Preventive effects of ZPDC glycoprotein (24 kDa) on hepatotoxicity induced by mercury chloride in vitro and in vivo

Mercury is a potent environmental contaminant that exerts toxic effect on various vital organs in the human body. Recently, we isolated glycoprotein from Zanthoxylum piperitum DC (ZPDC), which has antioxidant and anticancer effects. In the present study, we determined the preventive effects of ZPDC glycoprotein on hepatic damage induced by mercury chloride (HgCl₂). We evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)], extracellular signal‐regulated kinase (ERK)1/2, p38 mitogen‐activated protein kinase (MAPK), cyclo‐oxygenase (COX‐2), inducible nitric oxide synthetase (iNOS), and activator protein (AP‐1) and the quantitative expressions of nuclear factor E2‐related factor (Nrf2), heme oxygenase (HO‐1), metallothionein (MT) and reduced glutathione (GSH) in mercury‐chloride‐exposed (50 μM and 10 mg/kg body weight) primary cultured hepatocytes and ICR mice, using biochemical assays, radioactivity and immunoblot analysis. The results demonstrated that ZPDC glycoprotein decreased the levels of LDH, ALT, HO‐1 and MT, whereas it increased the activities of hepatic antioxidant enzymes (SOD, CAT and GPx) and reduced GSH in mercury‐chloride‐exposed primary cultured hepatocytes. Also, it suppressed arachidonic acid release and expression of ERK, p38 MAPK, COX‐2, iNOS, AP‐1 and Nrf‐2 in primary cultured hepatocytes and ICR mice exposed to mercury chloride. Collectively, ZPDC glycoprotein may have potential applications to prevent hepatotoxicity induced by mercury chloride. Copyright © 2014 John Wiley & Sons, Ltd.     

The tetrodotoxin-insensitive sodium current in rat dorsal root ganglia is unlikely to involve the expression of the tetrodotoxin-resistant sodium channel,SkM2

Tetrodotoxin-insensitive (TTX-I) sodium currents have been recorded from newborn and adult rat sensory neurons, but the sodium channel gene(s) responsible for the TTX-I current are unknown. Because SkM2, one of six voltage-sensitive sodium channel genes cloned from rat, encodes the only cloned channel that is relatively resistant to tetrodotoxin, we sought to test whether the TTX-I current in rat sensory neurons is due to the SkM2 channel. We hypothesized that the TTX-I current might be generated from (1) an RNA splicing variant of SkM2, (2) post-translational modification of the SkM2 protein, or (3) interaction with altenate additional channel subunits. SkM2 mRNA expression was examined in newborn rat dorsal root ganglia (DRG) by RNase arotection assay. No SkM2 expression was detected. Therefore, we conclude that the TTX-I sodium current in DRG is unlikely to result from the expression of the SkM2 gene.     

Immunocytochemical localization of laminin in rat anterior pituitary cells in vivo and in vitro

Summary The distribution of laminin was investigated by immunocytochemistry in the rat anterior pituitary in vivo and in primary culture. It was localized by immunofluorescence and by immunoperoxidase in the basement membranes of the pituitary in vivo. In addition it was also found inside glandular cells both in vivo and in culture. The number of immunoreactive cells greatly varied depending on the technical approach used. It was always higher in primary cultures than in vivo. At the electron microscope level, a staining was observed on secretory granules, on rough endoplasmic reticulum cisternae as well as on the membrane of some Golgi saccules and vesicles. Such a localization, at the level of subcellular sites involved in the secretory process, suggests that these cells are able to synthesize and to export in vivo as well as in vitro this component of their basement membranes. This work was supported by grants from CNRS (Grant E.R. 89 and ATP “Pharmacologie des Récepteurs des Neuromediateurs”). Part of this work was performed at the EMBL (Heidelberg) during a short stay of C. Tougard (supported by an EMBO short term fellowship). EDITOR'S STATEMENT This paper documents the interesting observation that glandular cells from anterior pituitary contain laminin in their basement membranes and also apparently synthesize and secrete this extracellular matrix component. Gordon H. Sato     

Purification,immunological characterization and cDNA cloning of a 47 kDa glycoprotein secreted by carrot suspension cells

