Hierarchical Mg/Al hydrotalcite oxide hollow microspheres with excellent adsorption capability towards Congo red.

A novel citrate anion-intercalated Mg/Al layered double hydroxide (CA-LDH) is synthesized via a one-step hydrothermal process. The synthesized CA-LDH is a hollow flower-like microsphere composed of thin nanoflakes (10 nm in thickness). After calcination, the formed Mg/Al layered double oxide (CA-LDO) hollow microspheres possess a high specific surface area of 247.8 m2 g-1 and a high pore volume of 0.97 cm3 g-1, which endow them with excellent adsorption ability towards Congo red (CR). The maximum adsorption capacity of CR onto CA-LDO can reach up to 1883 mg g-1. The significantly improved adsorption capacity of CA-LDO can be attributed to its unique structures of hierarchical hollow microspheres, in which the hierarchical porous shell layer provides enough adsorption sites to anchor the dye molecules, and the hollow core can preserve the absorbed dye. This study provides a promising novel adsorbent which can be used for efficient water remediation.

[Identification of released population of Japanese flounder based on stable isotopes analysis]. / åŸºäºŽç¨³å®šåŒä½ç´ æŠ€æœ¯çš„å¢žæ®–æ”¾æµç‰™é²†ç¾¤ä½“é‰´åˆ«.

Japanese flounder (Paralichthys olivaceus) is an important releasing fish in the Yellow and Bohai Seas of China. The identification of wild and releasing population is the premise to evaluate the enhancement effects. In order to study the application of stable isotope in the identification of released P. olivaceus population, captured juveniles in the offshore releasing area of Qinhuangdao were distinguished into wild and released population using previous method (combination of morphology and molecular). Then, the contents of carbon and nitrogen stable isotope in muscle and otoliths (including the whole and the core region) were measured. The cultured population was set as control. The results showed that δ13C values (wild population: -17.19‰±0.73‰; released population: -17.10‰±0.61‰; cultured population: -20.75‰±0.07‰) and δ15N values (wild population: 11.81‰±0.38‰; released population: 11.62‰±0.48‰; cultured population: 8.64‰±0.60‰) of muscle and δ13C value (wild popu-lation: -4.47‰±0.35‰; released population: -4.63‰±0.29‰; cultured population: -6.59‰±0.58‰) of the whole otolith could be used to identify the cultured population, but could not be used to distinguish the wild from the released population. The δ13C value (wild population: -4.66‰±0.30‰; released population: -5.41‰±0.21‰; cultured population: -5.37‰±0.19‰) of the core region of otolith could be used to identify the wild popu-lation. The δ18O values of the whole and the core region of otolith from these three groups were overlapped and could not be used to distinguish different populations. Our results indicated that the δ13C value of the core area of otolith could be used to identify wild and released population, with application prospect in the identification of broodstocks participating in spawning migration. This study provided basic data and technical methods for evaluating early resources replenishment and the effects of Japanese flounder enhancement.

Comparative Functional Analysis in vitro of 2 COL4A5 Splicing Mutations at the Same Site in 2 Unrelated Alport Syndrome Chinese Families.

X-linked Alport syndrome (XLAS) is a common hereditary nephropathy caused by COL4A5 gene mutations. To date, many splice site mutations have been described but few have been functionally analyzed to verify the exact splicing effects that contribute to disease pathogenesis. Here, we accidentally discovered 2 COL4A5 gene splicing mutations affecting the same residue (c.2917+1G>A and c.2917+1G>C) in 2 unrelated Chinese families. In vitro minigene assays showed that the 2 mutations produced 3 transcripts in H293T cells: one with a 96-bp deletion in exon 33, one with exon 33 skipping, and one with exon 33-34 skipping. However, fragment analysis results showed that the main splicing effects of the 2 mutations were different, the c.2917+1G>A mutation mainly activated a cryptic donor splice site in exon 33 and resulted in the deletion of 96 bp in exon 33, while the c.2917+1G>C mutation mainly caused exon 33 skipping. Our findings indicate that different nucleotide substitutions at the same residue can cause different splicing effects, which may contribute to the variable phenotype of Alport syndrome.

