[Expression profile of circular RNA in inflammatory response in human bronchial epithelial cells induced by carbon black nanoparticles].

Objective: To explore the toxic effect of carbon black nanoparticles on human bronchial epithelial cells, and identify the differentially expressed circular RNA based on the full transcriptome high-throughput sequencing, so as to provide evidence for the development of biomarkers exposed to carbon black nanoparticles and their application on epigenetic toxicology. Methods: In June 2020, 16 HBE cells were treated with carbon black nanoparticles at concentrations of 20, 40 and 80 µg/ml, and 16 HBE cells without any intervention were used as the control group. The cytotoxicity of carbon black nanoparticles was detected by CCK8 and LDH experiments. Real-time quantitative fluorescent PCR (qRT-PCR) and ELISA were used to detect the changes of interleukin-6 (IL-6) and interleukin-8 (IL-6, IL-8) mRNA and protein levels of carbon black nanoparticles with concentration gradient after 72 h exposure. Western blot analysis was conducted to detect the expression levels of toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB (P-NF-κB), apoptosis-related speckled protein (ASC) and Caspase-1 associated with nuclear factor-κB. According to high-throughput sequencing results, differentially expressed Circrnas were screened and identified by qRT-PCR, and those with stable differentially expressed circrnas and the strongest association with the NF-κB pathway were selected for ring performance identification. Results: After being exposed to carbon black nanoparticles for 72 h, the activity of 16HBE cells decreased significantly (P<0.05), and the release of lactate dehydrogenase increased significantly (P<0.05). Compared with control group, mRNA expression levels of IL-6 and IL-8, protein levels of IL-6 and IL-8 were increased, and protein levels of TLR4, p-NF-κB, ASC and Caspase-1 were significantly up-regulated in 16 HBE cells of different concentrations, with statistical significance (P<0.05). Compared with the control group, a total of 492 differentially expressed circular Rnas (|log2 FC|>1) were detected. Among the 5 differentially expressed (P<0.05) circular Rnas, circ_002642 was selected as the object of subsequent research on circular Rnas, affter 72 hours of exposure to 80 µg/ml CBNPs, 16HBE cells showed signlficantly higher expression of circ_002642 (P<0.05) . Conclusion: Carbon black nanoparticles can induce differentially expressed circular RNAs associated with inflammatory response in human bronchial epithelial cells.

[Diagnostic value of FNDC5 in patients with subclinical diabetic cardiomyopathy].

Objective: To estimate the diagnostic value of fibronectin type Ⅲ-domain containing protein 5 (FNDC5) in subclinical diabetic cardiomyopathy. Methods: A total of 94 patients with type 2 diabetes (T2DM), who were hospitalized from April 2018 to June 2019 in the Third Affiliated Hospital of Soochow University, were enrolled in this study. Patients were divided into T2DM with cardiac dysfunction (subclinical DCM) group (n=47) and T2DM without cardiac dysfunction (non-DCM) group (n=47) according to echocardiography and gated myocardial perfusion imaging results. Basic clinical data and serum FNDC5 level were compared between the two groups. Logistic regression analysis was used to establish predicting models and the diagnostic efficiency of established models was compared by ROC curve analysis. Results: Compared to non-DCM group, patients in subclinical DCM group were older, with longer duration of diabetes, and had higher levels of glycosylated hemoglobin A1c (HbA1c), total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) (all P<0.05). Serum FNDC5 level was significantly lower in subclinical DCM group than in non-DCM group (P<0.001). FNDC5 level was positively correlated with ventricular septal e'(r=0.451,P=0.005), mitral valve e'(r=0.291,P<0.001), the ratio of peak early diastolic trans-mitral flow velocity (E) to peak late diastolic trans-mitral flow velocity (A)(r=0.490,P=0.002), while negatively correlated with A(r=-0.399,P<0.001), the average ratio of E/e'(r=-0.490,P<0.001), tricuspid regurgitation velocity(r=-0.567,P<0.001), left atrial volume index(r=-0.491,P<0.001). Univariate ROC analysis showed that the diagnostic efficacy of FNDC5(AUC=0.940,95%CI 0.897-0.982)was superior to age(AUC=0.639,95%CI 0.523-0.752), diabetic duration(AUC=0.663,95%CI 0.555-0.772), HbA1c(AUC=0.740,95%CI 0.638-0.839), TG(AUC=0.661,95%CI 0.547-0.776), TC(AUC=0.675,95%CI 0.563-0.788)and LDL-C(AUC=0.644,95%CI 0.532-0.756). Model 1 was established with subclinical DCM as dependent variable, age, diabetic duration, TG, TC, LDL-C and HbA1c as independent variables. Model 2 was established by adding FNDC5 as independent variable on the basis of model 1. Diagnostic efficacy for subclinical DCM was compared between the two models by ROC analysis. The diagnostic efficiency was better with model 2 (AUC=0.980) than with model 1 (AUC=0.879, P<0.001). When sensitivity was set at 0.617, the specificity of model 2 was higher than that of model 1(0.979 vs. 0.936). When sensitivity was set at 0.532, the sensitivity of model 2 was higher than that of model 1 (1.000 vs. 0.915). Conclusions: Our findings suggest that serum FNDC5 could be used as a novel biomarker for the diagnosis of subclinical DCM.

