Probing the production of amidated peptides following genetic and dietary copper manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.     

Bivariate genome-wide association analyses of femoral neck bone geometry and appendicular lean mass

Objective

Femoral neck geometric parameters (FNGPs), such as periosteal diameter (W), cross-sectional area (CSA), cortical thickness (CT), buckling ratio (BR), and section modulus (Z), are highly genetically correlated with body lean mass. However, the specific SNPs/genes shared by these phenotypes are largely unknown.

Methods

To identify the specific SNPs/genes shared between FNGPs and appendicular lean mass (ALM), we performed an initial bivariate genome-wide association study (GWAS) by scanning ∼690,000 SNPs in 1,627 unrelated Han Chinese adults (802 males and 825 females) and a follow-up replicate study in 2,286 unrelated US Caucasians.

Results

We identified 13 interesting SNPs that may be important for both FNGPs and ALM. Two SNPs, rs681900 located in the HK2 (hexokinase 2) gene and rs11859916 in the UMOD (uromodulin) gene, were bivariately associated with FNGPs and ALM (p = 7.58×10⁻⁶ for ALM-BR and p = 2.93×10⁻⁶ for ALM-W, respectively). The associations were then replicated in Caucasians, with corresponding p values of 0.024 for rs681900 and 0.047 for rs11859916. Meta-analyses yielded combined p values of 3.05×10⁻⁶ and 2.31×10⁻⁶ for rs681900 and rs11859916, respectively. Our findings are consistent with previous biological studies that implicated HK2 and UMOD in both FNGPs and ALM. Our study also identified a group of 11 contiguous SNPs, which spanned a region of ∼130 kb, were bivariately associated with FNGPs and ALM, with p values ranging from 3.06×10⁻⁷ to 4.60×10⁻⁶ for ALM-BR. The region contained two neighboring miRNA coding genes, MIR873 (MicroRNA873) and MIR876 (MicroRNA876).

Conclusion

Our study implicated HK2, UMOD, MIR873 and MIR876, as pleiotropic genes underlying variation of both FNGPs and ALM, thus suggesting their important functional roles in co-regulating both FNGPs and ALM.     

Trastuzumab in the adjuvant treatment of HER2-positive early breast cancer patients: a meta-analysis of published randomized controlled trials

Background

Adjuvant trastuzumab therapy has yielded conflicting results for overall survival, concerns about central nervous system (CNS) metastasis, and questions about optimal schedule. Therefore, we carried out a meta-analysis to assess the benefits of concurrent or sequential trastuzumab with adjuvant chemotherapy for early breast cancer patients with HER2-positive tumors.

Methods

Computerized and manual searches were performed to identify randomized clinical trials comparing adjuvant chemotherapy with or without trastuzumab in HER2-positive early breast cancer patients. Odds ratios were used to estimate the association between the addition of trastuzumab to adjuvant chemotherapy and various survival outcomes. The fixed-effects or random-effects model was used to combine data.

Findings

With six eligible studies identified, this analysis demonstrated that patients with HER2-positive breast cancer derived benefit in disease-free survival, overall survival, locoregional recurrence and distant recurrence (all P<0.001) from the addition of trastuzumab to adjuvant chemotherapy, whereas trastuzumab did worse in CNS recurrence as compared to the control group (P = 0.018). Furthermore, concomitant use of trastuzumab significantly lowered the hazard of death (P<0.001) but bore a higher incidence of CNS recurrence (P = 0.010), while statistical significance failed to be discerned for either overall survival (P = 0.069) or CNS metastasis (P = 0.374) between the sequential and observation arms.

Conclusion

This analysis verifies the efficacy of trastuzumab in the adjuvant setting. Additionally, our findings indirectly corroborate the superiority of concurrent trastuzumab to sequential use and also illuminate that prolonged survival is the possible reason for the higher incidence of CNS with trastuzumab versus observation.     

Diphenyl difluoroketone: a potent chemotherapy candidate for human hepatocellular carcinoma

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was recently reported to inhibit proliferation of various cancer cells significantly. Here we try to determine the effect and mechanism of EF24 on hepatocellular carcinoma. 2 μM EF24 was found to inhibit the proliferation of PLC/PRF/5, Hep3B, HepG2, SK-HEP-1 and Huh 7 cell lines. However, even 8 μM EF24 treatment did not affect the proliferation of normal liver LO2 cells. Accordingly, 20 mg/kg/d EF24 inhibited the growth of the tumor xenografts conspicuously while causing no apparent change in liver, spleen or body weight. In addition, significant apoptosis and G(2)/M phase cell cycle arrest were found using flow cytometry. Besides, caspases and PARP activation and features typical of apoptosis including fragmented nuclei with condensed chromatin were also observed. Furthermore, the mechanism was targeted at the reduction of nuclear factor kappa b (NF-κB) pathway and the NF-κB-regulated gene products Bcl-2, COX-2, Cyclin B1. Our study has offered a strategy that EF24 being a therapeutic agent for hepatocellular carcinoma.     

