Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.     

Administration of recombinant bovine somatotropin (rbST) at the time of breeding in superovulated fertile and subfertile ewes

Two experiments were conducted to evaluate the effects of the administration of 125 mg rbST at the time of breeding on fertile and subfertile superovulated ewes – in terms of the ovulation rate, embryo yield and embryonic development. In addition, the effects of the treatment on the plasma progesterone concentrations and interferon-tau (IFNT) expression in the embryos were evaluated in the fertile animals. In the first experiment, carried out on fertile superovulated ewes, the treatment significantly increased the ovulation rate (p < 0.05) and shifted the embryo distribution towards more advanced stages of development (p < 0.01). Plasma progesterone concentrations increased faster in the treated ewes, being significantly different (p < 0.05) between groups at day 6 post-breeding. In the second experiment, embryonic development was more advanced in fertile, but not in subfertile superovulated ewes. The expression of IFNT was significantly (p < 0.01) decreased in blastocyst embryos obtained from rbST treated animals, compared with non-treated ewes. It is concluded that the administration of rbST to subfertile ewes at the time of breeding does not stimulate embryonic development as it does when administered to fertile animals.     

Environmental enrichment exerts anxiolytic effects in the Indian field mouse (Mus booduga)

Environmental enrichment (EE) is known to have behavioral and physiological anxiolytic effects in several animal models. However, it is as yet unclear how EE modulates behavior of wild animals and the underlying molecular mechanisms. The adult male field mouse Mus booduga (n = 42) captured at agricultural field, were housed in non-enriched standard condition (SC) for 7 days and considered as directly from wild (DW). Another two groups of mice were housed in either EE or SC for 30 days. Behavioral testing was carried out to assess their anxiety-like behavior in the elevated plus-maze (EPM). We found that on EPM, mice housed in EE display less anxiety like behavior when compared to mice housed in SC. Exposure to plus-maze did not increase the levels of corticosterone (CORT) in prefrontal cortex (PFC) and circulating CORT, and adrenocorticotropic hormone (ACTH) in the mice housed in EE but not in the mice housed in SC. We observed a trend in the EE induced inhibition of expression of corticotropin releasing hormone (CRH), glucocorticoid receptor (GR), E2 ubiquitin-conjugating enzyme (Ubc9) and steroid receptor coactivator-1 (SRC-1) mRNA levels, which are all known to be involved in the stress response signaling pathway. Our study suggests that EE exerts therapeutic and anxiolytic effects against stressors.     

Improving d-glucaric acid production from myo-inositol in E. coli by increasing MIOX stability and myo-inositol transport

d-glucaric acid has been explored for a myriad of potential uses, including biopolymer production and cancer treatment. A biosynthetic route to produce d-glucaric acid from glucose has been constructed in Escherichia coli (Moon et al., 2009b), and analysis of the pathway revealed myo-inositol oxygenase (MIOX) to be the least active enzyme. To increase pathway productivity, we explored protein fusion tags for increased MIOX solubility and directed evolution for increased MIOX activity. An N-terminal SUMO fusion to MIOX resulted in a 75% increase in d-glucaric acid production from myo-inositol. While our directed evolution efforts did not yield an improved MIOX variant, our screen isolated a 941 bp DNA fragment whose expression led to increased myo-inositol transport and a 65% increase in d-glucaric acid production from myo-inositol. Overall, we report the production of up to 4.85 g/L of d-glucaric acid from 10.8 g/L myo-inositol in recombinant E. coli.     

Mitochondrial response in the apical and lateral flower buds of the Hanfu apple to cold stress during the dormancy stage

In order to study the different physiological bases of cold tolerance in the apical flower buds (AFB) and the lateral flower buds (LFB) of the Hanfu apple (Malus domestica Borkh), we used 4-year-old grafted Hanfu plants as material and evaluated the physiological characteristics of mitochondria in the flower buds, such as electron transport chains (cytochrome pathway and alternative pathway), H₂O₂ content, mitochondrial membrane permeability transition (mPT), and MDA content. AFBs and LFBs showed different changes in total respiratory rate (V_t) during low-temperature stress, except that both reached the lowest V_ts at ?30 °C. The AFB V_t increased to a peak at ?25 °C and decreased sharply to its minimal value at ?30 °C, and then remained relatively low. In contrast, the LFB V_t decreased to its minimal value at ?30 °C and increased sharply to a peak at ?35 °C and then decreased again. In both AFBs and LFBs, the cytochrome pathway was still the main electron transport chain throughout the whole process, and the contributions of the cytochrome pathway (ρV_cyt/V_t) and of the alternative pathway (ρV_alt/V_t) showed similar tendencies to those of V_t as temperature changed. Changes in the AFB mPT were different from those of AFB V_t. LFB mPT zigzagged from peaks at ?25 °C and 35 °C. The H₂O₂ content of the LFBs increased from ?10 °C to ?30 °C, then decreased slightly from ?30 °C to ?35 °C, and then increased again. H₂O₂ content in AFBs went up steadily throughout the whole process. During the early stage of low-temperature treatment, before temperatures reached ?35 °C, LFB MDA content remained relatively low and later increased. MDA content in AFBs began to increase from the beginning of treatment. It can be concluded that the higher cold tolerance of LFBs relative to AFBs could be closely related to their higher V_t and ρV_alt/V_t, which may aid adaptations to stress by supplying energy and metabolic substrates under low-temperature stress conditions.     

