrimethylammonium bromide(DTAC). The adsorption mechanisms of these three kinds of amines on muscovite and quartz were studied by FT-IR spectrum analysis, contact angle measurement and molecular dynamics(MD) simulation. The results reveal that the separation of muscovite from quartz is feasible at strong acid pulp condition using amine collectors. TTA and DTAC show poorer collecting ability for flotation of the two minerals compared with DDA. Physical adsorption is found to be the main adsorption module of amine collectors on muscovite and quartz by FT-IR analysis. MD simulation results show a strong physical adsorption ability of DDA+ cation on muscovite and quartz(muscovite(001):-117.31 kJ/mol, quartz(100):-89.43 kJ/mol), while neutral DDA molecular can hardly absorb onto the surface of these two minerals. These findings provide a novel explanation for the flotation mechanism from the perspective of MD simulation.

Selective Affinity Separation of Yeast Alcohol Dehydrogenase by Reverse Micelles with Unbound Triazine Dye

The reversed micelles were formed with cationic cetyltrimethylammonium bromide (CTAB) as surfactant and n-hexanol as cosolvent in the CTAB (50mmol.L-1)/hexanol (15% by volume)/hexane system. Cibacron Blue 3GA (CB) as an affinity ligand in the aqueous phase was directly introduced to the reversed micelles with electrostatic interaction between anionic CB and cationic surfactant. High molecular weight (Mr) protein, yeast alcohol dehydrogenase (YADH, Mr = 141000) from baker's yeast, has been purified using the affinity reversed micelles by the phase transfer method. Various parameters, such as CB concentration, pH and ionic strength, on YADH forward and backward transfer were studied. YADH can be transferred into and out from the reversed micelles under mild conditions (only by regulation of solution pH and salt concentration) with the successful recovery of most YADH activity. Both forward and backward extractions occurred when the aqueous phase pH＞pI with electrostatic attraction between YADH and CTAB. The recovery of YADH activity and purification factor have been improved with addition of a small amount of affinity CB. The recovery of YADH activity obtained was ～99% and the purification factor was about 4.0-fold after one cycle of full forward and backward extraction. The low ionic strength in the initial aqueous phase might be responsible for the YADH transfer into the reversed micellar phase.

A New Pullulan-Producing Yeast and Medium Optimization for Its Exopolysaccharide Production

Yeast strain Y68 producing high level of pullulan was isolated from the phyton collected in Toulouse, France. This strain was identified to be Rhodotorula bacarum by BIOLOG analysis. This is the first report that pullulan was produced by Rhodotorula bacarum. The optimal medium (g L -1) for pullulan production by this strain was 80 glucose, 20 soybean cake hydrolysate, 5 K 2HPO 4, 1 NaCl, 0.2 MgSO 4·7H 2O, 0.6 (NH 4) 2SO 4, pH 7.0. Under this condition, 54 g L -1 pullulan was produced within 60 h at 30 ℃. Pullulan is a better starting material for producing marine prodrugs.

Farmer Participatory Breeding - The Foster Parent in the Breeding of Dioscorea spp. in Ghana

Yam （Dioscorea spp.）, has been aptly described as an orphan crop. It has suffered institutional neglect from time immemorial. Until this study there were no formally released yam varieties in Ghana. The farmer participatory breeding approach was adapted to fast-track the development and release of three new yam varieties in Ghana. This paper uses the breeding process as a case study and conducts a SWOT analysis to provide guidelines for breeding for crops in Low External Input Agriculture. It was faster than conventional breeding in varietal development. Scientific quality of Farmer Participatory Breeding is as good as conventional breeding. Farmer Participatory Breeding is a must for breeding yam in Ghana. Ultimately, three genotypes 2000/001, KUP2000/001 and TDr89/02665 were accepted and released as new varieties ＂CRIKukrupa＂, ＂CRIPona＂ and ＂Mankrong Pona＂ respectively in March 2005. Danger of omission of some promising genotypes in the course of varietal development and danger of opinion group cabalizing the group discussions were identified as potential threats to the approach. Effective research-extension-farmer linkage was identified as extremely vital to the success of the approach. Farmer participatory breeding process was also sustainable and more efficient than conventional breeding in varietal selection in yam breeding.

Optimization of Culture Components for Laccase Activity from Pleurotus Ostreatus P40 Using Response Surface Methodology

High enzymatic activity is required for laccase applications.Central composite design （CCD）-based response surface methodology （RSM） can effectively increase the enzymatic activity of Pleurotus ostreatus P40 in liquid substrate fermentation.Initial screening of the nutritional components was performed using a Plackett-Burman design.The variables,namely,bran,bagasse,Tween 80,and yeast extract,were found to have statistically significant effects on laccase activity.These variables were further optimized using CCD-based RSM.Optimal concentrations for the maximum laccase activity were 8.144 2 g/L bran,50 g/L bagasse,0.424 1 mL/L Tween 80,and 2.832 5 g/L yeast extract.Under optimized conditions,the maximum measured laccase activity reached 96 480 U/L,which was close to the predicted value （104 830 U/L） by RSM.Therefore,RSM can be used to optimize culture components for laccase activity from Pieurotus ostreatus P40.

Studies on the Chemical Constituents of Four Species of Fritillaria Plants

The bulbs of Fritillaria spp.(Liliaceae)are used in Chinese medicine as antipyretic,anti-tussive,expectorant,etc.In order to search for the bioactive constituents from this genus,thebulbs of four species of medicinal Fritillaria plants have been investigated.Thirteen compoundswere isolated from Fritillaria thunbergii Miq.,three of them were new compounds.Fourteencompounds from Fritillaria thunbergii var.chekiangensis Hsiao et K.C.Hsia collected inDongyang of Zhejiang Province,four of them are new compounds and another one was first iso-

Evaluation of the Nutritional Supplement VIUSID Vet Powder on the Productive Behaviour of Sows and Boars

The objective of this research was to evaluate the effect of administering VIUSID vet powder on the productive behaviour of sows and the quality of semen of boars. Two experiments were conducted on farms in the province of Sancti Spiritus, in the central region of Cuba. One was with sows, starting 21 days before parturition until weaning, and another was with boars in directed mounting. Each sow received 10 grams of VIUSID vet powder everyday, mixed with food, from 21 days before the probable date of farrowing and until weaning （33-day lactation period）. In the boars, the treatment consisted of administering 10 grams of VIUSID vet powder with feed, once a day for 6 weeks （week 1 to 6）; from week 7 onwards, the supplement was suspended. Results showed that, supplying VIUSID vet powder significantly improved the principal post-partum productivity indicators and significantly reduced diarrhoea and administration to boars in production for 6 weeks, which has improved spermatic motility and does not affect the other variables representative of the quality of the semen.

Occurrence and Antibiotic Susceptibility Profile of Streptococcus spp. Isolated from Ewe＇s Milk

The purpose of this study was to determine the occurrence and antimicrobial resistance of Streptococcus spp. isolated from fresh ewe＇s milk and consequently to draw conclusions concerning the health condition of the animal flocks and the current trends of antibiotic use in small ruminants. A total of 77 mountainous sheepfolds were investigated representing 10% of all transhumant sheep and goats flocks in the area. Duplicate milk samples were collected from January to February transferred to the laboratory and analyzed for the presence of Streptococcus spp. by using the serial dilution method and plating on the surface of Slanetz and Bartley agar. The isolates were tested against nine antimicrobial agents with the Kirby-Bauer disk diffusion method. The results showed that Streptococcus spp. occurred in 84.4% of the samples and in counts ranged between 1.8 log CFU/mL and 4.6 log CFU/mL. Concerning susceptibility, only 1.3% of the isolates were resistant to all antimicrobials while 27.3% were fully susceptible. Resistance to cefepime was the most common （93.1%） observation followed by resistance to ampicillin （82.1%）, ciprofloxacine （58.3%） and meropenem （51.9%）. The isolates were vancomycin susceptible at a rate of 83.6%. Multidrug resistance to at least three antibiotics was observed to be 22.1% of Streptococcus spp.. Given the prevalence of Streptococcus spp. and the elevated resistance of isolates to various antibiotics it is concluded that further examinations and even interventions are required in order to pertain the appropriate animal health and milk quality.

Comparison of Two Different Protocols for Induction of Parturition in Heifers with or without Estradiol Benzoate

Objectives of this study were to investigate the efficacy of dexamethasone in combination with estradiol benzoate in controlled induction of parturition in heifers, especially in the subjects of retained fetal membranes and dystocia caused by fetal oversize. A total number of 100 Holstein-Friesian heifers aged 24-25 months, mean weight 450 kg and body condition score of 3-4 from a dairy herd located in the suburb of Tabriz with similar nutrition and management systems were allocated at random into two groups. Group A （Control, n = 50） heifers, after passing the minimum 270 d of pregnancy were injected with 30 mg dexamethasone IM. Group B （Treatment, n = 50） heifers with the same period of pregnancy received 30 mg dexamethasone plus 20 mg estradiol benzoate IM on the same days of pregnancy. The overall durations of initial treatments to induction of parturition were （41.50 ~ 2.65） h in group A and （37.50 ：i： 1.27） h in group B. In group A, more dystocia cases were observed than in group B. After parturition, group A showed a higher percentage of retention of fetal membranes as well as the calf mortality and dystocia compared to group B. The differences between two groups were statistically significant （P 〈 0.05）. In conclusion, our results indicate that induction of parturition by estradiol benzoate and dexamethasone together will be more effective than dexamethasone alone because of the less retention of fetal membranes, easy calving and shorter time from induction to parturition.

Role of animal pole protuberance and microtubules during meiosis in sea cucumber Apostichopus japonicus oocytes

Fully grown oocytes of Apostichopus japonicus have a cytoplasmic protuberance where the oocyte attaches to the follicle. The protuberance and the oolamina located on the opposite side of the oocyte indicate the animal-vegetal axis. Two pre-meiotic centrosomes are anchored to the protuberance by microtubules between centrosomes and protuberance. After meiosis reinitiation induced by DTT solution, the germinal vesicle (GV) migrates towards the protuberance. The GV breaks down after it migrates to the oocyte membrane on the protuberance side. The protuberance then contracts back into the oocyte and the first polar body extrudes from the site of the former protuberance. The second polar body forms beneath the first. Thus the oocyte protuberance indicates the presumptive animal pole well before maturation of the oocyte.