Role of calcium-sensing receptor in regulating spontaneous activation of postovulatory aging rat oocytes.

Mechanisms for postovulatory aging (POA) of oocytes and for spontaneous activation (SA) of rat oocytes are largely unknown. Expression of calcium-sensing receptor (CaSR) in rat oocytes and its role in POA remain unexplored. In this study, expression of CaSR in rat oocytes aging for different times was detected by immunofluorescence microscopy, and western blotting and the role of CaSR in POA was determined by observing the effects of regulating its activity on SA susceptibility and cytoplasmic calcium levels. The results showed that CaSR was expressed in rat oocytes. Oocytes recovered 19 h post human chorionic gonadotropin (hCG) injection were more susceptible to SA and expressed more functional CaSR than oocytes recovered 13 h after hCG injection, although both expressed the same level of total CaSR protein. Treatment with CaSR antagonist significantly suppressed cytoplasmic calcium elevation and SA of oocytes. Activation of Na-Ca2+ exchanger with NaCl inhibited SA to a greater extent than suppression of CaSR with NPS-2143, suggesting that calcium sources other than CaSR-controlled channels contributed to the elevation of cytoplasmic calcium. Treatment with T- or L-type calcium channel blockers significantly reduced SA. Suppression of all calcium channels tested reduced SA to minimum. It is concluded that the level of CaSR functional dimer protein, but not that of the total CaSR protein, was positively correlated with the SA susceptibility during POA of rat oocytes confirming that CaSR is involved in POA regulation. Blocking multiple calcium channels might be a better choice for efficient control of SA in rat oocytes.

A tetramethylpyrazine piperazine derivate CXC137 prevents cell injury in SH-SY5Y cells and improves memory dysfunction of rats with vascular Dementia.

We investigated the effects of CXC137, a tetramethylpyrazine piperazine derivate, on cell damage induced by N-methyl-D-aspartate (NMDA) in human derived neuroblastoma cells (SH-SY5Y) and its effect on memory dysfunction of rats with vascular dementia. It was found that the presence of CXC137 increased SH-SY5Y cells viability by inhibition of cell apoptosis induced by NMDA. These effects of CXC137 were accompanied by increases of the antioxidant superoxide dismutase activity and the level of reduced glutathione, and a decrease of lipid peroxidation product, malondialdehyde. The presence of CXC137 also showed to produce strong inhibition of cellular lactate dehydrogenase leakage, cell apoptosis and intracellular calcium overload. In a vascular dementia rat model established by bilateral common carotid arteries occlusion, treatment with CXC137 from 2 to 35 day of post-operation significantly improves the motor performance, spatial learning and memory capability of rats in both the prehensile traction test and Morris water maze test, an effect that was companied by reductions of the animal glutamic acid levels and the degree of brain mitochondrial swelling. These results suggest that CXC137 can improve the memory dysfunction in dementia and thus has important therapeutic potential for the treatment of dementia.

MSTMP, a Stilbene Derivative, Protects SH-SY5Y Cells Against Oxidative Stress.

OBJECTIVE: the protective effects of a novel stilbene derivative, (e)-2-(3,4,5- trimethoxystyryl)-3,5,6-trimethylpyrazine (MStMp), on hydrogen peroxide (h2o2)-induced human derived neuroblastoma cell (Sh-Sy5y) damage and its molecular mechanisms were investigated. METHODS: Sh-Sy5y cells were exposed to 200 µmol.l-1 h2o2 for 12 h. the effect of MStMp on cell viability and apoptosis was assessed by 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Mtt) assay and flow cytometry method. the activities of lactate dehydrogenase (ldh), superoxide dismutase (Sod) and nitric oxide synthetase (noS) and the content of malondialdehyde (Mda), reduced glutathione (gSh) and nitric oxide (no) in cells were determined by commercial kits. the expressions of pro-apoptotic factor caspase-3, caspase-9 and inducible noS (inoS) were detected by Western blotting. intracellular formation of reactive oxygen species (roS) was assessed using 6-carboxy-2',7'-dichlorofluorescin diacetate (dCfh-da) fluorescent probe. RESULTS: MStMp increased the Sh-Sy5y cell viability by inhibition of cell apoptosis induced by h2o2. these effects were accompanied by an increase of Sod activity, gSh level, and a decrease of Mda content. Moreover, MStMp showed stronger effects on inhibition of ldh leakage, apoptotic cells, intracellular roS level and the expression of caspase-3 and caspase-9 than tMp. furthermore, MStMp induced a decrease of no level and the activity of inoS, tnoS in a time-dependent manner. CONCLUSIONS: MStMp prevents h2o2-induced cell injury through anti-oxidation and anti-apoptosis via roS-no pathway.

