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Objective: To investigate the feasibility of using human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) and demineralized bone matrix (DBM) scaffolds to repair critical-sized calvarial defects in athymic rats., Methods: Human UCB-MSCs were isolated, expanded and osteogenically induced in vitro. Osteogenic differentiation of UCB-MSCs was evaluated by Alizarin Red staining and measurement of calcium content respectively, and then the cells were seeded onto DBM scaffolds. Bilateral full-thickness defects (5 mm in diameter) of parietal bone were created in an athymic rat model. The defects were either repaired with UCB-MSC/DBM constructs (experimental group) or with DBM scaffolds alone (control group). Animals were harvested at 6 and 12 weeks post-implantation respectively, and defect repair was evaluated with gross observation, micro-CT measurement and histological analysis., Results: Micro-CT showed that new bone was formed in the experimental group at 6 weeks post-implantation, while no sign of new bone formation was observed in the control group. At 12 weeks post-transplantation, scaffolds had been degraded almost completely in both sides. It was shown that an average of (78.19 +/- 6.45)% of each defect volume had been repaired in experimental side; while in the control side, only limited bone formed at the periphery of the defect. Histological examination revealed that the defect was repaired by trabecular bone tissue in experimental side at 12 weeks, while only fibrous connection was observed in the control group., Conclusions: Tissue-engineered bone composed of osteogenically-induced human UCB-MSCs on DBM scaffolds could successfully repair the critical-sized calvarial defects in athymic rat models.

The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE , Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice.

Objective: To assess the protection against cisplatin-induced ototoxicity by adenovirus-mediated overexpression of the bcl-2 gene in cultured spiral ganglion cells (SGC)., Methods: SGC from P3 rats were cultured in vitro and exposed to adenovirus vector carrying green fluorescent protein gene (Ad-GFP), followed by immunocytochemical analysis for expression of the neuron-specific marker Neurofilament 200 (NF200) and detection under laser scanning confocal fluorescence microscope. Then, SGC were transduced by Ad-bcl-2 and the expression of human bcl-2 protein was evaluated by Western Blot. Finally, the cultures of SGC were divided into 4 groups: the group of Ad-bcl-2 transfection followed by cisplatin treatment, the group of Ad-GFP transfection followed by cisplatin treatment, the group of cisplatin treatment only and the untreated group. Cisplatin worked for 48 hours at a concentration of 2 microg/ml. Outcome measures included survival number of SGC and longest neurite length by using ImageJ software., Results: SGC were cultured successfully in vitro and transfected by adenovirus vector safely and efficiently. By Western Blot, human bcl-2 protein was expressed in the group after exposure to Ad-bcl-2, but not in the Ad-GFP transfected SGC. Cisplatin exposure resulted in shrinking of neuritis and pyknosis of cell body, even cell death. Expression of bcl-2 in the SGC provided a significant level of protection against cisplatin-induced SGC degeneration., Conclusions: Our results suggest that SGC can be transduced by adenovirus vector safely and efficiently in vitro. Adenovirus-mediated delivery of the bcl-2 gene attenuates cisplatin-induced SGC degeneration.

T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is preferentially expressed on Th1-helper type T-cells and functions to repress the Th1-mediated immune response. However, the role of Tim-3 during the inflammatory pathogenesis of asthma remains unclear. This study determines the expression level of Tim-3 in CD4+ T-cells within the peripheral blood and bronchoalveolar lavage fluid (BALF) isolated from a murine model of atopic asthma and explores the potential role of Tim-3 during the inflammatory response. Mice were randomly divided into normal control, asthma day 1, and asthma day 7 groups, and peripheral blood T lymphocytes and BALF cells were collected. The ratio of Tim-3+/CD4+ cells among the total CD4+cell populations from peripheral blood and BALF was determined by flow cytometry, and the expression of the Tim-3 mRNA was determined by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In contrast with the normal control group, the ratio of Tim-3+/CD4+:CD4+ cells and the level of Tim-3 mRNA in both the peripheral blood T lymphocytes and BALF cells among the asthma day 1 and asthma day 7 groups were significantly increased (p < 0.01), and those in the asthma day 7 group were higher than the asthma day 1 group (p < 0.05). There was also a positive correlation between the ratio of Tim-3+/CD4+:CD4+ detected in BALF and that the ratio detected in peripheral blood T lymphocytes (r = 0.84, p < 0.01). Therefore, the expression of Tim-3 is increased in CD4+ T-cells following airway challenge and likely affects asthma-induced inflammation by repressing the Th1-mediated immune response.

Background: The push-pull maneuver (PPM) can lead to loss of consciousness in pilots of high-performance aircraft. Modeling of the physical and physiological aspects of this phenomenon should allow improved countermeasures., Methods: A structurally based mechanistic computer model was developed to incorporate dynamic carotid baroreflex responses and detailed modeling of vessel segments for different anatomic regions. The model was used to predict the effect of the PPM on cardiovascular responses and the protection afforded by extended coverage anti-G suits (ECGS) and neck pressure., Results: The model was validated by comparing the simulation results with previously published experimental data obtained during centrifuge and tilt-table studies. Simulations of various PPM acceleration profiles indicated that +Gz tolerance was reduced in the presence of higher +Gz levels prior to the push phase, more -Gz levels during the push phase, and prolongation of the push phase. On the other hand, the onset rate for the two phases had only minor effects on +Gz tolerance. Model output suggested that improved protection could be provided by an ECGS with minimal inflation delay and a multilevel pressure schedule in which the leg bladders inflated to a higher pressure than the abdominal bladder. Modeling application of a 100-mmHg neck pressure during the push phase partly inactivated the carotid baroreflex, but induced only a small increase in tolerance., Conclusions: Mathematical modeling and simulation showed that +Gz tolerance for the PPM might be increased by improving the design and inflation schedule of the ECGS.

Objective: To investigate the effects of nicotine in combination with okadaic acid (OA) or taxol on bovine oocyte maturation and subsequent embryonic development., Design: Prospective randomized study., Setting: University research laboratory., Patient(s): Bovine ovaries, oocytes, and embryos., Intervention(s): Oocyte maturation and subsequent embryo development in the presence of nicotine in combination with okadaic acid and taxol., Main Outcome Measure(s): Haploid composition, cleavage rate, IVF and parthenogenetic embryo development, blastocyst cell number., Result(s): The results showed that nicotine and OA or nicotine and taxol significantly decreased oocyte maturation rates. Combinations of nicotine (10 or 50 micromol/L) and OA (0.01 or 0.05 micromol/L) did not affect oocyte haploid composition; however, taxol alone or combined with nicotine caused a decrease in haploid composition compared with control. Parthenogenetic activation of oocytes that were matured in nicotine, OA, or taxol resulted in blastocyst development rates of 12%-17%, which were not different from the control. Various combinations of nicotine and OA or nicotine and taxol significantly lowered (3%-8%) blastocyst development compared with control. The average cell number of blastocysts derived from nicotine + OA- and nicotine + taxol-treated oocytes was lower than all other treatment groups and control. For oocytes fertilized in vitro, oocytes matured in OA, taxol, or combinations of nicotine and OA or taxol-containing media resulted in significantly lower cleavage rates and blastocyst development compared with the control group and the 10 micromol/L nicotine treatment group. The IVF embryos cultured in nicotine + OA-containing medium had a significantly lower blastocyst development rate., Conclusion(s): Combinations of nicotine with OA or taxol adversely affect oocyte maturation and subsequently result in poor blastocyst development.