Identification of the CAD gene from Eucalyptus urophylla GLU4 and its functional analysis in transgenic tobacco.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in lignin biosynthesis. The genus Eucalyptus belongs to the family Myrtaceae, which is the main cultivated species in China. Eucalyptus urophylla GLU4 (GLU4) is widely grown in Guangxi. It is preferred for pulping because of its excellent cellulose content and fiber length. Based on GLU4 and CAD gene expression, a Eucalyptus variety low in lignin content should be obtained using transgenic technology, which could reduce the cost of pulp and improve the pulping rate, and have favorable prospects for application. However, the role and function of CAD in GLU4 is still unclear. In the present study, EuCAD was cloned from GLU4 and identified using bioinformatic tools. Subsequently, in order to evaluate its impact on lignin synthesis, a full-length EuCAD RNAi vector was constructed, and transgenic tobacco was obtained via Agrobacterium-mediated transformation. A significant decrease in CAD expression and lignin content in transgenic tobacco demonstrated a key role for EuCAD in lignin biosynthesis and established a regulatory role for RNAi. In our study, the direct molecular basis of EuCAD expression was determined, and the potential regulatory effects of this RNAi vector on lignin biosynthesis in E. urophylla GLU4 were demonstrated. Our results provide a theoretical basis for the study of lignin biosynthesis in Eucalyptus.

[Features of peripheral nerve injuries in workers exposed to vibration: an analysis of 197 cases].

Objective: To investigate the features of peripheral nerve injuries in workers exposed to vibration. Methods: A total of 197 male workers [median age: 34 years (21-50 years) ; median working years of vibration exposure: 7.3 years (1-20 years) ] engaged in grinding in an enterprise were enrolled. Their clinical data and electromyography results were analyzed to investigate the features of peripheral nerve impairment. Results: Of all workers, 96 (48.73%) had abnormal electromyography results. Of all workers, 88 (44.7%) had simple mild median nerve injury in the wrist, who accounted for 91.7% (88/96) of all workers with abnormal electromy-ography results. Six workers had ulnar nerve injury, superficial radial nerve injury, or/and superficial peroneal nerve injury and accounted for 6.3% of all workers with abnormal electromyography results. Of all workers, 88 had a reduced amplitude of median nerve sensory transduction, and 28 had slowed median nerve sensory transduction. A total of 46 workers were diagnosed with occupational hand-arm vibration disease and hospitalized for treatment. They were followed up for more than 4 months after leaving their jobs, and most of them showed improvements in neural electromyography results and returned to a normal state. Conclusion: Workers exposed to vibration have a high incidence rate of nerve injury in the hand, mainly sensory function impairment at the distal end of the median nerve, and all injuries are mild peripheral nerve injuries. After leaving the vibration job and being treated, most workers can achieve improvements and return to a normal state.

[Efficacy and safety of total neoadjuvant therapy versus neoadjuvant chemoradiotherapy in the treatment of locally advanced rectal cancer: a meta-analysis].