Matrix metalloproteinase-8 regulates dendritic cell tolerance in late polymicrobial sepsis via the nuclear factor kappa-B p65/ß-catenin pathway.

Background: Tolerogenic dendritic cells (DCs) are associated with poor prognosis of sepsis. Matrix metalloproteinases (MMPs) have been shown to have immunomodulatory effects. However, whether MMPs are involved in the functional reprogramming of DCs is unknown. The study aims to investigate the role of MMPs in sepsis-induced DCs tolerance and the potential mechanisms. Methods: A murine model of late sepsis was induced by cecal ligation and puncture (CLP). The expression levels of members of the MMP family were detected in sepsis-induced tolerogenic DCs by using microarray assessment. The potential roles and mechanisms underlying MMP8 in the differentiation, maturation and functional reprogramming of DCs during late sepsis were assessed both in vitro and in vivo. Results: DCs from late septic mice expressed higher levels of MMP8, MMP9, MMP14, MMP19, MMP25 and MMP27, and MMP8 levels were the highest. MMP8 deficiency significantly alleviated sepsis-induced immune tolerance of DCs both in vivo and in vitro. Adoptive transfer of MMP8 knockdown post-septic bone marrow-derived DCs protected mice against sepsis-associated lethality and organ dysfunction, inhibited regulatory T-cell expansion and enhanced Th1 response. Furthermore, the effect of MMP8 on DC tolerance was found to be associated with the nuclear factor kappa-B p65/ß-catenin pathway. Conclusions: Increased MMP8 levels in septic DCs might serve as a negative feedback loop, thereby suppressing the proinflammatory response and inducing DC tolerance.

[Research progress of dual inhibitors targeting HIV-1 reverse transcriptase and integrase].

Both reverse transcriptase (RT) and integrase (IN) play crucial roles in the life cycle of HIV-1, which are also key targets in the area of anti-HIV drug research. Reverse transcriptase inhibitors are involved in the most employed drugs used to treat AIDS patients and HIV-infected people, while one of the integrase inhibitors has already been approved by US FDA to appear on the market. Great achievement has been made in the research on both, separately. Recently, much more attention of medicinal chemistry researchers has been attracted to the strategies of multi-target drugs. Compounds with excellent potency against both HIV RT and IN, evidently defined as dual inhibitors targeting both enzymes, have been obtained through considerable significant exploration, which can be classified into two categories according to different strategies. Combinatorial chemistry approach together with high throughput screening methods and multi-target-based virtual screening strategy have been useful tools for identifying selective anti-HIV compounds for long times; Rational drug design based on pharmacophore combination has also led to remarkable results. In this paper, latest progress of both categories in the discovery and structural modification will be covered, with a view to contribute to the career of anti-HIV research.

Maternal-restraint stress increases oocyte aneuploidy by impairing metaphase I spindle assembly and reducing spindle assembly checkpoint proteins in mice.