This study was purposed to evaluate a method to discriminate the action loci of anticancer agents in G(2) and M phases of cell cycle. The meta-amsacrine (m-AMSA) and vinblastine (VBL), already known as G(2) and M phase arrest agent respectively, were used to induce the arrest of MOLT-4 cells at G(2) and M phases, the change of DNA content was detected by flow cytometry, the morphology of arrested cells was observed by confocal microscopy so as to find the arrest efficacy difference of 2 anticancer agents. As a result, the flow cytometric detection showed that the arrested MOLT-4 cells displayed the raise of peaks in G(2) and M phases, but flow cytometric detection alone can not discriminate the difference between them. The observation with confocal microscopy showed that the MOLT-4 cells arrested by m-AMSA displayed the morphologic features in G(2) phase, while the MOLT-4 cells arrested by VBL displayed the morphologic features in M phase. This observation with confocal microscopy is helpful to discriminate the difference between them. In conclusion, the combination of flow cytometry with confocal microscopy is one of the effective methods to discriminate the kind of G(2) or M phase arresting agent of anticancer drugs.

Four experiments were designed to examine the effects of colcemid, a microtubule assembly inhibitor, on the development of bovine nuclear transfer (NT) embryos in vitro and in vivo. Recipient oocytes matured at different times were exposed to colcemid. Approximately 80-93% of the exposed oocytes, with or without the first polar body (PB1), developed obvious membrane projections. In Experiment 1, oocytes matured for either 14-15 or 16-17 hr, treated with colcemid and used as recipient cytoplasm for NT resulted in over 40% blastocyst development. In Experiment 2, oocytes matured for 16-17 hr were treated with either 0.2 or 0.4 microg/ml colcemid for 2-3 or 5-6 hr, respectively. The percentages of blastocyst development (39-42%) were not statistically different among the different colcemid treatment groups, but were both higher (P < 0.05) than the control group (30%). Colcemid concentrations and length of colcemid treatment of oocytes did not affect their ability to support NT embryo development to the blastocyst and hatched blastocyst stages. Results from Experiment 3 indicate that semi-defined medium increases morula and blastocyst development of NT embryos derived from colcemid-treated oocytes under 5% CO2 in air atmosphere. In addition, cell numbers of blastocysts in colcemid-treated groups were numerically higher than the control groups. After embryo transfer, higher (P < 0.05) pregnant rates were obtained from the colcemid-treated group than the nontreated group. Five of 40 recipients (12.5%) which received embryos from colcemid-treated oocytes delivered healthy calves, significantly higher than those recipients (3.3%) that received embryos derived from nontreated oocytes.

The mol-ecule of the title compound, C(23)H(23)N(3), which was synthesized by the condensation reaction between 2,6-diacetyl-pyridine and 2-dimethyl-aniline, adopts an E configuration about both C=N imine bonds. The dihedral angles formed by the benzene rings with the pyridine ring are 89.68 (5) and 53.62 (6)°.

In the title compound, [ZnBr(2)(C(15)H(11)N(3))], the Zn(II) ion is five-coordinated by the three N atoms from a 2,2':6',2''-terpyridine ligand (terpy) and two bromide anions in a distorted trigonal bipyramidal configuration. Each mol-ecule is situated on a twofold rotational axis that passes through the Zn(II) ion and the central ring of the terpy ligand. In the crystal structure, aromatic π-π inter-actions between terpy ligands [centroid-centroid distances = 3.6265 (9) Å] link mol-ecules into stacks propagated in the [001] direction.

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets that leads to great economic losses in East Asia. It was reported that aminopeptidase N (APN) was the receptor for Transmissible gastroenteritis virus (TGEV), Human coronavirus 229E (HCoV-229E) and Feline coronavirus (FeCoV) which all belonged to group I coronavirus including PEDV. It was also confirmed previously that porcine aminopeptidase N (pAPN) could bind to PEDV, and anti-pAPN antibodies could inhibit the combination. To investigate whether pAPN was a receptor for PEDV, we transfected MDCK cells with porcine aminopeptidase (pAPN) cDNA and this enabled non-susceptible cells to support PEDV replication and serial viral propagation. Moreover, the infection was blocked by antibodies against pAPN, implying the critical role of pAPN during virus entry. In addition, immunofluorescence assays for detection of pAPN and PEDV antigens, together with neutralization assays using antibodies against pAPN, further confirmed the correlation between pAPN expression and viral replication in pAPN-transfected MDCK cells. These results indicated that pAPN is a functional receptor for PEDV.

Objective: To investigate the mechanism involved in aging process of immortalized human keratinocyte (HaCaT) and primary human epidermis keratinocyte of adults (HEKa) irradiated by ultraviolet B(UVB)., Methods: HEKa and HaCaT were repeatedly exposed to UVB at a subcytotoxic level. SA-beta-Gal staining was performed to evaluate the senescence state; flow cytometry was applied to detect the changes of apoptosis, necrosis and cell cycle. Intracellular levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by ELISA method. Western blot was performed to detect the expression pattern of redox protein p66Shc and RT-PCR was performed to determine the mRNA level of human telomerase reverse transcriptase (hTERT)., Result: Strong positive SA-beta-Gal staining was observed in both HEKa cell and HaCaT cells after UVB irradiation. Apoptosis rate increased from (1.81 +/-0.25)% to (4.43 +/-0.28)% and necrosis rate increased from (0.05 +/-0.01)% to (0.10 +/-0.03)% in HaCaT cell, but no marked arrest of cell cycle was observed during UVB irradiation. As a contrast, apoptosis rate of in HEKa cells significantly increased from (0.65 +/-0.05)% to (59.53 +/-2.35)%, and the necrosis rate in HEKa cells also reached (3.89 +/-0.24)%(P<0.05). Growth arrest in G0/G1 phase was also found in HEKa cells. In both cell lines, intracellular level of SOD decreased and MDA increased remarkably after UVB exposure, and an increased expression of p66Shc protein was also observed. High level of hTERT mRNA was detected in HaCaT cells and UVB exposure had little effect on its expression., Conclusion: The stress-induced premature senescence (SIPS) in HaCaT and HEKa cell lines by UVB irradiation might be closely associated with increased intracellular levels of oxidative stress, not related to the telomerase expression.

