放线菌素D和环已亚胺对伊贝母胚性愈伤组织中胚性细胞团和球形胚发生及大分子代谢动态的影响

伊贝母(F.pallidiflora Schrenk)胚性愈伤组织接种于NAA 1.0mg/L+6-BA2.0 mg/L的MS培养基上,在培养10天前可产生大量单细胞到多细胞胚性细胞团,培养10至15天,逐渐形成大量球形胚。利用这样一个实验体系,在培养0、1、2、3和4天后加入放线菌素D(AMD,20μg/ml)和环己亚胺(CHM,20μg/ml),继续培养至第6天,分析大分子代谢动态和观察胚性细胞团的形成情况;培养6和10天后加入同样浓度的AMD和CHM。继续培养至第15天,分析大分子代谢动态及观察球形胚形成情况。结果表明:(1)培养0、1、2、3和4天加入AMD的分别抑制胚性细胞团的100%、63%和45%,加入CHM的抑制100%、85%和75%,培养6和10天后加入CHM抑制球形胚的100%和75%;(2)DNA、RNA和蛋白质在胚性细胞团和球形胚形成时出现两个峰值,其中RNA变化剧烈,最早出现峰值。AMD和CHM分别抑制RNA和蛋白质的合成;(3)过氧化物酶同工酶带在胚性细胞团和球形胚形成过程中顺序表达,AMD和CHM分别在转录和转译水平上对其进行规律性抑制。根据以上结果,本文对伊贝母体细胞胚胎发生的机制进行了初步讨论。     

Expression of adiponectin in choroidal tissue and inhibition of laser induced choroidal neovascularization by adiponectin

The aim of this study was to investigate the role of adiponectin (APN) in a mouse model of laser induced choroidal neovascularization (CNV). We have shown by immunohistochemistry that the expression of APN, adiponectin receptor 1, adiponectin receptor 2 and T cadherin gradually increased from day 1 to day 7 post-laser in laser treated mice compared to controls. Recombinant APN (rAPN) was injected intraperitoneally (i.p., 25 microg/mouse) or intravitreally (2 microg/eye) in lasered mice. Another set of lasered mice received APN peptide via i.p. (75 microg/mouse) or intravitreal (30 microg/eye) route. Control mice received a similar treatment with PBS, control protein or control peptide after laser treatment. We found that in the i.p. and intravitreal injection of rAPN resulted in 78% and 68% inhibition respectively in the size of CNV complex compared to control mice. Similar results were observed when APN peptide was injected intravitreally or i.p. Treatment with rAPN or the peptide resulted in decreased levels of vascular endothelial growth factor. Thus, APN inhibited choroidal angiogenesis and may have therapeutic implications in the treatment of wet age related macular degeneration.     

Myometrial‐derived CXCL12 promotes lipopolysaccharide induced preterm labour by regulating macrophage migration,polarization and function in mice

Preterm birth is a major contributor to neonatal mortality and morbidity. Infection results in elevation of inflammation‐related cytokines followed by infiltration of immune cells into gestational tissue. CXCL12 levels are elevated in preterm birth indicating it may have a role in preterm labour (PTL); however, the pathophysiological correlations between CXCL12/CXCR4 signalling and premature labour are poorly understood. In this study, PTL was induced using lipopolysaccharide (LPS) in a murine model. LPS induced CXCL12 RNA and protein levels significantly and specifically in myometrium compared with controls (3‐fold and 3.5‐fold respectively). Highest levels were found just before the start of labour. LPS also enhanced the infiltration of neutrophils, macrophages and T cells, and induced macrophage M1 polarization. In vitro studies showed that condition medium from LPS‐treated primary smooth muscle cells (SMC) induced macrophage migration, M1 polarization and upregulated inflammation‐related cytokines such as interleukin (IL)‐1, IL‐6 and tumor necrosis factor alpha (TNF‐α). AMD3100 treatment in pregnant mice led to a significant decrease in the rate of PTL (70%), prolonged pregnancy duration and suppressed macrophage infiltration into gestation tissue by 2.5‐fold. Further, in‐vitro treatment of SMC by AMD3100 suppressed the macrophage migration, decreased polarization and downregulated IL‐1, IL‐6 and TNF‐α expression. LPS treatment in pregnant mice induced PTL by increasing myometrial CXCL12, which recruits immune cells that in turn produce inflammation‐related cytokines. These effects stimulated by LPS were completely reversed by AMD3100 through blocking of CXCL12/CXCR4 signalling. Thus, the CXCL12/CXCR4 axis presents an excellent target for preventing infection and inflammation‐related PTL.     