Effect of different iron loads on serum and tissue biochemical parameters and liver hepcidin mRNA abundance of neonatal piglets

Iron (Fe) is an essential and important trace element for animals. In order to study its metabolism and relationship with hepcidin, piglet models of Fe-deficiency and Fe-overload were established by intramuscular injection with different doses of Fe-dextran (150 mg Fe/ml) within 1 week of age. Twelve piglets were divided into three groups of four animals: deficiency, regular and overload group, receiving 0 ml, 1 ml and 6 ml Fe-dextran, respectively. The piglets were euthanised at the age of 7 days for analysis. The results showed that the Fe-concentrations in liver, spleen and serum of piglets in the overload group were higher than in the regular and deficiency groups (p < 0.05). In the overload group, several serum biochemical parameters, e.g. globulin, total bilirubin, total cholesterol, high density lipoprotein (HDL), malondialdehyde, glutathione peroxidase (GPx), peroxidase and xanthine oxidase were higher, while alkaline phosphatase (AKP) and triglycerides were lower, compared with the regular group (p < 0.05). The serum concentrations of AKP, total bilirubin and peroxidase in the deficiency group were lower, while HDL and GPx were higher, compared with the regular group (p < 0.05). Hepcidin mRNA abundance was 131 times lower in the liver of piglets with Fe-deficiency, and 7 times higher in the overloaded group than that in the regular group (p < 0.05). In conclusion, Fe-overload and deficiency would influence Fe-metabolism, serum biochemical indexes, oxidation state and hepcidin mRNA abundance in piglet liver.     

A human cancer-predisposing polymorphism in Cdc25A is embryonic lethal in the mouse and promotes ASK-1 mediated apoptosis

Background

Failure to regulate the levels of Cdc25A phosphatase during the cell cycle or during a checkpoint response causes bypass of DNA damage and replication checkpoints resulting in genomic instability and cancer. During G1 and S and in cellular response to DNA damage, Cdc25A is targeted for degradation through the Skp1-cullin-β-TrCP (SCF^β-TrCP) complex. This complex binds to the Cdc25A DSG motif which contains serine residues at positions 82 and 88. Phosphorylation of one or both residues is necessary for the binding and degradation to occur.

Results

We now show that mutation of serine 88 to phenylalanine, which is a cancer-predisposing polymorphic variant in humans, leads to early embryonic lethality in mice. The mutant protein retains its phosphatase activity both in vitro and in cultured cells. It fails to interact with the apoptosis signal-regulating kinase 1 (ASK1), however, and therefore does not suppress ASK1-mediated apoptosis.

Conclusions

These data suggest that the DSG motif, in addition to its function in Cdc25A-mediated degradation, plays a role in cell survival during early embyogenesis through suppression of ASK1-mediated apoptosis.

     

Preparation of cyclodextrin chiral stationary phases by organic soluble catalytic 'click' chemistry

We describe an effective and simple protocol that uses click chemistry to attach native β-cyclodextrin (β-CD) to silica particles, resulting in a chiral stationary phase (CCNCSP) that can be used for the enantioseparation of chiral drugs by high-performance liquid chromatography (HPLC). Starting from β-CD, the CCNCSP is prepared in several steps: (i) reaction of β-CD with 1-(p-toluenesulfonyl)-imidazole to afford mono-6-toluenesulfonyl-β-CD; (ii) azidolysis of mono-6-toluenesulfonyl-β-CD in dimethylformamide to give mono-6-azido-β-CD (N(3)-CD); (iii) reaction of cuprous iodide with triphenylphosphine to form an organic soluble catalyst CuI(PPh(3)); (iv) preparation of alkynyl-modified silica particles; and (v) click chemistry immobilization of N(3)-CD onto alkynyl-modified silica to afford the desired chiral stationary phase. Synthesis of the stationary phase and column packing takes ～1 week.     

Glycemic variability in abdominally obese men with normal glucose tolerance as assessed by continuous glucose monitoring system

The purpose of this study was to observe both the glycemic variability in abdominally obese men with normal glucose tolerance (NGT) and the relationship between glycemic variability and early atherosclerosis. This case‐control study included 23 abdominally obese men (waist circumference (WC) ≥90 cm) and 23 nonabdominally obese men (WC <90 cm) with NGT who were between 20 and 50 years of age. All subjects were of the Han ethnicity. The cases and controls were age‐matched. A continuous glucose monitoring system (CGMS) was used in this study. With the CGMS, the standard deviation of blood glucose (SDBG) and the mean amplitude of glucose excursion (MAGE) were calculated to estimate glycemic variability. The carotid intima‐media thickness (CIMT) was used as a surrogate marker of early atherosclerosis. Mean blood glucose (MBG) (6.13 ± 0.94 vs. 5.55 ± 0.87 mmol/l), SDBG (0.89 ± 0.34 vs. 0.64 ± 0.24 mmol/l), MAGE (2.05 ± 0.83 vs. 1.57 ± 0.52 mmol/l), and CIMT (0.73 ± 0.12 vs. 0.67 ± 0.05 mm) were significantly higher in the abdominally obese men than in the nonabdominally obese men (P < 0.05). WC positively correlated with MBG (r = 0.302, P = 0.041), SDBG (r = 0.362, P = 0.013), MAGE (r = 0.302, P = 0.041), and CIMT (r = 0.487, P = 0.001). CIMT did not correlate with MBG (r = 0.206, P = 0.169), SDBG (r = 0.114, P = 0.450), and MAGE (r = 0.085, P = 0.574). After multivariate analysis, WC was still significantly associated with MBG (β = 0.025, P = 0.041), SDBG (β = 0.010, P = 0.013), MAGE (β = 0.019, P = 0.042), and CIMT (β = 0.008, P = 0.022). This study demonstrates that glycemic variability is increased in abdominally obese men with NGT. A relationship between glycemic variability and atherosclerosis was not observed in this study and requires further investigation.