Studies in both humans and animals suggest detrimental effects of psychological stress on reproduction. Although our recent study shows that maternal-restraint stress diminishes oocyte developmental potential, the mechanism behind this effect is unknown. This prompted us to study the potential role of maternal-restraint stress in the genesis of aneuploidy during meiosis I. At 24 h after equine chorionic gonadotropin injection, mice were subjected to restraint stress for 24 h. After the restraint, some mice were killed to recover immature oocytes for in vitro maturation, while others were injected with human chorionic gonadotropin to recover in vivo matured oocytes. Analysis on chromosome complements of both mature oocytes and parthenotes confirmed that maternal restraint increased aneuploidy in both in vivo and in vitro matured oocytes and that the percentage of aneuploid oocytes were three times higher in the earlier matured oocytes than in the later matured ones. Further observations indicated that maternal restraint 1) impaired metaphase I (MI) spindle assembly while inhibiting MAPK activities, 2) accelerated progression of anaphase I while down-regulating the expression of spindle assembly checkpoint (SAC) proteins, and 3) induced intraoocyte oxidative stress. The following possible model was proposed to explain the results. Maternal-restraint stress increased oocyte aneuploidy by impairing MI spindle assembly and decreasing the SAC. Whereas abnormal spindles would affect centromere attachments, a reduction in SAC would accelerate the anaphase I progression. Failure of centromere attachment, together with the hastened anaphase, would result in nondisjunction of the unattached chromosomes. Furthermore, maternal-restraint stress might also impair spindle assembly and SAC function by inducing intraoocyte oxidative stress, which would then reduce MAPK activity, a critical regulator of microtubule assembly and the establishment and maintenance of the SAC during oocyte maturation.

[The application of structural optimization strategies in drug design of HIV NNRTIs].

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) play an important roles in the prevention and treatment of AIDS. NNRTIs can specifically target at HIV reverse transcriptase (RT) and have the advantages of high potency and low toxicity, which make them a research focus for a long time. In the guidance of structural optimization strategies (bioisosterism, molecular hybridization and scaffold hopping) in medicinal chemistry, structural modification to lead compounds can be carried out to design new compounds with different levels, which will improve the efficiency of drug discovery and decrease the cost of drug development. It is an effective way to find new NNRTIs. In this review, we will expatiate on the application of different levels of structural optimization strategies in the NNRTIs structural modification with concrete examples.

Mechanism of tetramethylpyrazine analogue CXC195 inhibition of hydrogen peroxide-induced apoptosis in human endothelial cells.

A tetramethylpyrazine analogue, CXC195, was synthesized by the Boekelheide reaction, in which the second methyl group of tetramethylpyrazine (TMP) was replaced with (4,4'-fluorine) diphenyl-methyl-1-piperazidine, the active group of flunarizine. We have observed protective effects of CXC195 on vascular endothelial cell survival under oxidative stress in previous study. The aim of the present study was to investigate the effects of CXC195 against apoptosis induced by hydrogen peroxide in human umbilical vein endothelial cells (HUVECs). Accordingly, a biochemical approach to elucidate the apoptotic signal pathways was attempted. HUVECs were exposed to 150 muM H(2)O(2) for 12 h, resulting in an increase of apoptotic cells assessed by the nuclear staining assay and flow cytometry. Mitochondrial membrane potential was detected by retention of rhodamine123. The concentration of free intracellular calcium was determined by fura-2/AM fluorometry. Co-incubation with CXC195 reduced the percentage of apoptotic cells and inhibited the loss of mitochondrial membrane potential and intracellular calcium overload induced by H(2)O(2). Induction of p53, the activation of caspase-3 by H(2)O(2) which accompanying downregulation of bcl-2, was blocked by CXC195. In addition, CXC195 clearly improved phosphorylation levels of the antiapoptotic extracellular signal-regulated kinase-1/2 (ERK1/2) in cells undergoing oxidative damage. Moreover, CXC195 showed stronger effects on inhibition of apoptotic cells and loss of mitochondrial membrane potential and activation of phosphorylated ERK1/2 than TMP. These results suggest that CXC195 prevents reactive oxygen species-induced apoptosis through inhibition of the mitochondria-dependent caspase-3 pathway and ERK pathway to show a better beneficial effect in protecting endothelial cells than TMP.

1,2,3-thiadiazole thioacetanilides. Part 2: Synthesis and biological evaluation of a new series of 2-{[4-(3,4-dichlorophenyl)-1,2,3-thiadiazol-5-yl]sulfanyl}acetanilides as HIV-1 inhibitors.

As part of our studies to discover new HIV-1 reverse transcriptase inhibitors, a series of 3,4-dichlorophenyl substituted 1,2,3-thiadiazole thioacetanilide (TTA=[(1,2,3-thiadiazole-5-yl)sulfanyl]acetanilide) derivatives were synthesized, and in vitro anti-HIV activity was evaluated. The results revealed that nearly half of the compounds show moderate-to-good inhibitory potency against HIV-1. In particular, compound 7f is highly potent, with an EC(50) value of 0.95+/-0.33 microM. The preliminary structure-activity relationship among the newly synthesized congeners is discussed.

