Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels in Arabidopsis Pollen Tubes

Potassium (K⁺) influx into pollen tubes via K⁺ transporters is essential for pollen tube growth; however, the mechanism by which K⁺ transporters are regulated in pollen tubes remains unknown. Here, we report that Arabidopsis thaliana Ca²⁺-dependent protein kinase11 (CPK11) and CPK24 are involved in Ca²⁺-dependent regulation of the inward K⁺ (K+_in) channels in pollen tubes. Using patch-clamp analysis, we demonstrated that K+_in currents of pollen tube protoplasts were inhibited by elevated [Ca²⁺]_cyt. However, disruption of CPK11 or CPK24 completely impaired the Ca²⁺-dependent inhibition of K+_in currents and enhanced pollen tube growth. Moreover, the cpk11 cpk24 double mutant exhibited similar phenotypes as the corresponding single mutants, suggesting that these two CDPKs function in the same signaling pathway. Bimolecular fluorescence complementation and coimmunoprecipitation experiments showed that CPK11 could interact with CPK24 in vivo. Furthermore, CPK11 phosphorylated the N terminus of CPK24 in vitro, suggesting that these two CDPKs work together as part of a kinase cascade. Electrophysiological assays demonstrated that the Shaker pollen K+_in channel is the main contributor to pollen tube K+_in currents and acts as the downstream target of the CPK11-CPK24 pathway. We conclude that CPK11 and CPK24 together mediate the Ca²⁺-dependent inhibition of K+_in channels and participate in the regulation of pollen tube growth in Arabidopsis.     

IL-15Rα deficiency leads to mitochondrial and myofiber differences in fast mouse muscles

The purpose of this study was to determine mitochondrial changes in fast muscles from interleukin-15 receptor alpha knockout (IL-15RαKO) mice. We tested the hypothesis that fast muscles from IL-15RαKO mice would have a greater mitochondrial density and altered internal structure compared to muscles from control mice. In fast muscles from IL-15RαKO mice, mitochondrial density was 48% greater with a corresponding increase in mitochondrial DNA content. Although there were no differences in the relative size of isolated mitochondria, internal complexity was lower in mitochondria from IL-15RαKO mice. These data support an increase in mitochondrial biogenesis and provide direct evidence for a greater density and altered internal structure of mitochondria in EDL muscles deficient in IL-15Rα.     

The Preparation and Performance of a New Polyurethane Vascular Prosthesis

We investigated the performance of small-caliber polyurethane (PU) small-diameter vascular prosthesis generated using the electrospinning technique. PU was electrospun into small-diameter, small-caliber tubular scaffolds for potential application as vascular grafts. We investigated the effects of electrospinning conditions (solution concentration, mandrel rotation speed) on the microstructure and porosity of the scaffolds for the purpose of preparing scaffolds with optimum microstructures and properties. We evaluated the mechanical properties of the scaffolds by tensile tests and the cytotoxicity of the PU small-diameter, small-caliber PU synthetic vascular graft by the MTT assay. The adhesion of endothelial cells to the PU scaffold was characterized by Hoechst staining and fluorescence microscopy, and we measured endothelial cell proliferation on the PU scaffold by the CCK-8 assay. We analyzed the prosthesis microstructure and endothelial cell morphology using scanning electron microscopy. With increasing PU concentration in the electrospinning solution, the fiber diameter of the vascular graft increased and the porosity decreased. In addition, with increasing electrospinning time, the wall thickness increased and the porosity decreased. We found that regular fiber orientation can be obtained by adjusting the rotation speed of the mandrel. Cell proliferation was not inhibited as the small-caliber PU synthetic vascular grafts showed little cytotoxicity. The endothelial cells had faster adherence to the PU scaffolds than to the PTFE surface during the initial contact. After prolonged cell culture, significantly higher endothelial cell proliferation rate was observed in the PU scaffold groups than the PTFE group. We obtained small-caliber PU vascular grafts with optimal fiber arrangement, excellent mechanical properties, and optimal biocompatibility by optimizing the electrospinning conditions. This study provides in vitro biocompatibility data that is helpful for the clinical application of the PU small-diameter, small-caliber PU vascular grafts.     

