A novel phosphoprotein is induced during bone marrow commitment to dendritic cells

Dendritic cells (DCs) play an important role in vertebrate immunity, but little is known of the molecular events associated with their development from bone marrow (BM). This report describes induction of a signature protein marking BM commitment to DCs. Using a standard procedure, DCs were generated from BM by cultivation in vitro. Appropriate phenotypic monitoring was done primarily by immunofluorescence, and polyclonal antibody reagents were developed against immature DC lysates. Using one specific antibody reagent, we identified, purified, and sequenced a unique cytosolic phosphoprotein DP58 that occurs within 30 min during BM commitment to DCs. Its sequence matches with a computationally predicted Riken cDNA (GenBank Accession No. XP_138799), and a specific anti-DP58 peptide antibody was developed for further characterization. The study suggests that DP58 induction signals distinct pathway(s) leading to early DC progenitors that may be generated and propagated for a short period in vitro.     

Elevation of anα(1,3) fucosyltransferase activity correlated with apoptosis in the human colon adenocarcinoma cell line,HT-29

We studied changes in the carbohydrate expression following apoptotic cell death induced by treatment with interferon (IFN)- and anti-Fas antibody using human colon adenocarcinoma HT-29 cells. An apoptotic cell death of HT-29 accompanied with typical DNA fragmentation was observed when the cells were cultured sequentially with IFN- and anti-Fas antibody. In flow cytometric analyses, the expression of Le^x and Le^y antigen was strongly and slightly enhanced, respectively, on the cell surface in accordance with the apoptosis. When the fucosyltransferase (Fuc-T) activities of the lysates from the treated cells were examined relative to those from untreated cells, a 2.5-fold increase of (1,3)-Fuc-T activities and a slight increase of (1,2)-Fuc-T activities were observed, but little or no increase of (1,4)-Fuc-T activity was detected. In Northern blot analyses using probes for Fuc-T III, IV, V, VI and VII genes, strong RNA messages for Fuc-T III, V and/or VI and a weak RNA message for Fuc-T IV were detected in the untreated HT-29 cells. On the other hand, in the treated cells, the messages for Fuc-T III, V and/or VI were found to almost disappear and the 2.3 kb message for Fuc-T IV was observed to elevate 2.8-fold. Therefore, we suggest that the strongly increased expression of Le^x antigen found on the HT-29 cell surface might be involved in the process of apoptosis, and that the enhancement of the antigen expression seems to result from the increased activity of (1,3)-Fuc-T encoded mainly by the Fuc-T IV gene.Abbreviations Bn Benzyl - EDTA ethylenediaminetetraacetic acid (sodium salt) - SDS sodium dodecyl sulfate - D-PBS Dulbecco's phosphate buffered saline (metal free) - FITC fluorescein isothiocyanate - AIDS acquired immune deficiency syndrome - FACS fluorescence-activated cell sorter - IFN interferon - dNTP deoxynucleosides-triphosphate - PCR polymerase chain reaction - kb kilobase(s) - bp base pair(s)     

Long-Term Expression of Fos-Related Antigen and Transient Expression of ΔFosB Associated with Seizures in the Rat Hippocampus and Striatum

Abstract: Systemic administration of kainic acid (KA), an analogue of glutamic acid, causes limbic seizures and pathophysiological changes in adult rats that are very similar to human temporal lobe epilepsy. One of the earliest changes in gene expression after treatment with KA is the induction of immediate-early genes. The fos and jun families are frequently studied immediate-early genes that are induced by KA. Several groups, including ours, have recently reported that a 35-kDa Fos-related antigen (FRA) is induced for a protracted time by various stimuli. It has been suggested that this FRA is ΔFosB, which has a molecular mass of ∼35 kDa. The present study characterizes the long-term expression of FRA and ΔFosB after systemic treatment with KA. Immunocytochemistry and western blot analysis using an antibody that cross-reacts with all known FRAs showed that a 35-kDa FRA was induced at high levels in both the hippocampus and striatum for up to 1 month by KA. A semi-quantitative PCR analysis showed that ΔFosB was induced by KA, but its expression lasted for only 6 h. This result was also verified by northern blot analysis. These results suggested that the 35-kDa FRA with long-term elevated levels seen with western blot analysis and immunocytochemistry is a new species of the FRA and not ΔFosB. The long-term expression of FRA in both the hippocampus and striatum may be associated with the pathophysiological changes after KA administration.     