Objective: To investigate the application of tissue-engineering bone with ADSCs (adipose-derived stem cells) and coral scaffold for repairing of cranial bone defect in canine., Methods: Autologous ADSCs isolated from canine subcutaneous fat were expanded, osteogenically induced, and seeded on coral scaffolds. Bilateral full-thickness defects (20 mm x 20 mm) of parietal bone were created (n = 7). The defects were either repaired with ADSC-coral constructs (experimental group) or with coral alone (control group). Radiological, gross, biomechanical and histological observations were done to evaluate the bone regeneration., Results: Three-dimensional CT scan showed that new bones were formed in the experimental group at 12 weeks after implantation, while coral scaffolds were partially degraded in the control group. By radiographic analysis at 24 weeks post-transplantation, it showed that an average repair percentage of each defect was (84.19 +/- 6.45)% in experimental group, and (25.04 +/- 18.82)% in control group (P < 0.01). The maximum compression loading was (73.45 +/- 17.26) N in experimental group, and (104.27 +/- 22.71) N in control group (P <0.01). Histological examination revealed that the defect was repaired by typical bone tissue in experimental group, while only minimal bone formation with fibrous connection in the control group., Conclusions: The tissue-engineering bone with autologous osteogenic ADSCs and scaffold could successfully repair the cranial defects in canine models.

The special process and special structure which bring organelle during the spermiogenesis of Japanese eel, Anguilla japonica were observed by scanning electron microscope and transmission electron microscope. The process which spermatoblast became sperm including four special stages, the early stage, the middle stage, the later stage and the spermic stage, then came into being a normal mature sperm. In the early stage, cell nucleus became long form gradually by the oval form. In one side of the cell nucleus, there was a big and special globoid structure dyeing lower, account for 1/3 of cell nucleus cubage. It contains a little of deep dyeing grain form and the lines form material, the outside is wrapped by plasmalemma separated with the cell nucleus, the outside of that structure and cell nucleus still lay a plasmalemma. The spermiogenesis of early stage did not form independent centriolar complex and mitochondria. In the middle stage, the cell nucleus presented a long form with the globoid structure on the top of the nucleus, and the down side had no globoid structure where the flagellum primordium appears. The globoid structure changed with the spermiogenesis. The inner part of the globoid differentiated a centriolar complex and mitochondria step by step. The lysosomes distributed in the medium segment of the cell nucleus obviously. In the late stage, the cell nucleus was similar with the shape of eyebrow or crescent. The centriolar complex released from the globoid structure, then became an independent structure. There were mitochondria which had not become the independent structure still in the globoid structure. Under the karyon, there was flagellum primordium where sent a rather long flagellum. The flagellum formed a typical "9+0" microtubular structure at that time. The spermatozoa in this phase has movable ability. In the spermic stage, the cell nucleus was round in shape. The centriolar complex was inside implantation fossa. Mitochondria were under karyon. And under the mitochondria was the central space of the sleeve. The flagellum formed a typical "9+2" microtubular structure at that time. The spermatozoa of Japanese eel, A. japonica became complete mature spermatozoa must pass through four phases for abnormality.

This study was purposed to investigate the biological effect of vinblastine (VLS), usually known as inductor of mitotic arrest, on MOLT-4 of ALL cells and to evaluate its significance. The cell arrest in M phase and/or cell apoptosis were induced by treatment of MOLT-4 cells with 0.05 microg/ml VLS for 0 - 12 hours; the DNA histogram was detected by flow cytometry; the morphological changes of cells were observed by confocal microscopy; the cell cycle distribution, cell apoptosis and morphological changes of cells before and after arrest were analyzed by using arrest increasing rate (AIR), arrest efficiency (AE), apoptosis rate (AR) and morphologic parameters respectively. The results indicated that the cell arrest did not accompanied by significant increase of apoptosis rate; the DNA histogram of cell arrest showed dynamic change of cell cycle in time-dependent manner; the arrest efficiency could be quantified. The cell arrest at M phase was accompanied by cell stack in S phase, the cell proliferation rate dropped after cell arrest occurred. The cells arrested at M phase possessed of characteristic morphologic features in cell mitosis. It is concluded that the vinblastine can solely induce arrest of MOLT-4 cells at M phase. This study provides experimental basis for further investigating the relation of cell cycle arrest to apoptosis, mechanism of checkpoint and development of new anticancer drugs.

Objective: To study the chemical constituents of Swertia mussotii., Methods: The constituents were isolated by various column chromatography methods, and their structures were identified by physico-chemical properties and spectral analysis., Results: Eleven compounds were isolated and identified as 1,3, 8-trihydroxy-7-methoxyxanthone (I), 2,8-dihydroxy-1,6-dimethyoxyxanthone (II), 1,8-dihydroxy-2,6-dimethoxyxanthone (III), 1,2,8-trimethoxyxanthone (IV), 1,3,5,6-tetrohyroxyxanthone (V), 1,8-dihydroxy-3,7-dimethoxyxanthone (VI), beta-daucosterol (VII), clerosterol 3beta-O-[6'-o-hydro-benzene-beta-D-glucoside] (VIII), ursolicacid (IX), 3beta,28-dihydroxylup-20 (29) -ene (X), erythrocentaurin (XI)., Conclusion: Compounds VIII, IX and X are isolated from Swertia mussotii for the first time.

Exhaustive dihydroxylation of the pair of cyclooctadienols consisting of 4 and 5, which are available in enantiomerically pure form from d-glucose, resulted in the formation of two diastereomeric tetraols in each case. The difference in polarity of the 6/7 and 8/9 pairs facilitated their chromatographic separation. Ensuing acetylation and PMB deprotection allowed for the assignment of relative (and ultimately absolute) stereochemistry to the resulting monohydric alcohols on the basis of J(HH) analysis of their (1)H NMR spectra. The highly functionalized exomethylenecyclooctanes 14-17, which were derived by periodinane oxidation and Wittig olefination, were further elaborated by hydroboration and global deprotection. The eight members of the cyclooctanose family of carbasugars and their precursor intermediates consistently showed patterns of J(HH) values in line with the contiguous stereochemical relationships. Also assayed was their specific inhibitory behavior toward glycosidases.

Bovine somatic cell nuclear transfer (SCNT) efficiency remains very low despite a tremendous amount of research devoted to its improvement over the past decade. Frequent early and mid-gestational losses are commonly accompanied by placental abnormalities. A transcription factor, activating protein AP-2gamma, has been shown to be necessary for proper placental development in the mouse. We first evaluated the expression of the gene coding for AP-2gamma (Tfap2c) in several bovine fibroblast donor cell lines and found it was not expressed. Subsequently we determined the expression profile of Tfap2c in oocytes and various stages of preimplantation in vitro fertilized (IVF) embryos. Tfap2c was undetectable in oocytes and early embryos, and was detectable at relatively high levels in morula and blastocyst IVF embryos. The lack of expression in oocytes and donor cells means Tfap2c must be induced in the zygote at the morula stage in properly reprogrammed embryos. SCNT embryos expressed Tfap2c at the eight-cell stage, 2 days earlier than control embryos. Control embryos first expressed Tfap2c at the morula stage, and at this stage Tfap2c was significantly lower in the SCNT embryos. No differences in expression were detected at the blastocyst stage. To determine whether Tfap2c was properly reprogrammed in the placenta of SCNT pregnancies, we evaluated its expression in cotyledons and caruncles of SCNT and control pregnancies between days 55 and 90 gestation. Expression of Tfap2c in caruncles significantly increased between days 55 and 90, while expression in cotyledons was relatively consistent over that same period. Expression levels in SCNT tissues were not different from controls. This data indicates Tfap2c expression is altered in early preimplantation SCNT embryos, which may have developmental consequences resulting from genes influenced by Tfap2c, but expression was not different at the blastocyst stage and in placentomes.

