Attachment of fluorescent metal chelates to macromolecules using “bifunctional” chelating agents

The use of “bifunctional” chelating agents to covalently attach stable chelates of terbium and europium to human serum albumin results in products whose lanthanide fluorescence may be studied easily at micromolar concentrations with standard instrumentation. The lanthanide ions may be added specifically and quantitatively to the protein-bound chelating groups in 0.1 M citrate, pH 6.5. The use of these reagents should greatly reduce ambiguities in the determination of distances between sites on macromolecules by energy transfer measurements.     

Peripheral Afferent Mechanisms Underlying Acupuncture Inhibition of Cocaine Behavioral Effects in Rats

Administration of cocaine increases locomotor activity by enhancing dopamine transmission. To explore the peripheral mechanisms underlying acupuncture treatment for drug addiction, we developed a novel mechanical acupuncture instrument (MAI) for objective mechanical stimulation. The aim of this study was to evaluate whether acupuncture inhibition of cocaine-induced locomotor activity is mediated through specific peripheral nerves, the afferents from superficial or deep tissues, or specific groups of nerve fibers. Mechanical stimulation of acupuncture point HT7 with MAI suppressed cocaine-induced locomotor activity in a stimulus time-dependent manner, which was blocked by severing the ulnar nerve or by local anesthesia. Suppression of cocaine-induced locomotor activity was elicited after HT7 stimulation at frequencies of either 50 (for Meissner corpuscles) or 200 (for Pacinian corpuscles) Hz and was not affected by block of C/Aδ-fibers in the ulnar nerve with resiniferatoxin, nor generated by direct stimulation of C/Aδ-fiber afferents with capsaicin. These findings suggest that HT7 inhibition of cocaine-induced locomotor activity is mediated by A-fiber activation of ulnar nerve that originates in superficial and deep tissue.     

Variants of BORIS minisatellites and relation to prognosis of prostate cancer

BORIS, a member of the cancer-testis antigen family with testis specific expression, showed abnormal expression in various cancer types including prostate cancer. In our previous work, we identified the polymorphic minisatellite, BORIS-MS2, located in the promoter region of BORIS. BORIS-MS2 was revealed as a polymorphic minisatellite that contains seven alleles each with different numbers of the repeat unit. We assessed the association between the allelic variation of BORIS-MS2 and prostate cancer by a case-control study. Using a PCR-based method, 315 cancer-free male controls and 648 cases with prostate cancer were genotyped. There was no significant difference observed in the overall distribution of this minisatellite, which indicates that this polymorphism is not responsible for prostate cancer susceptibility in the Korean population. Additionally, two new rare alleles (9 and 19 copies) were detected in this study, which were identified only in cancer subjects. When we compared the clinicopathological information with the Jewett-Whitmore system of prostate cancer classification, the frequency of rare allele cases in the higher grade was significantly higher than in the total prostate group (P = 0.032). The higher grades in the Jewett-Whitmore system were associated with a poor prognosis. Therefore, this result suggests that the rare alleles of BORIS-MS2 may be used as a marker of poor outcome in prostate cancer patients.     

Arrest defective 1 regulates the oxidative stress response in human cells and mice by acetylating methionine sulfoxide reductase A

