Supplementation with vascular endothelial growth factor during in vitro maturation of porcine cumulus oocyte complexes and subsequent developmental competence after in vitro fertilization

The aim of the present study was to investigate whether the effects of vascular endothelial growth factor (VEGF) on porcine cumulus oocyte complexes (COCs) and subsequent blastocyst formation following in vitro fertilization are attributable to improved fertilization and cytoplasmic maturation. Porcine COCs were cultured for 42 h in TCM199 medium with 5 ng/mL human recombinant VEGF, and the resultant metaphase II oocytes were fertilized in vitro. COCs without VEGF supplementation served controls. Supplementation with VEGF during in vitro maturation (IVM) significantly (P < 0.05) improved the blastocyst formation rate and total cell number (46.7 ± 3.1% and 82.8 ± 6.7, respectively) compared with controls (32.5 ± 3.4% and 64.1 ± 5.6, respectively). On day 2, the percentage of four-cell stage embryos was significantly higher in the VEGF-matured group (49.1 ± 2.7%) than in the control (33.1 ± 5.8%), and the percentage of two-cell stage embryos was significantly higher in the control group (10.4 ± 1.4%) than in the VEGF-matured group (6.6 ± 0.9%). At 10 h after the onset of in vitro fertilization (IVF), oocytes with two pronuclei were considered as monospermically or normally fertilized, and oocytes with more than two pronuclei were considered as polyspermically fertilized. Monospermy was significantly higher in VEGF-matured oocytes (47.2 ± 4.3%) than in the control (20.0 ± 2.4%), and polyspermy and sperm penetration per oocyte were significantly higher in the control group (54.4 ± 3.8% and 2.3 ± 0.1, respectively) than in the VEGF-matured oocytes (43.9 ± 3.6% and 1.8 ± 0.1, respectively). Supplementation with VEGF during IVM significantly (P < 0.05) improved male pronuclear formation as compared with the control (91.1 ± 1.9 vs 74.4 ± 3.8%). Type III cortical granule distribution in oocytes was more common in VEGF-matured oocytes (78.0%) than in the control (52.1%). These results suggest that VEGF supplementation during IVM enhanced the developmental potential of porcine IVF embryos through higher male pronuclear formation and higher monospermic fertilization rates as a consequence of improved cytoplasmic maturation.     

Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif

The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C′/D′ RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C′/D′ RNP despite its inability to bind the K-loop, thus indicating the importance of protein–protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs.     

Expression of gonococcal transferrin-binding protein 1 causes Escherichia coli to bind human transferrin.

The gene for gonococcal transferrin-binding protein 1 (TBP1) was cloned behind an inducible promoter in Escherichia coli. The resultant strain was capable of binding human transferrin with the same specificity as that of the gonococcus. E. coli expressing TBP1 did not internalize transferrin-bound iron or grow on transferrin as a sole iron source.     

Modelling and Characterization of Glial Fibrillary Acidic Protein

Glial Fibrillary Acidic Protein (GFAP) is an intermediate-filament (IF) protein that maintains the astrocytes of the Central Nervous System in Human. This is differentially expressed during serological studies in inflamed condition such as Rheumatoid Arthritis (RA). Therefore, it is of interest to glean molecular insight using a model of GFAP (49.88 kDa) due to its crystallographic nonavailability. The present study has been taken into consideration to construct computational protein model using Modeller 9.11. The structural relevance of the protein was verified using Gromacs 4.5 followed by validation through PROCHECK, Verify 3D, WHAT-IF, ERRAT and PROVE for reliability. The constructed three dimensional (3D) model of GFAP protein had been scrutinized to reveal the associated functions by identifying ligand binding sites and active sites. Molecular level interaction study revealed five possible surface cavities as active sites. The model finds application in further computational analysis towards drug discovery in order to minimize the effect of inflammation.     

The type III secretion system needle,tip, and translocon

Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.     

Impact of hyperglycemia on the expression of GLUT1 during oral carcinogenesis in rats

Molecular Biology Reports - On the background of the epidemiological link between diabetes and oral cancer, the present study aimed to analyze the potential involvement of selected glucose...     

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter,ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.