Nephrotoxicity assessments of acetaminophen during zebrafish embryogenesis

We used a green fluorescent kidney line, Tg(wt1b:GFP), as a model to access the acetaminophen (AAP)-induced nephrotoxicity dynamically. Zebrafish (Danio rerio) embryos at different developmental stages (12–60 hpf) were treated with different dosages of AAP (0–45 mM) for different time courses (12–60 h). Results showed that zebrafish embryos exhibited no evident differences in survival rates and morphological changes between the mock-treated control (0 mM) and 2.25 mM AAP-exposure (12–72 hpf) groups. In contrast, after higher doses (22.5 and 45 mM) of exposure, embryos displayed malformed kidney phenotypes, such as curved, cystic pronephric tube, pronephric duct, and a cystic and atrophic glomerulus. The percentages of embryos with malformed kidney phenotypes increased as the exposure dosages of AAP increased. Interestingly, under the same exposure time course (12 h) and dose (22.5 mM), embryos displayed higher percentages of severe defects at earlier developmental stage of exposure (12–24 hpf), whereas embryos displayed higher percentages of mild defects at later exposure (60–72 hpf). With an exposure time course less than 24 h of 45 mM AAP, no embryo survived by the developmental stage of 72 hpf. These results indicated that AAP-induced nephrotoxicity depended on the exposure dose, time course and developmental stages. Immunohistochemical experiments showed that the cells' morphologies of the pronephric tube, pronephric duct and glomerulus were disrupted by AAP, and consequently caused cell death. Real-time RT-PCR revealed embryos after AAP treatment decreased the expression of cox2 and bcl2, but increased p53 expression. In conclusion, AAP-induced defects on glomerulus, pronephric tube and pronephric duct could be easily and dynamically observed in vivo during kidney development in this present model.     

Vitellogenesis in Bufo arenarum: Identification,characterization and immunolocalization of high molecular mass lipovitellin during oogenesis

Vitellogenin (Vtg), a large lipoglycophosphoprotein, is the most important precursor of the yolk proteins, and the major source of nutrients for the developing embryo in oviparous species. After its uptake by the oocytes, Vtg is converted into lipovitellins (high and light) and phosvitin, which are deposited into crystalline yolk platelets. We describe here the presence of two high molecular mass lipovitellin isoforms in Bufo arenarum mature oocytes with masses of 113 and 100 kDa, respectively. The amino acid sequence analysis of p113 and p100 peptides showed a high sequence homology between both polypeptides and the complete reported sequences of Xenopus laevis vitellogenin. Using specific antibodies, we determined that the Vtg uptake begins early during oogenesis, at the previtellogenic stage, and continues until oocytes have reached their mature status. In addition, we found that large endocytic vesicles mediate Vtg uptake in stage I oocytes, and that the size of the endocytic vesicles declines with oogenesis progression. In terms of the Vtg protein trafficking, we detected the Vtg precursor (190 kDa) in the liver of estradiol-injected females. Finally, we propose a subclassification of B. arenarum stage II oocytes into three physiologically and morphologically distinct periods (early, mid and late).     

Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 °C while the other was transported at 37 °C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 °C in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto–orcein staining.Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI + MII) than the oocytes from anestrual ovaries in the 37 °C group (p < 0.05). However, oocytes harvested from anestrual ovaries transported at 4 °C had the highest maturation (MI + MII) rate, and the difference between anestrual and luteal ovary groups was significant (p < 0.05). The oocytes from anestrual ovaries transported at 4 °C have significantly higher maturation rates than those transported at 37 °C (p < 0.0001). However, the transport temperature (37 or 4 °C) did not significantly affect the maturation (MI + MII) rates of oocytes harvested from the luteal (p = 0.61) and follicular (p = 0.48) stage ovaries.It can be concluded from this study that (1) both transport temperature and transport temperature × estrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 °C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries.     