Objective: To systematically evaluate the efficacy and safety of total neoadjuvant therapy (TNT) in the comprehensive treatment of locally advanced rectal cancer. Methods: Literatures were screened from PubMed, Embase, Web of Science, Cochrane Library, CBM, Wanfang Data, VIP and CNKI from the inception date to May 2021 to collect the randomized controlled clinical trials (RCTs) of TNT followed by total mesorectal excision (TME) versus neoadjuvant chemotherapy (nCRT) followed by TME in the treatment of locally advanced rectal cancer. The data of overall survival, disease-free survival, R0 radical resection rate, pathological complete response (pCR) rate, T downstaging rate, the incidence of adverse events ≥ grade III, including neutropenia, nausea and vomiting, diarrhea, radiation dermatitis and nervous system toxicity, and the morbidity of complications within postoperative 30 days of the two groups were extracted from the included literatures. Review Manager 5.3 software was utilized for statistical meta-analysis. Results: Nine RCTs were finally enrolled including 2430 patients. Meta-analysis results showed that compared with nCRT group, patients in TNT group had longer overall survival (HR=0.80, 95%CI: 0.65-0.97, P=0.03) and higher pCR rate (RR=1.73, 95%CI: 1.44-2.08, P<0.01) with significant differences. Besides, there were no significant differences between two groups in disease-free survival (HR=0.86, 95%CI:0.71-1.05, P=0.14), R0 radical resection rate (RR=1.02, 95%CI: 0.99-1.06, P=0.17) and T downstaging rate (RR=1.04, 95%CI: 0.89-1.22, P=0.58) between two groups. In terms of treatment safety, the incidence of adverse events ≥ grade III (RR=1.09, 95%CI: 0.70-1.70, P=0.70) and morbidity of complications within postoperative 30 days (RR=1.07, 95%CI: 0.97-1.18, P=0.19) did not significantly differ between two groups. Conclusions: In the treatment of locally advanced rectal cancer, TNT may bring more survival benefits than nCRT and does not increase the incidence of adverse events and postoperative complications. Therefore, TNT could be used as a recommended treatment for patients with locally advanced rectal cancer.

DNA rearrangement and constitutive expression of the interleukin 6 gene in a mouse plasmacytoma.

To study the potential involvement of IL-6 in the development of plasmacytomas, a number of plasmacytoma lines were analyzed for alterations in the IL-6 locus. A DNA rearrangement due to the insertion of an intracisternal A particle retrotransposon 18 bp 5' of the transcriptional start site was detected in the cell line MPC11. The IL-6 gene is constitutively expressed in MPC11, suggesting the involvement of IL-6 in the development of certain myeloma/plasmacytomas according to the "autocrine growth hypothesis".

Analysis of restriction fragment length polymorphism in lymphokine genes of normal and autoimmune mice.

Analysis of RFLP has been employed in lymphokine genes of autoimmune and normal mice. No polymorphism could be detected in the loci containing IL-2, IL-2 receptor, IL-5, and IFN-gamma in NZB, NZW, BxSB, and MRL/lpr mice when compared with normal mice. Allelic forms were identified in the IL-1 alpha gene of BALB/c and in the IL-4 gene of NZW. The frequency of the Bam HI RFLP in the TNF-alpha gene of NZW which has been proposed to be associated with the development of autoimmune disease in (NZB x NZW)F1 mice has been analyzed in a number of different inbred strains and in wild mice. Since the same allele is inherited in most autoimmune, healthy laboratory and wild mice the TNF-alpha gene does not seem to be one of the causal agents that contributes to the development of autoimmunity in (NZB x NZW)F1 mice.

Tumor suppression after tumor cell-targeted tumor necrosis factor alpha gene transfer.

