Canonical Wnts and BMPs cooperatively induce osteoblastic differentiation through a GSK3β-dependent and β-catenin-independent mechanism

Both BMPs and Wnts play important roles in the regulation of bone formation. We examined the molecular mechanism regulating cross-talk between BMPs and Wnts in the osteoblastic differentiation of C2C12 cells. Canonical Wnts (Wnt1 and Wnt3a) but not non-canonical Wnts (Wnt5a and Wnt11) synergistically stimulated ALP activity in the presence of BMP-4. Wnt3a and BMP-4 synergistically stimulated the expression of type I collagen and osteonectin. However, Wnt3a did not stimulate ALP activity that was induced by a constitutively active BMP receptor or Smad1. Noggin and Dkk-1 suppressed the synergistic effect of BMP-4 and Wnt3a, but Smad7 did not. Overexpression of β-catenin did not affect BMP-4-induced ALP activity. By contrast, inhibition or stimulation of GSK3β activity resulted in either stimulation or suppression of ALP activity, respectively, in the presence of BMP-4. Taken together, these findings suggest that BMPs and canonical Wnts may regulate osteoblastic differentiation, especially at the early stages, through a GSK3β-dependent but β-catenin-independent mechanism.     

Segmental copy-number gain within the region of isopentenyl diphosphate isomerase genes in sporadic amyotrophic lateral sclerosis

Aims: Sporadic amyotrophic lateral sclerosis (SALS) seems to be a multifactorial disease, the pathogenesis of which may involve both genetic and environmental factors. The present study aims at identifying a possible genetic change that confers risk for SALS. Methods: We performed whole-genome screening of a copy-number variation (CNV) using a CNV beadchip, followed by real-time quantitative polymerase chain reaction (qPCR) and region-targeted high-density oligonucleotide tiling microarray. Results: Within the 40-kb region on 10p15.3 subtelomere, which harbours two genes encoding isopentenyl diphosphate isomerase 1 (IDI1) and IDI2, we found a segmental copy-number gain in a large proportion of SALS patients. qPCR analysis demonstrated the copy-number gain in 46 out of 83 SALS patients, as compared with 10 out of 99 controls (p = 4.86 × 10⁻¹¹, Odds Ratio 10.8); subsequent tiling microarray validated qPCR results and elucidated the fine structure of segmental gains. Conclusions: A segmental copy-number gain in the IDI1/IDI2 gene region may play a significant role in the pathogenesis of SALS.     

Microbial conversion of 5-sulfoisophthalic acid into 5-hydroxyisophthalic acid by Ochrobactrum anthropi S9

5-Hydroxyisophthalic acid-producing microorganisms were isolated from enrichment cultures using 5-sulfoisophthalic acid as a sulfur source. One bacterium, Ochrobactrum anthropi S9, had the highest 5-sulfoisophthalic acid-degrading activity, and stoichiometrically formed 5-hydroxyisophthalic acid, a raw material for polymer synthesis. Under optimum culture conditions, 1.3 mM 5-hydroxyisophthalic acid accumulated in the medium by 60 h. The addition of Na₂SO₄, l-methionine or l-cysteine at 2 mM inhibited the conversion of 5-sulfoisophthalic acid. O. anthropi S9 cells converted 5-sulfoisophthalic acid, benzenesulfonic acid, 3-sulfobenzoic acid, 4-aminobenzenesulfonic acid, naphthalene-1-sulfonic acid and naphthalene-2-sulfonic acid into the corresponding hydroxylated compounds.     

Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook

There is increasing interest in production of transportation fuels and commodity chemicals from lignocellulosic biomass, most desirably through biological fermentation. Considerable effort has been expended to develop efficient biocatalysts that convert sugars derived from lignocellulose directly to value-added products. Glucose, the building block of cellulose, is the most suitable fermentation substrate for industrial microorganisms such as Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. Other sugars including xylose, arabinose, mannose, and galactose that comprise hemicellulose are generally less efficient substrates in terms of productivity and yield. Although metabolic engineering including introduction of functional pentose-metabolizing pathways into pentose-incompetent microorganisms has provided steady progress in pentose utilization, further improvements in sugar mixture utilization by microorganisms is necessary. Among a variety of issues on utilization of sugar mixtures by the microorganisms, recent studies have started to reveal the importance of sugar transporters in microbial fermentation performance. In this article, we review current knowledge on diversity and functions of sugar transporters, especially those associated with pentose uptake in microorganisms. Subsequently, we review and discuss recent studies on engineering of sugar transport as a driving force for efficient bioconversion of sugar mixtures derived from lignocellulose.     

Xylitol production by recombinant Corynebacterium glutamicum under oxygen deprivation

Wild-type Corynebacterium glutamicum produced 0.6 g l⁻¹ xylitol from xylose at a productivity of 0.01 g l⁻¹ h⁻¹ under oxygen deprivation. To increase this productivity, the pentose transporter gene (araE) from C. glutamicum ATCC31831 was integrated into the C. glutamicum R chromosome. Consequent disruption of its lactate dehydrogenase gene (ldhA), and expression of single-site mutant xylose reductase from Candida tenuis (CtXR (K274R)) resulted in recombinant C. glutamicum strain CtXR4 that produced 26.5 g l⁻¹ xylitol at 3.1 g l⁻¹ h⁻¹. To eliminate possible formation of toxic intracellular xylitol phosphate, genes encoding xylulokinase (XylB) and phosphoenolpyruvate-dependent fructose phosphotransferase (PTS^fru) were disrupted to yield strain CtXR7. The productivity of strain CtXR7 increased 1.6-fold over that of strain CtXR4. A fed-batch 21-h CtXR7 culture in mineral salts medium under oxygen deprivation yielded 166 g l⁻¹ xylitol at 7.9 g l⁻¹ h⁻¹, representing the highest bacterial xylitol productivity reported to date.     

Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid l-alanine under oxygen deprivation

Corynebacterium glutamicum was genetically engineered to produce l-alanine from sugar under oxygen deprivation. The genes associated with production of organic acids in C. glutamicum were inactivated and the alanine dehydrogenase gene (alaD) from Lysinibacillus sphaericus was overexpressed to direct carbon flux from organic acids to alanine. Although the alaD-expressing strain produced alanine from glucose under oxygen deprivation, its productivity was relatively low due to retarded glucose consumption. Homologous overexpression of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the alaD-expressing strain stimulated glucose consumption and consequently improved alanine productivity. In contrast gapA overexpression did not affect glucose consumption under aerobic conditions, indicating that oxygen deprivation engendered inefficient regeneration of NAD⁺ resulting in impaired GAPDH activity and reduced glucose consumption in the alanine-producing strains. Inactivation of the alanine racemase gene allowed production of l-alanine with optical purity greater than 99.5%. The resulting strain produced 98 g l⁻¹ of l-alanine after 32 h in mineral salts medium. Our results show promise for amino acid production under oxygen deprivation.     

Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p

Peroxisomes require peroxin (Pex) proteins for their biogenesis. The interaction between Pex3p, which resides on the peroxisomal membrane, and Pex19p, which resides in the cytosol, is crucial for peroxisome formation and the post-translational targeting of peroxisomal membrane proteins (PMPs). It is not known how Pex3p promotes the specific interaction with Pex19p for the purpose of PMP translocation. Here, we present the three-dimensional structure of the complex between a cytosolic domain of Pex3p and the binding-region peptide of Pex19p. The overall shape of Pex3p is a prolate spheroid with a novel fold, the 'twisted six-helix bundle.' The Pex19p-binding site is at an apex of the Pex3p spheroid. A 16-residue region of the Pex19p peptide forms an α-helix and makes a contact with Pex3p; this helix is disordered in the unbound state. The Pex19p peptide contains a characteristic motif, consisting of the leucine triad (Leu18, Leu21, Leu22), and Phe29, which are critical for the Pex3p binding and peroxisome biogenesis.     

Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles

Background  

Bovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classify them by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in terms of healthy and atretic conditions by investigating mRNA localization of identified genes.