Microscopy, Electron, Transmission ; methods ; Negative Staining ; methods ; Viruses ; ultrastructure

To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.
Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; growth & development ; physiology ; ultrastructure ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Humans ; Virus Release ; Virus Replication

Multidrug resistance proteins (MRP2, ABCC2) may play a role in drug resistance in epilepsy by limiting gastrointestinal absorption and brain access of antiepileptic drugs (AEDs). We sought to investigate the effects of ABCC2 polymorphisms on plasma carbamazepine (CBZ) concentrations and pharmacoresistance in Chinese patients with epilepsy. ABCC2 rs717620, rs2273697, rs3740066 polymorphisms were genotyped by polymerase chain reaction amplification followed by restriction fragment length polymorphism analysis or direct automated DNA sequencing in 80 patients treated with CBZ monotherapy. There were no differences in CBZ maintenance doses or adjusted plasma CBZ concentrations among the ABCC2 rs717620, rs2273697 and rs3740066 genotypic groups. No associations between all the studied genotypes and haplotypes involving the three SNPs of ABCC2 and CBZ resistance were observed in this patient cohort. These results suggest that ABCC2 polymorphisms may not contribute to interindividual variabilities in CBZ daily maintenance doses, plasma concentrations, and treatment efficacy.
Epilepsy

Glutamatergic synapses are common excitatory chemical connections in mammalian central nervous system. At these synapses, most of baseline synaptic transmission is mediated by glutamate AMPA receptors. NMDA receptors that are sensitive to voltage-dependent magnesium blockade selectively contribute to activity-dependent synaptic plasticity. However, inhibition of NMDA receptors by systemic or local administration of NMDA receptor antagonists produced significant effects on different physiological functions that are not believed to depend on NMDA receptor related synaptic plasticity. Here we show that NMDA receptors contribute to synaptic responses in the anterior cingulate cortex (ACC), a region important for cognitive and other brain functions. The contribution of NMDA receptors became more prominent when synapses are stimulated at higher frequencies. Furthermore, at temperatures more close to physiological brain temperatures, more NMDA receptor mediated responses were recorded as compared to the room temperature. These data suggest a new function for NMDA receptors in the ACC as important postsynaptic receptors involved in synaptic transmission, in particular when cells are firing at high frequencies.
Animals ; Excitatory Postsynaptic Potentials ; physiology ; Gyrus Cinguli ; physiology ; Male ; Mice ; Mice, Inbred C57BL ; Neuronal Plasticity ; physiology ; Receptors, N-Methyl-D-Aspartate ; physiology ; Synapses ; physiology ; Synaptic Transmission ; physiology

Adenovirus type 40 and 41 (Ad40, Ad41), which belong to human adenovirus subgroup F, are called fastidious adenoviruses due to their property of poor growth in cultured cell lines in vitro The effect of expression of exogenous E1B55K in Hep2 on Ad41 replication in this cell line was investigated. E1B55K gene was amplified by PCR with DNA extracted from Ad41-positive feces supernatant as template. Eukaryotic expression plasmid (pcDNA3) carrying E1B55K was constructed, purified, and transferred into Hep2 cell. Expression of E1B55K in G418-resistant clones was assayed by RT-PCR, and one clone named as Hep2-E1B4#4 could produce more Ad41 progenies when compared with other clones by the method of inducing complete cytopathic effect (CPE) in 293 cells. Infection of equivalent Ad41 caused more significant cytopathic effect (CPE) in Hep2-E1B#4 than that in the control cells of Hep2 or Hep2-DNA3, also suggesting enhanced viral replication in Hep2-E1B#4. The titer of Ad41 was further determined by method of immunocytochemical staining, and semi-quantity PCR was employed to compare the copy number of Ad41 genome DNA. The results showed that the yield of Ad41 in Hep2-E1B#4 was more than 9 times of that in control cells when equal amount of seed viruses were incubated, and the copy number of Ad41 genome increased 4 times in the raw extract from the infected Hep2-E1B#4 when compared with that from control cells. In conclusion, E1B55K gene transfer improved the ability of Hep2 in packaging Ad41, and the Hep2-E1B#4 cell line, which expressed E1B55K constitutively, would be helpful in isolation, cultivation and amplification of Ad41.
Adenovirus E1B Proteins ; genetics ; metabolism ; Adenoviruses, Human ; genetics ; Cell Line, Tumor ; Gene Expression ; Humans ; Immunohistochemistry ; Reverse Transcriptase Polymerase Chain Reaction ; Virus Replication ; genetics