A 47 kDa glycoprotein, termed EP4, was purified from carrot cell suspension culture medium. An antiserum raised against EP4 also recognized a protein of 45 kDa that was ionically bound to the cell wall. EP4 was detected in culture media from both embryogenic and non-embryogenic cell lines and was found to be secreted by a specific subset of non-embryogenic cells. Secretion of the 47 kDa glycoprotein by embryogenic cells was not evident. The 45 kDa cell wall-bound EP4 protein was specific for non-embryogenic cells and was shown by immunolocalization to occur in the walls of clustered cells, with the highest levels in the walls separating adjacent cells. In seedlings, EP4 proteins were mainly found in roots. EP4 cDNA was cloned by screening a cDNA library with an oligonucleotide derived from an EP4 peptide sequence. The EP4 cDNA sequence was found to be 55% homologous to ENOD8, an early nodulin gene from alfalfa.DLO Centre for Plant Breeding and Reproduction Research (CPRO-DLO)     

Differential expression and localization of calmodulin-dependent phosphodiesterase genes during ontogenesis of chick dorsal root ganglion

The level and characteristics of 3'-5'-cyclic nucleotide phosphodiesterase (PDE) activity in chick dorsal root ganglion (DRG) extracts of 5-day posthatching chicken (P5) and E10 and E18 embryos were studied. At all stages, PDE activity is stimulated by calcium and calmodulin. A 5-fold increase in basal cAMP and cGMP PDE activity is evident from E10 to E18, while from E18 to P5 basal PDE activity remains constant. Ion exchange chromatography elution profile indicates that PDE1 isoforms represent the bulk of the PDE activity present. Inhibition studies were performed in order to distinguish the activity due to PDE1A, B and C. Western blot analysis using anti-mammalian PDE1A, B and C specific antibodies was also performed. Densitometric analysis of the stained bands reveals that PDE1B and PDE1C display a prominent increase between day 10 and day 18 of development (eight- and 3.6 fold, respectively) while a more limited increase (1.6- and 1.5-fold) is observed between E18 and P5; on the other hand PDE1A shows continuously increasing levels throughout development. Immunohistochemical analysis was performed with isoform specific antibodies used for western blot analysis. PDE1A immunoreactivity is found in the cytoplasm and fibers of several neurons differing in size and distributed throughout the ganglion. PDE1B staining is evident on all neurons, however, fibers appear very faintly labelled. All neurons appear stained by PDE1C antibody, although the intensity of immunostaining is always heterogeneous in different neuronal populations: no staining was evident on fibers or in non-neural cells. The distinct spatial and temporal expression patterns of PDE1 isoforms may indicate their different physiological roles in developing and mature chick DRG.     

Parallel modification of adenosine extracellular metabolism and modulatory action in the hippocampus of aged rats

The neuromodulator adenosine can be released as such, mainly activating inhibitory A1 receptors, or formed from released ATP, preferentially activating facilitatory A2A receptors. We tested if changes in extracellular adenosine metabolism paralleled changes in A1/A2A receptor neuromodulation in the aged rat hippocampus. The evoked release and extracellular catabolism of ATP were 49-55% lower in aged rats, but ecto-5'-nucleotidase activity, which forms adenosine, was 5-fold higher whereas adenosine uptake was decreased by 50% in aged rats. The evoked extracellular adenosine accumulation was 30% greater in aged rats and there was a greater contribution of the ecto-nucleotidase pathway and a lower contribution of adenosine transporters for extracellular adenosine formation in nerve terminals. Interestingly, a supramaximal concentration of an A1 receptor agonist, N6-cyclopentyladenosine (250 nM) was less efficient in inhibiting (17% in old versus 34% in young) and A2A receptor activation with 30 nM CGS21680 was more efficient in facilitating (63% in old versus no effect in young) acetylcholine release from hippocampal slices of aged compared with young rats. The parallel changes in the metabolic sources of extracellular adenosine and A1/A2A receptor neuromodulation in aged rats further strengthens the idea that different metabolic sources of extracellular adenosine are designed to preferentially activate different adenosine receptor subtypes.     

Enhanced neuronal regeneration by retinoic acid of murine dorsal root ganglia and of fetal murine and human spinal cord in vitro

Summary This study demonstrates that retinoic acid (RA), an active metabolite of vitamin A, can act to enhance regeneration of neurites, at physiologic concentrations, in vitro. Explanted fragments of mouse dorsal root ganglia (DRG) and mouse and human spinal cord (SC) were maintained, in vitro, for periods up to 11 d. Murine DRG neurons were exposed to RA concentrations ranging from 100 μM to 1 nM, whereas neurons within murine and human SC explants were exposed to 10 μM to 10 nM RA. Results show that RA significantly (P<0.001) increases mean neurite length but not neurite number. Specifically, murine DRG neurons showed increases in mean neurite length of 30.7% with individual explants showing increases of up to 133.5%. Murine and human SC showed mean enhancements of 43.4 and 58.1%, respectively, but did so at lower concentrations of RA. The results indicate that RA may play a potentially critical role in neuronal regeneration.     