The tumor necrosis factor alpha (TNF-alpha) gene was introduced by retroviral gene transfer into the TNF-alpha-insensitive tumor cell line J558L. Production of 40 pg/ml TNF-alpha by clone J2T12 consistently did not change the growth rate in vitro, but drastically suppressed tumor growth when injected into syngeneic BALB/c mice. Within 2 wk, 90% of the mice inoculated with J558L cells developed a tumor, but none of the mice injected with J2T12 did so. Within the observation period (greater than 3 mo), 60% of the mice inoculated with J2T12 did not develop a tumor. In the other 40% of the mice, tumor manifestation was significantly delayed. Mice injected simultaneously with J2T12 cells and an anti-TNF-alpha monoclonal antibody developed tumors similar to parental J558L cells. Similarly, the tumor-suppressive effects of TNF-alpha were abolished, e.g., by injection of an anti-type 3 complement receptor (CR3) monoclonal antibody that is known to prevent migration of inflammatory cells. These results and the observation of tumor-infiltrating macrophages suggest that lack of tumorigenicity of J2T12 cells is due to the TNF-alpha secretion by the tumor cells and that TNF-alpha acts indirectly by a mechanism that involves chemotactic recruitment and activation of cells, predominantly of macrophages. In contrast, the tumor growth was not affected when, instead of TNF-alpha, interleukin 6 was expressed by J558L cells. Together, our results support the concept of tumor cell-targeted cytokine gene transfer as a tool for cancer treatment, and particularly demonstrate that extremely low doses of TNF-alpha produced by tumor cells are sufficient to inhibit tumor growth without detectable side effects.

Morphology and CO adsorption on platinum supported on thin Fe(3)O(4)(111) films.

Nucleation, growth and thermal stability of Pt particles supported on well ordered Fe(3)O(4)(111) thin films grown on Pt(111) were studied by scanning tunnelling microscopy (STM) and temperature programmed desorption (TPD) of CO. STM studies showed that Pt grows through the formation of single-layer islands that coalesce at high coverage. Vacuum annealing at 600 K caused Pt sintering and the formation of extended two-dimensional (2D) islands one and two layers in thickness at sub-monolayer coverage. Well faceted, three-dimensional (3D) Pt nanoparticles formed by annealing to temperatures above 800 K were encapsulated by an FeO(111) monolayer. These results were rationalized in terms of the high adhesion energy for Pt on iron oxide surfaces. CO TPD studies showed that 2D structures, formed at 600 K, exhibit much lower CO adsorption capacity as compared to the Pt(111) single crystal surface. This effect has been tentatively assigned to lattice expansion in the Pt 2D islands leading to weakening of the Pt-CO bond due to reduction of the [Formula: see text] back-donation.

The vanadium (IV) compound rescues septo-hippocampal cholinergic neurons from neurodegeneration in olfactory bulbectomized mice.

The bilateral olfactory bulbectomy (OBX) mouse exhibits neurodegeneration of cholinergic neurons in the medial septum with concomitant cognitive deficits. Consistent with our previous observations, choline acetyltransferase (ChAT) protein levels in the medial septum decreased by 43.5% 2 weeks after OBX without changes in glutamic acid decarboxylase-65 (GAD65) levels. Interestingly, levels of the vesicular acetylcholine transporter (VAChT), which is localized at cholinergic neuron terminals, decreased both in hippocampal CA1 and CA3 regions following OBX. Confocal microscopy showed that VAChT expression was more severely reduced in CA3 14 days after OBX compared with CA1. Intriguingly, chronic treatment with a vanadium (IV) compound, VO(OPT) [bis(1-N-oxide-pyridine-2-thiolato)oxovanadium(IV)] (0.5-1 mg as vanadium (V)/kg/day, i.p.), significantly rescued cholinergic neurons in the medial septum in a dose-dependent manner. VO(OPT) treatment also prevented decreased VAChT immunoreactivity both in CA1 and CA3 regions in the hippocampus. Consistent with these findings, an impaired hippocampal long-term potentiation (LTP) and memory deficits seen in OBX mice were significantly prevented by VO(OPT) treatment. Taken together, OBX induces neurodegeneration of septo-hippocampal cholinergic neurons and impairment of memory-related behaviors. The neuroprotective effect of VO(OPT) could lead to novel therapeutic strategies to ameliorate cognitive deficits associated with cholinergic neuron degeneration in Alzheimer's disease and other neurodegenerative disorders.