ACE inhibition actively promotes cell survival by altering gene expression

We tested the effect of ACE inhibition on the survival of bovine retinal (REC) and choroidal (CEC) endothelial cells (EC) in culture. The ACE inhibitor captopril delayed the apoptotic tube collapse of REC on Matrigel for >15 days. Captopril treatment of confluent monolayers (2-8 weeks) followed by slow starvation (2-4 weeks) increased EC viability by approximately 200%. Two-week captopril exposures were sufficient to confer maximal protection. Only vehicle-treated EC demonstrated apoptotic features such as membrane blebbing and DNA laddering. By RT-PCR, the starvation marker p202 was upregulated only in starved cells. In REC, captopril upregulated the pro-survival proteins mortalin-2, uPA, and uPAR while downregulating the anti-growth sprouty-4 and tPA. In CEC, captopril also upregulated tPA and its inhibitor PAI-1. Amiloride (uPA inhibitor) blocked the captopril-induced increase in EC survival, secondary sprouting, and invasion in Matrigel. The pro-survival effects of captopril involve the reprogramming of genes involved in cell survival and immortalization.     

Remediation of AMD Contaminated Soil by Two Types of Reeds

Acid mine drainage (AMD) adversely impacts many regions in the world. The interactions among citric acid (CA), rhizosphere bacteria and metal uptake in different types of Phragmites australis cultured in spiked AMD contaminated soil were investigated. Compared with non-contaminated reeds cultured under the same conditions, wild reeds harvested from a contaminated site accumulated more metals into tissues. Rhizosphere iron oxidizing bacteria (Fe(II)OB) enhanced the development of Fe plaque but had no significant impact on the formation of Mn and Al plaque on the root surface of either reeds. Plaque may restrain the accumulation of Fe and Mn into tissues of reeds. CA inhibited the growth of Fe(II)OB, reduced the formation of metal plaque and significantly elevated metal accumulations into both underground and aboveground biomass of reeds. The concentrations of Fe, Al and Mn were higher in belowground organs than aboveground tissues. The roots contained 0.28 ± 0.01 mg/g Mn, 3.09 ± 0.51 mg/g Al, 94.47 ± 5.75 mg/g Fe, while the stems accumulated 0.19 ± 0.01 mg/g Mn, 1.34 ± 0.02 mg/g Al, 10.32 ± 0.60 mg/g Fe in wild reeds cultured in soil added with 33,616 ppm CA. Further field investigations may be required to study the effect of CA to enhance phytoremediation of metals from real AMD contaminated sites.     

Microperimetric Biofeedback in AMD Patients

To analyse biofeedback training by microperimeter MP-1 (Nidek Technologies) on patients with Age Related Maculopathy (AMD). We enrolled 15 patients (10 female and 5 male) and examined total of 27 eyes with AMD. All the patient underwent 10 training sessions of 10 min for each eye, performed once a week using the MP-1 biofeedback examination. Statistical analysis was performed using Student’s t-test. p values less than 0.05 were considered statistically significant. All patients displayed an improvement in visual acuity, fixation behaviour, retinal sensitivity an reading speed. The mean character size value improved from 36.4 to 11.7; this result was statistically significant (p = 0.031). A biofeedback examination using the MP-1 microperimeter can help the brain to memorize the final fixation location by increasing attention modulation, thereby providing an efficient preferred retinal locus for visual tasks in patients with macular disease and central scotoma.     

