Roles of rootstocks and scions in aluminum-tolerance of <Emphasis Type="Italic">Citrus</Emphasis>

Four scion-rootstock combination [i.e., X/X and X/SP, ‘Xuegan’ (Citrus sinensis) grafted on ‘Xugan’ and ‘Sour pummelo’ (Citrus grandis), respectively, and SP/X and SP/SP, ‘Sour pummelo’ grafted on ‘Xuegan’ and ‘Sour pummelo’, respectively] plants were treated for 18 weeks with 0 (?Al) or 1.2 mM AlCl₃·6H₂O (+Al). Thereafter, leaf, stem and root concentrations of phosphorus and aluminum (Al), leaf and root levels of organic acids (OAs), Al-induced release of OA anions (i.e., malate and citrate), photosynthesis and chlorophyll a fluorescence (OJIP) transients were measured. Al-induced decrease of photosynthesis and damage of photosynthetic electron transport chain were less pronounced in X/X and X/SP leaves than in SP/SP and SP/X leaves, which might be related with the higher Al-induced root efflux of OA anions and leaf P concentration. C. sinensis rootstock alleviated the influences of Al-toxicity on leaf photosynthetic electron transport chain by enhancing Al-induced release of root OA anions, hence lessening Al-induced photosynthesis inhibition in SP/X plants, while the reverse was the case for C. grandis rootstock in X/SP plants. In conclusion, the tolerance of grafted Citrus plants to Al depends on the scion as well as rootstock genotype, and the scion-rootstock interaction.     

Extreme Levels of HbA1c Increase Incident ESRD Risk in Chinese Patients with Type 2 Diabetes: Competing Risk Analysis in National Cohort of Taiwan Diabetes Study

Background

Whether HbA1c is a predictor of end-stage renal disease (ESRD) in type 2 diabetes patients remains unclear. This study evaluated relationship between HbA1c and ESRD in Chinese patients with type 2 diabetes.

Methods

Patients aged ≥ 30 years who were free of ESRD (n = 51 681) were included from National Diabetes Care Management Program from 2002–2003. Extended Cox proportional hazard model with competing risk of death served to evaluate association between HbA1c level and ESRD.

Results

A total of 2613 (5.06%) people developed ESRD during a follow-up period of 8.1 years. Overall incidence rate of ESRD was 6.26 per 1000 person-years. Patients with high levels of HbA1c had a high incidence rate of ESRD, from 4.29 for HbA1c of  6.0%–6.9% to 10.33 for HbA1c ≥ 10.0% per 1000 person-years. Patients with HbA1c < 6.0% particularly had a slightly higher ESRD incidence (4.34 per 1000 person-years) than those with HbA1c  of 6.0%–6.9%. A J-shaped relationship between HbA1c level and ESRD risk was observed. After adjustment, patients with HbA1c < 6.0% and ≥ 10.0% exhibited an increased risk of ESRD (HR: 1.99, 95% CI: 1.62–2.44; HR: 4.42, 95% CI: 3.80–5.14, respectively) compared with those with HbA1c of 6.0%–6.9%.

Conclusions

Diabetes care has focused on preventing hyperglycemia, but not hypoglycemia. Our study revealed that HbA1c level ≥ 7.0% was linked with increased ESRD risk in type 2 diabetes patients, and that HbA1c < 6.0% also had the potential to increase ESRD risk. Our study provides epidemiological evidence that appropriate glycemic control is essential for diabetes care to meet HbA1c targets and improve outcomes without increasing the risk to this population. Clinicians need to pay attention to HbA1c results on diabetic nephropathy.     

Copper uptake and translocation in chicory ( Cichorium intybus L. cv. Grasslands Puna) and tomato (Lycopersicon esculentum Mill. cv. Rondy) plants grown in NFT system. I. Copper uptake and distribution in plants

The uptake and distribution of copper was examined in chicory (Cichorium intybus L. cv. Grasslands Puna) and tomato (Lycopersicon esculentumMill. cv. Rondy) plants grown in a Nutrient Film Technique System (NFT) with addition of 0.05, 5, 10 and 20 mg Cu L^-1. Biomass production of shoots and roots of both chicory and tomato was strongly depressed by Cu concentrations higher than 5 mg Cu L^-1 in the rooting media. Although Cu concentrations in both shoots and roots of both species increased with increasing Cu concentrations in the rooting media, the increase in roots was very much greater than that in shoots, in which the range of concentrations was small. A large proportion of total Cu uptake was retained by roots except when plants were grown in solution Cu concentrations of 0.05 mg Cu L^-1. Copper retention by roots limited Cu translocation to xylem and shoots. Copper adsorption by the root appears to buffer against increases of Cu in the rooting media. A cupric-sensitive electrode used in conjunction with total Cu analysis by graphite furnace atomic absorption spectrophotometry (GFAAS) indicated that more than 99.6% of total Cu in xylem sap was in a complexed form. Large differences between measured and predicted Cu accumulation by shoots of tomato (0.134–0.243 mg Cu plant^-1, 0.660–4.274 mg Cu plant^-1, respectively) and chicory (0.095–0.203 mg Cu plant^-1, 0.626–1.620 mg Cu plant^-1, respectively) suggest that some xylem transported Cu is recirculated to roots via the phloem. This revised version was published online in June 2006 with corrections to the Cover Date.     

Impaired thymic export and apoptosis contribute to regulatory T-cell defects in patients with chronic heart failure

Objective

Animal studies suggest that regulatory T (T_reg) cells play a beneficial role in ventricular remodeling and our previous data have demonstrated defects of T_reg cells in patients with chronic heart failure (CHF). However, the mechanisms behind T_reg-cell defects remained unknown. We here sought to elucidate the mechanism of T_reg-cell defects in CHF patients.

Methods and Results

We performed flow cytometry analysis and demonstrated reduced numbers of peripheral blood CD4⁺CD25⁺FOXP3⁺CD45RO⁻CD45RA⁺ naïve T_reg (nT_reg) cells and CD4⁺CD25⁺FOXP3⁺CD45RO⁺CD45RA⁻ memory T_reg (mT_reg) cells in CHF patients as compared with non-CHF controls. Moreover, the nT_reg/mT_reg ratio (p<0.01), CD4⁺CD25⁺FOXP3⁺CD45RO⁻ CD45RA⁺CD31⁺ recent thymic emigrant T_reg cell (RTE-T_reg) frequency (p<0.01), and T-cell receptor excision circle levels in T_reg cells (p<0.01) were lower in CHF patients than in non-CHF controls. Combined annexin-V and 7-AAD staining showed that peripheral T_reg cells from CHF patients exhibited increased spontaneous apoptosis and were more prone to interleukin (IL)-2 deprivation- and CD95 ligand-mediated apoptosis than those from non-CHF individuals. Furthermore, analyses by both flow cytometry and real-time polymerase chain reaction showed that T_reg-cell frequency in the mediastinal lymph nodes or Foxp3 expression in hearts of CHF patients was no higher than that of the non-CHF controls.

Conclusion

Our data suggested that the T_reg-cell defects of CHF patients were likely caused by decreased thymic output of nascent T_reg cells and increased susceptibility to apoptosis in the periphery.     

Effect of mild intermittent hypoxia on glucose tolerance, muscle morphology and AMPK-PGC-1alpha signaling

The main goal of this study was to investigate the long-term effect of daily 8-hour mild intermittent hypoxia (14-15% O2) on glucose tolerance and muscle morphology of Sprague-Dawley rats. The involvement of AMPK-PGC-1alpha-VEGF signaling pathways in the skeletal muscle was also determined during the first 8 hours of hypoxia. We found that mRNA levels of VEGF and PGC-1alpha were significantly increased above control after 8-h mild hypoxia without a change in AMPK phosphorylation. After 8 weeks of mild intermittent hypoxia treatment, plasma glucose and insulin levels in oral glucose tolerance test (OGTT), epididymal fat mass, and body weight were significantly lower compared to the control group. While soleus muscle weight was not changed, capillary and fiber densities in the hypoxia group were 33% and 35% above the control suggesting reorganization of muscle fibers. In conclusion, our data provide strong evidence that long-term mild intermittent hypoxia decreases the diffusion distance of glucose and insulin across muscle fibers, and decreases adiposity in rats. These changes may account for the improved glucose tolerance observed following the 8-week hypoxia treatment, and provides grounds for investigating the development of a mild non-pharmacological intervention in the treatment of obesity and type 2 diabetes.     

Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain

Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using 1?N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.     

Proteomic characterization of human milk whey proteins during a twelve-month lactation period

Human milk is a rich source of bioactive proteins that support the early growth and development of the newborn. Although the major components of the protein fraction in human milk have been studied, the expression and relative abundance of minor components have received limited attention. We examined the expression of low-abundance proteins in the whey fraction of human milk and their dynamic changes over a twelve-month lactation period. The low-abundance proteins were enriched by ProteoMiner beads, and protein identification was performed by liquid chromatography tandem mass spectrometry. One hundred and fifteen proteins were identified, thirty-eight of which have not been previously reported in human colostrum or milk. We also for the first time described differences in protein patterns among the low-abundance proteins during lactation. These results enhance our knowledge about the complexity of the human milk proteome, which constitutes part of the advantages to the breast-fed infant.     