miR-212-5p exerts tumor promoter function by regulating the Id3/PI3K/Akt axis in lung adenocarcinoma cells.

microRNAs may function as oncogenes or tumor suppressor genes that play crucial roles in human carcinogenesis and cancer development. Growing evidence revealed that the tumor suppressor Id3 is involved in tumor progression, carcinogenesis, and the tumor microenvironment. We identified miR-212-5p as a negative posttranscriptional modulator of Id3. Dual luciferase reporter assay was used to verify that Id3 is a direct target gene of miR-212-5p. Id3 was lowly expressed and miR-212-5p was highly expressed in non-small-cell lung cancer (NSCLC) tissues and cells. In addition, we found that NSCLC patients having a higher level of miR-212-5p expression had a shorter survival time. Besides this, miR-212-5p could directly target Id3 and reduce its expression. miR-212-5p overexpression significantly accelerated cell proliferation, migration, and invasion by reversing the effects of Id3. Id3 overexpression by silencing miR-212-5p expression suppressed phosphatidylinositol 3 kinase (PI3K)/Akt activity and consequently promoted apoptosis and inhibited cell proliferation in lung cancer cells. Consistent with the in vitro results, a xenograft mouse model was used to validate the fact that miR-212-5p could promote tumorigenesis by targeting Id3 and activate the PI3K/Akt pathway in vivo as well. Taken together, the present results indicated that miR-212-5p may be involved in progression of NSCLC through the PI3K/Akt signaling pathway by targeting Id3.

[Meta-analysis on the Therapeutic Effect of Acupuncture at Meridian Sinew for Spastic Paralysis After Stroke].

OBJECTIVE: To review systematically the clinical effects of spastic paralysis after stroke treated with acupuncture at meridian sinew ("Jingjin", musculotendon). METHODS: "Meridian sinew" "stroke" and "spasm" were taken as the key words to retrieve from the Chinese National Knowledge Infrastracture Database (CNKI), Chongqing VIP Chinese Science and Technology Periodical Database (VIP), Chinese Biomedical Library (CBM), Wanfang Data, PubMed and the Cochrane Library. The Cochrane"risk of bias" tool was used to conduct the methodological quality evaluation to the literature. RevMan 5.3 software was adopted for Meta-analysis. RESULTS: Totally, 13 papers were included, with 820 patients involved. In reference to Cochrane Reviewers' Handbook 5.0.2, the randomized controlled trial (RCT) risk of bias was assessed and it was unclear for all of the 13 papers. The results of Meta-analysis showed that the clinical effect was improved with acupuncture at meridian sinew as compared with normal acupuncture technique[①total effective rate:OR=3.86, 95% CI (2.67,5.57), Z=7.20, P<0.00001; ②modified Ashworth spasm scale:OR=4.54, 95% CI (2.91,7.10), Z=6.64, P<0.00001; ③evaluation of limb motor function with Fugl-Meyer score:MD=4.18, 95% CI (-0.59,8.94), Z=1.72, P=0.09>0.05]. The publication bias of included papers was not obvious and therefore it could be neglected in the impact on the combined effect size. CONCLUSIONS: Acupuncture at meridian sinew is effective in the treatment of spastic paralysis after stroke. The total clinical effect and the improvement in muscular tone with acupuncture at meridian sinew are better than those with normal acupuncture technique. The quality of the included literature is not high generally. Hence, it is necessary to have more clinical studies with high-quality and strict design.

Moxibustion regulates inflammatory mediators and colonic mucosal barrier in ulcerative colitis rats.

