Cytological and biochemical analysis of COF1, an Arabidopsis mutant of an ABC transporter gene

In transposon-tagged lines of Arabidopsis we found two new mutants, cof1-1 and cof1-2 (cuticular defect and organ fusion), that show the phenotype of wilting when grown in soil, organ fusion of rosette leaves and infertility. Toluidine blue testing and scanning electron microscopy observation revealed that these mutants had cuticular defects in the stems and adult leaves, but not in cotyledones. Transmission electron microscopy observation revealed thinner cuticle layers in the mutants, and cuticular materials interspersed between the two fused epidermal layers were observed in the mutant rosette leaves. These two mutants had a transposon insertion in the coding regions of WBC11, which was classified as a member of ABC transporter genes in Arabidopsis. WBC11 showed high sequence similarity to CER5 (also called WBC12), which was involved in cuticular lipid export. Gas chromatographic analysis revealed that C29 alkane extracted from the stem surface of cof1 mutants was reduced whereas C29 ketone was accumulated, which was different from the case of cer5 mutants. While cer5 mutants had fairly normal morphology, cof1 mutants had pleiotropic phenotypes so that COF1/WBC11 could have important roles different from those of CER5/WBC12. We also found that C29 alkane was accumulated in the intracellular extract of cof1 mutants, suggesting a function for WBC11 in the direct transport of lipid molecules. Pollen observation showed that mutant pollen grains were irregularly shaped. The function of COF1/WBC11 in lipid transport for the construction of cuticle layers and pollen coats for normal organ formation is discussed.     

Postoperative cataract cases among atomic bomb survivors: radiation dose response and threshold

Recent evidence argues against a high threshold dose for vision-impairing radiation-induced cataractogenesis. We conducted logistic regression analysis to estimate the dose response and used a likelihood profile procedure to determine the best-fitting threshold model among 3761 A-bomb survivors who underwent medical examinations during 2000-2002 for whom radiation dose estimates were available, including 479 postoperative cataract cases. The analyses indicated a statistically significant dose-response increase in the prevalence of postoperative cataracts [odds ratio (OR), 1.39; 95% confidence interval (CI), 1.24-1.55] at 1 Gy, with no indication of upward curvature in the dose response. The dose response was suggestive when the restricted dose range of 0 to 1 Gy was examined. A nonsignificant dose threshold of 0.1 Gy (95% CI, <0-0.8) was found. The prevalence of postoperative cataracts in A-bomb survivors increased significantly with A-bomb radiation dose. The estimate (0.1 Gy) and upper bound (0.8 Gy) of the dose threshold for operative cataract prevalence was much lower than the threshold of 2-5 Gy usually assumed by the radiation protection community and was statistically compatible with no threshold at all.     

Effects of Decreased Occlusal Loading during Growth on the Mandibular Bone Characteristics

Background

Bone mass and mineralization are largely influenced by loading. The purpose of this study was to evaluate the reaction of the entire mandibular bone in response to decreased load during growth. It is hypothesized that decreased muscular loading will lead to bone changes as seen during disuse, i.e. loss of bone mass.

Methods and Findings

Ten 21-day-old Wistar strain male rats were divided into two groups (each n=5) and fed on either a hard- or soft-diet for 11 weeks. Micro-computed tomography was used for the investigation of bone mineralization, bone volume, bone volume fraction (BV/TV) and morphological analysis. Mandibular mineralization patterns were very consistent, showing a lower degree of mineralization in the ramus than in the corpus. In the soft-diet group, mineralization below the molars was significantly increased (p<0.05) compared to the hard diet group. Also, bone volume and BV/TV of the condyle and the masseter attachment were decreased in the soft-diet group (p<0.05). Morphological analysis showed inhibited growth of the ramus in the soft-diet group (p<0.05).

Conclusion

Decreased loading by a soft diet causes significant changes in the mandible. However, these changes are very region-specific, probably depending on the alterations in the local loading regime. The results suggest that muscle activity during growth is very important for bone quality and morphology.     

Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors

Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation.     

Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB) Affect Protein Function

Whooping cough due to Bordetella pertussis is increasing in incidence, in part due to accumulation of mutations which increase bacterial fitness in highly vaccinated populations. Polymorphisms in the pertussis toxin, ptxA and ptxB genes, and the pertactin, prn genes of clinical isolates of Bordetella pertussis collected in Cincinnati from 1989 through 2005 were examined. While the ptxA and prn genotypes were variable, all 48 strains had the ptxB2 genotype; ptxB1 encodes glycine at amino acid 18 of the S2 subunit of pertussis toxin, while ptxB2 encodes serine. We investigated antigenic and functional differences of PtxB1 and PtxB2. The S2 protein was not very immunogenic. Only a few vaccinated or individuals infected with B. pertussis developed antibody responses to the S2 subunit, and these sera recognized both polymorphic forms equally well. Amino acid 18 of S2 is in a glycan binding domain, and the PtxB forms displayed differences in receptor recognition and toxicity. PtxB1 bound better to the glycoprotein, fetuin, and Jurkat T cells in vitro, but the two forms were equally effective at promoting CHO cell clustering. To investigate in vivo activity of Ptx, one μg of Ptx was administered to DDY mice and blood was collected on 4 days after injection. PtxB2 was more effective at promoting lymphocytosis in mice.     