Nuclear factor kappaB nuclear translocation upregulates c-Myc and p53 expression during NMDA receptor-mediated apoptosis in rat striatum.

Nuclear factor kappaB (NF-kappaB) appears to participate in the excitotoxin-induced apoptosis of striatal medium spiny neurons. To elucidate molecular mechanisms by which this transcription factor contributes to NMDA receptor-triggered apoptotic cascades in vivo, rats were given the NMDA receptor agonist quinolinic acid (QA) by intrastriatal infusion, and the role of NF-kappaB in the induction of apoptosis-related genes and gene products was evaluated. QA administration induced time-dependent NF-kappaB nuclear translocation. The nuclear NF-kappaB protein after QA treatment was comprised mainly of p65 and c-Rel subunits as detected by gel supershift assay. Levels of c-Myc and p53 mRNA and protein were markedly increased at the time of QA-induced NF-kappaB nuclear translocation. Immunohistochemical analysis showed that c-Myc and p53 induction occurred in the excitotoxin-sensitive medium-sized striatal neurons. NF-kappaB nuclear translocation was blocked in a dose-dependent manner by the cell-permeable recombinant peptide NF-kappaB SN50, but not by the NF-kappaB SN50 control peptide. NF-kappaB SN50 significantly inhibited the QA-induced elevation in levels of c-Myc and p53 mRNA and protein. Pretreatment or posttreatment with NF-kappaB SN50, but not the control peptide, also substantially reduced the intensity of QA-induced internucleosomal DNA fragmentation. The results suggest that NF-kappaB may promote an apoptotic response in striatal medium-sized neurons to excitotoxic insult through upregulation of c-Myc and p53. This study also provides evidence indicating an unique signaling pathway from the cytoplasm to the nucleus, which regulates p53 and c-Myc levels in these neurons during apoptosis.

A fatal chronic ketamine poisoning.

A few papers in the literature reported incident deaths by acute ketamine poisoning. In this paper, we report an unusual homicide caused by chronic ketamine poisoning. The victim was a 34-year old married woman with no previous medical history (except as reported herein) who died in her own home. The court investigation revealed that she was chronically poisoned by her husband over a period of about one year in an act of homicide. Determination of ketamine concentrations in autopsy specimens was carried out with gas-chromatography/mass spectrometry (GC-MS). The results showed that ketamine concentration was 21 microg/mL in gastric contents, 3.8 microg/mL in blood and 1.2 microg/mL in urine. The most striking forensic findings were cardiac muscle fibrosis and hyaline degeneration of small arteries in victim's heart, the pathological features of ketamine poisoning previous reported only in animal studies.

DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX.

DRAM1 (DNA damage-regulated autophagy modulator 1) is a TP53 target gene that modulates autophagy and apoptosis. We previously found that DRAM1 increased autophagy flux by promoting lysosomal acidification and protease activation. However, the molecular mechanisms by which DRAM1 regulates apoptosis are not clearly defined. Here we report a novel pathway by which DRAM1 regulates apoptosis involving BAX and lysosomes. A549 or HeLa cells were treated with the mitochondrial complex II inhibitor, 3-nitropropionic acid (3NP), or an anticancer drug, doxorubicin. Changes in the protein and mRNA levels of BAX and DRAM1 and the role of DRAM1 in BAX induction were determined. The interaction between DRAM1 and BAX and its effect on BAX degradation, BAX lysosomal localization, the release of cathepsin B and cytochrome c by BAX and the role of BAX in 3NP- or doxorubicin-induced cell death were studied. The results showed that BAX, a proapoptotic protein, was induced by DRAM1 in a transcription-independent manner. BAX was degraded by autophagy under basal conditions; however, its degradation was inhibited when DRAM1 expression was induced. There was a protein interaction between DRAM1 and BAX and this interaction prolonged the half-life of BAX. Furthermore, upregulated DRAM1 recruited BAX to lysosomes, leading to the release of lysosomal cathepsin B and cleavage of BID (BH3-interacting domain death agonist). BAX mediated the release of mitochondrial cytochrome c, activation of caspase-3 and cell death partially through the lysosome-cathepsin B-tBid pathway. These results indicate that DRAM1 regulates apoptosis by inhibiting BAX degradation. In addition to mitochondria, lysosomes may also be involved in BAX-initiated apoptosis.

Rotenone impairs autophagic flux and lysosomal functions in Parkinson's disease.

BACKGROUND: Rotenone is an environmental neurotoxin that induces accumulation of α-synuclein and degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc), but the molecular mechanisms are not fully understood. We investigated whether rotenone induced impairment of autophagic flux and lysosomal functions. METHODS: Autophagy flux, accumulation of α-synuclein, lysosomal membrane integrity and neurodegeneration were assessed in the rotenone-treated rat model and PC12 cells, and the effects of the autophagy inducer trehalose on rotenone's cytotoxicity were also studied. RESULTS: Rotenone administration significantly reduced motor activity and caused a loss of tyrosine hydroxylase in SNpc of Lewis rats. The degeneration of nigral dopaminergic neurons was accompanied by the deposition of α-synuclein aggregates, autophagosomes and redistribution of cathepsin D from lysosomes to the cytosol. In cultured PC12 cells, rotenone also induced increases in protein levels of α-synuclein, microtubule-associated protein 1 light chain 3-II, Beclin 1, and p62. Rotenone increased lysosomal membrane permeability as evidenced by leakage of N-acetyl-beta-d-glucosaminidase and cathepsin D, the effects were blocked by reactive oxygen species scavenger tiron. Autophagy inducer trehalose enhanced the nuclear translocation of transcription factor EB, accelerated the clearance of autophagosomes and α-synuclein and attenuated rotenone-induced cell death of PC12 cells. Meanwhile, administration of trehalose to rats in drinking water (2%) decreased rotenone-induced dopaminergic neurons loss in SNpc. CONCLUSIONS: These studies indicate that the lysosomal dysfunction contributes to rotenone's neurotoxicity and restoration of lysosomal function could be a new therapeutic strategy for Parkinson's disease.

Downregulation of stereotyped behavior and production of latent locomotor behaviors in mice treated continuously with quinpirole.

D1 and D2 dopamine receptors mediate different behavioral effects in animals. We have attempted to selectively modulate dopamine-mediated behaviors by continuously administering D2 agonists to mice. Acute administration of the D2 agonist quinpirole caused dose-related stereotyped effects; no locomotor or grooming behavior was apparent. Continuously infusing quinpirole with implanted Alzet minipumps initially produced stereotyped behavior, but this stereotypy decreased by 2 hours, and was completely absent from 3 hours to 6 days of infusion. Within 1 day after implanting quinpirole, there appeared a significant locomotor behavior that continued for the 6 days of infusion. Acute challenge doses of quinpirole given either 6 days after infusing quinpirole or 12 hours after removing the quinpirole implant failed to elicit any stereotyped behavior and did not alter the locomotor behavior. By contrast, acute challenge injections with the D1 agonist SKF 38393 administered 12 hours after removing the quinpirole implant produced a characteristic grooming response. In an attempt to learn the mechanism of the behavioral effects induced by the continuous administration of quinpirole, the actions of relatively selective D1 and D2 antagonists were examined. The D2 antagonist sulpiride inhibited the stereotypy but not the locomotion induced by quinpirole. By contrast, the D1 antagonist Sch 23390 blocked the locomotion but not the stereotypy induced by quinpirole. These results indicate that chronic administration of quinpirole causes two separate behavioral effects: (1) a downregulation of stereotypy, perhaps by downregulating D2 receptors; and (2) the development of locomotion, which appears to involve both the downregulation of D2 receptors and an activation of D1 dopaminergic mechanisms.

Neuronal localization and modulation of the D2 dopamine receptor mRNA in brain of normal mice and mice lesioned with 6-hydroxydopamine.

