Caveolin-1 sensitizes rat pituitary adenoma GH3 cells to bromocriptine induced apoptosis

Background  

Prolactinoma is the most frequent pituitary tumor in humans. The dopamine D₂ receptor agonist bromocriptine has been widely used clinically to treat human breast tumor and prolactinoma through inhibition of hyperprolactinemia and induction of tumor cell apoptosis, respectively, but the molecular mechanism of bromocriptine induction of pituitary tumor apoptosis remains unclear. Caveolin-1 is a membrane-anchored protein enriched on caveolae, inverted flask-shaped invaginations on plasma membranes where signal transduction molecules are concentrated. Currently, caveolin-1 is thought to be a negative regulator of cellular proliferation and an enhancer of apoptosis by blocking signal transduction between cell surface membrane receptors and intracellular signaling protein cascades. Rat pituitary adenoma GH3 cells, which express endogenous caveolin-1, exhibit increased apoptosis and shrinkage after exposure to bromocriptine. Hence, the GH3 cell line is an ideal model for studying the molecular action of bromocriptine on prolactinoma.     

Observation of reproductive cycle of female yellow-margined box turtle (Cuora flavomarginata) using radiography and ultrasonography

This study presents a combination of radiography and ultrasonography to observe the reproductive cycle of 24 captive female yellow-margined box turtles in Taiwan. Radiography was mainly used to monitor clutch size, whereas ultrasonography was applied to detect changes in the follicles throughout the year. The observation of the 24 female turtles was performed from April 2007 to June 2008. Their average carapace length was 16.62 ± 1.66 cm and their average body weight was 812 ± 164.98 g. The mean clutch size was three (87 eggs/29 clutches) and the reproductive frequency was 95.83% (23/24). Double clutches were detected in 79.2%, and 20.8% had single clutches. Ovulation occurred from March through August, and the average follicular diameter was 2.16 ± 0.18 cm. Follicles entered the latent period in October (at 1.54 ± 0.26 cm), and vitellogenesis of the next reproductive cycle began in November. Using radiography, the eggshell could be detected on the ninth day after ovulation. The average period of the single clutch group was 6.9 weeks (range 5.1-8.5 weeks). In the double clutch group, the average period of the first clutch was 5.5 weeks (range 4-7.8 weeks) and that of the second clutch was 5.2 weeks (range 4-7.8 weeks). This study has advanced the understanding of reproductive physiology of yellow-margined box turtle and established a valuable and practical model for comparative study of the reproductive physiology of other chelonians.     

Human albumin prevents 6-hydroxydopamine-induced loss of tyrosine hydroxylase in in vitro and in vivo

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.     

Lack of modulatory effect of simvastatin on indoxyl sulfate-induced activation of cultured endothelial cells

AimsEndothelial dysfunction is a common manifestation of chronic kidney disease (CKD). The protein-bound uremic toxins have emerged as important factors associated with cardiovascular disease and the outcome of CKD. The effect of indoxyl sulfate (IS) on endothelial cells remains unclear.Main methodsHuman umbilical endothelial cells (HUVEC) were incubated using IS at two concentrations: 100 μM and 1000 μM over two periods of time: 16 and 48 h. HUVEC were also pre-treated with simvastatin to examine its effect. RT-PCR was used to assess changes in the gene expression of intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), Monocyte chemotactic protein-1 (MCP-1), E-selectin, and angiotensin receptor type 1 (AT1R). Protein abundance of the investigated molecules was assessed by immunoblotting.Key findingsTreatment with 100 μM IS for 16 h induced a 2-fold increase in the expression of ICAM-1, VCAM-1, and MCP-1. At a concentration of 1000 μM, there was a 2–3-fold increase. An extended treatment period at low concentrations was associated with a 2–3 fold increase and the increase of ICAM-1 and VCAM-1 was more prominent under high concentration. Results of immunoblotting confirmed an increase in the abundance of ICAM-1, VCAM-1 and MCP-1. No significant change was noted in E-selectin and AT1R according to concentration or treatment duration. Pre-treatment with simvastatin did not alter IS-induced changes.SignificanceIS increased the expression of adhesion molecules of endothelial cells exhibiting a concentration and duration dependent pattern. Simvastatin did not demonstrate any effect on IS-associated endothelial activation.     