In this study, 68 group A streptococcus (GAS) isolates associated with two outbreaks of acute glomerulonephritis (AGN) in China were analyzed by emm typing. A total of 11 different emm types were identified. Analysis of emm type distribution suggested that AGN outbreaks in two counties were caused by emm60.1- and emm63.0-type GAS. These two types were further characterized by pulsed-field gel electrophoresis, multilocus sequence typing, sof sequence typing, and PCR-based identification of streptococcal pyrogenic exotoxin A, B, and C (speA, speB, and speC) genes. In antimicrobial susceptibility tests, all outbreak strains were resistant to erythromycin and tetracycline, and the rates of resistance of nonoutbreak strains to the two antibiotics were 63.6% and 90.9%. This study is also the first to report a nephritogenic M63 GAS strain.

Objective: To study the effect on bioactive components of Xuesaitong injection in the process of ultra-filter by orthogonal test., Methods: The change of the contents of active components was examined by HPLC method, using notoginsennoside R1 , ginsennoside Rg1, ginsennoside Rb1 and ginsennoside Rd as the mark components. The effect of the transmissivity of active components was studied with four different membrane parameters (such as pore diameter of membrane, operational pressure, the temperature and the concentration of the officinal liquid) by orthogonal test., Results: In the process of ultra-filter the transmissivity of notoginsennoside R, and ginsennoside Rg, had the same characteristics. Both of the average transmissivities of these two components were in the range of 45% to 95%. The concentration of the officinal liquid showed significant influence. The transmissivities of ginsennoside Rb, and ginsennoside Rd in the process of ultra-filiter had the same characteristic. The average transmissivities of these two components were in the range of 2 8%-62% and 1.8%-45%, respectively. The pore diameter of membrane and the temperature showed significant influence. The concentration of the officinal liquid also showed significant influence on the transmissivity of ginsennoside Rd., Conclusion: This study provides an external direction for the preparation of Xuesaitong injection.

Objective: To investigate the survival of neural stem cell (NSC) infected by recombinant adenovirus with GFP (Ad-GFP) and the expression of GFP in normal rat cochlea and their potential effect on auditory function and cochlea structures via round window transplantation., Methods: In comparison with the normal rats without any transplantation (Group III), normal rat cochleae were transplanted with NSC infected with Ad-GFP (Group I) or the artificial perilymph (Group II) via round windows. Auditory functions were monitored by thresholds of auditory brain stem responses (ABR); the cochlea structures were examined by HE staining; survivals of implanted NSC were determined by the expression of GFP; survivals of hair cells were accessed by whole mount preparation., Results: Neither at pre-transplantation nor at post-transplantation, there were significant differences in the click-ABR thresholds in rats between Group I and Group II (P > 0.05). There were no significant differences in these values before and after transplantation in the same rats from each group. After transplantation, the cochlea structures were normal in both Group I & Group II. Grafted NSC was visualized by the GFP expression in every turn of the cochlea in all animals of Group I. There were no significant differences in the loss of outer hair cell (OHC) among three groups. The inner hair cell (IHC) and most OHC were normal in every turns of cochleae of all groups., Conclusions: The embryonic NSC infected with Ad-GFP could survive and express the GFP gene in normal rat cochlea after transplantation via round window, which had not obvious affection to auditory function and inner ear pathology of rat cochlea.

Objective: To investigate HIV survival time and it's influencing factors among former commercial blood and plasma donors engaged in unsafe blood donation practices in China., Methods: HIV/AIDS cases from 8 counties (districts) in 4 provinces confirmed prior to January 24, 2006 related with former commercial blood and plasma donors were selected and data regarding infection, AIDS progression, death, and influencing factors were retrospectively collected., Results: In 530 cases of HIV infection, 334 (63.0%) cases had developed AIDS, 168 (50.3%) had received antiretroviral therapy (ART), and 152 (29.0%) had died. For the 530 cases, there was an average (10.1 +/- 1.8) years of observation from time of infection. Among 166 AIDS patients not receiving ART, average survival was 9.1 years (95% CI: 9.1 - 9.4), with an 8 year survival rate of 52.0%. Among 168 AIDS patients receiving ART, average survival was 12.1 years (95% CI: 11.9 - 12.3), with a 12-year survival rate of 80.0%. In 3 years of ART, average survival was longer in the treatment group as compared to the no treatment group with a hazard ratio for death of 12.2. Univariate analysis showed a significant difference (P < 0.05) in AIDS patient average survival based on gender, age, location, ART status, and baseline CD(4)(+) T cells count. Results from multivariate COX-regression showed that highly active ant iretroriral therapy (HAART) was the strongest protective factor for prolonging AIDS patients' survival (HR = 13.3, P = 0.00)., Conclusion: Although there are many factors influencing AIDS patients survival, intervention with HAART is the principle measure to prolong survival and decrease the risk of death.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is one of the most common congenital anomalies in humans. The pathogenesis of nsCL/P involves both genetic and environmental factors. On the basis of linkage data suggesting that 14q21-24 is one of the chromosomal regions that affects nsCL/P and data locating the BMP4 gene to 14q22-23, we performed a case-control study to evaluate whether BMP4 538T/C polymorphism, resulting in an amino acid change of Val/Ala (V152A) in the polypeptide, is associated with nsCL/P in a Chinese children population. Genotypes of 184 patients with nsCL/P and 205 controls were detected using a PCR-RFLP strategy. The results showed significant differences in the genotype and allele distribution of 538T/C polymorphisms of the BMP4 gene among the cases and controls. The 538C allele carriers were associated with a significantly increased risk of nsCL/P as compared with the noncarriers (odds ratio = 1.52; 95% confidence interval, 1.13-2.03; p = 0.005). Hence, our results support the hypothesis that this polymorphism contributes to risk of nsCL/P, which suggests that BMP4 538T/C polymorphisms could be used as genetic susceptibility markers of nsCL/P.

By using multilocus sequence analysis, five Borrelia valaisiana-related strains isolated from rodents and ticks in southwestern China were eventually classified as a new genospecies of B. burgdorferi sensu lato rather than B. valaisiana. The finding explained the differences in transmission cycle and phenotype between B. valaisiana strains from Europe and B. valaisiana-related strains from eastern Asia.