AIM: To observe the efficacy and mechanism of grain-sized moxibustion at different acupoints in a rat model of ulcerative colitis (UC). METHODS: Sprague-Dawley rats were randomly divided into control, UC model, grain-sized moxibustion at a single acupoint (CV 12), grain-sized moxibustion at two acupoints (CV 12 and CV 4), grain-sized moxibustion at three acupoints (CV 12, CV 4, and ST 36), and medication groups (n = 8/group). The UC model was established by enema of trinitrobenzene sulfonic acid. Direct moxibustion was used once a day for 7 d. Disease activity index (DAI) was evaluated before and after the treatment. Morphologic changes of intestinal tissue were observed under an optical microscope. The expression of tumor necrosis factor (TNF)-α and p38 mitogen-activated protein kinase (p38MAPK) in colonic tissue was detected using Western blot, and the levels of occludin and zonula occludens-1 (ZO-1) mRNAs were detected using reverse transcription PCR. RESULTS: Compared with the control group, the intestinal mucosae were incomplete in the model group, glandular structures were irregular, and submucosae were edematous, hyperemic, and infiltrated with inflammatory cells. The DAI scores and expression of TNF-α and p38MAPK were increased significantly in the model group compared to controls (Ps < 0.01), while the mRNA levels of occludin and ZO-1 were reduced significantly (Ps < 0.01). Compared with the model group, colonic mucosa and the arrangement of glands were complete and regular in the treatment groups. DAI scores and the expression of TNF-α and p38MAPK were reduced significantly in moxibustion groups compared to controls (Ps < 0.01), while the mRNA levels of occludin and ZO-1 were increased significantly (Ps < 0.01). The improvements in the above indices in the three acupoints group and the medication group were superior to those in the single and two acupoints groups (all P < 0.05). CONCLUSION: Reduction of TNF-α and p38MAPK and increased expression of occludin and ZO-1 in colonic tissue represent a potential mechanism for improved intestinal mucosal tissue repair with grain-sized moxibustion.

Eye Acupuncture Treatment for Stroke: A Systematic Review and Meta-Analysis.

There were applications of eye acupuncture for stroke patients. Unfortunately, similar to many other Traditional Chinese Medicine (TCM) treatments, it lacks comprehensive evaluation and system review for its effect and safety. Objective. This study is a systematic review to appraise the safety and effectiveness of eye acupuncture for stroke. Methods. "Eye acupuncture therapy" in eleven databases was searched by randomized controlled trials and quasi-randomized controlled trials. The search activity was ended in April 2014. The data were extracted and assessed by three independent authors. Rev Man 5.0 software was used for data analysis with effect estimate presented as relative risk (RR) and mean difference (MD) with a 95% confidence interval. Results. Sixteen trials (1120 patients) were involved with generally poor methodological quality. The study indicated that when eye acupuncture was combined with western medicine compared to western medicine, there was a significant difference in the areas of mental state, swallow function, and NDS. When eye acupuncture was combined with western medicine and rehabilitation compared to western medicine and rehabilitation, there was significant difference in the changes of SSS, FMA, and constipation symptoms evaluation. No adverse events or side effects have been reported. Conclusions. The current evidence is insufficient and the rigorously designed trials are warranted.

[Effect of Electroacupuncture Stimulation of "Neiguan" (PC 6) on Expression of Myocardial Chloride Channel-related Genes, PKC and PKG Proteins in Myocardial Ischemia Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Neiguan" (PC 6), etc. on expression levels of myocardial chloride (CL-) channel-related genes and intracellular protein kinase C (PKC) protein in myocardial ischemia (M) rats. METHODS: Seventy SD rats were randomly divided into control group (n = 10), model group (n = 15) , Neiguan (PC 6) group (n = 15), Lieque (LU 7) group (n = 15) and non-acupoint group (n = 15). The MI model was established by i. p. of isoproterenol (ISO, a sympathomimetic beta adrenergic agonist). Electroacupuncture stimulation was applied to bilateral "Neiguan" (PC 6), "Lieque" (LU 7), or non-acupoint [the mid-point between "Tianshu" (ST 25) and "Shenque" (CV 8)] for 15 min, once a day for 7 days. Quantitative RT-PCR was employed to detect the expression levels of cystic fibrosis transmembrane conductance regulator (CFTR, a CL-channel) mRNA and chloride channel calcium-activated 1 (CLCa 1, a member of the family of calcium-activated chloride channels, CLCa) mRNA in the left cardiac ventricle tissue, and Western blot was used to detect the expression level of myocardial PKC protein of the left ventricle. RESULTS: Compared with the control group, the expression levels of myocardial PKC protein, and CLCa 1 and CFTR genes were significantly increased in the model group (P<0.05). In comparison with the model group, the expression levels of myocardial PKC protein, and CFTR mRNA and CLCa 1 mRNA in the Neiguan group, and PKC protein and CLCa 1 mRNA in the Lieque and non-acupoint groups, as well as CFTR mRNA in the Lieque group were notably down-regulated (P<0.05). No significant change was found in the expression of CFTR mRNA in the non-acupoint group (P>0.05), and no significant differences were found between Neiguan and Lieque groups in the expression levels of PKC protein (P>0.05). The effects of "Neiguan" (PC 6) were obviously superior to those of non-acupoint in down-regulating myocardial PKC protein, CLCa 1 mRNA and CFTR mRNA (P<0.05). CONCLUSION: EA stimulation of "Neiguan" (PC 6) can down-regulate the expression of myocardial PKC protein, CFTR and CLCa 1 genes in Ml rats, which may contribute to its effect in protecting rnyocardium from ischemic injury.

