基于23S／5SrDNA序列的柑橘黄龙病病原菌亚洲种系统发育研究

根据柑橘黄龙病亚洲种23S/5S的DNA序列设计一对引物对不同地理来源的6个柑橘黄龙病样品DNA进行扩增,扩增片段大小均为1 654 bp包括一个假定细胞壁水解酶假基因(putative cell wall hydrolase pseudogene)和5S rRNA 基因.序列同源性分析结果表明;6个柑橘黄龙病病原菌样品与柑橘黄龙病病原菌亚洲种Sihui样品的同源性为99%,然而与土壤杆菌,布鲁氏菌,根瘤菌,中华根瘤菌,巴通体菌和中慢生根瘤菌的同源性只有89%～95%,说明在23S/5S rDNA序列上黄龙病病原菌亚洲种与α变形菌纲根瘤菌目的其他病原菌相差较大.对黄龙病病原菌亚洲种种内的23S/5S rDNA序列进行比较分析,结果发现黄龙病病原菌亚洲种种内之间putative cell wall hydrolase pseudogene和5S rRNA的基因序列非常保守,但不同地理来源的柑橘黄龙病样品碱基序列间确实存在差异,差异的大小与地理的远近无关.利用简约法对黄龙病病原菌亚洲种及α变形菌纲其它病原菌的23S/5S rDNA序列构建的系统发育树显示黄龙病病原菌亚洲种单独聚为一类,其他细菌聚为另一类,该结果与基于rplJ基因及16S rRNA基因的DNA序列构建的分子系统进化树结果一致.     

A Plausible Microtubule-Based Mechanism for Cell Division Orientation in Plant Embryogenesis

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Immunohistochemical study of a membrane skeletal molecule, protein 4.1G, in mouse seminiferous tubules

Protein 4.1 families have recently been established as potential organizers of an adherens system. In the adult mouse testis, protein 4.1G (4.1G) localized as a line pattern in both basal and adluminal compartments of the seminiferous tubules, attaching regions of germ cells and Sertoli cells. By double staining for 4.1G and F-actin, their localizations were shown to be different, indicating that 4.1G was localized in a region other than the basal and apical ectoplasmic specializations, which formed the Sertoli–Sertoli cell junction and Sertoli–spermatid junction, respectively. By electron microscopy, immunoreactive products were seen exclusively on the cell membranes of Sertoli cells, attaching to the various differentiating germ cells. The immunolocalization of cadherin was identical to that of 4.1G, supporting the idea that 4.1G may be functionally interconnected with adhesion molecules. In an experimental mouse model of cadmium treatment, in which tight and adherens junctions of seminiferous tubules were disrupted, the 4.1G immunostaining in the seminiferous tubules was dramatically decreased. These results indicate that 4.1G may have a basic adhesive function between Sertoli cells and germ cells from the side of Sertoli cells.     

MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src

Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex.     

Genome-Wide Association Study Identifies Variants in PMS1 Associated with Serum Ferritin in a Chinese Population

Only a small proportion of genetic variation in serum ferritin has been explained by variant genetic studies, and genome-wide association study (GWAS) for serum ferritin has not been investigated widely in Chinese population. We aimed at exploring the novel genetic susceptibility to serum ferritin, and performed this two stage GWAS in a healthy Chinese population of 3,495 men aged 20–69 y, including 1,999 unrelated subjects in the first stage and 1,496 independent individuals in the second stage. Serum ferritin was measured with electrochemiluminescence immunoassay, and DNA samples were collected for genotyping. A total of 1,940,243 SNPs were tested by using multivariate linear regression analysis. After adjusting for population stratification, age and BMI, the rs5742933 located in the 5′UTR region of PMS1 gene on chromosome 2 was the most significantly associated with ferritin concentrations (P-combined = 2.329×10⁻¹⁰) (β = −0.11, 95% CI: −0.14, −0.07). Moreover, this marker was about 200kb away from the candidate gene SLC40A1 which is responsible for iron export. PMS1 gene was the novel genetic susceptibility to serum ferritin in Chinese males and its relation to SLC40A1 needs further study.     