A novel oligonucleotide probe was designed, characterized and utilized to study the distribution and modulation of the mRNA encoding the D2 dopamine receptor in the brain of the mouse. Using in situ hybridization histochemistry, the highest levels of the D2 receptor mRNA were found in regions of the brain containing the cell bodies and the terminal projection fields of the nigrostriatal, mesolimbic and mesocortical dopaminergic systems. Particularly high levels of the D2 receptor mRNA were found in substantia nigra pars compacta, ventral tegmental area, caudate-putamen and olfactory tubercle. This distribution generally paralleled that of the D2 dopamine receptor. Some areas, not usually associated with dopaminergic systems, also contained significant levels of the D2 receptor mRNA signal. These areas included the hippocampus, certain thalamic nuclei, the inferior colliculus and the spinal trigeminal nucleus of the medulla and spinal cord. Lesioning the corpus striatum with 6-hydroxydopamine had little effect on the level of the D2 receptor mRNA in the striatum but greatly reduced the hybridization signal in the substantia nigra pars compacta and ventral tegmental area. Similarly, lesioning the substantia nigra, nearly abolished the signal in the pars compacta but failed to substantially alter the D2 receptor mRNA signal in the striatum. These results suggest that the D2 receptor mRNA in the substantia nigra pars compacta was localized largely to dopaminergic cell bodies, the terminal projections of which lie in the striatum and codes for D2 autoreceptors and that the D2 receptor mRNA of the striatum is in non-dopaminergic cell bodies that are intrinsic to the striatum and probably codes for post-synaptic D2 receptors. Further, the evidence that lesions of striatum and substantia nigra induced with 6-hydroxydopamine greatly reduced the D2 receptor mRNA signal in the substantia nigra, without concomitantly increasing the D2 receptor mRNA in the striatum, suggests that the increase in dopamine receptor binding in the striatum that is ipsilateral to the lesion with 6-hydroxydopamine and the enhanced behavioral sensitivity to dopaminergic agonists, cannot be accounted for solely by an increase in D2 receptor mRNA.

D2 dopamine receptor messenger RNA is altered to a greater extent by blockade of glutamate receptors than by blockade of dopamine receptors.

To study further the molecular mechanisms by which glutamate and dopamine interact to regulate the functions of the basal ganglia, the effects of persistently inhibiting dopamine receptors and glutamate N-methyl-D-aspartate receptors on the density of D1 and D2 dopamine receptors and on the level of their transcripts were examined in mouse brain. To block dopamine receptors, mice were treated with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline once daily for two and six days, or were treated with fluphenazine-N-mustard once daily for five days. To block N-methyl-D-aspartate receptors, mice were treated with dizocilpine by continuous infusion with osmotic mini-pumps for two and six days. The density of D1 and D2 dopamine receptors was measured by receptor autoradiography, and the level of D1 and D2 dopamine receptor messenger RNA was measured by in situ hybridization histochemistry. The results showed that N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline blocked about 90% of both D1 and D2 dopamine receptors, but had no significant effect on the level of either D1 or D2 dopamine receptor messenger RNA. Fluphenazine-N-mustard, which was as effective as N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline in blocking D2 dopamine receptors but had little effect on D1 dopamine receptors, also had no significant effect on the level of D1 and D2 dopamine receptor messenger RNAs. By contrast, continuously infusing dizocilpine significantly decreased the levels of D2 dopamine receptor messenger RNA in striatum, nucleus accumbens and olfactory tubercle. Dizocilpine also caused small decreases in the density of D2 dopamine receptors, but only in posterior striatum was this decrease statistically significant. Dizocilpine slightly and transiently decreased the levels of D1 dopamine receptor messenger RNA in striatum but had no significant effect on the density of D1 dopamine receptors in any region examined. This study demonstrates that persistent blockade of D1 and D2 dopamine receptors has relatively little effect on the levels of D1 and D2 dopamine receptor messenger RNA, but that blockade of N-methyl-D-aspartate receptors produces a rapid and profound decrease in the levels of D2 dopamine receptor messenger RNA and a smaller decrease in the density of D2 dopamine receptors. These results suggest that N-methyl-D-aspartate receptors play an important role in the expression of D2 dopamine receptors in basal ganglia.

Antisense oligodeoxynucleotide inhibits D2 dopamine receptor-mediated behavior and D2 messenger RNA.

