dTULP,the Drosophila melanogaster Homolog of Tubby,Regulates Transient Receptor Potential Channel Localization in Cilia

Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.     

The N-end rule proteolytic system in autophagy

The N-end rule pathway is a cellular proteolytic system that utilizes specific N-terminal residues as degradation determinants, called N-degrons. N-degrons are recognized and bound by specific recognition components (N-recognins) that mediate polyubiquitination of low-abundance regulators and selective proteolysis through the proteasome. Our earlier work identified UBR4/p600 as one of the N-recognins that promotes N-degron-dependent proteasomal degradation. In this study, we show that UBR4 is associated with cellular cargoes destined to autophagic vacuoles and is degraded by the lysosome. UBR4 loss causes multiple misregulations in autophagic pathways, including an increased formation of LC3 puncta. UBR4-deficient mice die during embryogenesis primarily due to defective vascular development in the yolk sac (YS), wherein UBR4 is associated with a bulk lysosomal degradation system that absorbs maternal proteins from the YS cavity and digests them into amino acids. Our results suggest that UBR4 plays a role not only in selective proteolysis of short-lived regulators through the proteasome, but also bulk degradation through the lysosome. Here, we discuss a possible mechanism of UBR4 as a regulatory component in the delivery of cargoes destined to interact with the autophagic core machinery.     

Si‐Encapsulating Hollow Carbon Electrodes via Electroless Etching for Lithium‐Ion Batteries

Remarkable improvements in the electrochemical performance of Si materials for Li‐ion batteries have been recently achieved, but the inherent volume change of Si still induces electrode expansion and external cell deformation. Here, the void structure in Si‐encapsulating hollow carbons is optimized in order to minimize the volume expansion of Si‐based anodes and improve electrochemical performance. When compared to chemical etching, the hollow structure is achieved via electroless etching is more advanced due to the improved electrical contact between carbon and Si. Despite the very thick electrodes (30 ～ 40 μm), this results in better cycle and rate performances including little capacity fading over 50 cycles and 1100 mA h g^?1 at 2C rate. Also, an in situ dilatometer technique is used to perform a comprehensive study of electrode thickness change, and Si‐encapsulating hollow carbon mitigates the volume change of electrodes by adoption of void space, resulting in a small volume increase of 18% after full lithiation corresponding with a reversible capacity of about 2000 mA h g^?1.     

Sox2 Level Is a Determinant of Cellular Reprogramming Potential

Epiblast stem cells (EpiSCs) and embryonic stem cells (ESCs) differ in their in vivo differentiation potential. While ESCs form teratomas and efficiently contribute to the development of chimeras, EpiSCs form teratomas but very rarely chimeras. In contrast to their differentiation potential, the reprogramming potential of EpiSCs has not yet been investigated. Here we demonstrate that the epiblast-derived pluripotent stem cells EpiSCs and P19 embryonal carcinoma cells (ECCs) exhibit a lower reprogramming potential than ESCs and F9 ECCs. In addition, we show that the low reprogramming ability is due to the lower levels of Sox2 in epiblast-derived stem cells. Consistent with this observation, overexpression of Sox2 enhances reprogramming efficiency. In summary, these findings suggest that a low reprogramming potential is a general feature of epiblast-derived stem cells and that the Sox2 level is a determinant of the cellular reprogramming potential.     

Fine-Needle Aspirates CYFRA 21-1 is a Useful Tumor Marker for Detecting Axillary Lymph Node Metastasis in Breast Cancer Patients

Introduction

To assess whether the value of CYFRA21-1 in the aspirates of ultrasonography-guided fine-needle aspiration biopsy (US-FNAB) can contribute to improving the performances of US-FNAB in the diagnosis of axillary lymph node (LN) metastasis in breast cancer patients.

Methods

US-FNAB was performed in 156 axillary LNs in 152 breast cancer patients (mean age: 51.4 years, range: 17–92 years). Concentrations of CYFRA21-1 were measured from washouts of the syringe used during US-FNAB. Tumor marker concentrations, US-FNAB, intraoperative sentinel node biopsy (SNB), and surgical pathology results were reviewed and analyzed. For comparison, the values of CEA and CA15-3 were also measured from washouts.