CEP131 indicates poor prognosis and promotes cell proliferation and migration in hepatocellular carcinoma

Centrosomal proteins have been implicated in the progression of human diseases. CEP131 plays important roles in centrosome duplication and genome stability, but its role in cancers remains largely unknown. Here, we showed that CEP131 expression was increased in hepatocellular carcinoma (HCC), compared to the paracarcinoma tissues, at both mRNA and protein levels. High CEP131 expression was closely associated with tumor size (P = 0.020), tumor capsule (P = 0.043), TNM stage (P = 0.007) and tumor differentiation (P = 0.019). Furthermore, patients with high expression of CEP131 were accompanied with worse overall and disease-free survivals in our and TCGA cohorts consisting of a total of 802 cases. The prognostic value of CEP131 was further confirmed by stratified survival analysis. Multivariate cox regression model indicated that CEP131 was an independent factor for overall survival (hazard ratio = 1.762, 95% confident interval: 1.443–2.151, P < 0.001). In vitro data demonstrated that nucleophosmin (NPM) physically bound to CEP131 and maintained its protein stability. Overexpression of CEP131 in HCC cell lines enhanced cell proliferation and migration, whereas the knockdown of CEP131 led to the opposite phenotypes. Further studies demonstrated that CEP131 exhibited oncogenic activity via activation of PI3K/AKT signaling pathway. Taken together, our findings suggest CEP131 serves as a potential prognostic biomarker in HCC, and functions as an oncogene in this deadly disease.     

TnaA,an SP-RING Protein,Interacts with Osa,a Subunit of the Chromatin Remodeling Complex BRAHMA and with the SUMOylation Pathway in Drosophila melanogaster

Tonalli A (TnaA) is a Drosophila melanogaster protein with an XSPRING domain. The XSPRING domain harbors an SP-RING zinc-finger, which is characteristic of proteins with SUMO E3 ligase activity. TnaA is required for homeotic gene expression and is presumably involved in the SUMOylation pathway. Here we analyzed some aspects of the TnaA location in embryo and larval stages and its genetic and biochemical interaction with SUMOylation pathway proteins. We describe that there are at least two TnaA proteins (TnaA₁₃₀ and TnaA₁₂₃) differentially expressed throughout development. We show that TnaA is chromatin-associated at discrete sites on polytene salivary gland chromosomes of third instar larvae and that tna mutant individuals do not survive to adulthood, with most dying as third instar larvae or pupae. The tna mutants that ultimately die as third instar larvae have an extended life span of at least 4 to 15 days as other SUMOylation pathway mutants. We show that TnaA physically interacts with the SUMO E2 conjugating enzyme Ubc9, and with the BRM complex subunit Osa. Furthermore, we show that tna and osa interact genetically with SUMOylation pathway components and individuals carrying mutations for these genes show a phenotype that can be the consequence of misexpression of developmental-related genes.     

Nonmuscle myosin II-B (myh10) expression analysis during zebrafish embryonic development

Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes (myh9, myh10, and myh14) encoding different nonmuscle myosin heavy chains. The three NM II isoforms share a very similar molecular structure and play important roles in a variety of fundamental biological processes. NM II-B (myh10) has been shown to be essential for the formation of mouse neural system and heart. But so far the complete knowledge for its expression in developing zebrafish embryos is lacking. In current study, we proved the conservation of zebrafish NM II-B in vertebrate evolution by in silicon analysis. Afterwards the NM II-B (myh10) expression was demonstrated to initiate after gastrulation stage. At 20 hpf, the expression is mainly restricted in central nervous system (CNS). It was maintained and expanded to sensor organ including eye, otic vesicle, and olfactory bulb at 36 hpf and later. We also detected myh10 mRNA hybridization signal in 48 hpf zebrafish heart. In addition, we investigated myh9a and myh9b mRNA distribution in zebrafish developing embryos. It was shown that myh10 and myh9 have distinct expression pattern, with myh9s not in neural system but in epidermis, enveloping layer (EVL). Our study provides new insight into the NM II expression and the use of this model organism to tackle future studies on the role of NM II in embryo development.     

Identification of pre-zygotic reproductive and morphological barriers limiting controlled crossed seed production of triploid Acacia mearnsii

A study of the reproductive biology of black wattle (Acacia mearnsii de Wild) was conducted, specifically with regard to the compatibility of the diploid × tetraploid cross to produce a triploid variety, together with identifying ways of increasing triploid seed production. Fluorescent microscopy was used to determine pollination and fertilisation rates and to identify any pre-zygotic barriers at the stigma, style and ovary. Both diploid and tetraploid families were tested as maternal parents to establish if this was imperative to producing a triploid. Morphological measurements were documented in order to determine any incompatibilities in the cross to produce a triploid. The in vivo results showed that successful fertilisation of the ovary was possible whether one used a diploid or tetraploid maternal parent. When the maternal parent was a tetraploid, however, the pollination rate (polyads adhering to the stigma) and ovary fertilisation rates were significantly (p < 0.05) greater. Morphological measurements and observations also revealed that tetraploid floral parts were significantly (p < 0.05) larger than the diploids. The morphological size differences between the diploid and tetraploid polyads and pistils did not appear to influence the fertilisation of the ovaries and thus did not pose any identifiable barrier in the cross to produce a triploid. When considering the results from the cross to produce a triploid (2n × 4n or 4n × 2n), the diploid polyads were significantly (p < 0.05) more vigorous and suitable in fertilising the tetraploid ovaries as opposed to the reverse. Possible pre-zygotic barriers at the stigma, style or ovary were investigated and the only area that could be identified limiting seed production was within the ovary.