A comparison between antisense P75NTR oligonucleotides and neurotrophic factors in promoting the survival of postnatal sensory neurons in vitro

Summary The 75-kDa low-affinity neurotrophin receptor (p75^NTR) has been shown in previous reports to mediate neuronal cell death in vitro and in vivo under certain circumstances. Antisense oligonucleotides directed against p75^NTR promote the survival of nerve growth factor-deprived dorsal root ganglia sensory neurons in vitro (Barrett, G.; Bartlett, P., Proc. Natl. Acad. Sci. USA 91:6501–6505; 1994) and axotomized dorsal root ganglia sensory neurons in vivo (Cheema, S. S.; Barrett, G. L.; Bartlett, P. F., J. Neurosci. Res. 46:239–245; 1996). In this study we compared the neuroprotective effects of antisense p75^NTR oligonucleotides with two neurotrophic factors, namely nerve growth factor (NGF) and leukemia inhibitory factor, on cultured sensory neurons derived from postnatal day 7 and 14 rat dorsal root ganglia. After 3 d in culture, treatment with the neurotrophic factors had significant survival effects on sensory neuron cultures compared to treatment with basal medium (control). However, after 6 and 9 d in culture these rescue effects were not apparent. In contrast, antisense p75^NTR oligonucleotides rescued significantly higher numbers of dorsal root ganglia sensory neurons after 6 and 9 d in culture than treatment with neurotrophic factors, sense oligonucleotides, and basal medium. Furthermore, antisense p75^NTR oligonucleotides rescued trkA-, B-, and C-expressing neurons, while NGF and leukemia inhibitory factor targeted primarily the trkA-positive neurons. These findings suggest that antisense-based strategies that inhibit gene expression of cytotoxic molecules are more efficient at preventing postnatal sensory neuronal death in vitro than treatment with individual neurotrophic factors.     

Microarray analyses of lncRNAs and mRNAs expression profiling associated with diabetic peripheral neuropathy in rats

Diabetic peripheral neuropathy (DPN) is considered to be the most frequent neuropathic complication of diabetes, and severely affects the quality of life of patients. Long noncoding RNAs (lncRNAs) participate in various pathophysiological processes and associate with many diseases. However, the exact impact of lncRNAs on DPN remains obscure. To discover a potential connection, a microarray study was conducted to analyze the expression profiling of lncRNAs and messenger RNAs (mRNAs) in dorsal root ganglia (DRG) from streptozotocin-induced diabetic rats with DPN. As a result, 983 lncRNAs and 1357 mRNAs were aberrantly expressed compared with control samples. Using bioinformatics analyses, we identified 558 Gene Ontology terms and 94 Kyoto Encyclopedia of Genes and Genomes pathways to be significantly enriched. Additionally, the signal-net analysis indicated that integrin receptors, including Itgb3, Itgb1, Itgb8, and Itga6, might be important players in network regulation. Furthermore, the lncRNA-mRNA network analysis showed dynamic interactions between the dysregulated lncRNAs and mRNAs. This is the first study to present an overview of lncRNA and mRNA expressions in DRG tissues from DPN rats. Our results indicate that these differentially expressed lncRNAs may have crucial roles in pathological processes of DPN by regulating their coexpressed mRNAs. The data may provide novel targets for future studies, which should focus on validating their roles in the progression of DPN.     

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime,and K048) in vivo detected by biochemical and histochemical techniques

Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.     

