De novo transcriptome analysis of tobacco seedlings and identification of the early response gene network under low-potassium stress.

Tobacco is an economically important crop, and its potassium content can greatly affect the quality of tobacco leaves. However, the molecular mechanism involved in potassium starvation in tobacco has not been elucidated to date. In this study, Illumina (Solexa) sequencing technology was used to analyze the transcriptome of tobacco seedlings under low-potassium stress for 6, 12, and 24 h. After analysis, 107,824 assembled unigenes were categorized into 57 GO functional groups, and 31,379 unigenes (29.08%) were clustered into 25 COG categories. A total of 9945 genes were classified into 233 KEGG pathways, and 15,209 SSRs were found among the 107,824 unigenes. Between the two samples, 1034 genes were differentially expressed. Twelve randomly selected gene expression levels were analyzed by quantitative RT-PCR, and the results were highly consistent with those obtained by Solexa sequencing. Our results provide a comprehensive analysis of the gene-regulatory network of tobacco seedlings under low-potassium stress.

Identification of low potassium stress-responsive proteins in tobacco (Nicotiana tabacum) seedling roots using an iTRAQ-based analysis.

Potassium is one of the three main mineral nutrients, and is vital for leaf growth and the quality of tobacco (Nicotiana tabacum) plants. In recent years, the isobaric tags for relative and absolute quantitation (iTRAQ) method has been one of the most popular techniques for quantitative proteomic analysis. In this study, we used iTRAQ to compare protein abundances in the roots of control and low potassium-treated tobacco seedlings, and found that 108 proteins were differentially expressed between the two treatments. Of these, 34 were upregulated and 74 were downregulated, and 39 (36%) were in the chloroplasts. Kyoto Encyclopedia of Genes and Genomes pathway enrichment results suggested that metabolic pathways were the dominant pathways (10 upregulated and 14 downregulated proteins). Ten proteins involved in the pyruvate metabolism pathway increased their expression levels, and 17 upregulated proteins were enriched in the ribosomes category. To evaluate correlations between protein and gene transcript abundances, the expression patterns of 12 randomly chosen genes were examined. A quantitative real-time polymerase chain reaction revealed that the 12 genes were induced after low potassium treatment for 3, 6, 12, and 24 h. Our results demonstrate that low potassium levels affect protein profiles in tobacco roots.

Proteome analysis of tobacco leaves reveals dynamic changes in protein expression among different cultivation areas.

The leaves of tobacco plants were used to analyze differences in protein content of tobacco grown in the four main flue-cured tobacco-producing areas of Sichuan Province, China. An improved protein extraction method, isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gel electrophoretic separation, was used to extract and separate total protein from tobacco leaves. Proteomic maps with relatively high resolution and repeatability were produced. At isoelectric points 4 to 7 and molecular weight ranging from 20-100 kDa, we detected 1032, 1030, 1019, and 1011 clearly visible protein spots in tobacco leaves from the four study areas. Proteome comparison between these protein spots showed that 119 spots with a greater than 2-fold change in expression quantity contributed to the variation in expression. Of which, 115 were successfully identified and annotated. According to the annotation results, these proteins participate in photosynthesis, energy metabolism, mineral nutrition, terpene metabolism, defensive reaction, and other physiological and biochemical processes. This study preliminarily explains the effects of ecological conditions on the physiological metabolism of tobacco leaves and how such effects directly or indirectly contribute to tobacco leaf quality.

Variation of genomic DNA methylation in the nitrate reductase gene of sibling tobacco (Nicotiana tabacum) cultivars.

To better understand genomic DNA methylation in sibling plant cultivars, methylation-sensitive amplification polymorphism analysis was used to investigate two sibling tobacco cultivars, Yunyan85 and Yunyan87, and their two parents, K326 and Yunyan No. 2. Differences in the degree of genomic DNA methylation were found among the four tobacco cultivars. Compared with parents, the two sibling cultivars had fewer methylated sites. Twenty-nine methylation-sensitive amplification polymorphism fragments that exhibited methylation alteration in the four tobacco cultivars were recovered and sequenced. BLAST (nucleotide BLAST) searches showed that two of the 29 sequences have 99% similarity with nucleotides 1442-1694 of the nia-1 gene and the other 27 sequences contain GC, CAAT or TATA box. The nitrate reductase genes from Yunyan87, K326 and Yunyan No. 2 were found to be identical; however, the third intron of the nitrate reductase gene from Yunyan85 was different compared to the third introns of Yunyan87, K326 and Yunyan No. 2. We conclude that methylation alteration of promoter regions could be responsible for the different phenotypes in tobacco and that introns of the nitrate reductase gene can vary as a result of intra-species crossing in tobacco.

