Quantitative expression of regulatory and differentiation-related genes in the key steps of human hematopoiesis: The LeukoStage Database.

Differentiation during hematopoiesis leads to the generation of many cell types with specific functions. At various stages of maturation, the cells may change pathologically, leading to diseases including acute leukemias (ALs). Expression levels of regulatory molecules (such as the IKZF, GATA, HOX, FOX, NOTCH and CEBP families, as well as SPI-1/PU1 and PAX5) and lineage-specific molecules (including CD2, CD14, CD79A, and BLNK) may be compared between pathological and physiological cells. Although the key steps of differentiation are known, the available databases focus mainly on fully differentiated cells as a reference. Precursor cells may be a more appropriate reference point for diseases that evolve at immature stages. Therefore, we developed a quantitative real-time polymerase chain reaction (qPCR) array to investigate 90 genes that are characteristic of the lymphoid or myeloid lineages and/or are thought to be involved in their regulation. Using this array, sorted cells of granulocytic, monocytic, T and B lineages were analyzed. For each of these lineages, 3-5 differentiation stages were selected (17 stages total), and cells were sorted from 3 different donors per stage. The qPCR results were compared to similarly processed AL cells of lymphoblastic (n=18) or myeloid (n=6) origins and biphenotypic AL cells of B cell origin with myeloid involvement (n=5). Molecules characteristic of each lineage were found. In addition, cells of a newly discovered switching lymphoblastic AL (swALL) were sorted at various phases during the supposed transdifferentiation from an immature B cell to a monocytic phenotype. As demonstrated previously, gene expression changed along with the immunophenotype. The qPCR data are publicly available in the LeukoStage Database in which gene expression in malignant and non-malignant cells of different lineages can be explored graphically and differentially expressed genes can be identified. In addition, the LeukoStage Database can aid the functional analyses of next-generation sequencing data.

Mesophyll conductance to CO(2) transport estimated by two independent methods: effect of variable CO(2) concentration and abscisic acid.

Mesophyll conductance (g(m)) and stomatal conductance (g(s)) are two crucial components of the diffusive limitation of photosynthesis. Variation of g(m) in response to CO(2) concentration was evaluated by using two independent methods based on measurements of variable electron transport rate (J) and instantaneous carbon isotope discrimination, respectively. Both methods of g(m) estimation showed a very similar shape of the g(m)/C(i) relationship, with an initial increase at low substomatal CO(2) concentrations (C(i)), a peak at 180-200 micromol mol(-1) C(i), and a subsequent decrease at higher C(i). A good correlation was observed between values of g(m) estimated from the two methods, except when C(i) <200 micromol mol(-1), suggesting that the initial increase of g(m) at low C(i) was probably due to unreliable estimates over that range of C(i). Plants were also treated with abscisic acid (ABA), which induced a reduction in g(s) without significantly affecting the rate of photosynthesis, g(m) or the photosynthetic capacity. The present results confirm, using two independent methods, that g(m) is strongly sensitive to C(i), and that the relationship between g(s) and g(m) is not conservative, differing between control and ABA-treated plants.

CD44 and CD27 delineate B-precursor stages with different recombination status and with an uneven distribution in nonmalignant and malignant hematopoiesis.

The expression of CD27 and CD44 correlate with the genotype of B-precursor acute lymphoblastic leukemia (ALL). Based on the expression of these antigens, we identified counterparts of TEL/AML1(pos) and TEL/AML1(neg) leukemic cells in nonmalignant bone marrow. Although CD27 is known as a marker of mature memory B cells, we recently showed that CD27 is also expressed by malignant and nonmalignant B precursors. Here, we show that CD27 and CD44 delineate stages of B-precursor development. Well-established differentiation markers showed that the developmental sequence starts from undetermined progenitors, expressing CD44. Upon B-lineage commitment, cells gain CD27 and lose CD44. The CD27(pos)CD44(neg) (CD27 single positive, 27SP) cells are the earliest stage within CD10(pos)CD19(pos) B precursors and express RAG-1 and TDT. These cells correspond to TEL/AML1(pos) ALL (1/4 pediatric B-precursor ALL). The development follows to CD27/CD44 double-positive (27/44DP) stage, 44SP stage and CD27/CD44 double-negative (27/44DN) stage. Before exit to periphery, CD44 is reexpressed. The 27/44DP cells are mostly large and profoundly suppress RAG-1. Despite their presumably high proliferation potential, 27/44DP cells rarely dominate in leukemia. At 44SP stage, which corresponds to TEL/AML1(neg) leukemias, RAG-1 is reexpressed and Ig light chain gene starts to be rearranged.

Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis.