Objective: To investigate the main components of inner ear antigens inducing autoimmune Meniere's disease (AIMD) in guinea pigs., Methods: The guinea pigs were immunized with isologous crude inner ear antigens (ICIEAg). Then, the hearing function was measured with auditory brainstem response (ABR), the vestibular function was measured with electronystagmography (including spontaneous nystagmus and caloric test), and inner ear histopathological changes were observed by inner ear celloidin section with haematoxylin-eosin staining and observed under light microscope. According to these results, the AIMD-model animals from non-AIMD-model ones were distinguished. The special antibodies against ICIEAg in sera were measured with ELISA. The antigen-antibody reactions against different components of ICIEAg were detected by Western blotting with sera of AIMD and non-AIMD guinea pigs respectively. Then, we analysed the contrast between them and found the main components of the ICIEAg that were positive reaction in AIMD guinea pigs and negative reaction in non-AIMD guinea pigs., Results: The result of ELISA demonstrated that the sera of both the AIMD and non-AIMD guniea pigs contained the special antibodies against ICIEAg after immunized with ICIEAg. The difference of the amount of antibody against ICIEAg between AIMD guinea pig group and non-AIMD guinea pig group was not significant. Western blotting assay showed only the sera of AIMD guinea pig contained the antibodies against the specific antigens with the molecular of 68 000, 58 000, 42 000 and 28 000., Conclusions: ICIEAg contain many different components, the AIMD might only happen in the guinea pigs in which the special immunization against the main components that could induce this kind of disorder appeared. The inner ear antigens with molecular of 68 000, 58 000, 42 000 and 28 000 might be the main components inducing AIMD in guinea pigs.

Two novel bifunctional amine-thiourea organocatalysts 1 and 2, which both bear central and axial chiral elements, have been developed to promote enantioselective Michael reaction between 1,3-dicarbonyl compounds and nitro olefins. The catalyst 2 afforded the desired products with good levels of enantioselectivity (up to 96% ee), showing clearly that two chiral elements of 2 are matched, and enhance the stereochemical control.

Objective: To determine the PD-L1 expression levels in circulating dendritic cells(DCs) of patients with HBeAg positive chronic hepatitis B, and to investigate the effects of anti-PD-L1 antibody on DCs stimulating capacity of allogeneic lymphocytes., Methods: DCs were separated and induced from 22 HBeAg positive chronic hepatitis B patients (CHB), 8 acute resolved hepatitis B patients (AHB) and 10 healthy blood donors. PD-L1 and PD-L2 expression in DCs were determined using real-time RT-PCR and flow cytometry. The potential of circulating DCs on the proliferation of allogeneic T cells was detected and a specific monoclonal antibody against PD-L1 was used in alternative experiments. Serum HBV-DNA titers were measured using real-time PCR, and HBV markers and liver function were also evaluated., Result: The expression of PD-L1 but not PD-L2 was upregulated in circulating DCs of CHB patients, compared to AHB patients and healthy controls (both P<0.01). CHB patients with greater than 106 copies /ml of serum HBV DNA loads had a higher level of PD-L1 in circulating DCs than those with less than 106 copies/ml (P<0.05), and the high expression of PD-L1 in DCs was positively correlated with the plasma viral load. Moreover, the potential of circulating DCs from CHB patients was significantly decreased compared with healthy controls or AHB patients, while the blockade of PD-L1 using anti-PD-L1 monoclonal antibody increased the ability of DCs on the proliferation of allogeneic T cells in vitro., Conclusion: High expression of PD-L1 on circulating DCs may be associated with T cell exhaustion and persistent high levels of HBV DNA replication in chronic hepatitis B patients.

The present study was designed to investigate the effects of nicotine on development of bovine embryos derived from parthenogenetic activation (PA) and in vitro fertilization (IVF). Nicotine caused disfigured secondary meiotic spindle structures and affected embryonic development in a dose-dependent manner. Concentrations at 0.01-0.5 mM resulted in cleavage and blastocyst rates similar to the controls for both PA and IVF embryos. Nicotine at 2.0 and 4.0 mM significantly decreased the cleavage rates and none of the embryos developed beyond the 16-cell stage. Nicotine might disrupt the polymerization of microfilaments leading to impaired chromosome alignment or segregation, and induce the formation of polynuclei with a variety of abnormal nuclear structures such as 2-6 nuclei, 2-4 metaphase plates, 2-4 sets of anaphase/telophase plates, and the co-existence of polynuclei and 2-4 sets of anaphase/telophase plates. Nicotine adversely affected blastocyst chromosomal composition. Fifty-six to 70% of the IVF blastocysts and 71-88% of the PA blastocysts were polyploid and/or mixoploid after culture in 0.2-1.0 mM nicotine-containing media, which were higher (P < 0.05 or P < 0.01) than the controls. Cell numbers of the nicotine-cultured blastocysts were significantly lower than the control. In conclusion, nicotine induced disfigured spindles and irregular chromosome alignment and possibly impaired cytokinesis, which lead to decreased quality of the yielded blastocysts., ((c) 2007 Wiley-Liss, Inc.)

Objective: To investigate the effect of interleukin-6 (IL-6) and calcium sensing receptor (CASR) gene polymorphisms on bone mass accrual in Chinese adolescent girls., Methods: A total of 228 premenarche Chinese girls (9-11.5 years) were recruited for a 2-year study. Bone mineral densities (BMD) and Bone mineral contents (BMC) in the total body, total left hip including femoral neck, trochanter, intertrochanter and Ward's triangle area, and lumbar spine (L1-L4) were measured by DEXA. The -634C/G polymorphism of IL-6 gene was detected by PCR-restriction fragment length polymorphism and A986S polymorphism of CASR gene was detected by allele-special mutagenically separated amplication-PCR., Results: There were 176 available subjects when the 2-year study was completed. The -634C/G polymorphism of IL-6 gene and A986S polymorphism of CASR gene were significantly associated with bone mass accrual after adjusting the potential confounding factors. Girls with CC genotype of IL-6 -634G/C gene had higher percentage of increase in BMD of total body (P= 0.027) and femoral trochanter (P= 0.028) than those with CG+ GG genotypes. Girls with AA genotype of CASR gene had greater percentage of increase in L1-L4 lumbar spine BMC (P= 0.022) and Ward's triangle area BMD (P= 0.049) than their AS+ SS counterparts. Subjects with G allele of IL-6 gene in combination with S allele of CASR gene had less increase in BMC of femoral neck and L1-L4 lumbar spine., Conclusion: Adolescent girls carrying the G allele of IL-6 gene at -634C/G and S allele of CASR gene at A986S could potentially be the risk population of lower bone mass accrual.