Mifepristone acts as progesterone antagonist of non-genomic responses but inhibits phytohemagglutinin-induced proliferation in human T cells.

BACKGROUND: Progesterone is an endogenous immunomodulator that suppresses T cell activation during pregnancy. The stimulation of membrane progesterone receptors (mPRs) would seem to be the cause of rapid non-genomic responses in human peripheral T cells, such as an elevation of intracellular calcium ([Ca(2+)](i)) and decreased intracellular pH (pH(i)). Mifepristone (RU486) produces mixed agonist/antagonist effects on immune cells compared with progesterone. We explored whether RU486 is an antagonist to mPRs and can block rapid non-genomic responses and the induction by phytohemagglutinin (PHA) of cell proliferation. METHODS: Human male peripheral T cell responses in terms of pH(i) and [Ca(2+)](i) changes were measured using the fluorescent dyes, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and fura-2, respectively. Expression of mPR mRNA was determined by RT-PCR analysis. Cell proliferation and cell toxicity were determined by [(3)H]-thymidine incorporation and MTT assay, respectively. RESULTS: The mRNAs of mPRalpha, mPRbeta and mPRgamma were expressed in T cells. RU486 blocked progesterone-mediated rapid responses including, the [Ca(2+)](i) increase and pH(i) decrease, in a dose related manner. RU486 did not block, but enhanced, the inhibitory effect of progesterone on PHA induced cell proliferation. RU486 alone inhibited proliferation induced by PHA and at >25 microM seems to be cytotoxic against resting T cells (P < 0.01). CONCLUSIONS: RU486 is antagonistic to the rapid mPR-mediated non-genomic responses, but is synergistic with progesterone with respect to the inhibition of PHA-induced cell proliferation. Our findings shine new light on RU486's clinical application and how this relates to the non-genomic rapid physiological responses caused by progesterone.

Propylnitrosourea-induced T-lymphomas in LEXF RI strains of rats: genetic analysis.

Oral administration of propylnitrosourea (PNU) in drinking water induces high incidence of lympho-haemopoietic malignancies in rats. Previously we reported that F344 strain rats were highly susceptible to T-lymphomas, and LE/Stm rats, to erythro- or myeloid leukaemias. For analysis of the genetic factors determining types of diseases, we have established LEXF recombinant inbred strains of rats comprising 23 substrains, each derived from intercross between F344 and LE/Stm rats. Rats of 23 LEXF substrains were given PNU, and the development of tumours was observed. The overall incidence of haemopoietic tumours ranged from 100% to 66.7%, and the fractions of T-lymphomas, from 100% to 4%, showing a continuous spectrum. Based on the genetic profile published as a strain distribution pattern table for the LEXF, we screened the potential quantitative trait loci involved in determination of the types of disease and length of the latency period. Statistical calculation was performed using the Map Manager QT software developed by Manly. Four loci, on chromosome 4, 7, 10 and 18, were suggested to associate with the T-lymphoma susceptibility and three loci, on chromosome 1, 5 and 16, with the length of the latency period. These putative loci were further examined in backcross (F344 x LE)F1 x LE. Among seven loci suggested by the recombinant inbred study, three loci, on chromosome 5, 7 and 10, were significantly associated with T-lymphomas and another locus on chromosome 1, just weakly. These observations indicate that PNU-induced lymphomagenesis is a multifactorial genetic process involving a number of loci linked with susceptibility and resistance.

Bone marrow pre-B-1 (Bomb1): a quantitative trait locus inducing bone marrow pre-B-cell expansion in lymphoma-prone SL/Kh mice.

Abnormalities of regulatory genes in early B-cell development often lead to lymphomagenesis. Our previous study showed that there is an abnormal transient expansion of bone marrow (BM) pre-B cells in lymphoma-prone SL/Kh strain mice. Such expansion is a genetic property of SL/Kh stem cells rather than BM microenvironments. Using the percentage of BP1+ B220+ pre-B cells in total BM lymphoid cells as a quantitative parameter, we studied the genetic control of BM pre-B cells in 159 F2 offspring of crosses between SL/Kh and NFS/N mice and 334 back-crosses to SL/Kh mice. A highly significant quantitative trait locus was identified on the distal segment of chromosome 3, showing logarithm of odds scores of 22.7 in the F2 cohort and 10.7 in back-cross mice. This quantitative trait locus, named bone marrow pre-B-1, colocalized with lymphoid enhancer factor-1, which encodes a high mobility group DNA-binding protein that is expressed in T and pre-B cells.