Anorexia Nervosa during Adolescence Is Associated with Decreased Gray Matter Volume in the Inferior Frontal Gyrus

Anorexia nervosa (AN) is an eating disorder characterized by the relentless pursuit to lose weight, mostly through self-starvation, and a distorted body image. AN tends to begin during adolescence among women. However, the underlying neural mechanisms related to AN remain unclear. Using voxel-based morphometry based on magnetic resonance imaging scans, we investigated whether the presence of AN was associated with discernible changes in brain morphology. Participants were 20 un-medicated, right-handed patients with early-onset AN and 14 healthy control subjects. Group differences in gray matter volume (GMV) were assessed using high-resolution, T1-weighted, volumetric magnetic resonance imaging datasets (3T Trio scanner; Siemens AG) and analyzed after controlling for age and total GMV, which was decreased in the bilateral inferior frontal gyrus (IFG) (left IFG: FWE corrected, p < 0.05; right IFG: uncorrected, p < 0.05) of patients with AN. The GMV in the bilateral IFG correlated significantly with current age (left IFG: r = -.481, p < .05; right IFG: r = -.601, p < .01) and was limited to the AN group. We speculate that decreased IFG volume might lead to deficits in executive functioning or inhibitory control within neural reward systems. Precocious or unbalanced neurological trimming within this particular region might be an important factor for the pathogenesis of AN onset.     

Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: role of reactive oxygen/nitrogen species

We have previously reported that antioxidant response element (ARE)-regulated genes, such as heme oxygenase 1 (HO-1), sequestosome 1 (SQSTM1), and NAD(P)H quinone oxidoreductase 1 (NQO1), are induced in human umbilical vein endothelial cells (HUVEC) upon exposure to laminar shear stress. In the present study, we have confirmed a critical role for NF-E2-related factor 2 (Nrf2) in the induction of gene expression in HUVEC exposed to laminar shear stress. Although the mRNA levels of Nrf2 were unchanged during exposure to shear stress, the protein levels of Nrf2 were markedly increased. Small interfering RNA (SiRNA) against Nrf2 significantly attenuated the expression of Nrf2-regulated genes such as HO-1, SQSTM1, NQO1, glutamate-cysteine ligase modifier subunit (GCLM), and ferritin heavy chain. Nrf2 was rapidly degraded in cells treated with cycloheximide under static conditions, but shear stress decreased the rate of Nrf2 degradation. Incubation with the thiol antioxidant N-acetylcysteine strongly inhibited both the Nrf2 accumulation and the expression of Nrf2-regulated genes such as HO-1, GCLM, and SQSTM1. Nitric oxide (NO) production was increased with the strength of shear stress but neither the inhibitor of endothelial NO synthase (eNOS) nor the siRNA against eNOS affected the expression of Nrf2-regulated genes. A xanthine oxidase inhibitor oxypurinol and the flavoprotein inhibitor diphenyleneiodonium, which inhibits NAD(P)H oxidase and mitochondrial respiratory chain, markedly suppressed the expression of these genes. Moreover, diphenylpyrenlphosphine, a reducing compound of lipid hydroperoxides, also significantly suppressed Nrf2-regulated gene expression. Taken together, these findings suggest that shear stress stabilizes Nrf2 protein via the lipid peroxidation elicited by xanthine oxidase and flavoprotein mediated generation of superoxide, resulting in gene induction by the Nrf2-ARE signaling pathway.     

Genetic and biochemical investigations on bacterial catabolic pathways for lignin-derived aromatic compounds

Lignins are the most abundant aromatic compounds in nature, and their decomposition is essential to the terrestrial carbon cycle. White rot fungi secreting phenol oxidases are assumed to be involved in the initial degradation of native lignin, whereas bacteria play a main role in the mineralization of lignin-derived low-molecular-weight compounds in soil. There are a number of reports on the degradation pathways for lignin-derived aromatic compounds, but their catabolism has not been enzymatically or genetically characterized. Sphingomonas paucimobilis SYK-6 is one of the best-characterized lignin-degrading bacteria. It can grow on a wide variety of lignin-related biaryls and monoaryls, including beta-aryl ether, biphenyl, diarylpropane, and phenylpropane. These compounds are degraded via the protocatechuate (PCA) 4,5-cleavage pathway or multiple 3-O-methylgallate (3MGA) catabolic pathways. In this review, the enzyme systems for beta-aryl ether and biphenyl degradation, O demethylation linked with one carbon metabolism, the PCA 4,5-cleavage pathway, and the multiple 3MGA catabolic pathways in SYK-6 are outlined.