There are several subtypes of dopamine receptors in the central nervous system which mediate the actions of dopamine in producing its diverse motor and behavioral effects. In this study we determined whether an antisense oligodeoxynucleotide directed to the mRNA encoding one of the subtypes of the dopamine receptor can inhibit a specific dopamine-mediated behavior. Accordingly, the effects of a phosphorothioate-modified antisense oligodeoxynucleotide targeted toward the D2 dopamine receptor mRNA (D2 antisense) was studied in mice with unilateral 6-hydroxydopamine-induced lesions of the corpus striatum. Rotational behavior in response to different agents, and the levels of D2 and D1 dopamine receptors and D2 and D1 dopamine receptor mRNAs in corpus striatum were then measured. In control mice, lesioning resulted in a contralateral rotational behavior in response to the D1 dopamine receptor agonist SKF 38393, the D2 dopamine agonist quinpirole, and the muscarinic cholinergic agonist oxotremorine. Lesioning also caused an increase in D2 dopamine receptor mRNA levels in the dorsolateral striatum. Intraventricular injections of the D2 antisense inhibited rotational behavior induced by quinpirole but not that induced by SKF 38393 or that induced by oxotremorine. Repeated administration of the D2 antisense significantly reduced the levels of the D2 dopamine receptor and D2 dopamine receptor mRNA in the dorsolateral but not the dorsomedial striatum. Similar treatment failed to significantly alter the levels of the D1 dopamine receptor or D1 receptor mRNA in dorsolateral or dorsomedial striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Co-stimulation of cyclic-AMP-linked metabotropic glutamate receptors in rat striatum attenuates excitotoxin-induced nuclear factor-kappaB activation and apoptosis.

Interactions between glutamatergic mechanisms mediated by receptors of the ionotropic and metabotropic classes in the central nervous system are complex and incompletely understood. To explore the consequences of these interactions on excitotoxicity, we examined the influence of group II and group III selective metabotropic glutamate receptor agonists on the N-methyl-D-aspartate-induced apoptotic destruction of GABAergic neurons in rat striatum. The intrastriatal administration of a group III metabotropic glutamate receptor agonist (amino-4-phosphonobutyric acid, 900-1800 nmol), but not of a group II agonist [(2S,1'S,2'S)-(carboxycyclopropyl)glycine, 100-1800 nmol] produced internucleosomal DNA fragmentation. Similarly, amino-4-phosphonobutyric acid (600 nmol) but not (2S,1'S,2'S)-(carboxycyclopropyl)glycine (100-1800 nmol) destroyed some striatal neurons as indicated by a loss of D1 dopamine receptors and 67,000 mol. wt glutamate decarboxylase (glutamate decarboxylase-67) messenger RNA. On the other hand, the intensity of internucleosomal DNA fragmentation induced by N-methyl-D aspartate (150 nmol) was substantially decreased by the intrastriatal co-administration of either (2S,1'S,2'S)-(carboxycyclopropyl)glycine or amino-4-phosphonobutyric acid (100-600 nmol). Both (2S, 1'S,2'S)-(carboxycyclopropyl)glycine and amino-4-phosphonobutyric acid also reduced the N-methyl-D-aspartate-induced loss of striatal D1 dopamine receptors by 67% and 68% (both P < 0.001), and glutamate decarboxylase-67 messenger RNA by 68% and 61%, respectively. Furthermore, both (2S,1'S,2'S)-(carboxycyclopropyl)glycine and amino-4-phosphonobutyric acid also attenuated the N-methyl-D-aspartate-induced decline in striatal IKB-alpha protein levels by 62% and 37%, as well as the increase in nuclear transcription factor nuclear factor-kappaB binding activity by 135% and 94% (both P < 0.001), and the subsequent rise in p53 and c-Myc protein levels. These results suggest that stimulation of cyclic-AMP-linked metabotropic glutamate receptors inhibits ionotropic glutamate receptor-mediated activation of apoptotic cascades involving IkappaB-alpha degradation and nuclear factor-kappaB nuclear translocation, as well as p53 and c-Myc induction. Certain selective metabotropic glutamate receptor agonists might thus find utility as adjuncts to N-methyl-D-aspartate antagonists in the protection against the neurotoxicity initiated by excessive ionotropic glutamate receptor stimulation.