The distribution and requirements of microtubules and microfilaments in bovine oocytes during in vitro maturation

Microtubules and microfilaments are major cytoskeletal components and important modulators for chromosomal movement and cellular division in mammalian oocytes. In this study we observed microtubule and microfilament organisation in bovine oocytes by laser scanning confocal microscopy, and determined requirements of their assembly during in vitro maturation. After germinal vesicle breakdown, small microtubular asters were observed near the condensed chromatin. The asters appeared to elongate and encompass condensed chromatin particles. At the metaphase stage, microtubules were observed in the second meiotic spindle at the metaphase stage. The meiotic spindle was a symmetrical, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Treatment with nocodazole did not inhibit germinal vesicle breakdown. However, progression to metaphase failed to occur in oocytes treated with nocodazole. In contrast, microfilaments were observed as a relatively thick uniform area around the cell cortex and overlying chromatin following germinal vesicle breakdown. Treatment with cytochalasin B inhibited microfilament polymerisation but did not prevent either germinal vesicle breakdown or metaphase formation. However, movement of chromatin to the proper position was inhibited in oocytes treated with cytochalasin B. These results suggest that both microtubules and microfilaments are closely associated with reconstruction and proper positioning of chromatin during meiotic maturation in bovine oocytes.     

Effect of dietary Platycodon grandiflorum on the improvement of insulin resistance in obese Zucker rats

The effect of dietary Platycodon grandiflorum on the improvement of insulin resistance and lipid profile was investigated in lean (Fa/-) and obese (fa/fa) Zucker rats, a model for noninsulin dependent diabetes mellitus. Dietary Platycodon grandiflorum feeding for 4 weeks resulted in a significant decrease in the concentration of plasma triglyceride in both lean and obese Zucker rats. Furthermore, dietary Platycodon grandiflorum markedly decreased both plasma cholesterol and fasting plasma insulin levels, and significantly decreased the postprandial glucose level at 30 min during oral glucose tolerance test in obese Zucker rats. Although there was no statistical significance, the crude glucose transporter 4 protein level of obese rats fed Platycodon grandiflorum tended to increase when compared with that of obese control rats. Therefore, the present results suggested that dietary Platycodon grandiflorum may be useful in prevention and improvement of metabolic disorders characterized by hyperinsulinemia states such as noninsulin dependent diabetes mellitus, syndrome X, and coronary artery disease.     

Nuclear magnetic resonance solution conformation of alpha-conotoxin AuIB, an alpha(3)beta(4) subtype-selective neuronal nicotinic acetylcholine receptor antagonist

The neuronal nicotinic acetylcholine receptors constitute a highly diverse group, with subtypes consisting of pentameric combinations of alpha and beta subunits. alpha-Conotoxins are a homologous series of small peptides that antagonize these receptors. We present the three-dimensional solution structure of alpha-conotoxin AuIB, the first 15-residue alpha-conotoxin known to selectively block the alpha(3)beta(4) nicotinic acetylcholine receptor subtype. The pairwise backbone and heavy-atom root mean square deviation for an ensemble of 20 structures are 0.269 and 0.720 A, respectively. The overall fold of alpha-conotoxin AuIB closely resembles that of the alpha4/7 subfamily alpha-conotoxins. However, the absence of Tyr(15), normally present in other alpha4/7 members, results in tight bending of the backbone at the C terminus and effectively renders Asp(14) to assume the spatial location of Tyr(15) present in other neuronal alpha4/7 alpha-conotoxins. Structural comparison of alpha-conotoxin AuIB with the alpha(3)beta(2) subtype-specific alpha-conotoxin MII shows different electrostatic surface charge distributions, which may be important in differential receptor subtype recognition.     

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol‐butanol‐ethanol fuel mixture

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone‐butanol‐ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol‐butanol‐ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab‐scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot‐scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab‐scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1083–1088, 2013     

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5′ end of exon 8, a 33 nt RNA from 3′ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.     

Opposing interactions between homothorax and Lobe define the ventral eye margin of Drosophila eye

Patterning in multi-cellular organisms involves progressive restriction of cell fates by generation of boundaries to divide an organ primordium into smaller fields. We have employed the Drosophila eye model to understand the genetic circuitry responsible for defining the boundary between the eye and the head cuticle on the ventral margin. The default state of the early eye is ventral and depends on the function of Lobe (L) and the Notch ligand Serrate (Ser). We identified homothorax (hth) as a strong enhancer of the L mutant phenotype of loss of ventral eye. Hth is a MEIS class gene with a highly conserved Meis-Hth (MH) domain and a homeodomain (HD). Hth is known to bind Extradenticle (Exd) via its MH domain for its nuclear translocation. Loss-of-function of hth, a negative regulator of eye, results in ectopic ventral eye enlargements. This phenotype is complementary to the L mutant phenotype of loss-of-ventral eye. However, if L and hth interact during ventral eye development remains unknown. Here we show that (i) L acts antagonistically to hth, (ii) Hth is upregulated in the L mutant background, and (iii) MH domain of Hth is required for its genetic interaction with L, while its homeodomain is not, (iv) in L mutant background ventral eye suppression function of Hth involves novel MH domain-dependent factor(s), and (v) nuclear localization of Exd is not sufficient to mediate the Hth function in the L mutant background. Further, Exd is not a critical rate-limiting factor for the Hth function. Thus, optimum levels of L and Hth are required to define the boundary between the developing eye and head cuticle on the ventral margin.