NITROGEN LIMITATION IN ISOCHRYSIS GALBANA (HAPTOPHYCEAE). II. RELATIVE ABUNDANCE OF CHLOROPLAST PROTEINS1

Using spectroscopic, biophysical and immunological techniques, we assayed the relative abundance often chloroplast proteins and protein complexes in the marine haptophyte, Isochrysis galbana Green, grown at nine steady-state dilution rates in nitrogen-limited chemostats. The proteins included Photosystem I reaction center (RCI) chlorophyll protein, CP1; Photosystem II reaction center (RC II) protein, D1; two chlorophyll a-binding apoproteins, CP 43 and CP 47; 33 KDa oxygen evolving protein, OEC 33; α subunit of coupling factor, CF1α; large (LSU) and small subunits (SSU) of ribulose 1,5-bisphosphate carboxylase, RuBisCO; the chlorophyll a/c/fucoxanthin protein complex, LHCP; and cytochrome b₆/f. Seven of the ten protein complexes are encoded in the chloroplast, two are encoded in the nucleus and one shares chloroplast and nuclear genomes. Over the range of dilution rates (0.96-0.18 d^?1) cell N decreased 42% and cellular chlorophyll a decreased 50%; however, the stoichiometric proportion of RC II: cytochrome b₆/f: RC I remained constant, averaging 1:3.3:0.8. In contrast, RuBisCO / PS II decreased by 58%. The light harvesting chlorophyll a/c/fucoxanthin protein complex increased relative to RC II; however, as cells became more nitrogen limited the fraction of total cell nitrogen contained in RuBisCO decreased from 21.3 to 6.7%, whereas that of the light harvesting complex remained relatively constant, averaging 6.8%. Our results generally support the hypothesis that in nitrogen limited cells, proteins encoded in the nuclear genome are synthesized preferentially over those encoded in the chloroplast.     

花粉高尔基囊泡类动蛋白的鉴定

在萌发的烟草花粉管顶端,有囊泡状的颗粒被牛脑动蛋白重链的单克隆抗体所识别。用蔗糖密度梯度离心法从榛木花粉中分离得到高尔基囊泡,体外免疫胶体金处理后可被标记。ＳＤＳ－聚丙烯酰胺凝胶电泳和免疫印迹表明,分子量为１００ｋＤ的多肽大量存在于高尔基囊泡,此多肽可与动蛋白单克隆抗体进行特异性反应,证明花粉高尔基囊泡上有关动蛋白,其重链分子量为１００ｋＤ。     

Immunoreactivity of the corpuscles of Stannius of the garpike,Lepisosteus osseus,to antisera against salmon and trout stanniocalcins

Stanniocalcin-immunoreactive cells were localized in the corpuscles of Stannius of a holostean fish, the garpike (Lepisosteus osseus), using antisera against salmon and trout stanniocalcins and the peroxidase-antiperoxidase and protein A-gold immunohistochemical methods. The stanniocalcin-immunoreactive cells were periodic acid-Schiff-positive, and antibody staining was abolished if the antiserum was preabsorbed with corpuscle homogenate. Immunocytochemistry revealed two reactive cell types in the glandular parenchyma, and immunoreactivity was confined to the secretory granules. Staining of the granules was also abolished when the antisera were blocked with crude corpuscle homogenate. When corpuscle extracts from garpike were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis, a single dense band was evident with a molecular weight of 68 kDa under non-reducing conditions, whereas three bands were observed (29, 31, and 34 kDa) under reducing conditions. Staining of all bands disappeared following preabsorption of the antiserum with salmon stanniocalcin, trout stanniocalcin, or garpike corpuscle extract. The results are compared with stanniocalcins from another extant holostean, the bowfin (Amia calva), and from more modern bony fishes, the teleosts.     

生物素标记贾第虫全基因组DNA探针的制备及其特异性敏感性测定

用生物素标记的贾第虫全基因组ＤＮＡ探针,在斑点杂交试验中显示高度的敏感性和特异性。用它可检出１０ｎｇ贾第虫ＤＡ,１０＾３个贾第虫滋养体或包囊,且不与阴道毛滴虫、溶组织内阿米巴、弓形虫和ＢＡＢＬ／ｃ小鼠肝细胞ＤＮＡ,以及贾第虫患者粪便上清液发生交叉反应。本探针可用于贾第虫病病原体检测和虫株鉴定研究。     

羊输卵管液中发情相关糖蛋白的性质研究

哺乳动物输卵管液为受精和早期受精卵的发育提供了一个理想的生理生化环境。人们对输卵管液的成分已进行了初步研究,其中发情相关糖蛋白（ＥＧＰ）是被研究的成分之一。ＥＧＰ仅存在在排卵核受精前后的输卵管液中。当受精卵进入子宫区,输卵管液中的ＥＧＰ就消失了。显然,ＥＧＰ是与哺乳动物的早期发育过程密切相关的。尽管人们对ＥＧＰ的确切生理功能尚不清楚,但作为第一步,首先弄清ＥＧＰ的理化性质是十分重要的。本文目的在于研究羊输卵管液中ＥＧＰ的理化性质。本文采用单向（１Ｄ）、双向（２Ｄ）ＳＤＳ－聚丙烯胺凝胶电泳（ＰＡＧＥ）和等电聚胶电泳（ＩＥＦ）,结合Ｗｃｓｔｅｒｎｂｌｏｔ技术对ＥＧＰ的性质进行了研究。由于单克隆抗体及花生凝集素（ＰｅａｎｕｔＡｇｇｌｕｔｉｎｉｎ）和ＥＧＰ相结合的专一性很高,所以不必事先对ＥＧＰ进行纯化就可直接进行分析,我们的结果证明羊ＥＧＰ包括两种蛋白质,一种是酸性蛋白,它的等电点为数４．５;另一种是碱性蛋白,其等电点是８．０;这两种蛋白亚基的分子量相同,都是１０ＫＤ。从不同物种间ＥＧＰ这些理化数据上比较,羊和狒狒的非常接近,但和其他动物的有很多不同。我们认为来自不同动物的ＥＧＰ可能是一类性质相同的蛋白质,具有相