Analysis of the pinocytic process in rat kidney II. Biochemical composition of pinocytic vesicles compared to brush border microvilli,lysosomes and basolateral plasma membranes

Pinocytic vesicles, brush border microvilli, lysosomes and basolateral plasma membranes were isolated from rat kidney cortex and their biochemical composition and membrane turnover compared. Pinocytic vesicles are devoid of marker enzymes of brush border microvilli, such as alkaline phosphatase and 5′-nucleotidase, and of lysosomes, such as acid phosphatase and β-glucuronidase. The protein pattern as revealed by polyacrylamide gel electrophoresis differs for all four membranes. Analysis of the phospholipid composition shows that pinocytic vesicles are rich in the negatively charged phospholipid phosphatidylserine and have a low content of sphingomyelin and phosphatidylethanolamine.[¹⁴C]guanido-arginine, [³H]fucose and $\text{myo-}\text{[}{}^3\text{H]inositol}$ were preferentially incorporated into the pinocytic vesicles. Using a double label technique with leucine also, evidence of a more rapid turnover of the pinocytic vesicle membrane proteins was obtained.The results suggest that pinocytic vesicles are not derived from the brush border microvillous membrane but are independent entities that are newly synthesized during the pinocytic process.     

The use of 13C-NMR for studies of wheat straw decomposition

13C-NMR was used to study the field decomposition of surface retained and incorporated wheat straw. Results showed decreasing proportions of straw carbon as carbohydrate and increasing proportions of aromatic compounds during straw decomposition. These changes were greater for the surface retained straw, however greater relative microbial contamination of incorporated straw may have affected results. The cost of 13C-NMR may lessen its role in studies of this nature.     

Different requirements for Ia-bearing accessory cells in mitogen versus antigen induction of human B-cell responses

The accessory cell requirements for the induction of proliferative and specific antibody responses of human lymphocytes stimulated with either antigen or mitogen were examined. An Ia-negative human myeloid tumor cell line, K562, could substitute for monocytes in the proliferation of monocyte-depleted lymphocytes in response to pokeweed mitogen (PWM) stimulation. K562 cells could also act as accessory cells in the PWM-induced anti-keyhole limpet hemocyanin (KLH) antibody synthesis of cells from a KLH-immunized donor. In contrast, only monocytes and not K562 cells could function as accessory cells in antigen-induced lymphocyte proliferation as well as in antigen-induced, antigen-specific antibody production. However, K562 cells, like monocytes, were able to positively and negatively regulate polyclonal immunoglobulin responses. Thus, Ia-bearing accessory cells can function in antigen-induced proliferation and antibody responses while non-Ia-bearing cells can function in mitogen-induced, but not anti-geninduced responses. These studies indicate a dichotomy in the nature of required accessory cells in antigen-induced versus mitogen-induced human lymphocyte responses and strongly suggest an obligatory role of Ia or an Ia-related molecule on accessory cells in antigen-induced responses of human lymphocytes.     

Application of partial restriction procedure in both shotgun and non-random strategies for nucleotide sequencing

Partial digestion of a target DNA fragment with 4-bp-recognition restriction enzymes followed by a forced ligation to an M13 vector was employed for the construction of a subfragment library. The library can be used for either shotgun or non-random nucleotide sequencing. Application of the partial digests generated with the 4-bp recognition restriction enzymes instead of DNase I in the improved non-random strategy for nucleotide sequencing (Li and Wu, 1987) made the procedure as easy as that of the random strategy. The library can also be used in shotgun nucleotide sequencing directly, and few self-ligated subfragments were found. The usefulness of this procedure was demonstrated by the sequencing of a goat 6.5-kb EcoRI fragment, which is located 5' to the globin gene.     