Induction of B-cell lymphoma in BALB/c nude mice with an ecotropic, B-tropic helper virus present in the murine AIDS virus stock.

The pathogenicities of the murine AIDS (MAIDS) virus complex (LP-BM5) and ecotropic helper virus (BM5eco) isolated from the complex to BALB/c nude mice were studied to elucidate the possible role of replication-competent helper virus in inducing the monoclonal outgrowth of lymphoid cells. Neither LP-BM5 nor BM5eco was pathogenic in adult BALB/c nude mice. However, B-cell lymphoma developed with a very high frequency when either virus was inoculated into newborn BALB/c nude (nu/nu) mice. The cells from the B-cell lymphoma were easily transplanted into nude mice. These results suggested that ecotropic helper virus in the MAIDS virus complex plays an important role in inducing the monoclonal outgrowth of lymphoid cells under immunodeficient conditions caused by defective virus.

Mixed phenotype lymphomas in thymectomized (SL/KhxAKR/Ms)F1 mice.

Lymphomagenesis in mice is determined both by genetic and epigenetic mechanisms. The inbred strain SL/Kh mice selectively develop pre-B lymphomas and AKR/Ms, T-lymphomas. In crosses between SL/Kh and AKR/Ms, an AKR-derived dominant gene Tlsm1 (Thymic lymphoma susceptible mouse-1) determines the type of lymphoma to be a T-lymphoma. As an approach to the role of Tlsm1, we studied the effect of thymectomy at 1 week of age in (SL/KhxAKR/Ms)F1 hybrids. In intact F1 mice, the predominant type of lymphoma was of T-lineage, whereas in thymectomized mice, it was an unusual mixed-phenotype lymphoma. They were basically CD5+ B-lymphomas with a rearranged immunoglobulin gene, but carried NK1 and Mac1 on the cell surface and large lysosomal granules in the cytoplasm. Histologically, the lymphoma consisted of large lymphoblastoid cells and infiltrated the spleen, lymph node and liver. Electron microscopy and histochemistry revealed numerous cytoplasmic granules containing acid phosphatase and lysozyme. These morphological features are suggestive of large granular lymphocytes. They expressed interleukin-4, perforin, and interferon-gamma. On transplantation, these lymphoma cells grew equally well in intact and thymectomized F1 recipients.

To be or not to be a T-lymphomas, that is determined by a dominant gene Tlsm-1 in mouse models.

A single dominant gene Tlsm-1 was found to determine the type of spontaneous lymphomas to be T in the cross between AKR/Ms and SL/Kh. Microsatellite analysis mapped Tlsm-1 at the position 61 cM from centromere of Chr. 7. Study of this segment of T-lymphoma prone AKXD Rl strains also showed association of Tlsm-1 with T-lymphomas. On the other hand, lymphoma latency was significantly shorten by a recessive gene lla of SL/Kh. By a quantitative trait analysis, lla was located in class II gene in MHC.

Expression of LECAM-1 and LFA-1 on pre-B lymphoma cells but not on preneoplastic pre-B cells in SL/KH mice.

The pre-B lymphoma-prone inbred strain SL/Kh mice showed a polyclonal expansion of BP-1+ pre-B cells in bone marrow early in life. Preneoplastic pre-B cells did not express adhesion molecules LECAM-1 and LFA-1, whereas neoplastic pre-B cells consistently expressed both molecules. There were two types of pre-B lymphomas in SL/Kh with distinct in vivo behavior. One infiltrated lymph nodes and spleen and another, predominantly bone marrow. However, lymphoma cells of both types expressed BP-1, LECAM-1 and LFA-1. Expression of these adhesion molecules on BP-1+ cells, therefore, may represent an important consequence of pre-B lymphomagenesis in SL/Kh strain, but is not sufficient to explain the in vivo behavior of the pre-B lymphoma cells.

[Effects of gonadal steroid hormones on hypothalamic vasopressin mRNA level in male and female rats].