Methionine sulfoxide reductase A (MSRA) protects proteins from oxidation, and also helps remove reactive oxygen species (ROS) by recovering antioxidant enzymes inactivated by oxidation. Although its functions have been investigated extensively, little is known about the mechanism by which MSRA is regulated. Arrest defective 1 (ARD1) is an enzyme that catalyzes not only N-terminal acetylation as a cotranslational modification but also lysine acetylation as a posttranslational modification. ARD1, which is expressed in most cell types, is believed to participate in diverse biological processes, but its roles are poorly understood. Given that MSRA was hunted in a yeast two-hybrid screen with ARD1 as the bait, we here investigated whether ARD1 is a novel regulator of MSRA. ARD1 was shown to interact with and acetylate MSRA in both cells and test tubes. It specifically acetylated the K49 residue of MSRA, and by doing so repressed the enzymatic function of MSRA. ARD1 increased cellular levels of ROS, carbonylated proteins and DNA breaks under oxidative stress. Moreover, it promoted cell death induced by pro-oxidants, which was attenuated in MSRA-deficient cells. When mice were exposed to hyperoxic conditions for 2 days, their livers and kidneys were injured and protein carbonylation was increased. The oxidative tissue injury was more severe in ARD1 transgenic mice than in their wild-type littermates. In conclusion, ARD1 has a crucial role in the cellular response to oxidative stress as a bona fide regulator of MSRA. ARD1 is a potential target for ameliorating oxidative injury or for potentiating ROS-producing anticancer agents.Aerobic respiration is essential for eukaryotic life because molecular oxygen participates in ATP production and various oxidative metabolic reactions.¹ When oxygen is used, reactive oxygen species (ROS) are inevitably generated and threaten life as harmful metabolites that damage macromolecules such as nucleic acids, lipids and proteins.^2,3 ROS also act as second messengers that promote cell proliferation or differentiation.^{4, 5, 6, 7} From a functional perspective, ROS act as a double-edged sword in determining cell fate, and the roles of ROS depend on cell contexts.⁸ A variety of cell metabolic reactions are regulated depending on the intracellular redox state, which reflects the balance between ROS-generating oxidases and ROS-scavenging antioxidants.⁹ Accordingly, knowledge about the redox-balancing mechanism will help us to better understand normal physiology and pathology.The sulfur atom of methionine is easily oxidized by ROS, with methionine being modified to methionine sulfoxide (MetO), which forms two enantiomers (S-sulfoxide and R-sulfoxide).¹⁰ When proteins are sulfoxidized at methionine residues, their functions become impaired or altered.¹¹ Therefore, MetO is not only a convincing biomarker for reflecting the extent of oxidative stress but also a pathogenic factor that contributes to oxidative stress-related diseases.¹² As MetO causes serious problems in life, the defense systems against MetO have been evolutionally conserved in prokaryotic and eukaryotic cells.¹³ One such system, methionine sulfoxide reductase (MSR), has a crucial role in preventing the accumulation of MetO, and includes two enzymes, methionine sulfoxide reductase A (MSRA) and MSRB, which reduce S-sulfoxide and R-sulfoxide, respectively.¹⁴Arrest defective 1 (ARD1) is an enzyme that catalyzes N-terminal acetylation of nascent peptides as a cotranslational modification and lysine acetylation as a posttranslational modification.¹⁵ In yeast and mammalian cells, ARD1 is known to have essential roles in cell growth and differentiation.^16,17 ARD1 has also been reported to control cell migration by acetylating myosin light chain kinase¹⁸ and to promote cancer growth by acetylating β-catenin or the androgen receptor.¹⁹ Considering that ARD1 is widely expressed in most mammalian cells,²⁰ it is expected that ARD1 has diverse functions beyond those mentioned above. To further understand the functions of ARD1, we sought novel targets of ARD1 using the yeast two-hybrid method and identified MSRA as an ARD1-interacting molecule. Furthermore, we tested the possibility that ARD1 determines cell fate under oxidative stress by regulating MSRA. This study may provide new insights into how MSRA is regulated and identifies ARD1 as a potential target for modulating the cellular response to oxidative stress.     

Efficiency Enhancement of Organic Solar Cells Using Hydrophobic Antireflective Inverted Moth‐Eye Nanopatterned PDMS Films

Poly‐dimethylsiloxane (PDMS) films with 2D periodic inverted moth‐eye nanopatterns on one surface are implemented as antireflection (AR) layers on a glass substrate for efficient light capture in encapsulated organic solar cells (OSCs). The inverted moth‐eye nanopatterned PDMS (IMN PDMS) films are fabricated by a soft imprint lithographic method using conical subwavelength grating patterns formed by laser interference lithography/dry etching. Their optical characteristics, together with theoretical analysis using rigorous coupled‐wave analysis simulation, and wetting behaviors are investigated. For a period of 380 nm, IMN PDMS films laminated on glass substrates exhibit a hydrophobic surface with a water contact angle (θ_CA) of ≈120° and solar weighted transmittance (SWT) of ≈94.2%, both significantly higher than those (θ_CA≈ 36° and SWT ≈ 90.3%) of bare glass substrates. By employing IMN PDMS films with a period of 380 nm on glass substrates for OSCs, an enhanced power conversion efficiency (PCE) of 6.19% is obtained mainly due to the increased short‐circuit current density (J_sc) of 19.74 mA cm^‐2 compared to the OSCs with the bare glass substrates (PCE = 5.16% and J_sc = 17.25 mA cm^‐2). For the OSCs, the device stability is also studied.     

Correlation of IGF1R expression with ABCG2 and CD44 expressions in human osteosarcoma

Osteosarcoma is the most common type of malignant bone tumors. Insulin Growth Factor 1 receptor (IGFR1) has been known as a prognostic factor for metastasis of osteosarcoma. ABC subfamily G member2 (ABCG2) is related to resistance to anti-cancer drug, and CD44 has a role in tumor growth and metastasis. The purpose of this study is to investigate the relationship among expression patterns of IGF1R, ABCG2, and CD44 in osteosarcoma. The expression levels of IGF1R, ABCG2, and CD44 proteins were determined in tissue arrays containing osteosarcoma tissues from 59 osteosarcoma patients. The expression pattern of IGF1R was highly correlated with the expression pattern of ABCG2 (r?=?0.88) in overall osteosarcoma patients. According to pathological types, the expression pattern of IGF1R showed the higher correlation with ABGC2 (r?=?0.90) and CD44 (r?=?0.61) in osteoblatic type than in chondroblastic type. According to gender with pathologic type, the correlation between the expression patterns of IGF1R and CD44 was higher in male with osteoblatic type than in female with osteoblatic type. Among different age groups, the 1–10 years age group showed higher correlation in IGF1R versus CD44 (r?=?0.90) and ABCG2 versus CD44 (0.80) than in other age groups. These results showed that the expression of IGF1R appears to be highly correlated with the expression of ABCG2 in osteosarcoma and with the expression of CD44 in osteosarcoma patients under age of 10, which suggests that ABCG2 and CD44 can be used as prognostic factors with IGF1R for specific prognosis and efficient treatment of osteosarcoma.     

