Mobilization of human lymphoid progenitors after treatment with granulocyte colony-stimulating factor

Hemopoietic stem and progenitor cells ordinarily residing within bone marrow are released into the circulation following G-CSF administration. Such mobilization has a great clinical impact on hemopoietic stem cell transplantation. Underlying mechanisms are incompletely understood, but may involve G-CSF-induced modulation of chemokines, adhesion molecules, and proteolytic enzymes. We studied G-CSF-induced mobilization of CD34+ CD10+ CD19- Lin- and CD34+ CD10+ CD19+ Lin- cells (early B and pro-B cells, respectively). These mobilized lymphoid populations could differentiate only into B/NK cells or B cells equivalent to their marrow counterparts. Mobilized lymphoid progenitors expressed lymphoid- but not myeloid-related genes including the G-CSF receptor gene, and displayed the same pattern of Ig rearrangement status as their bone marrow counterparts. Decreased expression of VLA-4 and CXCR-4 on mobilized lymphoid progenitors as well as multipotent and myeloid progenitors indicated lineage-independent involvement of these molecules in G-CSF-induced mobilization. The results suggest that by acting through multiple trans-acting signals, G-CSF can mobilize not only myeloid-committed populations but a variety of resident marrow cell populations including lymphoid progenitors.     

A morphological study of the Petunia integrifolia complex (Solanaceae)

BACKGROUND AND AIMS: Petunia inflata has been treated taxonomically in various ways: it has been described as an independent species, treated as a synonym of P. integrifolia, and also regarded as a subspecies of P. integrifolia. The present study was designed to resolve the ambiguity involving the P. integrifolia complex (P. integrifolia plus P. inflata). METHODS: Tentative identification (either integrifolia group or inflata group) was carried out in the field based on the observation of live specimens at the restricted type localities. The accuracy of the tentative identification was later tested with principal component and cluster analyses of data obtained by measuring 21 morphological characters on cultivated live specimens sourced from 113 natural populations of the P. integrifolia complex in Argentina, Brazil, Paraguay and Uruguay. KEY RESULTS: There was a clear, statistically significant gap between the morphological measurements of the two groups, ensuring the accuracy of identification carried out in the field except for a probable hybrid swarm. Previously, the condition of the pedicel in the fruiting state was considered an important character distinguishing between these two groups; however, the condition of the pedicel was rather variable in the integrifolia group. The two groups were found to have geographically distinct distributions: the integrifolia group occurred in southern regions, whereas the inflata group occurred in northern regions. CONCLUSIONS: Based on the available evidence, it is suggested that the two groups are allopatric species, P. integrifolia and P. inflata, in agreement with the opinion of Fries (1911).     

Alteration of renal adrenomedullin and its receptor system in the severely hypertensive rat: effect of diuretic

OBJECTIVE: We investigated the pathophysiological role of the renal adrenomedullin (AM) system, including the ligand, receptor, and amidating activity, in severe hypertensive rats. METHOD: We studied three groups: control Wistar Kyoto rats (WKY), spontaneously hypertensive stroke-prone rats (SHR-SP), and diuretic-treated SHR-SP. We measured AM-mature, active form, and AM-total (active form+inactive form) in plasma and renal tissues, and mRNA levels of AM and AM receptor system components such as calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein (RAMP) 2, and RAMP3 in renal tissues. RESULTS: SHR-SP had higher blood pressure, plasma neurohumoral factors, and lower renal function than WKY. SHR-SP had higher AM-mature and AM-total levels in plasma and renal tissues than WKY. Although the plasma AM-mature/AM-total ratio was similar in the two groups, AM-mature/AM-total ratio in renal tissues was higher in SHR-SP than in WKY. In addition, mRNA levels of AM in the renal cortex and medulla and the mRNA levels of CRLR, RAMP2, and RAMP3 in the renal cortex were higher in SHR-SP than in WKY. Chronic diuretic treatment decreased blood pressure and improved kidney function and neurohumoral factors, with reductions in plasma and renal AM system. CONCLUSION: Upregulation of circulating and renal AM system may modulate pathophysiology in SHR-SP.     

Effect of peroxisome proliferator-activated receptor-gamma ligands on the expression of retinoic acid-inducible gene-I in endothelial cells stimulated with lipopolysaccharide

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box protein family and designated as a putative RNA helicase. RIG-I is implicated in host defense and inflammatory reactions by regulating the expression of various genes. RIG-I is expressed in endothelial cells and upregulated with lipopolysaccharide (LPS). Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear hormone receptor and regulates gene expressions in response to its specific ligands. In the present study, we examined the effect of PPAR-gamma ligands on the LPS-induced RIG-I expression in cultured human umbilical vein endothelial cells (HUVEC). 15-Deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), a metabolite of PGD2, is a natural ligand for PPAR-gamma and known to modulate inflammatory reactions by regulating the expression of various genes in PPAR-gamma-dependent and -independent manners. LPS-induced RIG-I expression in HUVEC was inhibited by pretreatment of the cells with 15d-PGJ2 in time-and concentration-dependent manners. However, ciglitazone and bisphenol A diglycide ether, authentic and specific ligands for PPAR-gamma, did not affect the RIG-I expression. These results suggest that 15d-PGJ2 inhibits LPS-induced RIG-I expression through a mechanism independent on PPAR-gamma. 15d-PGJ2 may regulate inflammatory reactions, at least in part, by inhibiting the expression of RIG-I.     