Small hydrophobic hormones like steroids control many tissue-specific physiological responses in higher organisms. Hormone response is characterized by changes in gene expression, but the molecular details connecting target-gene transcription to the physiology of responding cells remain elusive. The salivary glands of Drosophila provide an ideal model system to investigate gaps in our knowledge, because exposure to the steroid 20-hydroxyecdysone (20E) leads to a robust regulated secretion of glue granules after a stereotypical pattern of puffs (activated 20E-regulated genes) forms on the polytene chromosomes. Here, we describe a convenient bioassay for glue secretion and use it to analyze mutants in components of the puffing hierarchy. We show that 20E mediates secretion through the EcR/USP receptor, and two early-gene products, the rbp(+) function of BR-C and the Ca2+ binding protein E63-1, are involved. Furthermore, we demonstrate that 20E treatment of salivary glands leads to Ca2+ elevations by a genomic mechanism and that elevated Ca2+ levels are required for ectopically produced E63-1 to drive secretion. The results presented establish a connection between 20E exposure and changes in Ca2+ levels that are mediated by Ca2+ effector proteins, and thus establish a mechanistic framework for future studies.

Genetic analysis of the Drosophila 63F early puff. Characterization of mutations in E63-1 and maggie, a putative Tom22.

The 63F early puff in the larval salivary gland polytene chromosomes contains the divergently transcribed E63-1 and E63-2 ecdysone-inducible genes. E63-1 encodes a member of the EF-hand family of Ca(2+)-binding proteins, while E63-2 has no apparent open reading frame. To understand the functions of the E63 genes, we have determined the temporal and spatial patterns of E63-1 protein expression, as well as undertaken a genetic analysis of the 63F puff. We show that E63-1 is expressed in many embryonic and larval tissues, but the third-instar larval salivary gland is the only tissue where increases in protein levels correlate with increases in ecdysone titer. Furthermore, the subcellular distribution of E63-1 protein changes dynamically in the salivary glands at the onset of metamorphosis. E63-1 and E63-2 null mutations, however, have no effect on development or fertility. We have characterized 40 kb of the 63F region, defined as the interval between Ubi-p and E63-2, and have identified three lethal complementation groups that correspond to the dSc-2, ida, and mge genes. We show that mge mutations lead to first-instar larval lethality and that Mge protein is similar to the Tom22 mitochondrial import proteins of fungi, suggesting that it has a role in mitochondrial function.

Light-chain fibroin of Galleria mellonella L.

The posterior section of Galleria mellonella silk glands contains two abundant mRNAs that are identical except for the non-coding tail, which includes either two (1.1 kb mRNA) or three (1.2 kb mRNA) consensus sequences for polyadenylation sites. The transcripts are 40% homologous in the coding as well as non-coding regions with the mRNA encoding light-chain fibroin (L-fibroin) in Bombyx mori; the deduced translation product shows 43% identity with the Bombyx L-fibroin peptide, with all three cysteines conserved. Amino acid analysis of the N-termini of Galleria silk proteins revealed that L-fibroin (25 kDa) occurs in two isoforms, the shorter one lacking the Ala-Pro dipeptide residue at its N-terminus. The 29 and 30 kDa Galleria silk proteins appear to be homologs of Bombyx silk component P25. The results suggest that evolutionary diversification of Galleria and Bombyx L-fibroins involves alternative polyadenylation and proteolytic processing sites.

[Levels of homovanillic acid and 5-hydroxyindoleacetic acid in the cerebrospinal fluid in patients with focal cerebral ischemia]. / Koncentrácia kyseliny homovanilovej a 5-hydroxyindoloctovej v CSL u pacientov s loziskovou ischémiou mozgu.

The investigated group comprised 35 patients where clinical examination revealed focal cerebral ischaemia in the area of the a. cerebri media. In the group of elderly patients above 60 years, as compared with controls, the homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA) concentration in cerebrospinal fluid was elevated. In the group of patients under 60 years no changes in the concentration of these metabolites were found. In older patients the increased level of metabolites in cerebrospinal fluid persisted also after three days following the attack, while in younger patients the elevated HVA and 5-HIAA levels were found only in specimens collected up to three days.

Tn1000 insertional mutagenesis of cloned repressor gene of the phage L: plasmid oligomerization in the presence of F'lac.

An ampicillin resistance plasmid carrying the cloned repressor gene cII of the L phage (Salmonella typhimurium) was conducted by F'lac into an F- recipient. Two types of plasmids were isolated from Apr transconjugants. The majority of plasmids were dimers with one copy of Tn1000 inserted, the minority being monomers with one copy of Tn1000. This proportion remained unaltered when we used the F'lac strain transformed with a monomeric form of the recombinant plasmid as a donor. An extensive oligomerization of pBR322-originating plasmids was proved in the presence of F'lac; its presumable relationship to transposition-related processes is suggested.

Hot spot for Tn1000 insertions in cloned repressor gene of the L phage.

Insertions of Tn1000 into a cloned fragment of the L-phage genome comprising the repressor gene were prepared. Repressor activities produced by plasmids with insertions were assayed in vivo. As a result, the repressor gene was localized within 0.5 kb near one end of the cloned fragment. Transposon insertions were nonrandomly clustered within the repressor gene and in its close vicinity. An analysis of supercoiled plasmid DNA with the nuclease S1 revealed no distortion of the secondary structure of DNA in this region.