Role of moxibustion in inflammatory responses during treatment of rat ulcerative colitis.

AIM: To investigate the efficacy of moxibustion in ulcerative colitis (UC) rats from morphological, immunological and molecular biological perspectives. METHODS: Thirty-two Sprague-Dawley rats were randomly assigned to a blank control group (normal rats, n = 6) and a model replication (MR) group (UC rats, n = 26). A UC model was established by 2,4,6-trinitrobenzenesulfonic acid/dextran sulfate sodium enema. Rats in the MR group were further randomly assigned to a 9-min moxibustion (9M) group (9 moxa-cone, n = 6), 6-min moxibustion (6M) group (6 moxa-cone, n = 6), 3-min moxibustion (3M) group (3 moxa-cone, n = 6), and a waiting list control (WLC) group (no moxibustion treatment, n = 6). Rats in the moxibustion treatment group were treated in 14 sessions over 28 d. Disease activity, local tissue morphology, serum level of interleukin (IL)-8 and IL-10, and expression of Toll-like receptor (TLR)9 as well as nuclear factor (NF)-κB p65 in colonic tissue were determined by disease activity index (DAI), hematoxylin and eosin staining, electron microscopy, enzyme-linked immunosorbent assay and Western blotting, respectively. RESULTS: DAI was lowest in the 9M group and highest in the WLC group. The differences in DAI between the moxibustion treatment (3M, 6M, 9M) and no treatment groups were significant for all one-to-one comparisons (0.60 ± 0.54 vs 1.20 ± 0.44, 0.60 ± 0.54 vs 1.80 ± 0.45, 0.60 ± 0.54 vs 3.0 ± 0.45, respectively, P < 0.05). Light and electron microscopy showed that the neatness of the glandular arrangement in colonic mucosal epithelia gradually increased in the WLC, 3M, 6M to 9M groups. IL-8 level successively decreased while IL-10 level increased from the WLC to 3M, 6M and 9M groups. The differences among these groups were significant for all comparisons (105.46 ± 8.75 vs 76.61 ± 3.58, 105.46 ± 8.75 vs 69.78 ± 1.87, 105.46 ± 8.75 vs 67.41 ± 1.84, respectively, P < 0.01 for IL-8; and 30.83 ± 1.29 vs 75.64 ± 1.90, 30.83 ± 1.29 vs 80.90 ± 3.16, 30.83 ± 1.29 vs 83.46 ± 2.37, respectively, P < 0.01 for IL-10), except comparison of 6M vs 9M. Expression of TLR9 and NF-κB p65 decreased in order: highest in the WLC group and lowest in the 9M group. In addition, the differences among the WLC, 3M, 6M and 9M groups were significant for all comparisons (0.492 ± 0.026 vs 0.380 ± 0.022, 0.492 ± 0.026 vs 0.355 ± 0.005, 0.492 ± 0.026 vs 0.327 ± 0.015, respectively, P < 0.05 for TLR9; and 0.436 ± 0.041 vs 0.326 ± 0.022, 0.436 ± 0.041 vs 0.293 ± 0.006, 0.436 ± 0.041 vs 0.265 ± 0.017, respectively, P < 0.05 for NF-κB p65). CONCLUSION: Moxibustion repairs damaged colonic mucosa, suppresses serum IL-8, activates serum IL-10 level, and decreases expression of TLR-9 and NF-κB p65 in UC rats.