Opposing roles of IL-10 in acute bacterial infection

Interleukin-10 (IL-10) is recognized as an anti-inflammatory cytokine that downmodulates inflammatory immune responses at multiple levels. In innate cells, production of this cytokine is usually triggered after pathogen recognition receptor (PRR) engagement by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patters (DAMPs), as well as by other soluble factors. Importantly, IL-10 is frequently secreted during acute bacterial infections and has been described to play a key role in infection resolution, although its effects can significantly vary depending on the infecting bacterium. While the production of IL-10 might favor host survival in some cases, it may also result harmful for the host in other circumstances, as it can prevent appropriate bacterial clearance. In this review we discuss the role of IL-10 in bacterial clearance and propose that this cytokine is required to recover from infection caused by extracellular or highly pro-inflammatory bacteria. Altogether, we propose that IL-10 drives excessive suppression of the immune response upon infection with intracellular bacteria or in non-inflammatory bacterial infections, which ultimately favors bacterial persistence and dissemination within the host. Thus, the nature of the bacterium causing infection is an important factor that needs to be taken into account when considering new immunotherapies that consist on the modulation of inflammation, such as IL-10. Indeed, induction of this cytokine may significantly improve the host’s immune response to certain bacteria when antibiotics are not completely effective.     

维生素E对体外培养的人脐静脉内皮细胞白介素-8表达的影响

目的探讨维生素E(α-生育酚)对人脐静脉内皮细胞(HUVEC)白介素-8(IL-8)表达的影响.方法体外培养HUVEC,将其随机分为正常对照组和实验组,实验组细胞在用激动剂脂多糖(LPS)刺激之前,分别给予0、10、20、30mg/Lα-生育酚,然后在6h、24h、48h三个时段,利用双抗体夹心酶联免疫吸附技术(ELISA)和原位杂交技术(ISH)检测各组细胞IL-8蛋白表达及mRNA表达水平.结果 (1)正常对照组IL-8有基础表达;(2)与正常对照组比较,ELISA和ISH的结果均显示LPS能够明显诱导IL-8高表达:蛋白表达增强约28倍(P<0.01),mRNA表达增强约4.3倍(P<0.01);(3)维生素E能够抑制LPS诱导的IL-8的表达:ELISA结果显示20mg/L-48hα-T处理组作用最强,IL-8蛋白表达减少72.7%(P<0.01);ISH结果显示20mg/L -24hα-T处理组作用最强,IL-8mRNA表达减少73.2%(P<0.01).结论维生素E可显著抑制LPS诱导的HUVEC高表达IL-8,表明维生素E可通过影响对动脉粥样硬化有重要作用的IL-8的表达来发挥其抗动脉粥样硬化作用.     

Water management practices and climate in ancient agriculture: inferences from the stable isotope composition of archaeobotanical remains

Carbon isotope discrimination (Δ¹³C) in charred grains from archaeological sites provides reliable information about water availability of ancient crops. However, as cereals are cultivated plants, they may reflect not only climatic fluctuations, but also the effect on water status of certain agronomic practices, such as sowing in naturally wet soils or irrigation. In this work, we propose a methodological approach to combine Δ¹³C data from different plant species, in order to discriminate between climate-derived and anthropogenic effects on ancient crops. We updated previous models for estimating water inputs from Δ¹³C of cereal grains of Hordeum vulgare and Triticum aestivum/durum, and we applied them to published data from several archaeological sites, including samples from the Neolithic to the present day in northeast and southeast Spain, as well as from the Neolithic site of Tell Halula (northwest Syria). We found an important decrease in water availability from the Neolithic to the present time in the three areas of study, especially clear for the two driest areas (southeast Spain and northwest Syria). Potential differences in water management practices between wheat and barley, as well as between cereal and legume crops (Vicia faba and Lens culinaris), are also discussed on the basis of the comparison of Δ¹³C values across several archaeological sites.     

