E3 ubiquitin ligase UBR5 modulates circadian rhythm by facilitating the ubiquitination and degradation of the key clock transcription factor BMAL1.

The circadian clock is the inner rhythm of life activities and is controlled by a self-sustained and endogenous molecular clock, which maintains a ~ 24 h internal oscillation. As the core element of the circadian clock, BMAL1 is susceptible to degradation through the ubiquitin-proteasome system (UPS). Nevertheless, scant information is available regarding the UPS enzymes that intricately modulate both the stability and transcriptional activity of BMAL1, affecting the cellular circadian rhythm. In this work, we identify and validate UBR5 as a new E3 ubiquitin ligase that interacts with BMAL1 by using affinity purification, mass spectrometry, and biochemical experiments. UBR5 overexpression induced BMAL1 ubiquitination, leading to diminished stability and reduced protein level of BMAL1, thereby attenuating its transcriptional activity. Consistent with this, UBR5 knockdown increases the BMAL1 protein. Domain mapping discloses that the C-terminus of BMAL1 interacts with the N-terminal domains of UBR5. Similarly, cell-line-based experiments discover that HYD, the UBR5 homolog in Drosophila, could interact with and downregulate CYCLE, the BMAL1 homolog in Drosophila. PER2-luciferase bioluminescence real-time reporting assay in a mammalian cell line and behavioral experiments in Drosophila reveal that UBR5 or hyd knockdown significantly reduces the period of the circadian clock. Therefore, our work discovers a new ubiquitin ligase UBR5 that regulates BMAL1 stability and circadian rhythm and elucidates the underlying molecular mechanism. This work provides an additional layer of complexity to the regulatory network of the circadian clock at the post-translational modification level, offering potential insights into the modulation of the dysregulated circadian rhythm.

New phorbol ester derivatives as potent anti-HIV agents.

Tigliane esters show many biological activities, including anti-HIV-1 activity. Our aim in this study was to establish structure-anti-HIV activity relationships for four series of tigliane-type diterpenoids. We synthesized and evaluated 29 new phorbol ester derivatives for anti-HIV activity and for cytotoxicity against human tumor cell lines. Among them, three derivatives, two phorbol-13-monoesters (5d and 5e) and a phorbol-12,13-diester (6a), showed significant anti-HIV activity. We found that better anti-HIV activity was often associated with a shorter acyl ester at C-13. Particularly, compounds with a phenyl ring in the ester side chain exhibited excellent anti-HIV activity and had good safety indexes. Due to its significant anti-HIV potency with a high selectivity index, phorbol-12,13-dicinnamoate (6a) was chosen as the potential candidate for further preclinical trials.

Elevated serum levels of TNF-α, IL-8, and ECP can be involved in the development and progression of bronchial asthma.

OBJECTIVE: This study aimed to explore the value of elevated serum levels of tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and eosinophil cationic protein (ECP) in the diagnosis of bronchial asthma (BA). METHODS: A total of 170 patients with BA (case group, 85 patients in acute attack and 85 patients in clinical remission) and 150 healthy individuals (control group) were enrolled in this study. Enzyme-linked immunosorbent assay and receiver operating characteristic (ROC) curves were calculated for the contents and diagnostic values of serum TNF-α, IL-8, and ECP in BA. RESULTS: Compared with the control group, patients in acute attack and clinical remission had higher TNF-α, IL-8, and ECP levels (p < 0.05). The serum level of TNF-α was positively correlated with IL-8 and ECP (p < 0.05). ROC curves showed that the diagnostic threshold value of IL-8 was 13.53 ng/ml, its area under the curve (AUC) was 0.87, its specificity was 99.3%, and its sensitivity was 57.6%. The diagnostic threshold value of TNF-α was 1.29 ng/ml with AUC being 0.94, specificity was 89.3%, and sensitivity was 83.5%. ECP showed 7.22 ng/ml diagnostic threshold value (AUC = 0.88, specificity = 74.0%, sensitivity = 86.5%). The FEV1/pre(%) and FEV1/FVC were negatively correlated and the Z5/pre(%) and resonance frequency (Fres) were positively correlated with the serum levels of TNF-α, IL-8, and ECP in patients in acute attack and in clinical remission (all p < 0.05). CONCLUSION: Our findings reveal that elevated serum levels of TNF-α, IL-8, and ECP can be involved in the development and progression of BA.

[Effects of ketamine and alcohol on learning and memory impairment in mice].

