The cell body space occupied by the nucleus during the cell differentiation in human lymphocytic, granulocytic and erythroid cell lineages.

The present nuclear and cell body diameter measurements demonstrated size differences of the approximate cell space estimate occupied by the cell nucleus during the cell differentiation in lymphocytic, granulocytic and erythroid cell lineages. These lineages were used as convenient models because all differentiation steps were easily identified and accessible in diagnostic peripheral blood or bone marrow smears of blood donors (BDs), patients suffering from chronic lymphocytic leukemia (CLL), patients with chronic myeloid leukemia (CML) and refractory anemia (RA) of the myelodysplastic syndrome (MDS). The cell space occupied by the nucleus was constant and did not change during the cell differentiation in the lymphocytic cell lineages of BDs and CLL patients despite the decreased cell size. In contrary, the cell space occupied by the nucleus markedly decreased in differentiating cells of granulocytic and erythroid lineages of patients suffering from CML. In the erythroid cell lineage in patients with RA of MDS the small reduction of the cell space occupied by the nucleus during the differentiation was not significant. The measurements also indicated that in progenitor cells of all studied cell lineages nuclei occupied more than 70 % of the cell space. Thus, the nucleus-cytoplasmic morphological and functional equilibrium appeared to be characteristic for each differentiation step and each specific cell lineage.

To the Nuclear Region Occupied by Nucleolar Bodies in Human Leukaemic Myeloblasts of Kasumi 1 and K 562 Lineages.

Previous observation demonstrated that measured nucleolar and nuclear diameters and the resulting calculated ratio might facilitate estimation of the approximate size of the nuclear region occupied by the nucleolar bodies. The size of nuclear regions occupied by nucleolar bodies decreased during the differentiation and maturation of leukaemic lymphocytes, but was constant for each differentiation or maturation stage. The present study was undertaken to provide more information on the approximate size of the nuclear regions occupied by nucleolar bodies in leukaemic granulocytic progenitors. Myeloblasts of established Kasumi 1 and K 562 cell lineages originating from human myeloid leukaemias were convenient models for such study because they represented only one and early differentiation stage of granulocytic progenitors. According to the results, the maximal and mean nucleolar body : maximal and mean nuclear diameter ratios in myeloblasts without heavy nuclear alterations were stable and not markedly influenced by the anti-leukaemic treatment or aging. Thus, the roughly estimated size of nuclear regions occupied by nucleolar bodies in these cells appeared to be similar and stable regardless of aging or anti-leukaemic treatment. In contrast, the anti-leukaemic treatment or aging in such myeloblasts induced marked reduction of the nucleolar biosynthetic activity reflected by the decreased number of nucleolar fibrillar centres.

Proteins implicated in the increase of adhesivity induced by suberoylanilide hydroxamic acid in leukemic cells.

We have previously shown that suberoylanilide hydroxamic acid (SAHA) treatment increases the adhesivity of leukemic cells to fibronectin at clinically relevant concentrations. Now, we present the results of the proteomic analysis of SAHA effects on leukemic cell lines using 2-DE and ProteomLab PF2D system. Histone acetylation at all studied acetylation sites reached the maximal level after 5 to 10 h of SAHA treatment. No difference in histone acetylation between subtoxic and toxic SAHA doses was observed. SAHA treatment induced cofilin phosphorylation at Ser3, an increase in vimentin and paxillin expression and a decrease in stathmin expression as confirmed by western-blotting and immunofluorescence microscopy. The interaction of cofilin with 14-3-3 epsilon was documented using both Duolink system and coimmunoprecipitation. However, this interaction was independent of cofilin Ser3 phosphorylation and the amount of 14-3-3-ε-bound cofilin did not rise following SAHA treatment. SAHA-induced increase in the cell adhesivity was associated with an increase in PAK phosphorylation in CML-T1 cells and was abrogated by simultaneous treatment with IPA-3, a PAK inhibitor. The effects of SAHA on JURL-MK1 cells were similar to those of other histone deacetylase inhibitors, tubastatin A and sodium butyrate. The proteome analysis also revealed several potential non-histone targets of histone deacetylases.

The RNA content of nucleolar bodies is related to their size - a cytochemical study on human monocytes and lymphocytes in blood smears and blood cytospins.