免疫复合物型治疗性乙型肝炎疫苗有效成份佐剂吸附率检测初探

目的:探索免疫复合物型治疗性乙型肝炎疫苗"乙克"的主要有效成份HBsAg/抗-HBs复合物(IC)及游离HBsAg的佐剂吸附率检测方法。方法:氢氧化铝佐剂经不同转速离心后,用ICP-AES法检测上清中铝残留量。未吸附IC经不同转速离心后,用电化学发光法检测在各离心转速下上清中HBsAg和抗-HBs含量。用电化学发光法检测未吸附IC与同一批次的佐剂吸附IC的HBsAg含量,计算HBsAg回收率;检测佐剂吸附IC样品离心上清及未离心样品的HBsAg含量,计算吸附百分率及检测的精密度。用凯氏定氮法测定佐剂吸附免疫复合物离心上清与离心前样品蛋白含量。结果:氢氧化铝佐剂经6500r/min离心3min,上清中铝的残留量为0;未吸附IC经2000～9000r/min离心3min,所测上清中HBsAg与抗-HBs均未明显下降。佐剂吸附IC经解离后,HBsAg回收率为91.56%,表明经过解离后铝佐剂不会影响HBsAg含量的检测结果。佐剂吸附IC样品与离心上清比较,蛋白含量减少了3.2mg/mL,表明每毫克铝可吸附约2.54mg蛋白,对总蛋白的吸附率为13.5%。佐剂吸附IC及HBsAg的吸附百分率的重复性检测结果为99.87%±0.15%,CV为0.15%。结论:佐剂吸附的IC在6500r/min离心3min的条件下,上清中未被吸附的HBsAg和抗-HBs不会下沉,而氢氧化铝佐剂连同被吸附IC及HBsAg则被沉淀,且铝佐剂不会对HBsAg的测定造成干扰,故通过检测离心样品上清及未离心样品中的HBsAg含量,可有效测定铝吸附百分率,且精密度较好。     

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

Background

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Methodology/Principal Findings

Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.

Conclusion

Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.     

Tumor necrosis factor- \alpha induces caspase-independent cell death in human neutrophils via reactive oxidants and associated with calpain activity

Apoptosis mediated by caspase activation is important in the neutrophil homeostasis and resolution of tissue inflammation. Paradoxically, our previous study demonstrated that broad-spectrum caspase inhibition augmented tumor necrosis factor (TNF)-alpha-induced cell death in the human neutrophils. Therefore, we further explored the mechanisms related to the caspase-independent cell death in the neutrophils. The cell apoptosis/necrosis was determined by annexin V and propidium iodide dual staining in flow cytometry. Their morphological changes were observed under light microscopy. Fluorogenic substrates were used to measure the intracellular oxidative reactions and the activities of proteinases, calpains. Calpain inhibitors and antioxidants were used to elucidate the relationship of calpains and oxidants with the neutrophil cell death. Our results verified the caspase-independent cell death pathway in the zVAD-sensitized, TNF-alpha-stimulated neutrophils. Furthermore, the cell death was accompanied with increased calpain and oxidative activities in the cells. Calpain inhibitors, zLLY, as well as anti-oxidants, catalase and DMSO, were able to attenuate the cell death in the zVAD-sensitized, TNF-alpha-induced neutrophils. Pretreating the neutrophils with G-CSF or GM-CSF was not able to reduce the cell death. These results demonstrate that, in human neutrophils, TNF-alpha-induces a caspase-independent cell death signal, which is related to calpain and oxidative activities and cannot be rescued by the growth factor-related signaling mechanism.     

easyExon – A Java-based GUI tool for processing and visualization of Affymetrix exon array data

Background  

Alternative RNA splicing greatly increases proteome diversity and thereby contribute to species- or tissue-specific functions. The possibility to study alternative splicing (AS) events on a genomic scale using splicing-sensitive microarrays, including the Affymetrix GeneChip Exon 1.0 ST microarray (exon array), has appeared very recently. However, the application of this new technology is hindered by the lack of free and user-friendly software devoted to these novel platforms.     

The dynein light chain Tctex-1 has a dynein-independent role in actin remodeling during neurite outgrowth

Coordinated microtubule and microfilament changes are essential for the morphological development of neurons; however, little is know about the underlying molecular machinery linking these two cytoskeletal systems. Similarly, the indispensable role of RhoGTPase family proteins has been demonstrated, but it is unknown how their activities are specifically regulated in different neurites. In this paper, we show that the cytoplasmic dynein light chain Tctex-1 plays a key role in multiple steps of hippocampal neuron development, including initial neurite sprouting, axon specification, and later dendritic elaboration. The neuritogenic effects elicited by Tctex-1 are independent from its cargo adaptor role for dynein motor transport. Finally, our data suggest that the selective high level of Tctex-1 at the growth cone of growing axons drives fast neurite extension by modulating actin dynamics and also Rac1 activity.