OBJECTIVE: To study the effects of ketamine and alcohol on learning and memory in mice and its possible mechanism. METHODS: Forty mice were divided into 4 groups: normal control group, ketamine group, alcohol group, and alcohol plus ketamine group. Ketamine and alcohol were given by intraperitoneal injection and intragastric administration, respectively, 1 time per day, for 14 days. The ability of learning and memory in mice was tested by the method of step-down and Morris water maze. Acetylcholine (ACh) and 5-hydroxy tryptamine(5-HT) in mice brain tissue were analyzed for the possible mechanism. RESULTS: (1) Step-down: The treatment groups lessened the latency and added wrong times (P < 0.05). The number of errors in the combined treatment group significantly increased comparing with the single drug treatment group (P < 0.05). (2) Morris water-maze: The treatment groups prolonged the latency (P < 0.05), reduced the target quadrant activity time significantly (P < 0.05), and decreased the numbers of crossing the former platform significantly (P < 0.05). (3) Biochemical index determination: The concentrations of ACh and 5-HT in treatment groups decreased significantly (P < 0.05), showed a more decreasement comparing with the single drug treatment group. CONCLUSION: Ketamine has a synergistic effect with alcohol on learning and memory impairment in mice, which may be related to the common inhibitive effect on the ACh and 5-HT.

[Effects of ketamine on proliferation and apoptosis of pheochromocytoma cell].

OBJECTIVE: To explore the effect of ketamine on adrenal pheochromocytoma (PC12) cell proliferation inhibition and induction of apoptosis and its mechanism. METHODS: PC12 cells of rats were models for dopaminergic neuron. PC12 cells were cultured with ketamine at concentrations of 0.9, 1.2, 1.5, 1.8 and 2.1 mmol/L, respectively. The cell viability was measured by MTT method after incubation at 12, 24, 48 and 72h. Hoechst stain was used to observe the morphological changes of apoptosis. PC12 cells cultured after 48 h with different concentrations of ketamine were selected to detect apoptotic rate using flow cytometry and detect the expression of bax and bcl-2 proteins using Western blotting. RESULTS: For different concentrations of ketamine, vitality of PC12 cells significantly decreased with increase of the incubation time. Apoptosis was obviously observed using Hoechst staining. Flow cytometry showed that apoptosis rates significantly increased with increasing ketamine concentrations. CONCLUSION: Ketamine can inhibit the proliferation of PC12 cell by inducing apoptosis of the PC12 cell in a concentrations-dependent manner. The underlying mechanism may be related to promoting the expression of bax and inhibiting the expression of bcl-2 in the cells.

[The correlation between ketamine-induced schizophrenia-like signs in mice and the expressions of NRG1, ErbB4 mRNA].

OBJECTIVE: To explore the correlation between signs similar to schizophrenia in mice after ketamine administration and the expressions of NRG1 and ErbB4 mRNA in order to explain the possible pathogenesis of schizophrenia. METHODS: Fifty KM mice were randomly divided into 5 groups which were administered intraperitoneally with saline, clozapine and different dosages ketamine. The ketamine groups were administered intraperitoneally with low dosage (25 mg/kg), middle dosage (50 mg/kg) and high dosage (100 mg/kg) one time every day for 7 days. After administration of 100 mg/kg ketamine for 7 days, the clozapine group was introgastrically administered 20 mg/kg with clozapine one time every day for 7 days. The pathological changes of hippocampus neurons were observed by HE stain. The expressions of the NRG1 and ErbB4 mRNA in hippocampus were detected by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: In the group with high dosage of ketamine, the levels of NRG1 and ErbB4 mRNA were significantly lower than that of the group with saline. CONCLUSION: Ketamine may induce signs similar to schizophrenia in KM mice. The mechanism may be involved in the reduction of NRG1 and ErbB4 mRNA expression.

[Behavior study of ketamine-induced symptoms similar to schizophrenia in mice].

OBJECTIVE: To observe the symptoms similar to schizophrenia in mice after ketamine single or continuous injection and to evaluate the feasibility of schizophrenia model injected with different dose of ketamine. METHODS: A total of 40 male mice were randomly divided into 4 groups, which were injected intraperitoneally with physiological saline (control group), 25 mg/kg ketamine (low dose group), 50 mg/kg ketamine (middle dose group), and 100 mg/kg ketamine (high dose group) qd for 7 days continuously. The behavior changes of mice were observed. RESULTS: Hyperactivity, stereotyped behavior and ataxia (P < 0.01) were observed in high dose group after single injection. After continuous injection of ketamine for 7 days, the middle dose group showed hyperactivity, stereotyped behavior and ataxia (P < 0.05), stereotyped behavior and ataxia were more significant in high dose group (P < 0.01). CONCLUSION: Ketamine can induce the symptoms similar to schizophrenia in mice after single or continuous injection. The symptoms induced by high dose ketamine will be more prominent and stable after continuous injection.

[Advances in research of ketamine addiction mechanism].