Experiments were carried out in 10-11-week old gonadectomized male and female Sprague-Dawley rats. Dot-blot analysis and 3'-end digoxigenin-labeled 26 meroligonucleotide probe was used in detecting the mRNA level hypothalamic vasopressin (AVP). The basal hypothalamic AVP-mRNA level in the sham-operated intact males was 45% higher than that in the sham-operated intact females (P < 0.05). Plasma osmolality was also higher in the sham-operated intact males than in the sham-operated intact females (P < 0.05). The hypothalamic AVP-mRNA level in ovariectomized rats was 30% higher than that in sham-operated intact females (P < 0.05). Although the hypothalamic AVP mRNA level tended to be lower in castrated males than in sham-operated intact males, the difference was not statistically significant. The difference in plasma osmolality between gonadectomized males and females was statistically insignificant. In castrated males, hypothalamic AVP-mRNA level was decreased following sc injection of estradiol (P < 0.05), but testosterone, progesterone or a combination of estradiol and progesterone were without effect. In ovariectomized rats, sc injection of estradiol or a combination of estradiol and progesterone resulted in a decrease in hypothalamic AVP-mRNA level (P < 0.01), but progesterone or testosterone had no effect. The difference in plasma osmolality between gonadal steriod hormones-treated rats and vehicle-treated rats was not statistically significant. These findings indicate that gonadal steriod hormones can affect hypothalamic AVP-mRNA level in rats, through some central mechanism.

The origin of SL family mice.

The origin of SL family mice was studied by analyzing 100 microsatellite loci, the major histocompatibility complex, the Mx gene, murine leukemia provirus, and mammary tumor provirus. From the genetic profile of family members and their history, we assumed the existence of a proto-SL mouse, an ancestor of all SL family members. Many alleles were contributed to the proto-SL by the ancestors related to strains A2G and CF#1, and/or some wild mice. Among four existing family members, SL/Am and SL/Ni mice were almost identical and presumably closest to the proto-SL. The SL/Kh mouse was derived from a cross of the proto-SL and AKR mice, because SL/Kh mice inherited a considerable number of genes from AKR mice, the most outstanding of which were those of the provirus Emv-11 and Thy-1.1. The SL/QDj mice seemed to be a recombinant inbred strain between SL/Am and SL/Kh mice, because their alleles at all 100 microsatellite loci were shared by SL/Am or SL/Kh strains or both. All four SL family members shared the major histocompatibility complex haplotype q.

Two dominant host resistance genes to pre-B lymphoma in wild-derived inbred mouse strain MSM/Ms.

To explore possible host genes suppressing spontaneous B-lymphomagenesis in the mouse, expression of ecotropic murine leukemia virus (E-MuLV) and lymphoma development were observed in crosses between the pre-B lymphoma-prone SL/Kh and low-lymphoma strains of mice. E-MuLV expression was intensely inhibited in F1 hybrids with the strains either with the Fv-1b allele (BALB/C, C57BL/10, and A/J) or with the Fv-1nr allele (NZB). In these F1 mice, no lymphoma developed by 18 months of age. On the other hand, F1 hybrids with the strains with the Fv-1n allele [C3H/He, CBA/N, SJL, DBA/2, and MSM/Ms (hereafter referred to as MSM)], high or intermediate levels of E-MuLV expression were observed. Lymphoma incidence in these F1 hybrids, however, was low. This observation suggests the presence of non-Fv-1 dominant resistance genes in these strains. In an attempt to characterize such host genes, we analyzed crosses between SL/Kh mice and a wild mouse-derived inbred strain, MSM/Ms. The latter was susceptible to N-tropic virus expression, but (SL/Kh x MSM)F1 hybrids, did not develop and lymphomas. Of 60 SL/Kh x (SL/Kh x MSM)F1 hybrids, 14 B-lineage lymphomas, including 13 pre-B and 1 follicular center cell lymphoma, developed by 18 months of age. This was compatible with the hypothesis of two independently segregating dominant genes of MSM suppressing lymphomagenesis. By scanning all chromosomes for linkage of lymphoma susceptibility with polymorphic microsatellite loci, one significant linkage disequilibrium was found in the proximal segment of chromosome 17, containing D17MIT44 (map position 15.0) to D17MIT150 (position 33.3), and another linkage disequilibrium, in the midproximal segment of chromosome 18, containing D18MIT90 (map position 28.0) and D18MIT140 (37.0). All 13 pre-B lymphoma-bearing backcross mice were homozygous for SL/Kh-derived alleles at these loci. We named the gene on chromosome 17 Msmr1 (for MSM resistance 1) and that on chromosome 18 Msmr 2 (for MSM resistance 2).

[Study of the pisum sativum agglutinin receptors and DNA ploidy during carcinogenesis in esophageal epithelium by flow cytometry].

The relative mean contents of pisum sativum agglutinin receptors and DNA of human normal esophageal epithelium, its atypical hyperplasia as well as esophageal carcinoma were determined respectively by flow cytometry. The results revealed that the relative mean contents of pisum sativum agglutinin receptors which denote membrane composition and DNA which denote nuclear composition were increased gradually from normal, dysplastic and then malignant esophageal tissues. This suggested that during the neoplastic transformation, changes of the cell membrane component may correlate with changes of the cell nuclear component.