Phorbol 12-myristate 13-acetate prevents apoptosis in erythroleukemia K562 cells induced by some nucleosides

We studied the ability of phorbol 12-myristate 13-acetat to prevent erythroid differentiation and apoptosis in erythroleukemic K562 cells induced by cytidine, thymidine, and guanosine. The exposure of cancer cells to combinations of phorbol 12-myrsitate 13-acetate (100 nM) nucleosides for two days led to a loss of hemoglobin production (marker of erythroid differentiation) in cells and increased expression of monocyte-macrophage lineage associated surface antigen CD14. The treatment of K562 cells with nucleosides only was accompanied by the activation of caspase-3 and caspase-9, rather than caspase-6, increased fluorescence of ethidium bromide and DAPI upon binding to DNA, and apoptosis. Intracellular activation of caspase-6, inhibition of caspase-9, a markedly decreased activity of caspase-3 and of fluorescence of DNA-binding dyes, and inhibition of apoptosis were observed when the cells were treated with phorbol 12-myeristet 13-acetate combined with nucleosides.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 18–25.Original Russian Text Copyright © 2005 by Volkova, Malysheva, Nemova.     

Synthesis of a 13C-Labeled Tracer for Stream DOC: Labeling Tulip Poplar Carbon with 13CO2

Ecosystem tracer-level additions would benefit from a stable isotope-labeled source of complex organic molecules. We tested a method to label tree C with ¹³C and create a stable isotope tracer for stream dissolved organic carbon (DOC) using tulip poplar (Liriodendron tulipifera L.) seedlings. In 2000, seedlings were grown with 0.82 moles of ¹³CO₂ to assess the distribution and level of ¹³C enrichment in the tree tissues. In 2001, seedlings were grown with 25 times more ¹³CO₂ to generate tissues with a ¹³C signal strong enough for a ¹³C-DOC stream tracer addition. ¹³C enrichment in the trees varied in each year and by tissue age and type. Tissues formed during labeling (new) were more enriched in ¹³C than tissues established prior to the ¹³CO₂ injection (old). Stems were most enriched in ¹³C in both new and old tissues. A higher percentage of ¹³CO₂ was incorporated into seedlings in 2000 (59% ±1) than 2001 (43% ±0). Percent ¹³C incorporation among tree tissue types paralleled biomass distributions. Although tree C and ¹³C were equally soluble in both years, a greater percentage of tree C went into solution in 2001 (30%) than 2000 (20%). The water-soluble tree C accounted for approximately 12% of the injected ¹³CO₂ and had both humic and polysaccharide components. Results from a whole-stream ¹³C-DOC tracer addition demonstrated that tree C could be sufficiently labeled with ¹³CO₂ to create a stream DOC isotope tracer with some polymeric constituents.     

STAT6 ASODN对哮喘小鼠脾淋巴细胞影响的实验研究

目的研究STAT6反义寡核苷酸对哮喘小鼠脾淋巴细胞的影响作用。方法实验细胞分组:正常鼠空白组(A组)、正常鼠OVA组(B组)、哮喘空白组(C组)、哮喘OVA组(D组)、哮喘治疗组(E组)。正常设计并人工合成一段互补于小鼠STAT6 mRNA翻译起始区271-290的反义寡核苷酸片段,全链硫代修饰。用卵白蛋白和氢氧化铝复制哮喘模型,用淋巴细胞分离液分离脾淋巴细胞,进行体外培养并导入由阳离子脂质体转染剂Geneshuttle携带的反义寡核苷酸,观察反义寡核苷酸的转染对脾淋巴细胞STAT6蛋白表达水平及细胞培养上清中IL-4分泌水平的影响。免疫细胞化学观察脾淋巴细胞中STAT6蛋白的表达水平,同时采用酶联免疫吸附(ELISA)法测定脾细胞培养上清液中IL-4的浓度。结果D组细胞STAT6蛋白表达明显高于其余各组,均具有显著性差异(P均<0.01),STAT6 ASODN转染后,E组细胞该蛋白的表达量明显下降(P<0.01);D组脾淋巴细胞培养上清中IL-4分泌水平明显高于其余各组,均具有显著性差异(P均<0.01);STAT6 ASODN转染后,E组培养上清中IL-4分泌水平显著低于D组(P<0.01)。结论STAT6 ASODN可特异性抑制哮喘鼠脾淋巴细胞中STAT6蛋白的表达,并可特异性抑制脾淋巴细胞中IL-4的分泌,为反义基因技术治疗哮喘提供了依据。