[Effect of deep electroacupuncture stimulation of "Huantiao" (GB 30) on changes of function and nerve growth factor expression of the injured sciatic nerve in rats].

UNLABELLED: OBJECTIVE; To observe the effect of deep electroacupuncture (EA) stimulation of "Huantiao"(GB 30) on the functional and pathological changes and nerve growth factor (NGF) expression of the damaged sciatic nerve in rats, so as to study its mechanisms underlying reliving sciatica. METHODS: Forty-eight SD rats were randomly divided into normal, model, deep EA and shallow EA groups (n = 12 in each group). The sciatic nerve injury model was established by mechanical clamp of the sciatic nerve stem. For deep and shallow EA, the acupuncture needles were inserted into GB 30 about 16 mm and 7 mm, respectively. The EA treatment was given 20 min, once daily for 14 days. The evoked potentials of the injured sciatic nerve stem responding to electrical stimulation were recorded by using a biophysiological experimental system for calculating the motor conduction velocity. Pathological changes of the sciatic nerve were displayed by H. E. stain. The expression of NGF and Fos proteins was detected by immunohistochemistry. RESULTS: In comparison with the normal group, the conduction velocity and the amplitude of the evoked potentials of the sciatic nerve were significantly decreased in the model group (P < 0.05). Following EA, both conduction velocity and amplitude of the evoked potentials in the deep EA group, and the conduction velocity in the shallow EA group were considerably increased (P < 0.05). The therapeutic effects were significantly better in the deep EA group than that in the shallow EA group (P < 0.05). No significant differences were found between the shallow EA and model groups in the amplitude of evoked potentials (P > 0.05), and no significant changes of latencies of the evoked potentials inthe four groups (P > 0.05). In the model group, the disorganized nerve fibers axons, myelin and Schwann cells of the damaged sciatic nerve were found, which became milder in the EA groups particularly in the deep EA group. In regard to the NGF and Fos immunoactivity of the injured sciatic nerve, the expression levels of both NGF and Fos proteins were obviously higher in the model group than in the normal group (P < 0.05). After EA stimulation, NGF expression was further significantly up-regulated in both deep and shallow EA groups (P < 0.05), and Fos protein expression was notably down-regulated in the deep and shallow EA groups (P < 0.05). The expression of NGF was significantly higher and Fos protein was obviously lower in the deep EA group than in the shallow EA group (P < 0.05). CONCLUSION: Deep EA stimulation of GB 30 can improve the pathological changes and function of the injured sciatic nerve in the rat, which is closely associated with its effects in up-regulating NGF expression and down-regulating Fos expression. The deep EA is relatively better in the therapeutic effect. These facts may be one of the mechanisms of EA in relieving sciatica in clinic practice.

[Effects of osteogenic growth peptide to the proliferation and differentiation of bone marrow stromal cells].

OBJECTIVE: To investigate the effects of osteogenic growth peptide (OGP) to the proliferation and differentiation of cultured bone marrow stromal cells (BMSCs) of rats. METHODS: The SD rats (age 6 weeks) were sacrified, and the bone marrow stromal cells as the adherent stromal cell population were separated from the bone marrow culcure. The proliferation curves of bone marrow stromal cells which were conditioned cultured with four kind of different concentrations of osteogenic growth peptide were measured with the MTT method, and the osteogenesis markers including alkaline phosphatase and calcic deposition detected by histochemical staining. RESULTS: Osteogenic growth peptide at the concentration of 10(-10), 10(-11) mol/L promoted the proliferation of bone marrow stromal cells, while at the concentrations of 10(-8), 10(-9) mol/L suppressed the proliferation of bone marrow stromal cells. However,osteogenic growth peptide at the concentration of 10(-9), 10(-8) mol/L advanced the ratio of positive cells in alkaline phosphatase histochemical staining. CONCLUSION: Osteogenic growth peptide shows distinct effects on the proliferation and differentiation of bone marrow stromal cells depending on its concentration. Osteogenic growth peptide at the concentration of 10(-8), 10(-9) mol/L can promote bone marrow stromal cell differentiation to the osteogenic in vitro.

