Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells.

We have investigated by electrophoretic mobility shift assay (EMSA) the level of GATA-1 DNA-binding activity in nuclear extracts prepared from the human erythroleukaemic cell line, K562, after erythroid induction by hemin, sodium butyrate (NaB) or Trichostatin A or treatment with N -acetylcysteine (NAC). Relative to extract from untreated cells, GATA-1 binding activity increased markedly in all cases. However, immunoblot analysis revealed unchanged levels of GATA-1 protein after induction. Incubation of induced but not uninduced K562 extracts with phosphatase prior to EMSA weakened the binding activity, suggesting that the increase in GATA-1 binding following induction of K562 cells was a consequence of phosphorylation. When the mouse erythroleukaemic cell line MEL was induced with dimethylsulphoxide (DMSO), NaB or NAC, GATA-1 binding activity fell with DMSO, rose significantly with NaB and remained at about the same level in NAC-induced cells. In this case immunoblotting revealed that GATA-1 protein levels were in accord with the EMSA data. The DNA-binding activities of induced and uninduced MEL cell nuclear extracts were decreased by incubation with phosphatase, showing that phosphoryl-ation and DNA binding of GATA-1 are already optimalin these cells. The DNA-binding activity of affinity-purified GATA-1 from MEL cells was also reduced by phosphatase treatment, showing that phosphorylation/dephosphorylation is directly affecting the factor. Furthermore, when a comparison was made by EMSA of nuclear extracts prepared from K562 and MEL cells untreated or incubated with okadaic acid, a phosphatase inhibitor, GATA-1 binding was seen to increase with K562 cells, whereas with MEL cells there was no change in GATA-1 binding. Overall the results suggest that the level of GATA-1 phosphorylation increases after the induction of K562, but not MEL cells, where GATA-1 is already highly phosphorylated. Furthermore, phosphorylation increases the binding affinity of GATA-1 for a canonical binding site.     

Changes in the location of polyphenol oxidase in potato (Solanum tuberosum L.) tuber during cell death in response to impact injury: comparison with wound tissue

In order to elucidate the nature of the response of potato to impact injury at the biochemical level, changes in the location of the enzyme responsible for the discoloration, polyphenol oxidase, were determined using immunogold location with an antibody specific for potato tuber polyphenol oxidase. Tissue printing revealed that the enzyme was distributed throughout the tuber. Following impact injury, both tissue printing and quantitative electron microscopy indicated that there was no increase in the level of the enzyme although there was subcellular redistribution of polyphenol oxidase. This redistribution was first apparent at 12 h after impact, as determined by the use of confocal immunolocation, and coincided with loss of membrane integrity. These changes were examined in parallel with a number of stress-related parameters in both impact and wound responses. Wounding was accompanied by active gene expression and protein synthesis, leading to metabolic activity and tissue repair. In contrast, the bruising response was characterised by a limited active response and vital-staining methods indicated that after 16 h the tissue undergoes cell death. Received: 4 June 1998 / Accepted: 18 September 1998     

Kappa-chain constant-region gene sequences in genus Rattus: coding regions are diverging more rapidly than noncoding regions

We have determined the nucleotide sequence of a 1,200-base pair (bp) genomic fragment that includes the kappa-chain constant-region gene (C kappa) from two species of native Australian rodents, Rattus leucopus cooktownensis and Rattus colletti. Comparison of these sequences with each other and with other rodent C kappa genes shows three surprising features. First, the coding regions are diverging at a rate severalfold higher than that of the nearby noncoding regions. Second, replacement changes within the coding region are accumulating at a rate at least as great as that of silent changes. Third, most of the amino acid replacements are localized in one region of the C kappa domain--namely, the carboxy-terminal "bends" in the alpha-carbon backbone. These three features have previously been described from comparisons of the two allelic forms of C kappa genes in R. norvegicus. These data imply the existence of considerable evolutionary constraints on the noncoding regions (based on as yet undetermined functions) or powerful positive selection to diversify a portion of the constant-region domain (whose physiological significance is not known). These surprising features of C kappa evolution appear to be characteristic only of closely related C kappa genes, since comparison of rodent with human sequences shows the expected greater conservation of coding regions, as well as a predominance of silent nucleotide substitutions within the coding regions.      