Surface Properties and Corrosion Behavior of Co–Cr Alloy Fabricated with Selective Laser Melting Technique

We sought to study the corrosion behavior and surface properties of a commercial cobalt–chromium (Co–Cr) alloy which was fabricated with selective laser melting (SLM) technique. For this purpose, specimens were fabricated using different techniques, such as SLM system and casting methods. Surface hardness testing, microstructure observation, surface analysis using X-ray photoelectron spectroscopy (XPS) and electrochemical corrosion test were carried out to evaluate the corrosion properties and surface properties of the specimens. We found that microstructure of SLM specimens was more homogeneous than that of cast specimens. The mean surface hardness values of SLM and cast specimens were 458.3 and 384.8, respectively; SLM specimens showed higher values than cast ones in hardness. Both specimens exhibited no differences in their electrochemical corrosion properties in the artificial saliva through potentiodynamic curves and EIS, and no significant difference via XPS. Therefore, we concluded that within the scope of this study, SLM-fabricated restorations revealed good surface properties, such as proper hardness, homogeneous microstructure, and also showed sufficient corrosion resistance which could meet the needs of dental clinics.     

2,3,4′,5-Tetrahydroxystilbene-2-O-β-D-glucoside Suppresses Expression of Adhesion Molecules in Aortic Wall of Dietary Atherosclerotic Rats and Promonocytic U937 Cells

We sought to investigate whether TSG suppressed the ICAM-1/VCAM-1 expression in dietary atherosclerotic rats and in Ox-LDL-induced U937 cells. For this purpose, 60 male Sprague–Dawley rats were randomly-and-equally divided into six groups. Atherosclerosis was induced by feeding rats a hyperlipidemic diet. TSG (120, 60 or 30 mg/kg/day) was administered by oral gavage. Simvastatin (2 mg/kg/day) was administered as positive control whereas physiological saline (0.9 % NaCl) served as untreated control. After 12 weeks, rats were euthanized by ethyl carbonate (1,200 mg/kg) and aortic wall samples were collected. Besides, U937 cells were stimulated for 48 h by Ox-LDL (80 μg/mL) with and without TSG (120, 60, 30 μg/L) or simvastatin (100 μg/L). ICAM-1/VCAM-1 mRNA expression was determined by RT-PCR and protein expression was detected by immunohistochemistry and/or western blotting. The data show that ICAM-1/VCAM-1 mRNA/protein expression was significantly enhanced in atherosclerotic aortas compared with normal diet group. Ox-LDL-induced ICAM-1/VCAM-1 mRNA/protein expression in U937 cells. Importantly, TSG significantly inhibited ICAM-1/VCAM-1 expression in atherosclerotic aortas in a dose-dependent manner. TSG-pretreatment also inhibited ICAM-1/VCAM-1 expression in Ox-LDL-induced U937 cells. Therefore, we concluded that TSG suppressed the expression of adhesion (ICAM-1/VCAM-1) molecules both in vivo (in aortic wall of dietary atherosclerotic rats) and in vitro (U937 cells).     

A Strategy for the Molecular Diagnosis in Hemophilia A in Chinese Population

Hemophilia A is an x-linked recessive inherited bleeding disorder. So far, more than 1,885 disease-causing mutations of factor VIII gene have been identified. Clinic confers a great challenge for the molecular diagnosis. We aim to make a better strategy for the molecular diagnosis in Hemophilia A. First, factor VIII intron 22 inversion and intron 1 inversion mutations were detected using Inversion-PCR and double-tube multiple PCRs. And then, non-inversion mutations were analyzed by denaturing high performance liquid chromatography and/or direct sequencing. Novel mutations were further analyzed the conservation and 3D structures by a B domain deleted crystallographic model and bioinformatics. Finally, we can indirectly confirm the diagnosis by linkage analysis for the patients with the confusing diagnosis by the techniques mentioned above. Eleven patients with the factor VIII Inv 22 were found, and the remaining 16 patients were found with 11 different mutations, of which 3 was novel mutations affecting A1, B domains and splicing site. Moreover, the prenatal diagnosis was performed on 14 fetuses. Ten fetuses were successfully confirmed to be normal, 1 fetus to be a heterozygote with factor VIII c.3275–3276 ins A and 3 fetuses to be hemizygotes with factor VIII Inv 22 mutation.     

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus

The crustacean X-organ–sinus gland (XO–SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9 kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO–SG, raises new questions concerning the established mode of MIH action.