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.     

Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses

Arabidopsis thaliana plants with mutations in the genes encoding eukaryotic initiation factor (eIF4E) or isoform of eIF4E (eIF(iso)4E) were tested for susceptibility to Clover yellow vein virus (ClYVV), a member of the genus Potyvirus. ClYVV accumulated in both inoculated and upper uninoculated leaves of mutant plants lacking eIF(iso)4E, but not in mutant plants lacking eIF4E. In contrast, Turnip mosaic virus (TuMV), another member of the genus Potyvirus, multiplied in mutant plants lacking eIF4E but not in mutant plants lacking eIF(iso)4E. These results suggest the selective involvement of members of the eIF4E family in infection by potyviruses.     

Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.     

Analysis of the function of a hyperthermophilic endoglucanase from Pyrococcus horikoshii that hydrolyzes crystalline cellulose

A hyperthermophilic -1,4 endoglucanase was identified in Pyrococcus horikoshii, a hyperthermophilic archaeon. In order to clarify the function of the protein in detail, structural and catalytic site studies were performed using protein engineering. By removing some of the C-terminal sequence of the ORF of the endoglucanase (PH1171), two types of recombinant proteins were expressed from one ORF, using Escherichia coli. One exhibited endoglucanase activity, and the other did not. An SD-like sequence was identified in the ORF of the endoglucanase. By removing the SD-like sequence without changing the amino acid sequence of the endoglucanase, one recombinant endoglucanase was prepared effectively from E. coli. From the analysis of the N- and C-terminal regions of the ORF, this endoglucanase appears to be a secreted and membrane-binding enzyme of P. horikoshii. A mutation analysis of the endoglucanase, using the synthetic substrate, indicated that Glu342 is a candidate for the active center and plays a critical role in the activity of the enzyme. Additional catalytic amino acid residues were not found. These results indicate that the catalytic residue of the enzyme is different from that of typical family 5 endoglucanase, even though it has a high homology to the endoglucanase from Acidothermus celluloliticus. The activity of the enzyme, using carboxy methylcellulose and crystalline cellulose as the substrates, was increased, but not for a synthetic low-molecular substrate when a carbohydrate-binding module of chitinase from P. furiosus was added to the C-terminal region.     

Factors affecting production of transgenic rats by ICSI-mediated DNA transfer: effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA

Factors affecting the efficiency of producing transgenic rats by intracytoplasmic sperm injection (ICSI)-mediated DNA transfer were investigated. Epididymal spermatozoa from Sprague-Dawley (SD) rats were sonicated and/or frozen-thawed for cutting the tail and membrane disruption. The sperm heads were exposed for 1 min to different concentrations (0.02-2.5 microg/ml) of 3.0 kb enhanced green fluorescent protein (EGFP) DNA solution, and then microinjected into the denuded F1 hybrid (Donryu x LEW) rat oocytes. The optimal concentration of EGFP DNA solution was 0.1 microg/ml, as determined by the in vitro developmental competence into morulae/blastocysts of the ICSI oocytes and the EGFP expression of the resultant embryos. The efficiency of producing transgenic rat offspring (per transferred zygote) was 2.8%, 1.6%, and 3.3% in the oocytes injected with sonicated, frozen-thawed, and sonicated + frozen-thawed sperm heads, respectively. The founder transgenic rats carrying the EGFP gene transmitted their transgenes to their progeny according to the Mendelian fashion, suggesting the stable incorporation of the transgenes into the rat genomes. Four rat strains (F344, LEW, Donryu, and SD) were compared for their suitability as sperm/oocyte donors for the production of transgenic rats by ICSI with sonicated, frozen-thawed and solution of EGFP DNA-exposed sperm heads. The efficiency of producing transgenic rats in the SD strain (8.2%) was higher than that in the LEW strain (0.9%), while those in the F344 and Donryu strains (4.3%-4.4%) were intermediate. One plasmid DNA (Fyn, 5.0 kb) and two BAC DNA (BAC/Fyn, 208 kb; Svet1/IRES-Cre, 186 kb) were successfully introduced into the SD rat genomes via ICSI, with the producing efficiencies of 2.8%, 0.9%, and 2.4%, respectively.