Accumulations of Cyclic AMP in Adenine-Labeled Cell-free Preparations from Guinea Pig Cerebral Cortex: Role of α-Adrenergic and H1-Histaminergic Receptors

Norepinephrine, histamine, adenosine, glutamate, and depolarizing agents elicit accumulations of radioactive cyclic AMP from adenine-labeled nucleotides in particulate fractions from Krebs-Ringer homogenates of guinea pig cerebral cortex. The particulate fractions contain sac-like entities, which apparently are associated with a significant portion of the membranal adenylate cyclase. Particulate fractions from sucrose homogenates are a less effective source of such responsive entities. Activation of the adenine-labeled cyclic AMP-generating systems by norepinephrine is by means of alpha-adrenergic receptors, while activation by histamine is through H1- and H2-histaminergic receptors. Adenosine responses are potentiated by the amines and are antagonized by alkylxanthines. Glutamate and depolarizing agents appear to elicit accumulations of cyclic AMP via "release" of endogenous adenosine. It is proposed, based on the virtual absence of an alpha-adrenergic or H1-histaminergic response in the presence of a combination of potent adenosine and H2-histaminergic antagonists, that alpha-adrenergic and H1-histaminergic receptor mechanisms do not activate adenylate cyclase directly in brain slices or Krebs-Ringer particulate fractions, but merely facilitate activation by beta-adrenergic, H2-histaminergic, or adenosine receptors.     

Symbiotic relationships between soil fungi and plants reduce N2O emissions from soil

N₂O is a potent greenhouse gas involved in the destruction of the protective ozone layer in the stratosphere and contributing to global warming. The ecological processes regulating its emissions from soil are still poorly understood. Here, we show that the presence of arbuscular mycorrhizal fungi (AMF), a dominant group of soil fungi, which form symbiotic associations with the majority of land plants and which influence a range of important ecosystem functions, can induce a reduction in N₂O emissions from soil. To test for a functional relationship between AMF and N₂O emissions, we manipulated the abundance of AMF in two independent greenhouse experiments using two different approaches (sterilized and re-inoculated soil and non-mycorrhizal tomato mutants) and two different soils. N₂O emissions were increased by 42 and 33% in microcosms with reduced AMF abundance compared to microcosms with a well-established AMF community, suggesting that AMF regulate N₂O emissions. This could partly be explained by increased N immobilization into microbial or plant biomass, reduced concentrations of mineral soil N as a substrate for N₂O emission and altered water relations. Moreover, the abundance of key genes responsible for N₂O production (nirK) was negatively and for N₂O consumption (nosZ) positively correlated to AMF abundance, indicating that the regulation of N₂O emissions is transmitted by AMF-induced changes in the soil microbial community. Our results suggest that the disruption of the AMF symbiosis through intensification of agricultural practices may further contribute to increased N₂O emissions.     

颈髓损伤的常规MRI表现与DTI技术临床应用

目的：比较不同时期颈髓损伤的MRI表现及DTI的应用价值。方法：收集急性颈髓压迫病例15例、慢性颈髓压迫病例23例、颈髓慢性压迫合并急性压迫病例12例。15例健康志愿者作为对照组。进行常规MRI检查,应用DTI检查测量表现扩散系数（ADC）值和各向异性分数（FA）。比较各组间ADC值和FA值,并进行统计学分析。结果：急性颈髓迫病例,常规MRI显示颈髓增粗,呈等T1长T2信号;慢性颈髓压迫病例,9例呈长T1长T2信号,14例呈等T1长T2信号;慢性颈髓压迫并急性压迫病例颈髓明显增粗,呈等、长T1明显长T2信号。与对照组比较：急性颈髓压迫组的ADC值和FA值均明显降低,两组的差异有显著性;慢性颈髓压迫组的FA值降低,ADC值增高,两组的差异有显著性;慢性脊髓压迫合并急性脊髓压迫组ADC值与对照组比较无差异,FA值低于对照组。颈髓压迫各组间ADC值及FA值比较差异显著。结论：不同时期颈髓损伤常规MRI图像缺乏特异性,根据ADC值及FA值可判断颈髓损伤的时期。     

肿瘤干细胞研究进展及临床应用前景

肿瘤干细胞是肿瘤中具有自我更新能力和分化潜能,能产生异质性细胞的细胞。我们简要阐述了肿瘤干细胞存在的证据、分离技术及来源,并对以肿瘤干细胞为靶标的肿瘤治疗途径、临床应用前景和问题进行了综述。