TMPDP, a tetramethylpyrazine derivative, protects vascular endothelial cells from oxidation damage by hydrogen peroxide.

A novel ligustrazine derivative, tetramethylpyrazine diphenylmethyl piperazidine (TMPDP), prepared by hybridization and bioisosteric replacement of the molecular structure of TMP, was studied for its protective effects on oxidative damage of human umbilical vein endothelial cells (HUVECs) in response to hydrogen peroxide (H2O2). The antioxidative effect of TMPDP was assessed by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) test. Cell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione peroxidase (GSH) and the content of malondialdehyde (MDA) in cells were determined by commercial kits. The intracellular formation of reactive oxygen species (ROS) and the concentration of free intracellular calcium ([Ca2+]i) were determined using DCFH-DA assay and with fura-2/AM fluorimetry, respectively. Results showed that TMPDP had a moderate antioxidative effect against DPPH. Cell viability was decreased markedly by exposure to H2O2. Introduction of TMPDP, however, significantly increased cell viability, markedly reduced LDH release from cells and decreased lipid peroxidation in response to H2O2 treatment. These effects of TMPDP were accompanied by increased activity of the endogenous antioxidant enzymes, SOD and GSH, reduced production of ROS and reduced intracellular concentration of Ca2+. These results suggest that TMPDP protects HUVECs against oxidative damage by scavenging ROS and regulates intracellular calcium concentration. This might have important implications for the development of new agents for the effective treatment of vascular disease.

[The role of structural protein Gag and related gene (protein) in late stages of the HIV-1 replication cycle and the inhibitors].

The late stages of the HIV-1 replication cycle are important to the overall replication cycle. During the late stages, HIV-1 replication undergoes the processes of assembly, release, and maturation, resulting in the production of a mature virus particle capable of infecting a new target cell. The structural protein Gag and its related gene (protein) play a central role in these pathways. The different regions of Gag worked in concert to drive production of a mature infectious particle through protein-protein, protein-RNA and protein-lipid interactions. The designed drug aimed directly at these stages can efficiently block the maturation and infectivity of HIV-1. In this article, the role of structural protein Gag and related gene (protein) in late stages of the HIV-1 replication cycle and related inhibitors is reviewed.

[Progress in the study of HIV-1 Vif and related inhibitors].

Human immunodeficiency virus type 1 (HIV-1) viral infectivity factor (Vif), one of the accessory proteins, which is a small basic phosphoprotein, is essential for viral replication and pathogenesis. The best well-characterized function of Vif is its ability to neutralize the host cell antiviral factor, apolipoprotein B mRNA editing enzyme catalytic polypeptide like 3G (APOBEC3G), which makes the viral particles more infective. In addition, Vif can regulate the reverse transcription and the advanced stage of replication of the virus particle, as well as induce the termination of cell cycle at G2 stage and so on. The designed drug aimed directly at Vif can efficiently block the maturation and infectivity of HIV-1. In this review, the structure, function and especially the related inhibitors of Vif are reviewed.

Role of the HIV-1 positive elongation factor P-TEFb and inhibitors thereof.

Transcription is considered to be a crucial step in the replication cycle of HIV-1. Tat regulates an early step of transcription elongation. The positive elongation factor P-TEFb, a heterodimer containing a catalytic subunit (CDK9) and unique regulatory cyclins (CycT1), is required for HIV-1 Tat transcriptional activation. This is a potential target for new HIV-1 transcription inhibitors. Without P-TEFb, transactivation is restrained and only short transcripts are generated. All the P-TEFb inhibitors can suppress the HIV-1 transactivation process by inhibition of CycT1, CDK9 or their interaction. Several low-molecular-weight compounds such as flavopiridol, roscovitine and the human small nuclear RNA 7SK which have been showed to possess potent anti-HIV activity by interfering with P-TEFb functions are reviewed in this article.

Ligustrazine derivatives. Part 3: Design, synthesis and evaluation of novel acylpiperazinyl derivatives as potential cerebrocardiac vascular agents.