Generation and characterization of a zebrafish muscle specific inducible Cre line

Zebrafish transgenic lines provide valuable insights into gene functions, cell lineages and cell behaviors during development. Spatiotemporal control over transgene expression is a critical need in many experimental approaches, with applications in loss- and gain-of-function expression, ectopic expression and lineage tracing experiments. The Cre/loxP recombination system is a powerful tool to provide this control and the demand for validated Cre and loxP zebrafish transgenics is high. One of the major challenges to widespread application of Cre/loxP technology in zebrafish is comparatively small numbers of established tissue-specific Cre or CreERT2 lines. We used Tol2-mediated transgenesis to generate Tg(CrymCherry;-1.9mylz2:CreERT2) which provides an inducible CreERT2 source driven by muscle-specific mylz2 promoter. The transgenic specifically labels the trunk and tail skeletal muscles. We assessed the temporal responsiveness of the transgenic by screening with a validated loxP reporter transgenic ubi:Switch. Further, we evaluated the recombination efficiency in the transgenic with varying concentrations of 4-OHT, for different induction time periods and at different stages of embryogenesis and observed that higher recombination efficiency is achieved when embryos are induced with 10 μM 4-OHT from 10-somites or 24 hpf till 48 or 72 hpf. The transgenic is an addition to currently available zebrafish transgenesis toolbox and a significant tool to advance muscle biology studies in zebrafish.     

Correlation between body composition and risk factors for cardiovascular disease and metabolic syndrome

Body mass index (BMI) is an important diagnostic tool for determining obesity; however, while BMI reflects the influence of body height over body weight, it does not reveal body fat percentage (BF%). We explored whether BF% correlated with risk factors for cardiovascular disease and metabolic syndrome and whether metabolically obese, normal weight people were at risk for these diseases. A total of 2,867 healthy volunteers participated in this study. Blood pressure, height, weight, waist circumference, BMI, BF%, lipid profile, fasting glucose, uric acid, and lifestyle factors were collected from healthy subjects during their annual health examinations. In both males and females, BF% correlated positively with BMI and waist circumference. Participants were divided into three groups according to BF% and data were compared between groups. The results suggest that BF% correlates with risk factors for cardiovascular disease and metabolic syndrome for both men and women, and that BF% may be a useful predictor of risk, particularly in metabolically obese, normal weight individuals. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

Impacts of climate variability on tree demography in second growth tropical forests: the importance of regional context for predicting successional trajectories

Naturally regenerating and restored second growth forests account for over 70% of tropical forest cover and provide key ecosystem services. Understanding climate change impacts on successional trajectories of these ecosystems is critical for developing effective large‐scale forest landscape restoration (FLR) programs. Differences in environmental conditions, species composition, dynamics, and landscape context from old growth forests may exacerbate climate impacts on second growth stands. We compile data from 112 studies on the effects of natural climate variability, including warming, droughts, fires, and cyclonic storms, on demography and dynamics of second growth forest trees and identify variation in forest responses across biomes, regions, and landscapes. Across studies, drought decreases tree growth, survival, and recruitment, particularly during early succession, but the effects of temperature remain unexplored. Shifts in the frequency and severity of disturbance alter successional trajectories and increase the extent of second growth forests. Vulnerability to climate extremes is generally inversely related to long‐term exposure, which varies with historical climate and biogeography. The majority of studies, however, have been conducted in the Neotropics hindering generalization. Effects of fire and cyclonic storms often lead to positive feedbacks, increasing vulnerability to climate extremes and subsequent disturbance. Fragmentation increases forests’ vulnerability to fires, wind, and drought, while land use and other human activities influence the frequency and intensity of fire, potentially retarding succession. Comparative studies of climate effects on tropical forest succession across biogeographic regions are required to forecast the response of tropical forest landscapes to future climates and to implement effective FLR policies and programs in these landscapes.     

Derivation and characterization of human embryonic stem cell lines from poor quality embryos

Poor quality embryos discarded from in vitro fertilization （IVF） laboratories are good sources for deriving human embryonic stem cell （hESC） lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses （ICMs） could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods （P〉0.05）. As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.     

Mucosal Immunization of Lactating Female Rhesus Monkeys with a Transmitted/Founder HIV-1 Envelope Induces Strong Env-Specific IgA Antibody Responses in Breast Milk

We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission.