Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh-123). The sorted cells were then assayed for their in vivo spleen colony-forming ability (day -8, -12, and -16 CFU-S) and their ability to repopulate the bone marrow or spleen over a 13-day period with CFU-S-12, CFU-GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU-S-12 or CFU-GM, had low affinity for Rh-123. These cells showed minimal spleen colony-forming ability, and the ratio of MRA to CFU-S-12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU-S-16, CFU-S-12, and CFU-S-8 retained increasing amounts of Rh-123, respectively, and CFU-S-8 were almost exclusively found among cells with high Rh-123 affinity. These cells also included about half of all day-12 CFU-S, and the ratio of MRA to day-12 CFU-S was 0. The results show that MRA cells, SRA cells, CFU-S-16, CFU-S-12, and CFU-S-8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre-CFU-S.     

The radiosensitivity of hematopoietic stem cells from mice forming splenic colonies after 8 and 12 days following bone marrow cell transplantation (CFU-S-8 and CFU-S-12)]

The method of "macro-" and "microcolonies" was used to study the radiosensitivity of CFU-S that form "early" (8 days) and "late" (12 days) splenic colonies after transplantation of syngeneic bone marrow to fatally exposed mice: no significant differences were found. Median lethal doses (D0) for CFU-S-8 and CFU-S-12 were 1.03 and 1.13 Gy for "microtest" and 0.99 and 1.16 Gy for "microtest" respectively.     

Clonogenic hemopoietic cells (CFU-S) in mouse pre- and postnatal ontogeny

Content of three classes of clonogenic haemopoietic cells (CFU-S-7, CFU-S-11 and CFU-S-ep) was determined in haemopoietic organs of mouse during embryogenesis (10, 14 and 18 day) and postnatal ontogenesis (2, 3 and 7 day, 1, 2, 3 and 18 month). CFU-S-7 and CFU-S-11 that from big splenic colonies on 7th and 11th days of transplantation are present in liver, spleen and bone marrow at all developmental stages. However their concentration and CFU-S-7 CFU-S-11 ratio change in haemopoietic organs. CFU-S-ep that form small colonies on 11th day are observed before birth in liver and spleen and 1 week after birth there and also in bone marrow but are practically absent from haemopoietic organs of older animals. Thus, CFU-S compartment structure is characterized by definite ratio of its subpopulations. It seems to reflect functional state of haemopoietic system during development.     

The influence of some retarding agents NOS of dihydrothiazine-thiazoline rank on postradiational of recovery endogenous CFU-S-8 of mice

In this work the attempt to estimate a nitric oxide (NO*) role in regulation of the number of pool haemopoietic stem cells at the irradiated mice was made. With this purpose the number of new compounds from dihydrothiazine-thiazoline line was synthesized, their NO-inhibiting activity was investigated in vivo by the method of ESR-spectroscopy of spin trap and their influence on an output endogenous spleen colonies (CFU-S-8) after the total sublethal y-irradiation of mice in a doze of 6 Gy was also investigated. Was shown, that the tested compounds reduced the contents of NO* in a liver tissue of mice which have received an injection of nitric oxide synthesis inductor - lipopolysaccharide, and also increased an output CFU-S-8 forming endogenous colonies in the spleen of the irradiated mice. Received data testify to perceptivity of search radioprotective agents among NO* synthesis inhibitors.     

Distinct thresholds for CD8 T cell activation lead to functional heterogeneity: CD8 T cell priming can occur independently of cell division

To examine the bases for CD8 T cell functional heterogeneity, we analyzed responses to partial vs full agonist Ag. An extended period of interaction with APCs was required to set the threshold required for cell division in response to partial as compared with full agonist Ag. Acquisition of cytolytic function was restricted to the divided T cell population. In contrast, the threshold for commitment to produce IFN-gamma and express some activation markers appeared lower and independent of cell division. Indeed, we characterized a T cell population stimulated in response to the partial agonist that was committed to produce IFN-gamma, but failed to divide or secrete IL-2. Importantly, this activated nondivided population behaved as "primed" rather than "anergized," indicating 1) that priming of CD8 T cells may be induced by suboptimal stimulation independent of cell division and 2) that encounter with Ag does not always induce a complete differentiation program in naive CD8 T cells, as previously reported.     