Analgesic effect of Photobiomodulation Therapy: An in vitro and in vivo study

Laser therapy, also known as Photobiomodulation (PBM) is indicated to reduce pain associated with different pathologies and applied using protocols that vary in wavelength, irradiance and fluence. Its mechanisms of action are still unclear and possibly able to directly impact on pain transmission, reducing nociceptor response. In our study, we examined the effect of two specific laser wavelengths, 800 and 970 nm, extensively applied in the clinical context and known to exert important analgesic effects. Our results point to mitochondria as the primary target of laser light in isolated dorsal root ganglion (DRG) neurons, reducing adenosine triphosphate content and increasing reactive oxygen species levels. Specifically, the 800 nm laser wavelength induced mitochondrial dysregulation, that is, increased superoxide generation and mitochondrial membrane potential. When DRG neurons were firstly illuminated by the different laser protocols and then stimulated with the natural transient receptor potential cation channel subfamily V member 1 (TRPV1) ligand capsaicin, only the 970 nm wavelength reduced the calcium response, in both amplitude and frequency. Consistent results were obtained in vivo in mice, by subcutaneous injection of capsaicin. Our findings demonstrate that the effect of PBM depends on the wavelength used, with 800 nm light mainly acting on mitochondrial metabolism and 970 nm light on nociceptive signal transmission.     

Induction of stress response and differential expression of 70 kDa stress proteins by sodium fluoride in hela and rat brain tumor 9l cells

We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr-phosphatases but not to Tyr-phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali-resistant 65-kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat-shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat-shock protein70 (the glucose-regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well-characterized regulatory elements revealed the activation of the heat-shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF-κB, AP-2, and CRE-like elements were detected in NaF-treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed. J. Cell. Biochem. 69:221–231, 1998. © 1998 Wiley-Liss, Inc.     

Differential expression of esterase and MDH isozymes during in vitro culture in maize (Zea mays L.)

Isozyme patterns of esterase and malate dehydrogenase were analyzed at different stages of in vitro culture of immature embryos and glumes of Zea mays L. viz. explant, callus formation, root formation and shoot formation. Significant changes in isoenzyme patterns of esterase and MDH were observed besides the appearance of specific and new isozymes. Specific fast migrating isozymes were noted in differentiating calli of embryo and glume calli which were absent at other stages suggesting a possible association of these isozyme patterns with in vitro differentiation.     

Target specificity and size of avian sensory neurons supported in vitro by nerve growth factor,brain-derived neurotrophic factor,and neurotrophin-3

To obtain insight into which subpopulations of sensory neurons in dorsal root ganglia are supported by different neurotrophins, we retrogradely labeled cutaneous and muscle afferents in embryonic day 9 chick embryos and followed their survival in neuron-enriched cultures supplemented with either nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3). We found that NGF is a wide survival factor for subpopulations of both cutaneous and muscle afferents, whereas the survival effects of BDNF and NT-3 are restricted primarily to muscle afferents. We also measured soma size in each neurotrophic factor. These new data show that BDNF- and NT-3–dependent cells appear to be a mixture of two populations of neurons: one small diameter and the other large diameter. In contrast, based on size alone, NGF-dependent cells appear to be a single population of only small-diameter neurons. Thus, BDNF and NT-3 may have some new, previously unreported effects on small-diameter afferent neurons. © 1994 John Wiley & Sons, Inc. 1994 John Wiley & Sons, Inc.     

Effect of estrus-associated glycoprotein and tissue inhibitor of metalloproteinase-1 secreted by oviduct cells on in vitro bovine embryo development

The effect of two glycoproteins (estrus-associated glycoprotein [EGP] and tissue inhibitor of metalloproteinase [TIMP-1]) secreted by bovine oviduct cells on in vitro bovine embryo development was assessed. A first set of experiments was conducted to determine whether the embryotrophic activity of the bovine oviduct-conditioned medium (BOCM) was correlated with the presence of EGP or TIMP-1. EGP and TIMP-1 were detected in BOCM, supporting the development of 22% zygotes to the blastocyst stage, as well as in BOCM yielding a low blastocyst rate (3–4% blastocysts). These glycoproteins do not seem to be necessary for bovine embryo development up to the blastocyst stage in our BOCM. In a second experiment, zygotes were cultured in modified synthetic oviduct fluid (mSOF) supplemented with different concentrations (0.5, 5, 50, and 500 μg/ml) of purified bovine EGP. In the third experiment, since purified bovine TIMP-1 was not available, zygotes were cultured in BOCM depleted of TIMP-1 by immunoprecipitation treatment. Adding EGP to mSOF, or removing TIMP-1 from BOCM, did not affect bovine embryo development up to the blastocyst stage, or mean number of cells per blastocyst after 8 days of culture. The results indicate that, under our culture conditions, EGP and TIMP-1 do not play an important role in sustaining bovine embryo development, and do not influence blastocyst quality, assessed in terms of total number of cells per embryo. Mol. Reprod. Dev. 46:527–534, 1997. © 1997 Wiley-Liss, Inc.