[Reconstruction of embryo using an improved nuclear transfer method].

Previous methods used for nuclear transplantation were further investigated to develop a method that was both easy to carryout and did not require any special apparatus, such as Piezoimpact or Spindle-View. Following the puncture of zona pellucida with two holes by injection pipette that contained donor nuclei or cells, the injection pipette was pulled back to the perivitelline space while the negative pressure was increased in the holding pipette until the polar body and karyoplasm were wiped off completely. Then a reconstructed embryo was completed by the direct injection of the donor nucleus or cell without pulling out the injection pipette. 200 oocytes were manipulated using this method and it cost about 40 seconds with nucleus injection and about 30 seconds with cell injection to complete a reconstructed embryo. The success rates were 62.6% and 86. 0%, respectively, and enucleation rate was about 73.3% validated by Hoechst 33342. Using this method, the nucleus was completely eliminated and another was injected using the microscope and micromanipulator. Moreover, the efficiency of nuclear transplantation and survival rate of reconstructed embryos were greatly improved. Furthermore, it is very easy to manipulate and popularize in practice.

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells.

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.

Efficient differentiation of embryonic stem cells into neurons in glial cell-conditioned medium under attaching conditions.

Embryonic stem (ES) cells can differentiate into neurons in vitro, which provides hope for the treatment of some neurodegenerative diseases through cell transplantation. However, it remains a challenge to efficiently induce ES cells to differentiate into neurons. Here, we show that murine ES cells can efficiently differentiate into neurons when cultured in glial cell-conditioned medium (GCM) under attaching conditions without the formation of embryoid bodies. In comparison with murine embryonic fibroblast-conditioned medium, we found that GCM has a positive effect on limiting the generation of non-neuronal cells, such as astrocytes. In addition, compared with suspension conditions, attaching conditions delay the differentiation process of ES cells.

Inhibitory role of transcription factor COUP-TFII in expression of hTERT in HeLa cells.

OBJECTIVE: To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity. METHODS: The full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint. The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA. RESULTS: COUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter. Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity. CONCLUSION: The human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.

Macrophage-associated erythropoiesis and lymphocytopoiesis in mouse fetal liver: ultrastructural and ISH analysis.

To elucidate the process of fetal liver hematopoiesis, the relationships between stroma and hematopoietic cells involved in maturation were investigated. Cultured mouse fetal liver explants were established for morphological analysis of the interactions between fetal liver stroma and hematopoietic cells ex vivo. Fetal liver stroma comprised epithelial cells and macrophages, which occupied most of the culture surface. Macrophages proliferated extensively in primary culture, but almost disappeared after 3 passages. Close morphological and functional relationships were established between macrophages and hemopoietic cells, whereas epithelial cells did not interact with blood cells. Scanning electron microscopy revealed that macrophages were in close contact with erythroblasts and formed a three-dimensional network. In each erythroblastic island, 2-3 lymphocytes were also in contact with the macrophages; erythroblasts, lymphocytes and macrophages formed close, firm associations through their cytoplasmic membranes. This cell orientation was confirmed by transmission electron microscopy of fetal liver in vivo. In situ hybridization revealed that the macrophages expressed jagged-1, an important ligand of the Notch signaling system in hematopoiesis.

Viral replication modulated by synthetic peptide derived from hepatitis B virus X protein.

