Regulation of the G2/M phase of the cell cycle by sperm associated antigen 8 (SPAG8) protein

Sperm associated antigen 8 (SPAG8), a testis‐specific protein produced during male germ cell differentiation, was isolated from a human testis expression library using antibodies found in the serum obtained from an infertile woman. It was found to have a close functional relationship with microtubules. In this study, we generated a stably expressing SPAG8 CHO‐K1 cell line. Immunofluorescence confocal microscopy showed that SPAG8 was concentrated at the microtubule‐organizing center (MTOC) during prophase. As the cells progressed into metaphase, it co‐localized with α‐tubulin on the spindle. In anaphase, it was detected on both astral microtubules and mid‐zone. Following cytokinesis, SPAG8 resumed its localization on the MTOC. Meanwhile, flow cytometry analysis found that SPAG8 prolonged the G2/M phase of CHO‐K1 cells stably expressing SPAG8. Furthermore, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay showed that SPAG8 inhibited the proliferation of the stable cells. SPAG8 might be involved in the regulation of cell cycle by changing the phosphorylation level of Tyr15 on cdc2. These results suggest that SPAG8 might play a role in cell division during spermatogenesis. Copyright © 2009 John Wiley & Sons, Ltd.     

Sitagliptin attenuates sympathetic innervation via modulating reactive oxygen species and interstitial adenosine in infarcted rat hearts

We investigated whether sitagliptin, a dipeptidyl peptidase‐4 (DPP‐4) inhibitor, attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in post‐infarcted normoglycemic rats, focusing on adenosine and reactive oxygen species production. DPP‐4 bound adenosine deaminase has been shown to catalyse extracellular adenosine to inosine. DPP‐4 inhibitors increased adenosine levels by inhibiting the complex formation. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline or sitagliptin in in vivo and ex vivo studies. Post‐infarction was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle‐treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with sitagliptin significantly increased interstitial adenosine levels and attenuated oxidative stress. Sympathetic hyperinnervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis and western blotting and real‐time quantitative RT‐PCR of NGF. Arrhythmic scores in the sitagliptin‐treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed a similar effect of erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (an adenosine deaminase inhibitor) to sitagliptin on attenuated levels of superoxide and NGF. Furthermore, the beneficial effects of sitagliptin on superoxide anion production and NGF levels can be reversed by 8‐cyclopentyl‐1,3‐dipropulxanthine (adenosine A₁ receptor antagonist) and exogenous hypoxanthine. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation via adenosine A₁ receptor and xanthine oxidase‐dependent pathways, which converge through the attenuated formation of superoxide in the non‐diabetic infarcted rats.     

Design and selection of trap color for capture of the tea leafhopper,Empoasca vitis,by orthogonal optimization

The tea leafhopper, Empoasca vitis (Göthe) (Hemiptera: Cicadellidae), is a major pest of the tea plant, Camellia sinensis (L.) O. Kuntze (Theaceae). In this study, the RGB color model was used to describe the colors of sticky traps. The most effective color for attraction of E. vitis was investigated by orthogonal optimization. The selected color was verified in tea gardens and the most effective height for positioning of color sticky traps for capturing tea leafhoppers was investigated. After the determination of the effect of the three color parameters and their interactions by orthogonal optimization, the color gold (RGB: 255, 215, 0) was selected as the most effective color to trap tea leafhoppers. In tea gardens, more leafhoppers were captured using gold sticky traps (RGB: 226, 204, 4) than using commercially available yellow sticky traps. The most effective height of gold sticky traps for trapping leafhoppers was 40–60 cm above the tea canopy. Few lady beetles were captured at this height. We conclude that the orthogonal optimization method is a convenient and efficient method to screen digitally generated colors for attracting and trapping of pests.     

Synthesis and characterization of new porphyrazine-Gd(III) conjugates as multimodal MR contrast agents

Magnetic resonance imaging (MRI) has long been used clinically and experimentally as a diagnostic tool to obtain three-dimensional, high-resolution images of deep tissues. These images are enhanced by the administration of contrast agents such as paramagnetic Gd(III) complexes. Herein, we describe the preparation of a series of multimodal imaging agents in which paramagnetic Gd(III) complexes are conjugated to a fluorescent tetrapyrrole, namely, a porphyrazine (pz). Zinc metalated pzs conjugated to one, four, or eight paramagnetic Gd(III) complexes are reported. Among these conjugates, Zn-Pz-8Gd(III) exhibits an ionic relaxivity four times that of the monomeric Gd(III) agent, presumably because of increased molecular weight and a molecular relaxivity that is approximately thirty times larger, while retaining the intense electronic absorption and emission of the unmodified pz. Unlike current clinical MR agents, Zn-Pz-1Gd(III) is taken up by cells. This probe demonstrates intracellular fluorescence by confocal microscopy and provides significant contrast enhancement in MR images, as well as marked phototoxicity in assays of cellular viability. These results suggest that pz agents possess a new potential for use in cancer imaging by both MRI and near-infrared (NIR) fluorescence, while acting as a platform for photodynamic therapy.     

A novel testis-specific GTPase serves as a link to proteasome biogenesis: functional characterization of RhoS/RSA-14-44 in spermatogenesis

Most Rho family GTPases serve as key molecular switches in a wide spectrum of biological processes. An increasing number of studies have expanded their roles to the spermatogenesis. Several members of Rho family have been confirmed to be essential for mammalian spermatogenesis, but the precise roles of this family in male reproduction have not been well studied yet. Here we report a surprising function of an atypical and testis-specific Rho GTPase, RSA-14-44 in spermatogenesis. Featured by unique structural and expressional patterns, RSA-14-44 is distinguished from three canonical members of Rho cluster. Thus, we define RSA-14-44 as a new member of Rho GTPases family and rename it RhoS (Rho in spermatogenic cells). RhoS associates with PSMB5, a catalytic subunit of the proteasome, in a series of stage-specific spermatogenic cells. More importantly, RhoS does not directly modulate the cellular proteasome activity, but participates in regulating the stability of "unincorporated" PSMB5 precursors. Meanwhile, our data demonstrate that the activation of RhoS is prerequisite for negatively regulating the stability of PSMB5 precursors. Therefore, our finding uncovers a direct and functional connection between the Rho GTPase family and the pathway of proteasome biogenesis and provide new clues for deciphering the secrets of spermatogenesis.     

Plasma-mediated release of morphine from synthesized prodrugs

Two morphine prodrugs (‘PDA’ and ‘PDB’) were synthesized and the kinetics of esterase-mediated morphine release from these prodrugs were determined when incubated with plasma from different animal species. Morphine was rapidly released from PDA by all species plasma with the maximum reached within 5–10 min; the released morphine was biologically active as determined by an in vitro cAMP assay. The morphine was released from PDB at a slower and species-dependent rate (mouse > rat > guinea pig > human). Morphine’s release from PDB appeared to be mediated by carboxyl esterases as the release was inhibited by the carboxyl esterase inhibitor benzil. PDA nor PDB induce cytotoxicity in the neuronal cell lines SK-NSH and SH-SY5Y. The carboxyl and amino functional moieties present on the linker portions of PDA and PDB, respectively, may facilitate their conjugation to nanoparticles to tailor morphine pharmacokinetics and specific targeting. These studies suggest the potential clinical utility of these prodrugs for morphine release at desired rates by administration of their mixture at selected ratios.