A transgene expression system for the marine microalgae Aurantiochytrium sp. KRS101 using a mutant allele of the gene encoding ribosomal protein L44 as a selectable transformation marker for cycloheximide resistance

In the present study, we established a genetic system for manipulating the oleaginous heterotrophic microalgae Aurantiochytrium sp. KRS101, using cycloheximide resistance as the selectable marker. The gene encoding ribosomal protein L44 (RPL44) of Aurantiochytrium sp. KRS101 was first identified and characterized. Proline 56 was replaced with glutamine, affording cycloheximide resistance to strains encoding the mutant protein. This resistance served as a novel selection marker. The gene encoding the Δ12-fatty acid desaturase of Mortierella alpina, used as a reporter, was successfully introduced into chromosomal DNA of Aurantiochytrium sp. KRS101 via 18S rDNA-targeted homologous recombination. Enzymatic conversion of oleic acid (C18:1) to linoleic acid (C18:2) was detected in transformants but not in the wild-type strain.     

Metabolic engineering of Corynebacterium glutamicum to produce GDP-l-fucose from glucose and mannose

Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5′-diphosphate (GDP)-l-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-d-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-l-fucose from GDP-d-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-l-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-d-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-l-fucose at the specific rate of 0.11 mg g cell^?1 h^?1. The specific GDP-l-fucose content reached 5.5 mg g cell^?1, which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.     

Vascular Calcifying Progenitor Cells Possess Bidirectional Differentiation Potentials

Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFRα markers. Sca-1⁺ (Sca-1⁺/PDGFRα⁺ and Sca-1⁺/PDGFRα⁻) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1⁻ (Sca-1⁻/PDGFRα⁺ and Sca-1⁻/PDGFRα⁻) progenitor cells. Among Sca-1⁺ progenitor populations, Sca-1⁺/PDGFRα⁻ cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1⁺/PDGFRα⁺ cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor γ (PPARγ) agonist, Sca-1⁺/PDGFRα⁻ cells preferentially differentiated into osteoclast-like cells. Sca-1⁺ progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1⁺ calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1⁺ progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1⁺/PDGFRα⁻ cells treated with a PPARγ agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1⁺/PDGFRα⁻ cells into Apoe^−/− mice increased the severity of calcified atherosclerotic plaques. However, Sca-1⁺/PDGFRα⁻ cells in which PPARγ was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1⁺/PDGFRα⁻ cells mainly expressed osteocalcin; however, activation of PPARγ triggered receptor activator for nuclear factor-κB (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPARγ activation may be an option to reverse calcification.     

LORETA Current Source Density for Duration Mismatch Negativity and Neuropsychological Assessment in Early Schizophrenia

Introduction

Patients with schizophrenia elicit cognitive decline from the early phase of the illness. Mismatch negativity (MMN) has been shown to be associated with cognitive function. We investigated the current source density of duration mismatch negativity (dMMN), by using low-resolution brain electromagnetic tomography (LORETA), and neuropsychological performance in subjects with early schizophrenia.

Methods

Data were obtained from 20 patients meeting DSM-IV criteria for schizophrenia or schizophreniform disorder, and 20 healthy control (HC) subjects. An auditory odd-ball paradigm was used to measure dMMN. Neuropsychological performance was evaluated by the brief assessment of cognition in schizophrenia Japanese version (BACS-J).

Results

Patients showed smaller dMMN amplitudes than those in the HC subjects. LORETA current density for dMMN was significantly lower in patients compared to HC subjects, especially in the temporal lobes. dMMN current density in the frontal lobe was positively correlated with working memory performance in patients.

Conclusions

This is the first study to identify brain regions showing smaller dMMN current density in early schizophrenia. Further, poor working memory was associated with decreased dMMN current density in patients. These results are likely to help understand the neural basis for cognitive impairment of schizophrenia.     

Knockdown of the Sodium-Dependent Phosphate Co-Transporter 2b (NPT2b) Suppresses Lung Tumorigenesis

The sodium-dependent phosphate co-transporter 2b (NPT2b) plays an important role in maintaining phosphate homeostasis. In previous studies, we have shown that high dietary inorganic phosphate (Pi) consumption in mice stimulated lung tumorigenesis and increased NPT2b expression. NPT2b has also been found to be highly expressed in human lung cancer tissues. The association of high expression of NPT2b in the lung with poor prognosis in oncogenic lung diseases prompted us to test whether knockdown of NPT2b may regulate lung cancer growth. To address this issue, aerosols that contained small interfering RNA (siRNA) directed against NPT2b (siNPT2b) were delivered into the lungs of K-ras ^LA1 mice, which constitute a murine model reflecting human lung cancer. Our results clearly showed that repeated aerosol delivery of siNPT2b successfully suppressed lung cancer growth and decreased cancer cell proliferation and angiogenesis, while facilitating apoptosis. These results strongly suggest that NPT2b plays a role lung tumorigenesis and represents a novel target for lung cancer therapy.     

Time-course Changes in Immunoreactivities of Glucokinase and Glucokinase Regulatory Protein in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia–reperfusion (I–R). Five days after I–R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.