Antibody side chain conformations are position‐dependent

Side chain prediction is an integral component of computational antibody design and structure prediction. Current antibody modelling tools use backbone‐dependent rotamer libraries with conformations taken from general proteins. Here we present our antibody‐specific rotamer library, where rotamers are binned according to their immunogenetics (IMGT) position, rather than their local backbone geometry. We find that for some amino acid types at certain positions, only a restricted number of side chain conformations are ever observed. Using this information, we are able to reduce the breadth of the rotamer sampling space. Based on our rotamer library, we built a side chain predictor, position‐dependent antibody rotamer swapper (PEARS). On a blind test set of 95 antibody model structures, PEARS had the highest average χ₁ and accuracy (78.7% and 64.8%) compared to three leading backbone‐dependent side chain predictors. Our use of IMGT position, rather than backbone ϕ/ψ, meant that PEARS was more robust to errors in the backbone of the model structure. PEARS also achieved the lowest number of side chain–side chain clashes. PEARS is freely available as a web application at http://opig.stats.ox.ac.uk/webapps/pears .     

Protective effect of the phosphodiesterase III inhibitor cilostazol on amyloid β-induced cognitive deficits associated with decreased amyloid β accumulation

Alzheimer’s disease (AD), which is characterized by progressive cognitive impairment, is the most common neurodegenerative disease. Here, we investigated the preventive effect of a phosphodiesterase III inhibitor, cilostazol against cognitive decline in AD mouse model. In vitro studies using N2a cells stably expressing human amyloid precursor protein Swedish mutation (N2aSwe) showed that cilostazol decreased the amyloid β (Aβ) levels in the conditioned medium and cell lysates. Cilostazol attenuated the expression of ApoE, which is responsible for Aβ aggregation, in N2aSwe. Intracerebroventricular injection of Aβ_25–35 in C57BL/6J mice resulted in increased immunoreactivity of Aβ and p-Tau, and microglia activation in the brain. Oral administration of cilostazol for 2 weeks before Aβ administration and once a day for 4 weeks post-surgery almost completely prevented the Aβ-induced increases of Aβ and p-Tau immunoreactivity, as well as CD11b immunoreactivity. However, post-treatment with cilostazol 4 weeks after Aβ administration, when Aβ was already accumulated, did not prevent the Aβ-induced neuropathological responses. Furthermore, cilostazol did not affect the neprilysin and insulin degrading enzymes involved in the degradation of the Aβ peptide, but decreased ApoE levels in Aβ-injected brain. In addition, cilostazol significantly improved spatial learning and memory in Aβ-injected mice. The findings suggest that a phosphodiesterase III inhibitor, cilostazol significantly decreased Aβ accumulation and improved memory impairment induced by Aβ_25–35. The beneficial effects of cilostazol might be explained by the reduction of Aβ accumulation and tau phosphorylation, not through an increase in Aβ degradation but via a significant decrease in ApoE-mediated Aβ aggregation. Cilostazol may be the basis of a novel strategy for the therapy of AD.     

Analysis of promoter methylation and polymorphic minisatellites of BORIS and lack of association with gastric cancer

BORIS is a member of the cancer-testis gene family that comprises genes normally expressed only in testis but abnormally activated in different malignancies. In this study, we examined the relation between BORIS expression and gastric cancer, which is the most common cancer in Korea. Abnormal BORIS expression in the patient's gastric cancer tissues was observed. We checked the methylation status of the gene in gastric cancer tissue, because the regulation by methylation in its CpG islands is well known for BORIS. However, there was no correlation between the methylation status and gene expression. Then, we focused on the minisatellites (variable number of tandem repeats) of BORIS as another possible regulator for this abnormal expression. Previously, we reported the characterization of BORIS-MS2 and determined the frequency of alleles in cancer patients. A case-control study was performed using DNA from 774 controls and 496 patients with gastric cancer. There was no significant difference observed in the overall distribution of minisatellite alleles. These results suggest that additional different regulators for the abnormal BORIS expression in gastric cancer may exist. Additionally, we performed a segregation analysis of BORIS-MS2 with genomic DNA obtained from two generations of five families and from three generations of two families. BORIS-MS2 alleles were transmitted through meiosis following Mendelian inheritance, which suggests that this polymorphic minisatellite could be a useful marker for paternity mapping and DNA fingerprinting.