Solution structure and RNA interactions of the RNA recognition motif from eukaryotic translation initiation factor 4B

Eukaryotic initiation factor 4B (eIF4B) is a multidomain protein with a range of activities that serves primarily to promote association of messenger RNA to the 40S ribosomal subunit during translation initiation. We report here the solution structure of the eIF4B RNA recognition motif (RRM) domain. It adopts a classical RRM fold, with a beta alpha beta beta alpha beta topology. The most striking difference with other RRM structures is in the disposition of loop 3, which connects the beta 2 and beta 3 strands and is implicated in RNA recognition. This loop folds down against the body of the RRM and exhibits restricted motion on a milli- to microsecond time scale. Although it contributes to a large basic patch on the RNA binding surface, it does not protrude out from the domain as observed in other RRM structures, possibly implying a different mode of RNA binding. On its own, the core RRM domain provides only a relative weak interaction with RNA targets and appears to require extensions at the N- and C-terminus for high-affinity binding.     

Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold

Polypyrimidine tract binding protein (PTB), an RNA binding protein containing four RNA recognition motifs (RRMs), is involved in both pre-mRNA splicing and translation initiation directed by picornaviral internal ribosome entry sites. Sequence comparisons previously indicated that PTB is a non-canonical RRM protein. The solution structure of a PTB fragment containing RRMs 3 and 4 shows that the protein consists of two domains connected by a long, flexible linker. The two domains tumble independently in solution, having no fixed relative orientation. In addition to the betaalphabetabetaalphabeta topology, which is characteristic of RRM domains, the C-terminal extension of PTB RRM-3 incorporates an unanticipated fifth beta-strand, which extends the RNA binding surface. The long, disordered polypeptide connecting beta4 and beta5 in RRM-3 is poised above the RNA binding surface and is likely to contribute to RNA recognition. Mutational analyses show that both RRM-3 and RRM-4 contribute to RNA binding specificity and that, despite its unusual sequence, PTB binds RNA in a manner akin to that of other RRM proteins.     

Knowledge,Practices, and Attitudes of Emergency Contraception among Female University Students in KwaZulu-Natal,South Africa

Objective

The purpose of this study is to investigate the knowledge, practices, and attitudes among female university students in South Africa regarding emergency contraceptives (EC).

Methods

A cross-sectional study was conducted among 582 female university students who were selected using multi-stage sampling techniques. Multivariate logistic regression analysis was used to find significant predictors for EC awareness.

Results

The average age of the female students was 20.9 years (SD = 3.0) and 57.2% were presently sexually active. Overall, 49.8% of the participants reported having heard about EC prior to the study. Regarding sexual activities among the female students, 53.2% reported to have sex, and 21.2% of the sexually experienced students used EC prior to the study. Regarding the effectiveness of EC, 29.5% students said it could be used up to 72 hours after unprotected sexual intercourse, and 8% said it could be used just before sex. About two-thirds (61.8%) would recommend the use of EC and 63.2% would use it if they needed. The multivariate analysis indicated that students who were older (>20 years), presently sexually active, and living with their parents were more likely to be aware of EC (p<0.05).

Conclusion

The students’ knowledge and utilization of EC were low. Health education and promotion should be targeted towards these students, and the EC services should be offered on campus.     

Akt‐ and Erk‐mediated regulation of proliferation and differentiation during PDGFRβ‐induced MSC self‐renewal

Understanding the mechanisms that direct mesenchymal stem cell (MSC) self‐renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet‐derived growth factor receptor β (PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF‐BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl‐2′‐deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT‐PCR, and by long‐term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR‐β expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E‐BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ‐induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self‐renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self‐renewal strategies.     

Follicle and oocyte morphology in ewes after treatment with insulin in the late follicular phase

Stress reduces fertility in ruminants. Various experimental models, such as insulin-induced hypoglycaemia, have been used to investigate the mechanisms involved, and have revealed abnormal LH profiles (both pulse and surge secretion). This disruption affects follicular function and it is proposed there may be negative consequences on subsequent oocyte morphology. Insulin (5 iu/kg), administered to ewes in the late follicular phase, induced hypoglycemia for 10 h, decreased estradiol concentrations for 8-12 h and delayed the LH surge by 15 h. Although the diameters of dominant follicles just before ovulation were not affected, granulosa cells had fewer pycnotic nuclei, less apoptosis and increased proliferation 16-17 h after the LH surge. Nevertheless, we did not observe gross ultra-structural differences in nuclear, cytoplasmic or cumulus maturity between oocytes from insulin-treated and control animals. This suggests that reduced LH pulsatility and a delay in the LH surge may only produce very subtle changes in gross oocyte morphology, imperceptible by electron microscopy.     