Ketamine is a phencyclidine derivative acting primarily as a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) excitatory glutamate receptors. As a common intravenous anaesthetic in clinic, it is also increasingly abused because of its hallucination and addiction effects. Based on the pharmacological and toxicologic characteristics of ketamine and the acknowledged addiction mechanism of other abused drugs, this article reviews the possible addiction mechanism of the ketamine in the aspects of its enhanced effects and reward systems, the anatomic structures, the related receptors and the individual differences.

[Receptor antagonist of NMDA and animal models of schizophrenia].

Schizophrenia is one of the common mental diseases. Because the mechanism of the schizophrenia is significantly complicated, the cause is still unknown. N-methyl-D-aspartate receptor antagonist can simulate the positive and negative symptoms, as well as the cognitive disorder of schizophrenia. Thus it has been widely used to establish the animal models of schizophrenia. The relationship of the three blocking agents of ion channels (phencyclidine, MK-801, ketamine) and the establishment of schizophrenia animal models is reviewed in this article.

[Expression of MMP-2 and its clinical significance in cervical squamous carcinoma].

OBJECTIVE: To investigate the relationship of the expression of matrix metalloproteinase-2 (MMP-2) with tumorigenesis, development and metastasis of cervical squamous carcinoma. METHODS: The expression and distribution of MMP-2 protein were detected by using immunohistochemical SP method. The active protein and mRNA of MMP-2 were determined by using gelatin Zymography and RT-PCR, respectively. The relationships between those indexes and the factors related to clinical pathology of cervical carcinoma were analyzed statistically. RESULTS: Immunohistochemical studies demonstrated that MMP-2 was expressed in 81.13% of squamous cell carcinomas (SCC), but was less frequently expressed in high-grade squamous intraepithelial lesions (HSIL, 47.62%, 10/21) and low-grade squamous intraepithelial lesions (LSIL, 10.00%, 1/10, P<0.01). In SCC, MMP-2 protein was expressed in 91.30%(21/23) of patients, which positively correlated with lymph node metastasis significantly (P<0.05). And the expression of MMP-2 was not significantly related to the pathological grade, or stage status. By direct analysis of enzyme activities we found that the gelatinolytic activity of MMP-2 was significantly higher in SCC [(5.81 +/- 2.17)x10(4)] than in HSIL [(2.28 +/- 0.83) x10(4), P<0.01] and LSIL [(1.94 +/- 0.71)x10(4), P<0.01]. Expression of MMP-2 mRNA was significantly higher in carcinoma(0.87 +/- 0.44) than in HSIL(0.46 +/- 0.22, P<0.01) and in LSIL(0.37 +/- 0.20, P<0.01). CONCLUSION: The positive expression of MMP-2 can be used to estimate the metastatic potentiality and help the adjuvant treatment. MMP-2 is related well with the occurrence, invasion and metastasis of cervical squamous carcinoma.

Application of correlation techniques in the analysis of corpus cavernosum electromyographic signals.

AIM: To establish an objective, easy-to-use and comprehensive method to analyze corpus cavernosum electromyographic signals (CC-potentials). METHODS: CC-potentials were recorded during flaccidity in 23 young healthy volunteers, with surface electrodes placed on the penile shaft bilaterally. Based on the correlation function of Matlab software, an application program for the analysis of CC-potentials was developed. Individual CC-potentials and their autocorrelation function were evaluated, yielding parameters amplitude (A), duration (D), and dominant frequency (DF). The cross-correlation function of both longitudinal and bilateral pairs of adjacent electrodes was calculated to assess the similarity and mutual delay of CC-potentials recorded simultaneously from different parts of the CC. The parameters derived were squared maximum cross-correlation coefficient (Rmax) and delay (tau). Based on the absolute value of tau and the corresponding inter-electrode distance, propagation velocity (PV) was calculated. RESULTS: The values of the parameters were determined automatically. No significant difference related to the locations of the electrodes for parameters A, D, and DF was detected. The cross-correlation showed that both longitudinal and bilateral CC-potential pairs had highly similar waveforms (the absolute values of Rmax were 0.80 +/- 0.05 and 0.87 +/- 0.06, respectively). PV of longitudinal pairs was estimated as 6.15 +/- 3.98 cm/s. CONCLUSION: The application program for correlation analysis of CC-potentials is a comprehensive and versatile method to analyze corpus cavernosum electromyographic recordings. Its objectiveness makes multi-center application possible.

Antitumor and immunomodulatory activity of a polysaccharide from fungus Coprinus comatus (Mull.:Fr.) Gray.

