Site and mechanism of behavioral tolerance to cocaine: A study of dopamine release in Wistar-Kyoto and spontaneously hypertensive rats

Wistar-Kyoto and spontaneously hypertensive rats received i.v. infusions of cocaine hydrochloride (60 mg/kg per day) for 3, 7, and 14 days, or saline for 7 days. Acute cocaine challenge (40 mg/kg, s.c.) was given to treated and control rats 24 hr after the termination of each infusion period. There were no strain differences in brain levels of cocaine during cocaine infusion, nor after cocaine challenges. There were no strain differences in resting levels of [³H]dopamine release. Release of [³H]dopamine decreased in nuclei accumbens of 7- and 14-day cocaine-infused animals. Release of [³H]dopamine was maximal in both brain regions 2 hr after acute cocaine challenge. After 14 days of cocaine infusion, cocaine challenge in both strains reduced [³H]dopamine release in the nucleus accumbens, but not in the striatum; the reduction being greater in Wistar-Kyoto rats. The behavioral tolerance which accompanies similar cocaine infusion regimens may be related to striatal tolerance to cocaine-induced dopamine release.     

GPD1L inhibits renal cell carcinoma progression by regulating PINK1/Parkin-mediated mitophagy

Few approaches have been conducted in the treatment of renal cell carcinoma (RCC) after nephrectomy, resulting in a high mortality rate in urological tumours. Mitophagy is a mechanism of mitochondrial quality control that enables selective degradation of damaged and unnecessary mitochondria. Previous studies have found that glycerol-3-phosphate dehydrogenase 1-like (GPD1L) is associated with the progression of tumours such as lung cancer, colorectal cancer and oropharyngeal cancer, but the potential mechanism in RCC is still unclear. In this study, microarrays from tumour databases were analysed. The expression of GPD1L was confirmed by RT–qPCR and western blotting. The effect and mechanism of GPD1L were explored using cell counting kit 8, wound healing, invasion, flow cytometry and mitophagy-related experiments. The role of GPD1L was further confirmed in vivo. The results showed that GPD1L expression was downregulated and positively correlated with prognosis in RCC. Functional experiments revealed that GPD1L prevented proliferation, migration and invasion while promoting apoptosis and mitochondrial injury in vitro. The mechanistic results indicated that GPD1L interacted with PINK1, promoting PINK1/Parkin-mediated mitophagy. However, inhibition of PINK1 reversed GPD1L-mediated mitochondrial injury and mitophagy. Moreover, GPD1L prevented tumour growth and promoted mitophagy by activating the PINK1/Parkin pathway in vivo. Our study shows that GPD1L has a positive correlation with the prognosis of RCC. The potential mechanism involves interacting with PINK1 and regulating the PINK1/Parkin pathway. In conclusion, these results reveal that GPD1L can act as a biomarker and target for RCC diagnosis and therapy.     

Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).     

广椭牛齿兰，中国兰科一新记录种

报道了在海南热带雨林国家公园内发现的兰科牛齿兰属中国一新记录种：广椭牛齿兰[Appendicula ovalis(Schltr.) J.J.Sm. ex Mansf.]。该种原分布于马来半岛、苏门答腊岛、爪哇岛、婆罗洲、苏拉威西岛,在形态上与国内记录的4种牛齿兰属植物均有较大差别,如茎中部具分枝、叶广椭圆形、单个花序仅着生1朵花、且唇瓣扭转具粉紫色斑块等特征。对产自海南的该种形态和生境进行了详细描述,并提供高清解剖照片。凭证标本保存于海南大学林学院教学标本馆(HUFB)。     

Novel (E)-3-(3-Oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides as Histone Deacetylase Inhibitors: Design,Synthesis and Bioevaluation

Herein, we report the design, synthesis and evaluation of novel (E)-3-(3-oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides ( 4 a – i , 7 a – g ) targeting histone deacetylases. Three human cancer cell lines were used to test the cytotoxicity of the synthesized compounds (SW620, colon; PC-3, prostate; NCI−H23, lung cancer); inhibitory activity towards HDAC; anticancer activity; as well as their impact on the cell cycle and apoptosis. As a result, compounds 4 a – i bearing the alkyl substituents seemed to be less potent than the benzyl-containing compounds 7 a – g in all biological assays. Compounds 7 e – f were found to be the most active HDAC inhibitors with IC₅₀ of 1.498±0.020 μM and 1.794±0.159 μM, respectively. In terms of cytotoxicity and anticancer assay, 7 e and 7 f also showed good activity with IC₅₀ values in the micromolar range. In addition, the cell cycle and apoptosis of SW620 were affected by compound 7 f in almost a similar manner to that of reference compound SAHA. Docking assays were carried out for analysis the binding mode and selectivity of this compound toward 8 HDAC isoforms. Overall, our data confirmed that the inhibition of HDAC plays a pivotal role in their anticancer activity.     

