Complete Chloroplast Genome Sequence of Holoparasite Cistanche deserticola (Orobanchaceae) Reveals Gene Loss and Horizontal Gene Transfer from Its Host Haloxylon ammodendron (Chenopodiaceae)

Background

The central function of chloroplasts is to carry out photosynthesis, and its gene content and structure are highly conserved across land plants. Parasitic plants, which have reduced photosynthetic ability, suffer gene losses from the chloroplast (cp) genome accompanied by the relaxation of selective constraints. Compared with the rapid rise in the number of cp genome sequences of photosynthetic organisms, there are limited data sets from parasitic plants.

Principal Findings/Significance

Here we report the complete sequence of the cp genome of Cistanche deserticola, a holoparasitic desert species belonging to the family Orobanchaceae. The cp genome of C. deserticola is greatly reduced both in size (102,657 bp) and in gene content, indicating that all genes required for photosynthesis suffer from gene loss and pseudogenization, except for psbM. The striking difference from other holoparasitic plants is that it retains almost a full set of tRNA genes, and it has lower dN/dS for most genes than another close holoparasitic plant, E. virginiana, suggesting that Cistanche deserticola has undergone fewer losses, either due to a reduced level of holoparasitism, or to a recent switch to this life history. We also found that the rpoC2 gene was present in two copies within C. deserticola. Its own copy has much shortened and turned out to be a pseudogene. Another copy, which was not located in its cp genome, was a homolog of the host plant, Haloxylon ammodendron (Chenopodiaceae), suggesting that it was acquired from its host via a horizontal gene transfer.     

miR-146a Polymorphism (rs2910164) Predicts Colorectal Cancer Patients’ Susceptibility to Liver Metastasis

miR-146a plays important roles in cancer as it directly targets NUMB, an inhibitor of Notch signaling. miR-146a is reportedly regulated by a G>C polymorphism (SNP; rs2910164). This polymorphism affects various cancers, including colorectal cancer (CRC). However, the clinical significance of miR-146a polymorphism in CRC remains unclear. A total of 59 patients with CRC were divided into 2 groups: a CC/CG genotype (n = 32) and a GG genotype (n = 27), based on the miR-146a polymorphism. cDNA microarray analysis was performed using 59 clinical samples. Significantly enriched gene sets in each genotype were extracted using GSEA. We also investigated the association between miR-146a polymorphism and miR-146a, NUMB expression or migratory response in CRC cell lines. The CC/CG genotype was associated with significantly more synchronous liver metastasis (p = 0.007). A heat map of the two genotypes showed that the expression profiles were clearly stratified. GSEA indicated that Notch signaling and JAK/STAT3 signaling were significantly associated with the CC/CG genotype (p = 0.004 and p = 0.023, respectively). CRC cell lines with the pre-miR-146a/C revealed significantly higher miR-146a expression (p = 0.034) and higher NUMB expression and chemotactic activity. In CRC, miR-146a polymorphism is involved in liver metastasis. Identification of this polymorphism could be useful to identify patients with a high risk of liver metastasis in CRC.     

Association between Smoking Status and Obesity in a Nationwide Survey of Japanese Adults

Objective

A positive association between the number of cigarettes smoked per day and obesity has been reported, whereas how other smoking-related indices, such as pack-years and duration of smoking, are related with obesity has been less investigated. We analyzed the age-adjusted cross-sectional association between smoking and obesity in a general Japanese population.

Methods

We used data from a nationwide epidemiological study of Japanese adults (N = 23,106). We compared the prevalence of obesity (defined as body mass index ≥ 25kg/m²) among groups classified by smoking behavior, pack-years, number of cigarettes per day, duration of smoking, and duration and time of smoking cessation.

Results

In men, current smokers had a lower odds ratio (OR) for obesity of 0.80 (95% confidence interval (CI): 0.72–0.88) compared to non-smokers, whereas past smokers had a higher OR of 1.23 (95% CI: 1.09–1.37) compared to current smokers. In women, there were no differences in obesity between the three groups classified by smoking behavior. However, in both sexes, the prevalence of obesity tended to increase with pack-years and the number of cigarettes per day, but not with duration of smoking in current and past smokers. Further, in male smokers, the risks for obesity were markedly higher in short-term heavy smokers compared with long-term light smokers, even with the same number of pack-years. Regarding the impact of smoking cessation, female past smokers who quit smoking at an age > 55-years had an elevated OR of 1.60 (95% CI:1.05–2.38) for obesity.

Conclusions

In a general Japanese population, obesity is progressively associated with pack-years and number of cigarettes per day, but not with the duration of smoking. When investigating the association between obesity and cigarette smoking, the daily smoking burden and the duration of smoking require to be independently considered.     

A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.     

Impairment in a negative regulatory system for TCR signaling in CD4+ T cells from old mice

To examine the involvement of lipid rafts in an age-associated decline in T cell function, we analyzed the effect of aging on the constituents of lipid rafts in resting mouse CD4(+) T cells. We found a pronounced, age-dependent reduction in PAG/Cbp, which is involved in the regulation of Src family kinases (SFKs) by recruiting Csk (a negative regulator of SFKs) to lipid rafts. This reduction is specific for T cells and is attributed, at least in part, to the reduction in its mRNA level. The reduction of PAG accompanies marked impairment in recruiting Csk to lipid rafts and a concomitant decrease in the inactive forms of SFKs. These findings indicate that old mouse CD4(+) T cells have a defect in a negative SFK regulatory system.     

Cloning and characterization of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite

The human malaria parasite (Plasmodium falciparum) possesses a plastid-derived organelle called the apicoplast, which is believed to employ metabolisms crucial for the parasite's survival. We cloned and studied the biochemical properties of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR), a redox system that potentially supplies reducing power to Fd-dependent metabolic pathways in malaria parasite apicoplasts. The recombinant P. falciparum Fd and FNR proteins were produced by synthetic genes with altered codon usages preferred in Escherichia coli. The redox potential of the Fd was shown to be considerably more positive than those of leaf-type and root-type Fds from plants, which is favourable for a presumed direction of electron flow from catabolically generated NADPH to Fd in the apicoplast. The backbone structure of P. falciparum Fd, as solved by X-ray crystallography, closely resembles those of Fds from plants, and the surface-charge distribution shows several acidic regions in common with plant Fds and some basic regions unique to this Fd. P. falciparum FNR was able to transfer electrons selectively to P. falciparum Fd in a reconstituted system of NADPH-dependent cytochrome c reduction. These results indicate that an NADPH-FNR-Fd cascade is operative in the apicoplast of human malaria parasites.     

Phosphorylation of adult type Sept5 (CDCrel-1) by cyclin-dependent kinase 5 inhibits interaction with syntaxin-1

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.     

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: Involvement of endogenous regucalcin

The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 M), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 M), an antagonist of calmodulin, or akadaic acid (10 M), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.