Two stages of the differentiation of HL-60 cells induced by 1α,25 dihydroxyvitamin D3; Commitment by 1α,25 dihydroxyvitamin D3 and promotion by DMSO

The differentiation of HL-60 cells induced by 1,25 dihydroxyvitamin D₃ was found to be separated into two stages, i.e. commitment and promotion. Most of the HL-60 cells were committed to monocyte/macrophage lineage by pretreatment with 1,25 dihydroxyvitamin D₃ (5–50 ng/ml) for 18–24 hr. The promotion in the second stage was inducer and lineage independent; treatment with 1.25% DMSO for 2 or 3 days promoted the differentiation of the committed HL-60 cells by 1,25 dihydroxyvitamin D₃ into monocyte/macrophage lineage, but not granulocyte lineage.Abbreviations used NEA nonspecific esterase activity - NBT nitroblue tetrazolium - DMSO dimethylsulfoxide - RA retinoic acid - TPA 12-O-tetradecanoylphorbol-13-acetate     

Effect of Continuous γ-Irradiation at Low Doses on Clonogenic Hemopoietic (CFU-S) and Stromal (CFU-F) Cells of Bone Marrow

We studied the effects of low doses of continuous -irradiation (Co⁶⁰, 10 days, mean daily dose power 1.5-2.0 mGy, total dose 15 mGy) on hemopoietic and stromal progenitor cells of murine bone marrow. The content of hemopoietic clonogenic cells representing a younger (CFU-S-11) and more mature (CFU-S-7) categories in the compartment of stem cells was determined in the bone marrow. The state of bone marrow stroma was estimated by the method of in vitro cloning according to the number of progenitor cells that form colonies of fibroblasts (CFU-F) and by the method of ectopic transplantation according to the capacity of stroma of organizing and building new hemopoietic territories. Continuous -irradiation at low doses, that were by one order of magnitude lower than those inducing hermesis, exerted a stimulating effect on both hemopoietic (CFU-S) and stromal (CFU-F) progenitor cells. The number of CFU-S in the compartment of stem cells of the bone marrow markedly increased and they formed larger hemopoietic territories but these cells appeared to create a qualitatively different microenvironment, which stimulated the proliferation of CFU-S.     

Postradiation recovery of hematopoiesis in mice affected by Neogen (IEW)

The effect of the synthetic peptide IEW (Neogen) with immunomodulating properties on postradiation recovery of haemopoiesis was investigated. We have shown that Neogen is a potential stimulator of haemopoiesis. The administration of Neogen after irradiation shortened duration of period of the recovery of the compartment of CFU-S-8 and the amount of bone marrow cells. The comparision of the effects of Neogen and GM-CSF (Leucomax) and G-CSF (Granocyte 34) have shown that the targets for these agents are probably different: polypotent CFU-S-for Neogen, and CFU-GM-for GM-CFS. Based on the results, we suggested the mechanism of Neogen effects on heamopoiesis.     

Distorted Segregations of RFLP Markers and Their Distribution on Chromosomes in an indica/japonica F2 Population of Rice (Oryza sativa L.)

he segregation ratio of RFLP markers in an F2 population from indica "Zhaiyeqing 8” and japonica "Jingxi 17' of rice (Oryza sativa L., 2n= 24) was studied using 54 RFLP markers distributed on 12 chromosomes. Distorted segregation was found in 25.9% of the marker tested, which was indicated by significant deviation from the expected Mendelian segregation ratio ( I: 2: 1) at 5% or 1% level. Among the three RFLP genotypes of the F2 population “Zhaiyeqing” 8 genotype was significantly more than the expected, and its gene frequency was up to 52.1 %. Three positions for distorted segregation were found on chromosome 3 (RG227-RG369), 7 (RG678-RG511-RG528) and 12 (RG463-RG323). These positions could be related to gametophyte loci responsible for the distortion.     

QTL mapping and epistatic interaction analysis of field resistance to sudden death syndrome (<Emphasis Type="Italic">Fusarium virguliforme</Emphasis>) in soybean

Key message

Two interactive quantitative trait loci (QTLs) controlled the field resistance to sudden death syndrome (SDS) in soybean. The interaction between them was confirmed.

Abstract

Sudden death syndrome (SDS), caused by Fusarium virguliforme, is a major disease of soybean [Glycine max (L.) Merr.] in the United States. Breeding for soybean resistance to SDS is the most cost-effective method to manage the disease. The objective of this study was to identify and characterize quantitative trait loci (QTLs) underlying field resistance to SDS in a recombinant inbred line population from the cross GD2422?×?LD01-5907. This population was genotyped with 1786 polymorphic single nucleotide polymorphisms (SNPs) using SoySNP6 K iSelect BeadChip and evaluated for SDS resistance in a naturally infested field. Four SDS resistance QTLs were mapped on Chromosomes 4, 8, 12 and 18. The resistant parent, LD01-5907, contributed the resistance alleles for the QTLs on Chromosomes 8 and 18 (qSDS-8 and qSDS-18), while the other parent, GD2422, provided the resistance alleles for the QTLs on Chromosomes 4 and 12 (qSDS-4 and qSDS-12). The minor QTL on Chromosome 12 (qSDS-12) is novel. The QTL on Chromosomes 8 and 18 (qSDS-8 and qSDS-18) overlapped with two soybean cyst nematode resistance-related loci, Rhg4 and Rhg1, respectively. A significant interaction between qSDS-8 and qSDS-18 was detected by disease incidence. Individual effects together with the interaction effect explained around 70% of the phenotypic variance. The epistatic interaction of qSDS-8 and qSDS-18 was confirmed by the field performance across multiple years. Furthermore, the resistance alleles at qSDS-8 and qSDS-18 were demonstrated to be recessive. The SNP markers linked to these QTLs will be useful for marker-assisted breeding to enhance the SDS resistance.

     

Tracing the progeny of the aortic hemangioblast in the avian embryo

A population of hematopoietic progenitors becomes committed within the embryo proper in the floor of the aorta (P-Sp/AGM in the mouse). In birds, this first aspect of intraembryonic hematopoiesis is prominent during embryonic day 3 (E3) as endothelium-associated "intra-aortic clusters." Between E6 and E8, diffuse hematopoiesis then occurs as "para-aortic foci" located in the dorsal mesentery ventral to the aorta. These foci are not associated with endothelium. Whether these two hematopoietic cell populations arise from distinct or common progenitors is not known. We could recently trace back the origin of intra-aortic clusters in the avian embryo by labeling aortic endothelial cells (EC) in vivo with acetylated low-density lipoproteins. This approach established the derivation of early intraembryonic hemopoietic cells from the endothelium, but did not indicate how long during ontogeny such a relationship may exist, since the progeny of EC labeled at E2 could be traced for 1-2 days at most. Here we report that, when E2 aortic ECs were infected prior to the formation of intra-aortic clusters with a nonreplicative LacZ-bearing retroviral vector, numerous cells were labeled in the para-aortic foci at E6. In contrast, when the retroviral vector was inoculated at E4 rather than E2, that is, after the disappearance of intra-aortic clusters, no cells in the para-aortic foci were labeled. Taken together, our results demonstrate that ECs from the aortic floor seed the two aspects of aorta-associated hemopoiesis and that these ECs with hemangioblastic potential are present only transiently in the aorta.     

The problem of the radiosensitivity of bone marrow cellular elements and assessments of the postradiation kinetics of myelopoiesis (an analysis based on data on the sequelae of the accident at the Chernobyl Atomic Electric Power Station]

On the basis of the data obtained from 61 cases of accidental exposure (0.1-12.5 Gy) at the Chernobyl A.P.S. the kinetics of acute radiation bone-marrow syndrome was analyzed and radiosensitivity of the entire spectrum of human granulocytic compartment cells was estimated. The radiosensitivity estimates were made by a "functional" criterion, developed by the authors, which was based on the comparative ability of irradiated and nonirradiated bone marrow cells of different maturity to produce peripheral blood neutrophils. Changes were found in physiology of myeloid cells during their maturation: the maturation mechanism, for cells of the committed pool, was "attached" to the division process, whereas these processes were independent for cells of the dividing and maturing pool. It is once again confirmed that the transit time of a maturing myeloid cell, to begin with the primarily committed one and to end with a peripheral blood neutrophil, is not constant and lasts normally for 32 days.     

Expression of CD7 on normal human myeloid progenitors.

Existence of biphenotypic leukemias co-expressing CD7 and CD34 has prompted the question of whether a similar population of cells is present in normal human bone marrow. As CD7 is considered to be a T cell-restricted Ag, the co-expression of CD7 with the "human stem cell Ag" CD34 may identify a bipotent stage within hemopoietic differentiation. Cells with this phenotype have previously been isolated from human thymus. In this report we provide evidence that human marrow mononuclear cells also contain a minor subpopulation of cells co-expressing CD7 and CD34. The CD7+/CD34+ cells were found to contain committed myeloid progenitors assayed both as CFU in semi-solid media and by their ability to produce granulocytes in long term marrow cultures. Expression of CD7 on myeloid committed progenitors was further confirmed in a C-mediated cytotoxic assay. We conclude that CD7 expression is not restricted to T cells but is also expressed during early stages of myeloid differentiation.     