Results

Among the 156 LNs, 75 (48.1%) were benign, and 81 (51.9%) were metastases. Mean concentrations of CYFRA21-1 were significantly higher in metastasis compared to benign LNs (P<0.001). US-FNAB combined to CYFRA21-1 showed significantly higher sensitivity, NPV, and accuracy compared to US-FNAB alone (all values P<0.05). All diagnostic indices of US-FNAB combined to CYFRA21-1 were significantly higher compared to US-FNAB combined with CEA or CA15-3 (all P<0.001). Of the 28 metastatic LNs which showed metastasis on SNB, CYFRA21-1 showed higher positive rate of 75.0% (CEA or CA15-3∶60.7%, P = 0.076).

Conclusion

Measuring CYFRA 21-1 concentrations from US-FNAB aspirates improves sensitivity, NPV, and accuracy of US-FNAB alone, and may contribute to reducing up to 75.0% of unnecessary intraoperative SNB. Compared to CEA or CA15-3, CYFRA21-1 shows significantly higher performances when combined to US-FNAB in the preoperative diagnosis of LN metastasis in breast cancer patients.     

Inhibitory effect of α-ketoglutaric acid on α-glucosidase: integrating molecular dynamics simulation and inhibition kinetics

Abstract

The inhibition of α-glucosidase is used as a key clinical approach to treat type 2 diabetes mellitus and thus, we assessed the inhibitory effect of α-ketoglutaric acid (AKG) on α-glucosidase with both an enzyme kinetic assay and computational simulations. AKG bound to the active site and interacted with several key residues, including ASP68, PHE157, PHE177, PHE311, ARG312, TYR313, ASN412, ILE434 and ARG439, as detected by protein–ligand docking and molecular dynamics simulations. Subsequently, we confirmed the action of AKG on α-glucosidase as mixed-type inhibition with reversible and rapid binding. The relevant kinetic parameter IC₅₀ was measured (IC₅₀ = 1.738?±?0.041?mM), and the dissociation constant was determined (K_{i Slope} = 0.46?±?0.04?mM). Regarding the relationship between structure and activity, a high AKG concentration induced the slight modulation of the shape of the active site, as monitored by hydrophobic exposure. This tertiary conformational change was linked to AKG inhibition and mostly involved regional changes in the active site. Our study provides insight into the functional role of AKG due to its structural property of a hydroxyphenyl ring that interacts with the active site. We suggest that similar hydroxyphenyl ring-containing compounds targeting key residues in the active site might be potential α-glucosidase inhibitors. Abbreviations AKG alpha-ketoglutaric acid

pNPG 4-nitrophenyl-α-d-glucopyranoside

ANS 1-anilinonaphthalene-8-sulfonate

MD molecular dynamics.

Communicated by Ramaswamy H. Sarma     

Time-course Changes in Immunoreactivities of Glucokinase and Glucokinase Regulatory Protein in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia–reperfusion (I–R). Five days after I–R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

Identification of a novel ligand binding site in phosphoserine phosphatase from the hyperthermophilic archaeon Thermococcus onnurineus

Phosphoserine phosphatase (PSP) catalyzes the final and irreversible step of L‐serine synthesis by hydrolyzing phosphoserine to produce L ‐serine and inorganic phosphate. Developing a therapeutic drug that interferes with serine production is of great interest to regulate the pathogenicity of some bacteria and control D ‐serine levels in neurological diseases. We determined the crystal structure of PSP from the hyperthermophilic archaeon Thermococcus onnurineus at 1.8 Å resolution, revealing an NDSB ligand bound to a novel site that is located in a fissure between the catalytic domain and the CAP module. The structure shows a half‐open conformation of the CAP 1 module with a unique protruding loop of residues 150–155 that possesses a helical conformation in other structures of homologous PSPs. Activity assays indicate that the enzyme exhibits marginal PSP activity at low temperature but a sharp increase in the k_cat/K_M value, approximately 22 fold, when the temperature is increased. Structural and biochemical analyses suggest that the protruding loop in the active site might be an essential component for the regulation of the activity of PSP from hyperthermophilic T. onnurineus. Identification of this novel binding site distantly located from the catalytic site may be exploited for the development of effective therapeutic allosteric inhibitors against PSP activity. © Proteins 2013. © 2012 Wiley Periodicals, Inc.