ARHI (DIRAS3)-mediated autophagy-associated cell death enhances chemosensitivity to cisplatin in ovarian cancer cell lines and xenografts

Autophagy can sustain or kill tumor cells depending upon the context. The mechanism of autophagy-associated cell death has not been well elucidated and autophagy has enhanced or inhibited sensitivity of cancer cells to cytotoxic chemotherapy in different models. ARHI (DIRAS3), an imprinted tumor suppressor gene, is downregulated in 60% of ovarian cancers. In cell culture, re-expression of ARHI induces autophagy and ovarian cancer cell death within 72 h. In xenografts, re-expression of ARHI arrests cell growth and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks, dormancy is broken and xenografts grow promptly. In this study, ARHI-induced ovarian cancer cell death in culture has been found to depend upon autophagy and has been linked to G1 cell-cycle arrest, enhanced reactive oxygen species (ROS) activity, RIP1/RIP3 activation and necrosis. Re-expression of ARHI enhanced the cytotoxic effect of cisplatin in cell culture, increasing caspase-3 activation and PARP cleavage by inhibiting ERK and HER2 activity and downregulating XIAP and Bcl-2. In xenografts, treatment with cisplatin significantly slowed the outgrowth of dormant autophagic cells after reduction of ARHI, but the addition of chloroquine did not further inhibit xenograft outgrowth. Taken together, we have found that autophagy-associated cancer cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells are still vulnerable to cisplatin-based chemotherapy.Autophagy has a well-defined role in cellular physiology, removing senescent organelles and catabolizing long-lived proteins.^{1, 2} Under nutrient-poor conditions, the fatty acids and amino acids produced by hydrolysis of lipids and proteins in autophagolysosomes can provide energy to sustain starving cells. Prolonged autophagy is, however, associated with caspase-independent type II programmed cell death. Although the mechanism of autophagy-associated cell death has not been adequately characterized, programmed necrosis or necroptosis has been implicated in some studies.^{3, 4}Given the ability to sustain or kill cells, the role of autophagy in cancer is complex and dependent on the context of individual studies. During oncogenesis in genetically engineered mice, reduced hemizygous expression of genes required for autophagy (BECN1, Atg4, ATG5, Atg7) can accelerate spontaneous or chemically induced tumor formation,^{5, 6} suggesting that autophagy can serve as a tumor suppressor. Other observations with established cancers suggest that autophagy can sustain metabolically challenged neoplasms, particularly in settings with inadequate vascular access.^{7, 8} Autophagy has also been shown to protect cancer cells from the lethal effects of some cytotoxic drugs.^{9, 10}Our group has found that cancer cell proliferation,^{11, 12, 13} motility,¹⁴ autophagy and tumor dormancy^{15, 16} can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), that is downregulated in 60% of ovarian cancers by multiple mechanisms,^{17, 18} associated with shortened progression-free survival.¹⁹ Ovarian cancer cell sublines have been developed with tet-inducible expression of ARHI. In cell culture, re-expression of ARHI induces autophagy and clonogenic ovarian cancer cell death within 72 h.¹⁶ In xenografts, re-expression of ARHI arrests cell growth, inhibits angiogenesis and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks of induction, dormancy is broken, vascularization occurs and xenografts grow promptly. Treatment of dormant xenografts with chloroquine (CQ), a functional inhibitor of autophagy, delays tumor outgrowth, suggesting that autophagy facilitates survival of poorly vascularized, nutrient-deprived ovarian cancer cells. The relevance of this model to human disease is supported by the recent observation that small deposits of dormant ovarian cancer found on the peritoneal surface at ‘second look'' operations following initial surgery and chemotherapy exhibit autophagy and increased expression of ARHI in >80% of cases.²⁰Ovarian cancer develops in >22 000 women each year in the United States.²¹ Over the past four decades, the 5-year survival has increased from 37% to ∼50% with optimal cytoreductive surgery and combination chemotherapy using taxane- and platinum-based regimens,^{21, 22} but long-term survival and cure stand at ∼30% for all stages, due, in large part, to the persistence and recurrence of dormant, drug-resistant ovarian cancer cells. For the past two decades, standard chemotherapy for ovarian cancer has included a combination of a platinum compound and a taxane. Carboplatin and cisplatin are alkylating agents that bind covalently to DNA producing intra- and inter-strand crosslinks that, if not repaired, induce apoptosis and cell death.^{23, 24} Our previous studies suggest that ∼20% of primary ovarian cancers exhibit punctate immunohistochemical staining for LC3, a biomarker for autophagy that decorates autophagosome membranes, whereas >80% of cancers that have survived platinum-based chemotherapy exhibit punctate LC3.²⁰ Consequently, autophagy might provide one mechanism of resistance to platinum-based therapy.In this report, we have explored mechanism(s) by which ARHI induces autophagy-associated cell death and enhances cisplatin cytotoxicity. Cisplatin has been found to trigger apoptosis by inducing caspase-3 activation and PARP cleavage in ovarian cancer cells.^{25, 26} We hypothesized that autophagy-associated cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells might still be vulnerable to platinum-based chemotherapy.