Cytosolic activation of cathepsins mediates parvovirus H-1-induced killing of cisplatin and TRAIL-resistant glioma cells

Gliomas are often resistant to the induction of apoptotic cell death as a result of the development of survival mechanisms during astrocyte malignant transformation. In particular, the overexpression of Bcl-2-family members interferes with apoptosis initiation by DNA-damaging agents (e.g., cisplatin) or soluble death ligands (e.g., TRAIL). Using low-passage-number cultures of glioma cells, we have shown that parvovirus H-1 is able to induce death in cells resistant to TRAIL, cisplatin, or both, even when Bcl-2 is overexpressed. Parvovirus H-1 triggers cell death through both the accumulation of lysosomal cathepsins B and L in the cytosol of infected cells and the reduction of the levels of cystatin B and C, two cathepsin inhibitors. The impairment of either of these effects protects glioma cells from the viral lytic effect. In normal human astrocytes, parvovirus H-1 fails to induce a killing mechanism. In vivo, parvovirus H-1 infection of rat glioma cells intracranially implanted into recipient animals triggers cathepsin B activation as well. This report identifies for the first time cellular effectors of the killing activity of parvovirus H-1 against malignant brain cells and opens up a therapeutic approach which circumvents their frequent resistance to other death inducers.     

Metal-catalyzed oxidation of the Werner syndrome protein causes loss of catalytic activities and impaired protein-protein interactions

Metal-catalyzed oxidation reactions target amino acids in the metal binding pocket of proteins. Such oxidation reactions generally result in either preferential degradation of the protein or accumulation of a catalytically inactive pool of protein with age. Consistently, levels of oxidized proteins have been shown to increase with age. The segmental, progeroid disorder Werner syndrome results from loss of the Werner syndrome protein (WRN). WRN is a member of the RecQ family of DNA helicases and possesses exonuclease and ATP-dependent helicase activities. Furthermore, each of the helicase and exonuclease domains of WRN contains a metal binding pocket. In this report we examined for metal-catalyzed oxidation of WRN in the presence of iron or copper. We found that WRN was oxidized in vitro by iron but not by copper. Iron-mediated oxidation resulted in the inhibition of both WRN helicase and exonuclease activities. Oxidation of WRN also inhibited binding to several known protein partners. In addition, we did not observe degradation of oxidized WRN by the 20 S proteasome in vitro. Finally, exposure of cells to hydrogen peroxide resulted in oxidation of WRN in vivo. Therefore, our results demonstrate that WRN undergoes metal-catalyzed oxidation in the presence of iron, and iron-mediated oxidation of WRN likely results in the accumulation of a catalytically inactive form of the protein, which may contribute to age-related phenotypes.     

(R)- and (S)-verapamil differentially modulate the multidrug-resistant protein MRP1

The multidrug-resistant protein MRP1 (involved in the cancer cell multidrug resistance phenotype) has been found to be modulated by racemic verapamil (through stimulation of glutathione transport), inducing apoptosis of human MRP1 cDNA-transfected baby hamster kidney 21 (BHK-21) cells and not of control BHK-21 cells. In this study, we show that the two enantiomers of verapamil have different effects on MRP1 activity. Only the S-isomer (not the R-isomer) potently induced the death of MRP1-transfected BHK-21 cells. The decrease in cellular glutathione content induced by the S-isomer, which was not observed with the R-isomer, was stronger than that induced by the racemic mixture, indicating that the R-isomer antagonized the S-isomer effect. Both enantiomers altered leukotriene C(4) and calcein transport by MRP1. Thus, the R-isomer behaved as an inhibitor, which was confirmed by its ability to revert the multidrug resistance phenotype toward vincristine. Molecular studies on purified MRP1 using fluorescence spectroscopy showed that both enantiomers bound to MRP1 with high affinity, with the binding being prevented by glutathione. Furthermore, conformational changes induced by the two enantiomers (monitored by sodium iodide accessibility of MRP1 tryptophan residues) were quite different, correlating with their distinct effects. (S)-Verapamil induces the death of potentially resistant tumor cells, whereas (R)-verapamil sensitizes MRP1-overexpressing cells to chemotherapeutics. These results might be of great potential interest in the design of new compounds able to modulate MRP1 in chemotherapy.     

Nitric oxide synthase immunoreactivity and NADPH-d histochemistry in the enteric nervous system of Sarda breed sheep with different PrP genotypes in whole-mount and cryostat preparations.

Until now, significant differences in the neurochemical pattern of enteric neurons have been demonstrated in all species studied; however, some strong similarities also occur across species, such as the occurrence of nitric oxide synthase immunoreactivity (NOS-IR) in inhibitory motor neurons to muscle. In consideration of the insufficient data regarding the enteric nervous system (ENS) of sheep, we investigated the myenteric plexus and submucosal plexus of the ovine ileum. Since the pivotal role of the ENS in the early pathogenesis of sheep scrapie, the "prototype" of prion diseases, has been suggested, we have focused our observations also on the host's PrP genotype. We have studied the morphology and distribution of NOS-IR neurons and their relationships with the enteric glia in whole-mount preparations and in cryostat sections. NOS-IR neurons, always encircled by glial processes, were located in both plexuses. Many NOS-IR fibers were seen in the circular muscle layer, in the submucosa, and in the mucosa. In the submucosa they were close to the lymphoid tissue. No differences in the distribution and percentage of NOS-IR fibers and neurons were observed among sheep carrying different PrP genotype, thus making unlikely their contribution in the determinism of susceptibility/resistance to scrapie infection.     