Glutamate is the major fast excitatory transmitter in the central nervous system. While normal synaptic transmission is mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors, N-methyl-D-aspartate (NMDA) receptors are thought to selectively contribute to plasticity. Genetically enhancing NMDA receptor functions enhances animal behavior in normal physiological learning and enhances their sensitivity in the case of tissue injury. One major mechanism for NMDA receptors is synaptic long-term potentiation (LTP). Here we present evidence that NMDA receptors not only contribute to normal synaptic responses induced by stimulation of local layer V or white matters, but also contribute to generation of action potentials induced by a depolarizing step applied to the soma. Calcium-calmodulin sensitive adenylyl cyclase 1 and cAMP signal pathways likely mediate these effects. Considering the importance of cingulate neurons in nociception and pain, our results provide a new mechanism for NMDA receptor contributing to neuronal synaptic transmission, spiking properties in forebrains, and possible forebrain-related behavioral nociceptive responses and pain.
Action Potentials ; Adenylyl Cyclases ; metabolism ; Animals ; Cyclic AMP ; metabolism ; Mice ; Prosencephalon ; physiology ; Receptors, N-Methyl-D-Aspartate ; physiology

OBJECTIVETo construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity.

METHODSPreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting.

RESULTSThe morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting.

CONCLUSIONChimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.

Epitopes ; chemistry ; genetics ; immunology ; Hepatitis B ; immunology ; virology ; Hepatitis B Core Antigens ; chemistry ; genetics ; immunology ; Hepatitis B Surface Antigens ; chemistry ; genetics ; immunology ; Hepatitis B virus ; chemistry ; genetics ; immunology ; Humans ; Neutralization Tests ; Protein Precursors ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology

OBJECTIVETo study the antigenic properties of mutant hepatitis B virus surface antigen, to understand the sensitivity of the commercially available HBsAg assays to the variants and to reduce the undetectability of the variants.

METHODSRecombinant eukaryotic expression plasmids for HBsAg. The recombinant eukaryotic expression plasmids pSS1adr, pSS1adw2, pSS1adw2- 145Arg, pSS1adr-126 Asn and pSS1adr-126Ser were transfected into COS-7 cells. HBsAg in the supernatants of transfected cells was detected by using different commercial ELISA kits.

RESULTSThe absorbance value of pSS1adr-126 Asn and pSS1adr-126Ser plasmids were similar to that of the wild type HBsAg, the absorbance value of pSS1adw2-145Arg plasmids was lower than that of the wild type HBsAg.

CONCLUSIONIt is estimated that the antigenicity of HBsAg mainly depended on the amino acid sequence of "a" antigen determinant and its conformation, so 145 amino acid substitutions led to the change of conformation and the antigenicity of variant HBsAg was lower than that of the wild type.

Animals ; COS Cells ; Cercopithecus aethiops ; Culture Media, Conditioned ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B Surface Antigens ; analysis ; genetics ; immunology ; Mutation ; Transfection ; Viral Envelope Proteins ; genetics

OBJECTIVETo investigate the protective effect and mechanism of curcumin derivatives B06 on myocardium from type 2 diabetic rats.

METHODSThirty-five male SD rats were randomly divided into 5 groups, normal control group (NC group), high fat group (HF group), high fat treatment group (FT group), diabetes mellitus group (DM group) and diabetes treatment group (DT group) (n = 7). The late four groups were fed with high fat food, after four weeks of high fat feeding, the rats from DM group and DT group were injected with low dosage of streptozocin intraperitoneally to induce diabetes mellitus, FT group and DT group were gavaged with curcumin derivatives B06 at the dosage of 0.2 mg/kg x d. The blood glucose and lipid were detected biochemically, blood insulin was assayed by ELISA and the insulin resistance index was calculated, the morphology of myocardium was observed by light and transmission electron microscopy, the protein expression of AMP-activated protein kinase alpha (AMPKalpha) and phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) in myocardium were tested by Western blot.

RESULTSThe level of blood glucose, lipid, insulin and the insulin resistance index were increased in HF group and DM group, but they were decreased after the treatment with B06. The expression of AMPKalpha and p-AMPKalpha were decreased, but they became increased after the treatment of B06. There were increased collagen fibers in interstitium and expansion of mitochondria in cytoplasm of myocardium from DM group, but they were ameliorated in B06 treatment group.

CONCLUSIONIt is suggested that B06 may relieve the damage of myocardium from type 2 diabetic rats and the increased expression of AMPKalpha and p-AMPKalpha may be involved in it.

AMP-Activated Protein Kinases ; metabolism ; Animals ; Blood Glucose ; Curcumin ; pharmacology ; Diabetes Mellitus, Experimental ; physiopathology ; Heart ; drug effects ; Insulin Resistance ; Male ; Myocardium ; pathology ; Rats ; Rats, Sprague-Dawley ; Streptozocin