AIM: A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We reported it here for the first time not only to highlight the biohazard issue and safety importance for viral peptide vaccine, but also to explore the effect of a fully conserved peptide on HBV replication within the carboxyl terminus of HBx. METHODS: We synthesized the fully conserved peptide (CP) with nine residues, FVLGGCRHK. HBV-producing 2.2.15 cells were treated with or without 3.5 microM CP for 36 hours. Quantitative detection of viral DNA was performed by real-time PCR. HBV antigens were determined by enzyme-linked immunoadsorbent assay (ELISA). Quantitative analyses of p53 and Bax proteins were based on immunofluorescence. Flow cytometry was performed to detect cell cycle and apoptosis. RESULTS: Both extracellular and intracellular copies of HBV DNA per ml were significantly increased after incubation with 3.5 microM of CP. HBsAg and HBeAg in the cultured medium of CP-treatment cells were as abundant as untreated control cells. CP influenced negatively the extracellular viral gene products, and 3.5 microM CP could significantly inhibit intracellular HBsAg expression. In response to CP, intracellular HBeAg displayed an opposite pattern to that of HBsAg, and 3.5 microM CP could efficiently increase the level of intracellular HBeAg. Flow cytometric analyses exhibited no significant changes on cell cycle, apoptosis, p53 and Bax proteins in 2.2.15 cells with or without CP. CONCLUSION: Together with the results generated from the synthetic peptide, we address that the conserved region, a domain of HBx, may be responsible for modulating HBV replication. As conserved peptides from infectious microbes are used as immunogens to elicit immune responses, their latent biological hazard for human beings should be evaluated.

Protective effects of transplanted and mobilized bone marrow stem cells on mice with severe acute pancreatitis.

AIM: To evaluate the protective effects of transplanted and mobilized bone marrow stem cells (BMSCs) on mice with severe acute pancreatitis (SAP) and to probe into their possible mechanisms. METHODS: A mouse model of SAP induced by intraperitoneal injections of L-arginine was employed in the present study. Two hundred female Balb/c mice weighing 18-22 g were randomly assigned into 4 groups. Group A was the stem cell mobilized group treated by injection of granulocyte-colony stimulating factor (G-CSF) into mice for 4 days at a dose of 40 microg.kg(-1).d(-1) before induction of SAP. Group B was the group of BMSCs transplantation, in which the mice were given the isolated BMSCs via the tail vein 4 days prior to induction of SAP. Group C served as the model control and only SAP was induced. The mice without induction of SAP in group D acted as the normal control. At the time of animal sacrifice at 24, 48 and 72 h after induction of SAP, blood samples were obtained and prepared to detect serum amylase, while the abdominal viscera were examined both grossly and microscopically for the observation of pathological changes. RESULTS: The mortality of mice in the model control, groups A and B was 34%, 8% and 10% respectively within 72 h after induction of SAP. The serum level of amylase in the model control was significantly increased at all time points after induction of SAP as compared with that of the normal control (P<0.05-0.01). When the mice were pretreated with BMSCs' transplantation or G-CSF injection, their serum level of amylase was significantly reduced at 48 h and 72 h after induction of SAP in comparison with that of the model control (P<0.05-0.01). In accordance with these observations, both gross and microscopic examinations revealed that the pathological changes of SAP in mice pretreated with BMSCs transplantation or G-CSF injection were considerably attenuated as compared with those in the model control at all observed time points. CONCLUSION: Both transplanted allogenic and mobilized autologous BMSCs can protect mouse pancreas from severe damage in the process of SAP.

Aggregate formation of hepatitis B virus X protein affects cell cycle and apoptosis.

AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses. METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography. Anti-HBx monoclonal antibody was developed for immunocytochemical detection. Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 cells. Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells. The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry. RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules. Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time. EGFP fluorescence in HBx expression cells was significantly decreased. CONCLUSION: Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase. Our data may provide helpful information to understand the biological effects of HBx aggregates on cells.

A quantitive evaluation of gap junctions and their morphological alteration during differentiation of amphibian notochord cells.

The early development of notochord cells may be divided into three phases according to the quantitative evaluation of gap junctions: late gastrula, neurula and from tailbud to tadpole. In late gastrula, the percentage of the area of gap junctions to total membrane is 0.054% and most of the gap junctions are small in size. During the stages of neurulation, the ratios of gap junctions to total membrane area increase and remain high (0.106-0.181 %), and the majority of the gap junctions are of medium and large size. The high ratios of gap junctions to membrane area during neurulation suggests that intercellular communication via gap junctions is important during this period. In the stages from tailbud to tadpole the ratios decrease and drop drastically to 0.001 % and most of the gap junctions found are small in size. It is in the last phase that gap junctions of altered configuration appear.