利用回交法与Wx基因分子标记辅助选择培育糯性小麦

以中国春糯性位点全套近等基因系为研究材料,对小麦Wx基因的6个STS标记和1个CAPS标记进行了筛选。改良PCR扩增条件以及产物检测方式后,从这些标记中筛选出3个标记,包括鉴定Wx-A1、Wx-D1位点的2个共显性STS标记和Wx-B1位点的1个显性STS标记,用于本研究中糯性小麦的分子标记辅助育种。在育种过程中,首先配制全糯材料“98Y1441”与推广品种“川育12”的杂交组合,采用籽粒碘染法从其F2种子中选择全糯基因型个体与回交亲本川育12杂交,如此反复自交、回交,历经数代异地加代繁殖得到BC5F2代回交改良群体。利用上述3个分子标记从该群体中筛选出了8种Wx基因型,经卡方检验,其分离比符合3对基因的分离比例,其中基因型为aabbdd的植株有2株,直链淀粉含量分别为1.81%和0.82%,为全糯小麦;基因型为AAbbdd, aabbDD的部分糯性植株各有1株,直链淀粉含量分别为15.24%和17.57%。研究中获得的BC5 F2代群体的农艺性状接近回交亲本,并明显优于全糯材料“98Y1441”,表明采用回交法与Wx基因分子标记辅助选择相结合,有助于培育高产、优质的全糯和部分糯小麦。     

MtDNA and Y-Chromosome Lineages in the Yakut Population

The structure of female (mtDNA) and male (Y-chromosome haplotypes) lineages in the Yakut population was examined. To determine mtDNA haplotypes, sequencing of hypervariable segment I and typing of haplotype-specific point substitutions in the other parts of the mtDNA molecule were performed. Y haplogroups were identified through typing of biallelic polymorphisms in the nonrecombining part of the chromosome. Haplotypes within haplogroups were analyzed with seven microsatellite loci. Mitochondrial gene pool of Yakuts is mainly represented by the lineages of eastern Eurasian origin (haplogroups A, B, C, D, G, and F). In Yakuts haplogroups C and D showing the total frequency of almost 80% and consisting of 12 and 10 different haplopypes, respectively, were the most frequent and diverse. The total part of the lineages of western Eurasian origin (Caucasoid) was about 6% (4 haplotypes, haplogroups H, J, and U). Most of Y chromosomes in the Yakut population (87%) belonged to haplogroup N3 (HG16), delineated by the T–C substitution at the Tat locus. Chromosomes of haplogroup N3 displayed the presence of 19 microsatellite haplotypes, the most frequent of which encompassed 54% chromosomes of this haplogroup. Median network of haplogroup N3 in Yakuts demonstrated distinct starlike phylogeny. Male lineages of Yakuts were shown to be closest to those of Eastern Evenks.     

水稻籼粳杂种生殖障碍的基因定位分析

籼稻(Oryza sativa L.ssp.indica)与粳稻(O.sativa ssp.japonica)杂交优势明显但存在生殖隔离。生殖障碍主要表现为胚囊败育、花粉败育、开花时花药不开裂和雌雄异熟。应用具有137个标记位点的籼、粳杂交(“窄叶青8号”/“京系17”)F_1花药培养获得的127个双单倍体(DH)群体构建的RFLP图谱,对控制籼、粳杂种小穗败育的基因座位进行了定位研究。结果在第1、3、4、5、6、7、8、12染色体上检测到10个基因座位,其中第3、12染色体上的2个不育基因位点stj-3和stj-12与同一杂交组合F_2分离群体中发现的异常分离热点处于相同的染色体区段。Ssj-6的基因加性效应为负值,有增加籼、粳亲和性的作用;其余的不育基因座位皆有增加籼、粳杂种不育性的作用。     

On the distribution of allele frequencies in a diffusion model

An expression is derived and values tabulated for the expected allele frequencies and their variances, arranged in decreasing order in a population, from the finite and infinite alleles diffusion model in Watterson (1976). The neutral model and also a model with heterozygote selection are considered. Some observed ABO blood group allele frequencies are compared with the tabulated expected frequencies in the neutral three allele model. This extends the results of Watterson and Guess (1977) who tabulate the expected value of the most common allele. One test of neutrality previously advocated is to consider the distribution of F, the population homozygosity, conditional on G, the product of allele frequencies. However it is shown here that for a large number of alleles, F and G are asymptotically independent, the test would not be a good one in this case. A limit theorem is derived for the distribution of allele frequencies in the neutral model when the mutation rate is large. In this case F is shown to be asymptotically normal. An inequality is derived for the probability that the oldest allele in a population is amongst the r most frequent types. An inequality is also found for the probability that a sample will only contain representatives of the r most frequent allele types in the population.     