A series of novel acylpiperazinyl Ligustrazine derivatives was designed, synthesized, and their protective effects on damaged ECV-304 cells and antiplatelet aggregation activities were evaluated. The results showed that compound E33 displayed most potential protective effects on the ECV-304 cells damaged by hydrogen peroxide, and compound E1 was found to be the most active antiplatelet aggregation agent. Structure-activity relationships were briefly discussed.

[Action of protein phosphatase-1 on Tat-dependent HIV-1 transcription and its related inhibitors].

Host cell protein phosphatase-1 (PP1) is an important regulator of human immunodeficiency virus-1 (HIV-1) transcription. PP1 is involved in the regulation of HIV-1 transcription, and dephosphorylates RNA polymerase II C-terminal domain (RNAPII CTD) or CycT1-dependent kinase 9 (CDK9) to increase Tat-dependent HIV-1 transcription. In this review, we discuss the action of PP1 in Tat-induced HIV-1 transcription and related to PP1 inhibitors.

[LEDGF/p75: a novel target for anti-HIV therapy and advances in the study of its related inhibitors].

LEDGF/p75 is a newly found cell cofactor, which plays an essential role in the integration of HIV-1 cDNA into host chromosomes. LEDGF/p75 tethers HIV integrase to chromatin, protects it from degradation, and strongly influences the genome-wide pattern of HIV integration. Depleting the protein from cells or over-expressing the integrase-binding domain of LEDGF/p75 blocks viral replication. The essential role of LEDGF/p75 in HIV-1 replication makes it a new target for anti-HIV-1 drug development. This article reviews the function of LEDGF/p75, LEDGF/p75-integrase interaction and LEDGF/p75 inhibitors.

[The action of TSG101 on HIV-1 budding and related inhibitors].

tsg101 gene is a newly found tumor suppressor gene whose product TSG101 has many important biological functions. Recent research of TSG101 has revealed that TSG101 aids HIV-1 budding from infected cells by attaching to Gag. HIV-1 budding is arrested in the cells with mutant TSG101 or without TSG101. So TSG101 would be a useful target for anti-HIV drug design. Now there is already some research on anti-HIV agents based on TSG101 structure. In this article the structure and function of TSG101 as well as the related inhibitors were reviewed.

[Recent progress in the study of HIV-1 transcription factor NF-kappaB and its inhibitors].

Human immunodeficiency virus type 1 (HIV-1) transcription is a crucial step in the viral replication cycle, which is considered to be a potential target for inhibition of HIV-1 replication. Among the factors involved in this step, the cellular protein nuclear factor NF-kappaB is the most powerful inducer of HIV-1 transcription. HIV-1 transcription is initiated by the binding of NF-kappaB to the enhancer region in the long terminal repeat (LTR) of HIV-1. Several compounds suppress HIV-1 transcription through the inhibition of NF-kappaB activation. The mechanisms of NF-kappaB in the transcription of HIV-1 and progress of the current inhibitors of NF-kappaB are reviewed.

[HIV-1 Rev and related inhibitors].

HIV-1 Rev is an indispensable regulatory factor of the virion protein expression. The interaction between Rev and RRE RNA accelerates the nuclear export of viral mRNA. The unspliced and singly spliced mRNA will be degraded in the absence of Rev, resulting in the interception of HIV-1 replication at the same time. The pivotal role that Rev plays in HIV-1 replication as a trans-acting factor makes it a new target in the research of AIDS drugs. In this review, the function of Rev, Rev-RRE interaction, as well as their related inhibitors are reported.

Synthesis and anti-HIV evaluation of the novel 2-(m-chlorobenzyl)-4-substituted-7-methyl-1, 1, 3-trioxo-pyrazolo[4, 5-e] [1, 2, 4]thiadiazines.

A novel series of 2-(m-Chlorobenzyl)-4-substituted-1, 1, 3-trioxo-2H, 4H-pyrazolo[4, 5-e][1, 2, 4] thiadiazines (7a-k) were synthesized, and evaluated for their anti-HIV replication in MT-4 cell cultures. Compound (7a) showed activity against HIV-1-induced cytopathicity, with an EC50 value of 45.6 microM, but none of the compounds exhibited inhibitory activity against HIV-2.