In our study, a water-soluble polysaccharide (CCPa-1) was successfully purified from the fruiting bodies of Coprinus comatus by DEAE-cellulose and Sepharose CL-6B column chromatography. The molecular weight was evaluated to be 53.6 kDa as determined by high performance size exclusion chromatography (HPSEC). Sugar composition analysis revealed that CCPa-1 consisted primarily of galactose, glucose and arabinose in a molar ratio of 6.6:1.2:2.2. CCPa-1 could not only inhibit the growth of sarcoma 180 (S180) tumor transplanted in mice, but also increase the relative spleen/thymus indexes and body weight of tumor bearing mice. Moreover, Con A- or LPS-induced splenocyte proliferation was also enhanced after CCPa-1 administration in tumor-bearing mice. Furthermore, CCPa-1 significantly enhanced the Con A- or LPS-induced splenocyte proliferation and increased the production of TNF-α and IL-2. All the data demonstrated that CCPa-1 had a potential application as natural antitumor agent with immunomodulatory activity.

Glaucocalyxin A activates FasL and induces apoptosis through activation of the JNK pathway in human breast cancer cells.

THis study was conducted to analyze the molecular mechanisms responsible for anti-proliferation effects of glaucocalyxin A in cultured MCF-7 and Hs578T breast cancer cells. The concentration that reduced cell viability to 50% (IC50) after 72 h treatment was derived and potential molecular mechanisms of anti-proliferation using the IC50 were investigated as changes in cell cycle arrest and apoptosis. Gene and protein expression changes related to apoptosis were investigated by semi-quantitative RT-PCR and western blotting, respectively. Involvement of phosphorylated mitogen-activated protein kinases and JNK signaling in regulation of these molecules was characterized by western blotting. Cell viability decreased in a concentration-dependent manner and the IC50 was determined as 1 µM in MCF-7 and 4 µM in Hs578T cell. Subsequently, we demonstrated that the GLA-induced MCF-7 and Hst578T cell death was due to cell cycle arrest at the G2/M transition and was associated with activation of the c-jun N-terminal kinase (JNK) pathway. We conclude that GLA has the potential to inhibit the proliferation of human breast cancer cells through the JNK pathway and suggest its application forthe effective therapy for patients with breast cancer.

[An expression element of a new controlling insulin growth factors binding protein-3(IGFBP-3)in senescent cells].

AIM: To study the influencing factors of the increased expression of the controlling insulin growth factors binding protein-3(IGFBP-3)in the senescent cells. METHODS: Northern blot was used to show of the differential expression of the IGFBP-3 gene in the young and senescent 2BS cells; The size of 2 kb human IGFBP-3 upstream sequence including the series of the 5'-UTR area was amplified by PCR, and four groups of IGFBP-3 promoter fragments of different lengths were obtained by enzyme digestion. The Effectene Transfection Reagent (Qiagen) kit was used to transfect the fragments into the young and senescent cells. The promoting activity of several groups of constructing gene fragments were evaluated. The area of the controlling transfection activity was determined. The enhancer element in the activity area was ascertained by superimposing the oligonucleotide gel blocking experiment. RESULTS: Compared with the young 2BS cells, the expression of the IGFBP-3 gene in the senescent 2BS cells was enhanced. There was a protein binding in the fragment site from site +59 to -58 of the IGFBP-3 enhancer. 5'-ccagcctgccaagcagcgtgccccggttgc-3' was the enhancer element of IGFBP-3. CONCLUSION: In the 30 bp fragment from site -37 to -8 of the IGFBP-3 gene upstream, there is a new IGFBP-3 enhancer element IEE, which plays a controlling role in the expression of IGFBP-3.

Characterization of a novel positive transcription regulatory element that differentially regulates the insulin-like growth factor binding protein-3 (IGFBP-3) gene in senescent cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) is a well documented growth inhibitor and pro-apoptotic factor. IGFBP-3 mRNA and its protein are overexpressed by senescent human diploid fibroblasts. However, the mechanism responsible for the up-regulation of its expression is still unclear. This report describes a novel transcriptional regulatory element, IGFBP-3 enhancer element (IEE), identified in the 5' untranslated region of the IGFBP-3 gene. This element differentially activates IGFBP-3 expression in senescent versus young fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes in senescent cell nuclear extracts compared with young cell nuclear extracts. Similar to young proliferative cells, young quiescent cells showed reduced binding activity; enhancement of this activity was specific to senescent cells and not an effect of cell cycle arrest. The DNase I footprint revealed the protein-binding core sequence within the IEE through which the protein binds the IEE. Site-directed mutagenesis within IEE abolished binding activity and selectively decreased IGFBP-3 promoter activity in senescent (but not young) cells. Furthermore, introduction of an IEE decoy suppressed the endogenous IGFBP-3 gene expression specifically in senescent cells. These results point to the IEE as being a positive transcription regulatory element that contributes to the up-regulation of IGFBP-3 during cellular senescence.