Plant-derived phenolic compounds impair the remediation of acid mine drainage using treatment wetlands

The use of wetlands to remediate acid mine drainage has expanded rapidly since the realisation that acid coal mine drainage running into natural sphagnum wetlands undergoes an increase in pH and a precipitation of metals. However, our study suggests that the inclusion of plants in the acid mine drainage treatment system may be questionable, due to inefficiencies caused by exudation of dissolved organic carbon (DOC), and in particular its phenolic constituents. They complex with iron, causing increased solubility, the exact opposite of what is required to facilitate amelioration. The addition of minewater to planted wetland mesocosms initially caused a decline in Fe concentrations, typically from over 1100 to a low of 75 mg L⁻¹. However, it increased higher than 300 mg L⁻¹ after 15 days. The rise in iron occurred concurrently with DOC and phenolic increases; 15-69 and 5-15 mg L⁻¹, respectively, for Eriophorum angustifolium. Removal of DOC by precipitation with calcium lowered the DOC abundance, but without a simultaneous decrease in iron concentration. The concentration of one fraction of the DOC, phenolic compounds, did not decline, and we propose that the Fe was complexed with that phenolic DOC pool. The proposal was confirmed by enzymic depletion of the phenolic compounds using phenol oxidase. Our findings suggest that phenolic complexation represents a potent constraint on wetland-based bioremediation of iron in acid mine drainage.     

Insulin-like growth factor-1 contributes to neovascularization in age-related macular degeneration

Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration and a leading cause of vision loss. Along with other angiogenic factors like vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and its receptor, IGF-1R, have been implicated in CNV. IGF-1 is produced in neurons and retinal pigment epithelium (RPE) but its targets and impact in CNV are not understood. IGF-1 immunoreactivity was abundant throughout surgically isolated human CNV tissues and RPE cells were immunopositive for IGF-1R. Cultured RPE cells obtained from CNV tissues expressed IGF-1R. IGF-1 stimulation of cultured cells from CNV tissues induced monophasic sustained rises in intracellular free Ca(2+). VEGF concentration in the medium of unstimulated RPE cell cultures from CNV tissues increased with time to a steady-state (8h) which was increased twofold by IGF-1 stimulation. Thus, in RPE cells IGF-1 stimulates the second messenger Ca(2+) and increases VEGF secretion which, in turn, induces neovascularization.     

Uncontrolled zinc- and copper-induced oligomerisation of the human complement regulator factor H and its possible implications for function and disease

Polymorphisms in factor H (FH), a major regulator of complement activation, and the accumulation of high zinc concentrations in the outer retina are both associated with age-related macular degeneration. FH is inhibited by zinc, which causes FH to aggregate. To investigate this, we quantitatively studied zinc-induced FH self-association by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation in conditions corresponding to physiological levels of FH and pathological levels of zinc in the outer retina. By scattering, FH at 2.8-7.0 μM was unaffected until [Zn] increased to 20 μM, whereupon the radius of gyration, R_G, values increased from 9 to 15 nm at [Zn] = 200 μM. The maximum dimension of FH increased from 32 to 50 nm, indicating that compact oligomers had formed. By ultracentrifugation, size-distribution analyses showed that monomeric FH at 5.57 S was the major species at [Zn] up to 60 μM. At [Zn] above 60 μM, a series of large oligomers were formed, ranging up to 100 S in size. Oligomerisation was reversed by ethylenediaminetetraacetic acid. Structurally distinct large oligomers were observed for Cu, while Ni, Cd and Fe showed low amounts of oligomers and Mg and Ca showed no change. Fluid-phase assays showed reduced FH activities that correlated with increased oligomer formation. The results were attributed to different degrees of stabilisation of weak self-dimerisation sites in FH by transition metals. The relevance of metal-induced FH oligomer formation to complement regulation and age-related macular degeneration is discussed.