Objective: To investigate the association between the polymorphism of vitamin D receptor (VDR) gene start codon (Fok I) and bone mass accrual, and assessing if such an association could be modified by physical activity in Chinese adolescent girls., Methods: A total of 228 premenrche Chinese girls (9-11.5-years-old) were recruited for 2-year study. Bone mineral densities (BMD) at the total body, total left hip (including femoral neck, trochanter, intertrochanteric and Ward's triangle area) and lumbar spine (L1-L4) were measured by dual energy X-ray absorptiometry. The Fok I polymorphism of VDR gene was detected with PCR-RFLP., Results: There remained 176 available subjects in our cohort when 2-year study was completed. No significant association was observed between Fok I polymorphism of VDR gene and percentage change in BMD at all sites. Girls with FF genotype had lower percentage change in total left hip BMD (THBMD) and femoral neck BMD (FNBMD) than girls with Ff + ff genotype only in low physical activity(< 1197 kJ/d), and physical activity was associated with percentage change in THBMD and FNBMD only in FF genotype group., Conclusion: The Fok I polymorphism of VDR gene should have significant interaction effect with physical activity on bone mass accrual in Chinese adolescent girls. Girls with FF genotype in low physical activity would be the potential risk population for low bone mass accrual, and high physical activity would be of benefit to gain higher bone mass accrual for girls with FF genotype.

Objective: To understand the relationship between the susceptibility to inflammatory bowel disease (IBD) and ATG16L1 gene single nucleotide polymorphism (SNP) site, rs2241880., Methods: Peripheral blood samples were collected from 80 IBD patients (including 40 with Crohn's disease and 40 with ulcerative colitis) and 50 healthy controls, and the genomic DNA was extracted from the white blood cells. Specific primers were designed according to the target gene sequence for PCR amplification of the target gene fragment, and the PCR products were purified followed by sequence analysis of the target region of ATG16L1 gene. The results of the sequence analysis were compared with the BenBank data to analyze the relationship between the allele gene polymorphisms and the susceptibility to Crohn's disease., Results: No significant differences were noted in the ATG16L1 gene SNP site rs2241880 polymorphisms among the patients with Crohn's disease, ulcerative colitis and the control subjects (Chi(2)=4.94, P=0.293)., Conclusion: ATG16L1 gene polymorphisms in the SNP site rs2241880 are not found to correlate to the susceptibility to Crohn's disease as reported in literature. The SNP site associated with Crohn's disease susceptibility identified in foreign populations does not seem to be identical with that in Chinese population.

Two new heterocyclics, named hirudonucleodisulfide A (1) and hirudonucleodisulfide B (2), were isolated from the dried material of Whitmania pigra Whitman. Their structures were elucidated by various spectroscopic means in combination with X-ray crystallographic analysis. A biological assay in vitro against anoxic injury demonstrated that 1 and 2 have moderately anti-anoxic activity with EC (50) values of 27.01 +/- 2.23 microg/mL and 19.54 +/- 1.53 microg/mL, respectively.

Objective: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP., Methods: The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP., Results: After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently., Conclusion: We have successfully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.

Objectives: To investigate the underlying mechanism of age related increase of pulmonary fibrosis incidence, Methods: The levels of pulmonary collagen and TGF-beta1 protein, MMP2,9 mRNA and TIMP 1,2,3 mRNA expression were measured by ELISA and realtime PCR., Results: As compared with adult rats [(756 +/- 160) pg/g vs (1 000 +/- 246) pg/g, P<0.05] the aged rats had more extracellular matrix components, increased TGF-beta1 protein level, lower (3.15 +/- 1.76 vs 0.17 +/- 0.13, P<0.01) MMP2 mRNA expression and TIMP 1,2,3 mRNA expressions (TIMP1 2.00 +/- 1.74 vs 0.11 +/- 0.06,TIMP2 7.60 +/- 2.51 vs 2.69 +/- 1.76, TIMP3 1.32 +/- 0.46 vs 0.29 +/- 0.16, P<0.01). No difference was observed in MMP9 (1.66 +/- 0.67 vs 1.74 +/- 0.87, P>0.05) mRNA expression., Conclusion: elevation in TGF-beta1 protein level, changes in the expression of MMP2/9 and decreased expression of TIMPS mRNA could be the underlying mechanism of age related increase of pulmonary incidence.

Objective: To evaluate the diagnostic accuracy in the assessment of coronary artery bypass grafts using 64 multi-slice computed tomography angiography (64-MSCTA) technology., Methods: There were 228 patients post coronary artery bypass grafting (CABG) underwent 64-MSCTA from July 2005 to April 2007. Thirty-one patients with 82 bypass grafts performed coronary angiography (CAG) because of angina or grafts lesion showed by 64-MSCTA., Results: All bypass grafts could be visualized by 64-MSCTA. Thirteen bypass graft occlusions and fourteen significant stenosis were detected by 64-MSCTA and confirmed by CAG. One venous grafts distal anastomosis was missed and another one was miss diagnosed as stenosis. One false negative and one false positive CT-finding resulted in a sensitivity of 93.3%, a specificity of 98.1%, a positive predictive value of 93.3%, a negative predictive value of 98.1% and an accuracy of 97.1% for grafts stenosis. As to the grafts lesion, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy for grafts occlusion were 96.4%, 98.1%, 96.4%, 98.1% and 97.6%, respectively., Conclusion: 64-MSCTA demonstrates high diagnostic accuracy in the assessment of graft patency and suitable for the follow-up of patients post CABG.

Spatial variations in dimethylsulfide (DMS) and dimethylsulfoniopropionate (DMSP) were surveyed in the surface microlayer and in the subsurface waters of the low productivity South China Sea in May 2005. Overall, average subsurface water concentrations of DMS and DMSP of dissolved (DMSPd) and particulate (DMSPp) fractions were 1.74 (1.00-2.50), 3.92 (2.21-6.54) and 6.06 (3.40-8.68) nM, respectively. No enrichment in DMS and DMSPp was observed in the microlayer. In contrast, the microlayer showed a DMSPd enrichment, with an average enrichment factor (EF, defined as the ratio of the microlayer concentration to subsurface water concentration) of 1.40. In the study area, none of the sulfur components were correlated with chlorophyll a. An important finding in this study was that DMS, DMSP and chlorophyll a concentrations in the surface microlayer were respectively correlated with those in the subsurface water, suggesting a close linkage between these two water bodies. The ratios of DMS:Chl-a and DMSPp:Chl-a showed a gradually increasing trend from North to South. This might be due to changes in the proportion of DMSP producers in the phytoplankton community with the increased surface seawater temperature. A clear diurnal variation in the DMS and DMSP concentrations was observed at an anchor station with the highest concentrations appearing during the day and the lowest concentrations during the night. The higher DMS and DMSP concentrations during daytime might be attributed to the light-induced increase in both algal synthesis and exudation of DMSP and biological production of DMS. The mean flux of DMS from the investigated area to the atmosphere was estimated to be 2.06 micromo lm(-2)d(-1). This low DMS emission flux, together with the low DMS surface concentrations was attributed to the low productivity in this sea.

The putative effect of nicotine on maturation and the chromosomal complement of bovine oocytes were investigated in the present study. Cumulus-enclosed oocytes were incubated in maturation medium with 0, 0.5, 1.0, 2.5, 5.0, and 10.0 mmol concentrations of nicotine. The results indicated that: (1) nicotine affected cumulus cell expansion in a dose-dependent manner and the perivitelline space failed to form when concentrations were equal to or greater than 5.0 mmol; (2) oocytes treated with 0.5 and 1.0 mmol nicotine concentrations resulted in maturation rates (83.3% and 85.9%, respectively) which was similar to the control (86.2%), whereas treatment with 2.5 and 5.0 mmol concentrations significantly decreased maturation rates to 70.2% and 26.7%, respectively; (3) nicotine at or over 2.5 mmol caused extremely irregular meiotic spindles and interrupted microfilament organization; (4) chromosomal analyses of oocytes with PB1 showed that oocytes derived from 0.5 and 1.0 mmol nicotine groups had haploid complements similar to the control (87-90%), but when the concentrations were increased to 2.5 and 5.0 mmol the haploid state was significantly reduced to around 70%; (5) oocytes at GVBD (germinal vesicle breakdown) and metaphase I stages were less affected by nicotine at 5.0 and 10.0 mmol concentrations than GV-stage oocytes; (6) maturation rates of the short-term nicotine-treated oocytes could be improved when subsequently incubated in normal maturation medium. Prolonged culture of nicotine-pretreated oocytes resulted in self-activation and some oocytes formed 1 or 2 pronuclei. In conclusion, nicotine affects bovine oocyte cumulus cell expansion, maturation rate, and chromosomal complement in a dose-dependent and an oocyte-stage-dependent manner.

We assessed the main and interaction effects of interleukin-6 and estrogen receptor gene polymorphisms on bone mass accrual in Chinese adolescent girls. A total of 228 premenarche Chinese girls (9-11.5 years old) were recruited for a 2-year follow-up study. Bone mineral density (BMD) at the total body, lumbar spine (L1-L4), and total left hip were measured by dual-energy X-ray absorptiometry at baseline and follow-up. The -174G/C and -634C/G polymorphism of IL-6 gene, and PvuII and XbaI polymorphisms of the estrogen receptor (ER)-alpha gene, were determined. The -634C/G polymorphism of the IL-6 gene and PvuII polymorphism of ER-alpha gene were significantly associated with bone mass accrual after adjusting the potential confounding factors. Girls with pp genotype of ER-alpha gene had greater percentage accrual in BMD of total body (P = 0.010) and femoral intertrochanter (P = 0.038) than their PP and Pp counterparts. Girls with CC genotype of IL-6 -634G/C gene had higher percentage accrual in BMD of total body (P = 0.032) and femoral trochanter (P = 0.048) than their CG + GG counterparts. Significant interaction effects of IL-6 -634C/G polymorphism and ER-alpha PvuII polymorphism were observed on percentage change in BMD of total left hip (P = 0.009) and femoral intertrochanter (P = 0.007). The genotype CC (IL-6 -634C/G) x pp (ER-alpha PvuII) was associated with greater BMD accrual than other genotype combination in Chinese adolescent girls. We found that the IL-6 -634C/G and ER-alpha PvuII polymorphism were significantly associated with BMD accrual and that they have an interactional effect on BMD accrual in Chinese adolescent girls.

Reprogramming impairment of DNA methylation may be partly responsible for the low efficiency in somatic cell nuclear transfer. In this study, bovine fibroblast cells were transfected with enhancer green fluorescence protein (eGFP), and then treated with a histone-deacetylase inhibitor, trichostatin A (TSA). The results showed that the effect of TSA on transfected cells was dose dependent. When the TSA concentration was over 5 ng/ml, cell proliferation was significantly inhibited. The majority of the cells died when TSA reached 100 ng/ml (P < 0.01). The number of cells in the S phase was significantly decreased in the 5- to 50-ng/ml TSA-treated groups, while the majority of the cells were at the G0/G1 phases. The number of eGFP-expressed cells were approximately twofold higher in 25-ng/ml (30.5%) and 50-ng/ml (29.5%) TSA groups than the control (15.0%). Reduced DNA methylation and improved histone acetylation were observed when the cells were treated with 10 to 50 ng/ml of TSA. Transfer of the TSA-treated cells to enucleated recipient oocytes resulted in similar cleavage rates among the experimental groups and the control. Cells treated with 50 ng/ml of TSA resulted in significantly lower blastocyst development (9.9%) than the other experimental and the control groups (around 20%). Analysis of the putative blastocysts showed that 86.7% of the embryos derived from TSA-treated cells were eGFP positive, which was higher than that from untreated cells (68.8%). In conclusion, treatment of transfected cells with TSA decreased the genome DNA methylation level, increased histone acetylation, and eGFP gene expression was activated. Donor cells with reduced DNA methylation did not improve subsequent cloned embryo development; however, transgene expression was improved in cloned embryos.

The growth and development of larval Amur sturgeon Acipenser schrenckii could be divided into two stages, i.e., pre-larval or free embryonic stage [from hatching (0 day of age and 10.17 +/- 0.63 mm of body length) to initial feeding (9 days of age and 18.93 +/- 0.74 mm of body length)], and post-larval stage [from initial feeding to juvenile stage (38 days of age and 41.89 +/- 5.09 mm of body length)]. During pre-larval stage, some organs related to feeling, feeding, breathing and swimming developed and differentiated rapidly; and during post-larval stage, dorsal, lateral and ventral scuta appeared, and the fish morphologically developed into juvenile. The growth characteristics of larval A. schrenckii presented allometric patterns, i.e., the growth inflexion points of eye diameter, mouth width, tail fin length, and pectoral fin length appeared in the ages of 2 days, 8-9 days, 10 days, and 11 days, respectively. Before the growth inflexion points, the organs grew fast; while after the growth inflexion points, the growth slowed down and approximately appeared isometric. The organs of larval A. schrenckii had the characteristics of congruous and rapid development. With the development of the organs related to feeling, feeding, breathing and swimming, larval A. schrenckii obtained the ability of escaping from predators and feeding, and consequently, its survival chance increased.

The effects of nicotine on nuclear maturation and meiotic spindle dynamics of bovine oocytes and subsequent embryonic development were investigated. Maturation rates (85%-94%) derived from nicotine treatments at 0.01 to 1.0 mM were similar to the control (86%), but significantly decreased at 2.0 to 6.0 mM. Haploid complements of metaphase II oocytes in 0.01 to 1.0 mM nicotine (approximately 90%) were similar to the control, while lower (ranged from 63% to 76%, P < 0.05 or P < 0.01) haploid oocytes were observed in the 2.0 to 6.0 mM nicotine groups. The majority of the PB1-free oocytes derived from 3.0 to 6.0 mM nicotine treatments were diploidy (2n = 60). Spindle microtubules changed from characteristically being asymmetrical in the controls to being equally distributed into two separate chromosome groups in the nicotine treatments. Nicotine disorganized the microfilament organization and inhibited the movement of anaphase or telophase chromosomes to the cortical area. The inhibited two chromosome groups became two spindles that either moved close in proximity or merged entirely together resulting in diploidy within the affected oocyte. Nicotine treatment significantly reduced the rate of cleavage and blastocyst development after parthenogenetic activation. Diploidy and cell number were drastically reduced in the resultant blastocysts. In conclusion, nicotine can alter the normal process of bovine oocyte meiosis and affects subsequent embryonic development., ((c) 2007 Wiley-Liss, Inc.)

Objective: To analyze the expression levels of PD-1 (program death factor-1) in peripheral T cells from patients infected with HBV, and to investigate its relationship with HBV serological markers., Methods: A total of 65 HLA-A2+ subjects, including 31 patients with chronic hepatitis B (CHB), 9 with acute resolved hepatitis B (AHB), 15 with HBV related liver cirrhosis (LC) and 10 healthy blood donators, were enrolled. The expression of PD-1 in peripheral T cells and PD-1 ligands PD-L1 and PD-L2 in PBMCs were determined by relative quantitative real-time PCR. The serum HBV markers, HBV DNA load and liver function were also measured., Results: Taken the PD-1 and PD-ligands expression in normal controls as a baseline level, the expression of PD-1 and PD-L1 from CHB patients was significantly increased, while the expression of PD-L2 was relatively low in all groups. In CHB patients, the PD-1 expression in peripheral T cells from patients with high viral load was much higher than that from those with low viral load or from normal controls. And the PD-1 expression level positively correlated with serum HBV DNA load (r=0.41, P<0.01) but not with serum ALT level., Conclusion: Long-term exposure to HBV antigens in CHB patients may increase the expression of PD-1 in T cells and thus leads to the virus persistent infection.

Objective: To study the feasibility of enforcing immunization certificate check before children enroll in primary schools or kindergartens in Guizhou Province., Methods: Quantitative and qualitative studies were conducted. The multi-stage and cluster sampling approach was adopted for the quantitative part of the study. A questionnaire was designed and 996 children and their keepers were interviewed. Principals, doctors or teachers of the primary schools, directors and child care nurses of kindergarten, and staff of immunization agencies were invited to take part in 12 focus group discussions; meanwhile, face-to-face individual in-depth interviews with 16 officials of the Health, Education and Governmental Departments at various levels were conducted., Results: The total number of subjects was 996. 16.7% of the children in the study completed all the procedures of the National Immunization Programme. 34.3% of them had immunization certificates while the remainder 44.7% registered in immunization agencies. Factors, including the migrant children, doubt about vaccine efficiency, mother's occupation and educational background, knowledge of the National Immunization Programme on targeted vaccines, played an important role in obtaining or not immunization certificates. 95% of the keepers interviewed thought the immunization certificates were useful; 94.8% of them considered the check was critical while only 3.6% of them thought it unnecessary. The first reason from those who found it unnecessary was that they feared that repeated immunization might affect their children's health. The second reason was the cost of immunization, which some of them could not afford to pay. However, the Health Department expressed a favorable attitude to the checking scheme. Though the Education Department agreed that the scheme was essential, they worried that it would affect the enrollment rate., Conclusion: In spite of the difficulty in administering immunization certificate check, the effort would be rewarding for raising the immunization coverage rate among the children in Guizhou Province.

Objective: Liver regeneration occurs through hepatocytes after acute liver injury. However, severe liver injury activates bipotential oval cells from canals of Hering which can differentiate into hepatocytes and biliary epithelial cells. Most models of oval cell activation have employed potential carcinogens to inhibit hepatocyte replication in the face of a regenerative stimulus. Oval cells must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette transporters are cytoprotective efflux pumps that may contribute to the protection of these cells. The aim of this study was to determine the ABC transporter expressions in hepatic oval cells., Methods: A rat model was established by feeding 2-acetylaminofluorene combined with partial hepatectomy to activate hepatic oval cells. Oval cells were isolated and purified using selective enzymatic digestion and density gradient centrifugation from the heterogeneous hepatic cell population. The expressions of ABC transporter gene, including MDR1, MRP1 and Bcrp1, in isolated hepatic oval cells and hepatocytes were measured by quantitative real-time reverse transcription-polymerase chain reaction and those in rat liver tissues were measured by immunohistochemistry., Results: Compared to those in the rat hepatocytes, mRNA expressions of the genes encoding MDR1, MRP1 and Bcrp1 were increased up to 9-, 1.5- and 13.8-folds in hepatic oval cells. Immunohistochemical staining of rat liver slides demonstrated that the expression of MDR1 proteins was found around periportal areas, and Bcrp1 protein was found located on cell membranes., Conclusion: Hepatic oval cells express high levels of the ABC transporter gene that may have cytoprotective functions during severe hepatotoxicity.

Micro-computed tomography (MicroCT) is an emerging technique in the last years which can achieve three dimensional X-ray photographs with resolution higher than 10microm. It can provide nondestructive assessment and analysis for bone, tooth and biomaterial samples. According to the operation experience, combined with recent publications, this paper summarized the application of microCT in the field of dental research.

In order to identify potential biochemical markers that can be used as indicators for phase change, the dynamics of polyphenolic compounds across apple seedlings (Malus domestica, Jonathan x Golden Delicious) were analyzed in this study by high performance liquid chromatography. Precocious flowering was induced by foliar sprays of plant growth regulators. Qualitative changes in the concentration of polyphenols were observed at node nos. 50, 80 and 120. Spontaneous and induced flowering was found at node nos. 122 and 77. It was reasonable to conclude that node no. 77 represented the point of transition between the juvenile phase and the adult vegetative phase, which was marked by the presence of phloridzin in the buds. The disappearance of myricitrin in the bark and the absence of caffeic acid in the aboveground tissues were qualitative markers of the reproductive phase, which was reached at node no. 122.

Integrins facilitate attachment of cells to the extra-cellular matrix, often binding the arginine-glycine-aspartic acid tri-peptide motif, thus facilitating cell migration, mediating cell-cell adhesion, linking the extracellular matrix (ECM) with cytoskeletal elements, and acting as signaling molecules. Adhesion activates signaling mechanisms that regulate integrin function, cytoskeletal assembly, cell behavior, and protein synthesis. Immunofluorescence was used to determine the presence of integrin alpha and beta subunits on the surface of bovine oocytes using a panel of monoclonal antibodies (mAbs) specific for alphaL, alphaM, alphaX, alphaV, alpha2, alpha4, alpha6, beta1, beta2, and beta3 antigens, with multiple antibodies for each subunit. Confocal microscopy indicated the presence of alphaV, alpha6, alpha4, alpha2, ss1, and ss3 integrin subunits on the plasma membrane of bovine oocytes. The presence of these subunits was verified by RT-PCR analysis using primers designed based on known gene sequences of bovine integrin subunits, or by using sequence information using bovine expressed sequence tags (EST) compared with known human and murine integrin subunit gene sequence information. Previously unpublished sequence information for bovine alpha6 and beta3 integrins was determined. The presence of these integrin subunits on the bovine oocyte vitelline membrane supports the hypothesis that sperm-oocyte interactions in the bovine are mediated by integrins., (Copyright (c) 2006 Wiley-Liss, Inc.)