Differential production of IFN-gamma, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIV(+) subjects

BACKGROUND: Interferon gamma is a cytokine that plays a central role in immunity, and is physiologically secreted by T and NK cells under appropriate stimuli during the immune response. By means of flow cytometry, we performed a single cell analysis of interferon gamma producing NK cells and their surface phenotype in normal and HIV(+) individuals that show several defects of cytokine production and cellular immunity. METHODS: PBMC or purified NK cells were stimulated for 1-12 h with PMA/ionomycin in the presence of monensin, subsequently stained for surface CD56 and CD3 or CD8, and for intracytoplasmic IFN-gamma, and analysed by flow cytometry. RESULTS: Our results show that CD56(+) NK cells are more efficient interferon gamma producers than T cells. Moreover, within the CD56(+) NK cell population, those that co-express low density CD8 are the best producers. Finally, we show that NK cells during HIV infection are more massively recruited to interferon gamma production than those from normal subjects. CONCLUSIONS: Both in the normal and HIV(+) subjects, a higher percentage of NK cells than T cells can produce IFN-gamma although differences can be identified within the NK cells subset in terms of IFN-gamma production. The production of IFN-gamma is fully achievable in the HIV(+) subjects, which is consistent with their elevated plasmatic levels of the cytokine. The possibility that NK cells that produce interferon gamma could represent a functionally distinct population committed to the production of this cytokine, is discussed.     

Maternal Aggression and Litter Size in the Female House Mouse

It has been commonly argued that, in house mice, female post-partum fighting against a male intruder functions to protect the offspring from infanticide. The aim of this study was to test the hypothesis that maternal aggression is actually related to pup defence and, specifically, according to parental investment theory, that its intensity should increase with litter size. 60 nulliparous albino female mice were mated and randomly assigned to four experimental groups in which litters were culled at birth to 0, 4, 8, or 12 pups, respectively. On day 8 after delivery all females were tested for maternal aggression against a stranger adult male conspecific (5-min exposure). No aggression occurred in the group in which all pups had been removed. In the other groups, the proportion of females displaying overt aggression increased with litter size. Several scores of female agonistic behaviour (proportion of females displaying overt aggression, total attacking time, frequency of tail rattling) were significantly higher for the females rearing 8 and 12 pups than for the females rearing 4 pups. Aggressive behaviour of females rearing 12 pups was not significantly higher than that of females rearing 8 pups. No male committed infanticide. These results support the hypothesis that rodent maternal aggression is strictly related to offspring defence and are consistent with the theoretical prediction that, the costs of the defence being equal and the gain in fitness increasing with litter size, the intensity of maternal defence of the young should increase with their number.     

Immunohistochemical and ultrastructural evidence for myelopoiesis in the scid/scid mouse thymus

The ultrastructure of scid mouse thymus (a small encapsulated epithelial mass within the precardial fat pad) is described. The epithelium did not form cortex or medulla and hence remained relatively undifferentiated. Small unmyelinated nerves innervated the capsule, the major blood vessels and were distributed between the epithelial cells. Fenestrated blood vessels were common. Thymocytes were not identified but numeous granulocytes, mast cells and some fibroblasts, macrophages and interdigitating cells were present. All stages of granulopoiesis were observed in scid thymus. A very small number of immunoreactive ER-MP58 cells indicated bone marrow derived myeloid precursor cells, and low numbers of ER-MP12⁺ and ER-MP20⁺ mononuclear cells indicated stages of myeloid cells committed to the granulocyte/macrophage lineage. Cells containing proliferating nuclear cell antigen (cells in G1, S and G2-M stage) were present throughout the thymic mass. BALB/c thymuses contained cortical foci of p53⁺ cells whereas in scid mice, p53 positive cells were scattered singly throughout the thymus. This study indicates that the presence of moderately extensive myelopoiesis within the scid mouse thymus has potential for the study of extramedullary hematopoiesis, and also is important to bear this function in mind when using the scid mouse as an immunological model for thymus reconstitution and for creating organoid cultures.