Impact of buserelin acetate or hCG administration on day 12 post-ovulation on subsequent luteal profile and conception rate in buffalo (Bubalus bubalis)

The present study investigated the impact of gonadotropic hormone administration on day 12 post-ovulation on subsequent luteal profile and conception rate in buffaloes. All the buffaloes (n = 48) were estrus synchronized by a synthetic analogue of prostaglandin F_2α (PGF_2α), administered 11 days apart, followed by insemination during mid to late estrus. To examine the effect of mid-luteal phase hormonal treatment, buffaloes were randomly divided into control (normal saline, n = 14), d12-BA (buserelin acetate, 20 μg, n = 17) and d12-hCG (hCG, 3000 IU, n = 17) groups. Ovaries were scanned on the day of induced estrus to measure the preovulatory follicle (POF) diameter and on days 5, 12, 16 and 21 post-ovulation to examine the alterations in corpus luteum (CL) diameter. On the day of each sonography, blood samples were collected for the estimation of plasma progesterone. In treatment groups, luteal profile (CL diameter and plasma progesterone) on day 16–21 post-ovulation was better (P < 0.05) as well as first service conception rate was higher (52.9% in each treatment group vs. 28.6%, P > 0.05) compared to controls. All the pregnant buffaloes exhibited higher (P < 0.05) plasma progesterone on various post-ovulation days than their respective non-pregnant counterparts. Treatment-induced accessory corpus luteum (ACL) formation was observed in 58.8 per cent and 70.6 per cent buffaloes of d12-BA and d12-hCG group, respectively, that also had higher (P < 0.05) plasma progesterone compared to controls. Compared to the spontaneous CL, the diameter of ACL was less (P < 0.05) in the treatment groups. In conclusion, buserelin acetate and hCG administration on day 12 post-ovulation leads to accessory CL formation, improves luteal profile and consequently increases conception rate in buffaloes.     

Crystal structural analysis of human serum albumin complexed with hemin and fatty acid

Background

Human serum albumin (HSA) is an abundant plasma protein that binds a wide variety of hydrophobic ligands including fatty acids, bilirubin, thyroxine and hemin. Although HSA-heme complexes do not bind oxygen reversibly, it may be possible to develop modified HSA proteins or heme groups that will confer this ability on the complex.

Results

We present here the crystal structure of a ternary HSA-hemin-myristate complex, formed at a 1:1:4 molar ratio, that contains a single hemin group bound to subdomain IB and myristate bound at six sites. The complex displays a conformation that is intermediate between defatted HSA and HSA-fatty acid complexes; this is likely to be due to low myristate occupancy in the fatty acid binding sites that drive the conformational change. The hemin group is bound within a narrow D-shaped hydrophobic cavity which usually accommodates fatty acid; the hemin propionate groups are coordinated by a triad of basic residues at the pocket entrance. The iron atom in the centre of the hemin is coordinated by Tyr161.

Conclusion

The structure of the HSA-hemin-myristate complex (PDB ID 1o9x) reveals the key polar and hydrophobic interactions that determine the hemin-binding specificity of HSA. The details of the hemin-binding environment of HSA provide a structural foundation for efforts to modify the protein and/or the heme molecule in order to engineer complexes that have favourable oxygen-binding properties.

     

Impact of pre-ovulatory follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in buffalo (Bubalus bubalis)

The present study was designed to investigate the impact of pre-ovulatory follicle (POF) diameter on the day of estrus on plasma estradiol concentration, subsequent luteal profile (corpus luteum, CL, diameter and plasma progesterone concentration) and conception rate in buffaloes. Twenty-eight buffaloes were synchronized with synthetic analogue of prostaglandin F(2α) (PGF(2α)) administered 11 days apart. Transrectal ultrasonography and jugular vein blood sampling was carried out on the day of estrus and on days 0 (day of ovulation), 5, 12, 16 and 21 post-ovulation. Out of 28 buffaloes, 11 (39.3%) were diagnosed pregnant on day 40 post-ovulation. Retrospective analysis revealed that the buffaloes getting pregnant had larger (p<0.05) POF diameter. In fact, all the buffaloes (n=5/5) having POF diameter between >14 and 16 mm conceived, whereas, conception rate in buffaloes with POF diameter between >12 and ≤14 mm (n=6/17) or <12 mm (n=0/6) was 35.3% and 0.0%, respectively. A positive correlation (r=0.57, p<0.05) was observed between POF diameter and plasma estradiol concentration at estrus. On day 5 post-ovulation, luteal profile was positively correlated (CL: r=0.34, p<0.05; plasma progesterone concentration: r=0.27, p>0.05) with POF diameter. Further, on the same day, plasma progesterone concentration was positively correlated (r=0.47, p<0.05) with CL diameter, however, this correlation was absent (r=0.05, p>0.05) during the subsequent luteal phase. Nevertheless, the post-ovulation luteal profile of pregnant buffaloes was higher (p<0.05) compared to non-pregnant counterparts. In conclusion, the diameter of POF in buffaloes has positive impact on plasma estradiol concentration at estrus, post-ovulation luteal profile and conception rate. The diameter of CL can be used as an indicator of luteal function at early but not at mid or late luteal phase of estrus cycle in buffaloes.