Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45(low) c-Kit(+) cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45(low) c-Kit(-) cells that showed a granulocyte morphology; CD45(high) c-Kit(low/-) that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45(low) c-Kit(+) cells from the AGM culture had the abilities to reproduce CD45(low) c-Kit(+) cells and differentiate into CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) cells, whereas CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) did not produce CD45(low) c-Kit(+) cells. Furthermore, CD45(low) c-Kit(+) cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45(low) c-Kit(+) cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.     

Screening and identification of novel isolate Streptomyces sp., NLKPB45 from Nellore costal region for its biomedical applications

Actinobacteria, which are the prolific producers of antibiotics and significant suppliers to the pharmaceutical industry, can produce a wide variety of bioactive metabolites. An actinomycete strain designated NLKPB45 was isolated from mangrove soils samples of Nellore coastal regions Andhra Pradesh and assessed for antibiotic production and activity against pathogenic bacteria. From a total of 9 mangrove soil samples, 143 acinomycetes were isolated. Among the isolated them 6 actinomycetes strains showed potential antibacterial activity against at two tested pathogens gram positive and gram negative bacteria E. coli and S. aureus. The potent strain NLKPB45 was identified by 16S gene isolation and sequencing to the Streptomyces genus. The ethyl acetate extracts also as shown excellent antimicrobial activity against Salmonella sp., staphylococcus aureus, E. coli, and B. subtilus were detected in both the supernatant extract samples from fermentations of culture NLKPB45. The anticancer activity of extracts in the HeLa with IC₅₀ value of 37.1924 μg/ml, MCF-7 IC₅₀ value of 40.9177 μg/ml and HT 29 IC₅₀ value of 43.3758 μg/ml.     

GM2 Promotes Ciliary Neurotrophic Factor-Dependent Rescue of Immortalized Motor Neuron-Like Cell (NSC-34)

We have examined whether ciliary neurotrophic factor (CNTF) can alter serum-free cell survival of immortalized motor neuron-like cells, which were established by fusing mouse neuroblasoma N18TG2 with mouse motor neurons. One of the cell lines, NSC-34 exhibited cell survival in the presence of CNTF. NSC-34 preserves the most characteristics of motor neurons, such as the formation of neuromuscular junctions on co-cultured myotube. GM2 ganglioside is characteristic of motor neurons, and expressed highly in NSC-34. When NSC-34 was cultured with exogenous GM2 ganglioside and CNTF, GM2 facilitated the cell survival effect of CNTF. In the addition, 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity was enhanced up to 3.9-fold by culture in the presence of CNTF. GM2 might be a functional modulator of CNTF in motor neurons. It might be presented to cell surface by its enzyme activation, and become a signal of early stage, when CNTF rescues motor neurons.     

Induction of a high-CO2-inducible, periplasmic protein, H43, and its application as a high-CO2-responsive marker for study of the high-CO2-sensing mechanism in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii can acclimate to a broad range of environmental CO(2) concentrations. We observed that the cells synthesized a specific 43 kDa protein, H43, in the periplasmic space under photoautotrophic high-CO(2) conditions. Under low-CO(2) conditions, H43 disappeared. However, H43 mRNA expression was observed even under heterotrophic low-CO(2) conditions when the cells were grown with 17.4 mM acetate in darkness. When the cells were treated with 4,4'-dithiocyanatostilbene-2,2'-disulfonate (DIDS) and mersalyl to modify cell surface proteins, H43 mRNA expression was strongly affected under both heterotrophic and photoautotrophic conditions. The H43 induction pattern in a mitochondrial respiration-deficient mutant dum-1 that lacks cytochrome c oxidase was the same, but the level was much lower than that in the wild type. Even under illumination, the dissolved CO(2) concentration in the culture rapidly increased slightly following the addition of acetate and dramatically increased even further by the inhibition of photosynthesis with DCMU. Radiotracer experiments with [U-(14)C]acetate revealed that (14)CO(2) release from cells was greater in darkness than in the light due to the great stimulation of internal CO(2) evolution, resulting in an increase in external CO(2) concentration. Strong light inhibited H43 induction and DCMU promoted the induction under photoheterotrophic low-CO(2) conditions. The results demonstrate that H43 is strictly regulated by a concentration of CO(2) resulting from respiration and photosynthesis. Our results suggest that Chlamydomonas induces high-CO(2)-responsive protein H43 by sensing the concentration of ambient CO(2) with the contribution of cell surface protein.     

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations

Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal⁴⁵Ca⁺⁺ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (⁴⁵Ca⁺⁺) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [¹²⁵I]iodopindolol ([¹²⁵I]IPIN). The suspensions had a significantly lower B _max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K _D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10⁻⁷ M isoproterenol resulted in a significant increase in⁴⁵Ca⁺⁺ exchange as early as 15 s. In contrast,⁴⁵Ca⁺⁺ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated⁴⁵Ca⁺⁺ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart. This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.     

Molecular characterization of PoGT8D and PoGT43B, two secondary wall-associated glycosyltransferases in poplar

Dicot wood is mainly composed of cellulose, lignin and glucuronoxylan (GX). Although the biosynthetic genes for cellulose and lignin have been studied intensively, little is known about the genes involved in the biosynthesis of GX during wood formation. Here, we report the molecular characterization of two genes, PoGT8D and PoGT43B, which encode putative glycosyltransferases, in the hybrid poplar Populus alba x tremula. The predicted amino acid sequences of PoGT8D and PoGT43B exhibit 89 and 75% similarity to the Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9, respectively, both of which have been shown to be required for GX biosynthesis. The PoGT8D and PoGT43B genes were found to be expressed in cells undergoing secondary wall thickening, including the primary xylem, secondary xylem and phloem fibers in stems, and the secondary xylem in roots. Both PoGT8D and PoGT43B are predicted to be type II membrane proteins and shown to be targeted to Golgi. Overexpression of PoGT43B in the irx9 mutant was able to rescue the defects in plant size and secondary wall thickness and partially restore the xylose content. Taken together, our results demonstrate that PoGT8D and PoGT43B are Golgi-localized, secondary wall-associated proteins, and PoGT43B is a functional ortholog of IRX9 involved in GX biosynthesis during wood formation.     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, ₂-microglobulin (₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/₂m dimers. Association of mouse MHC class I heavy chain with ₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with ₂m are different for each allele. While searching for the PLC binding site in the ₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, ₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse ₂m. Thus, there are allelic differences in the modes of binding of ₂m to the heavy chain of MHC class I.     

抑制T细胞增殖的抗CD45单克隆抗体的建立

目的寻找能调节T细胞功能的相关分子,进行与T细胞介导的自身免疫性疾病相关的研究。方法从BALB/c小鼠骨髓中收集树突状细胞,免疫Wistar大鼠,进行细胞融合,建立杂交瘤细胞系。筛选得到很多株能调节T细胞功能的杂交瘤细胞系,对其中一株最能抑制T细胞增殖的杂交瘤细胞系进行了进一步的深入研究。结果显示其目标分子是CD45,同时增殖实验结果显示该抗体能显著抑制T细胞增殖反应。结论抗CD45单克隆抗体能有效抑制T细胞增殖,有望将本抗体用于T细胞介导的自身免疫性疾病的相关预防及治疗中。