The Puccinia striiformis effector Hasp98 facilitates pathogenicity by blocking the kinase activity of wheat TaMAPK4

The obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst) employs virulence effectors to disturb host immunity and causes devastating stripe rust disease. However, our understanding of how Pst effectors regulate host defense responses remains limited. In this study, we determined that the Pst effector Hasp98, which is highly expressed in Pst haustoria, inhibits plant immune responses triggered by flg22 or nonpathogenic bacteria. Overexpression of Hasp98 in wheat (Triticum aestivum) suppressed avirulent Pst-triggered immunity, leading to decreased H₂O₂ accumulation and promoting P. striiformis infection, whereas stable silencing of Hasp98 impaired P. striiformis pathogenicity. Hasp98 interacts with the wheat mitogen-activated protein kinase TaMAPK4, a positive regulator of plant resistance to stripe rust. The conserved TEY motif of TaMAPK4 is important for its kinase activity, which is required for the resistance function. We demonstrate that Hasp98 inhibits the kinase activity of TaMAPK4 and that the stable silencing of TaMAPK4 compromises wheat resistance against P. striiformis. These results suggest that Hasp98 acts as a virulence effector to interfere with the MAPK signaling pathway in wheat, thereby promoting P. striiformis infection.     

ARK2 stabilizes the plus-end of microtubules and promotes microtubule bundling in Arabidopsis

Microtubule dynamics and organization are important for plant cell morphogenesis and development. The microtubule-based motor protein kinesins are mainly responsible for the transport of some organelles and vesicles, although several have also been shown to regulate microtubule organization. The ARMADILLO REPEAT KINESIN (ARK) family is a plant-specific motor protein subfamily that consists of three members (ARK1, ARK2, and ARK3) in Arabidopsis thaliana. ARK2 has been shown to participate in root epidermal cell morphogenesis. However, whether and how ARK2 associates with microtubules needs further elucidation. Here, we demonstrated that ARK2 co-localizes with microtubules and facilitates microtubule bundling in vitro and in vivo. Pharmacological assays and microtubule dynamics analyses indicated that ARK2 stabilizes cortical microtubules. Live-cell imaging revealed that ARK2 moves along cortical microtubules in a processive mode and localizes both at the plus-end and the sidewall of microtubules. ARK2 therefore tracks and stabilizes the growing plus-ends of microtubules, which facilitates the formation of parallel microtubule bundles.     

Correlation between fibronectin and its receptor in chick myoblast differentiation

Alterations in the amount of fibronectin and in the number of its receptors during myoblast differentiation of chicken embryo were investigated. The amount of fibronectin in the cell surface pool as measured by immunoblotting decreased during myogenesis To identify and characterize the fibronectin receptors on the myoblasts, the interactions of the 28,000 dalton (28 kDa) amino terminal fragment and 85,000 dalton (85 kDa) cell-binding fragment of fibronectin with my-oblasts were examined. The binding of the 28 kDa fragment was found to be time-dependent and reached a maximum level within 60 min. The unlabeled 28 kDa fragment inhibited the binding of the radioiodinated 28 kDa fragment, whereas the unlabeled 85 kDa fragment and antibody to integrin did not inhibit it, suggesting that the 28 kDa fragment interacts with the matrix assembly receptors but not with the cell adhesion receptors. There was a single class of 3.4 × 10⁵ binding sites per cell with an apparent dissociation constant of 1.4 × 10^?7 M on 30 hr old myoblasts. The specific binding of the radioiodinated 28 kDa fragment to myoblasts decreased as the fusion proceeded. This decrease of binding was consistent with the decrease in the amount of fibronectin. Furthermore, the levels of fibronectin and binding of the radioiodinated 28 kDa fragment in the fusion-blocked myoblasts by EGTA treatment appeared to remain constant. These results suggest that the decrease and/or loss of fibronectin during myoblast fusion is closely correlated with the alteration of fibronectin receptors and with the fusion of myoblasts.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.