猪卵母细胞c-mos基因表达以及RNA干扰

c-mos基因在动物卵母细胞减数分裂调控中起作用,但其作用机制目前仍不清楚。本实验通过RT-PCR、免疫荧光激光共聚焦检测方法检测了猪卵母细胞在体外成熟培养过程中c-mos基因在转录水平、翻译水平上的表达以及蛋白的分布,并应用注射小干扰RNA(siRNA)方法对其进行了RNA干扰(RNAi)研究。结果显示,猪卵母细胞在体外成熟培养过程中c-mos基因mRNA量逐渐增高,电激活后6h接近完全降解;MOS(c-mos基因蛋白产物)在GV卵母细胞生发泡中有一定量的表达,生发泡破裂(GVBD)前表达量增加且开始向卵母细胞胞质弥散,成熟培养44h未成熟卵母细胞中的MOS表达量要高于成熟卵母细胞,激活后6h核区MOS明显减少,但仍然有少量MOS分布于胞质中;成熟培养前干扰c-mos基因,所用三个siRNA都能成功敲低mRNA量,分别是同时期对照组mRNA量的0.08±0.03,0.11±0.06和0.20±0.06倍,干扰后虽然没有完全剔除MOS,但MOS量比同期卵母细胞有明显下降,仍可以引发成熟卵母细胞染色体解凝集。研究结果揭示了猪卵母细胞体外成熟及发育进程中c-mos基因在转录和翻译水平上的动态表达规律,建立了猪卵母细胞c-mos基因RNAi体系,为MOS在猪卵母细胞发育过程中的功能研究建立了重要的基础。     

A preliminary study of oscillating electromagnetic field effects on human spermatozoon motility

Some effects of extremely low frequency electromagnetic fields (ELF-EMFs) on human spermatozoa are reported. Significant increases in the values of the motility and of the other kinematic parameters have been observed when spermatozoa were exposed to an ELF-EMF with a square waveform of 5 mT amplitude and frequency of 50 Hz. By contrast, a 5 mT sine wave (50 Hz) and a 2.5 mT square wave (50 Hz) exposure did not produce any significant effect on sperm motility. The effects induced by ELF-EMF (50 Hz; 5 mT) during the first 3 h of exposure persisted for 21 h after the end of the treatment. These results indicate that ELF-EMF exposure can improve spermatozoa motility and that this effect depends on the field characteristics.     

Neuropeptidergic control of Octopus oviducal gland

The oviducal gland of the female of Octopus vulgaris lies about halfway along the oviduct. Progesterone and 17beta-estradiol receptors have been immunolocalized in the nuclei of the cells of the glandular compartment of previtellogenic glands. We also have evidence of FMRFamide-like and cGnRH-I-like immunoreactivity in the nerve endings that reach the oviducal gland. Moreover, we have recently shown APGWamide immunoreactivity in the glandular cells of the inner part of the oviducal gland. Here we report a review on these findings as well as our latest studies on the effect that neuropeptides may exert on the secretory activity of the oviducal gland. cAMP seems to be a possible second messenger involved in such a process. We discuss the findings of a neuropeptidergic action on the glandular cells of oviducal gland in a more complex frame of molecules, such as steroids, biogenic amines and neuromodulators, controlling the activity of the gland.     

Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade

Aβ vaccination as a therapeutic intervention of Alzheimer’s has many challenges, key among them is the regulation of inflammatory processes concomitant with excessive generation of free radicals seen during such interventions. Here we report the beneficial effects of melatonin on inflammation associated with Aβ vaccination in the central and peripheral nervous system of mice. Mice were divided into three groups (n = 8 in each): control, inflammation (IA), and melatonin-treated (IAM). The brain, liver, and spleen samples were collected after 5 days for quantitative assessment of plasma lipid peroxides (LPO), an oxidative stress marker, and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (Gpx). IA group mice have shown the elevated concentration of LPO significantly while there was a reduction at antioxidant enzyme levels. In addition, a significant (P < 0.05) reduction in neurotransmitters like dopamine (DA), 5-hydroxytryptamine (5-HT), and norepinephrine (NE) was also observed in the IA group mice. Nevertheless, their metabolites, such as homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) increased significantly (P < 0.05) as compared to control. Samples were further evaluated at microscopic level to examine the neuropathological changes by immunohistochemical methods. Melatonin treatment effectively reversed these above changes and normalized the LPO and antioxidant enzyme levels (P < 0.05). Furthermore, melatonin salvaged the brain cells from inflammation. Our Immunohistochemical findings in the samples of melatonin-treated animals (IAM group) indicated diminished expression of glial fibrillary acidic protein (GFAP) and nuclear factor kappa B (NfκB) than those observed in the IA group samples. Our results suggest that administration of melatonin protects inflammation associated with Aβ vaccination, through its direct and indirect actions and it can be an effective adjuvant in the development of vaccination in immunotherapy for Alzheimer’s disease (AD).