The present study was undertaken to provide more information on the relationship between the nucleolar size and RNA density. Mature monocytes circulating in human peripheral blood appeared to be very convenient for such study because they contain multiple nucleoli of various sizes in one and the same nucleus. In addition, nucleoli without perinucleolar chromatin represented by nucleolar bodies are easy to be visualized by a simple cytochemical procedure for RNA demonstration. The diameter and density of NoBs in specimens stained for RNA were determined by computer-assisted measurements of individual cells. According to the results, the nucleolar RNA content was apparently related to the nucleolar size because the RNA density of small and large NoBs was practically the same. In addition, the diameter measurements also indicated that one or two of several NoBs in one nucleus were dominant - larger - than the others. It should also be mentioned that the diameter and RNA density of NoBs were studied in monocytes in peripheral blood smears regardless of their limited number. Additional study on lymphocytes indicated that the preparation procedure of smears modified both diameter and density of the measured parameters less than the preparation of cytospins as demonstrated by lower variability of the resulting values. Thus, the observed differences between smears and cytospins also indicated that the specimen preparation procedures should always be considered during evaluation of the nucleolar size or staining intensity of nucleoli or cytoplasm due to the presence of RNA.

[NGAL--neutrophil gelatinase associated lipocalin in biochemistry, physiology and clinical praxis]. / NGAL-neutrofilní, s gelatinázou asociovaný lipokalin v biochemii, fyziologii a klinické praxi.

Neutrophil gelatinase associated lipocalin belongs to a family of small proteins, lipocalins, engaged in the transmembrane transportation of lipophylic substances. Originally isolated from specific granules of neutrophils, it was later located in bone marrow cells as well as lung, bronchial and colon epithelial cells. The expression of neutrophil lipocalin in epithelial cells and in body fluids considerably augments during the occurrence of inflammations and some cancers. A modulation of immunity response was thus suggested to be the main function of neutrophil lipocalin as well as the bacteriostatic effect originating from competition between neutrophil lipocalin and bacteria for siderophoric iron. Forming protective complexes with gelatinase B, the neutrophil lipocalin is implicated in regulatory processes of physiological and pathological rebuilding of tissues, mainly in the angiogenesis. The determination of neutrophil lipocalin levels in body fluids able to discriminate between bacterial and viral infections provides a powerful diagnostic tool. The examination of neutrophil lipocalin in the sera and urine of patients at risk of renal failure offers a very early marker of this acute state. Neutrophil lipocalin represents a sensitive non-invasive marker of renal ischemia and in patients with cystic fibrosis the marker of acute pulmonary exacerbation. Discussions have been conducted regarding the role of neutrophil lipocalin as an early marker of pancreatic cancer or of neutrophilic activation in severe cases of bowel diseases.

[Activation of the ribosomal gene during blast transformation of human lymphocytes]. / Aktivace ribozomálního genu v prubehu blastické transformace lidských lymfocytu.

BACKGROUND: Natural lectin, phytohemagglutinin, initiates the transformation of normally quiescent T lymphocytes into proliferating lymphoblast-like cells. Recently we have shown that the transformation is accompanied by strong promotion of ribosomal RNA synthesis and by phosphorylation of its activator, initiating factor UBF, both culminating in a synthetic phase of the first cell division cycle. In contrast we have revealed that the UBF gene was activated and its transcription culminated in the early G1 phase. We examined three possible delaying mechanisms: the kinetics of unwinding of rDNA chromatin, the kinetics of transcription of genes coding for the second initiating factor, SL1 complex, and the kinetics of the translation of UBF protein product. METHODS AND RESULTS: Up to 48 hrs following the addition of phytohemagglutinin to the growth medium, we monitored structural changes in the rDNA chromatin using indirect antiUBF immunofluorescence. The data indicated an increased number of separated transcriptional units during the G1 phase of the first cycle. In a time interval of up to 70 hrs we measured the mRNA levels of four constituents of SL1 complex: TAF110, TAF63, TAF48 and TBP using the RT-PCR method. We found a close correlation between the kinetics of the transcription of UBF and SL1 genes and the maximal rate in the early G1 phase. Using metabolic labelling with 35S methionine/cysteine we monitored the translation of UBF protein in PHA stimulated lymphocytes. The data suggested that UBF translation, starting in the S phase, paralleled chromosomal DNA replication. CONCLUSIONS: During the transformation of normal T lymphocytes into proliferating blast-like cells, the multicopy rDNA gene unwinds in the G1 phase of the first cycle forming individual transcriptional units. Genes coding for factors which initiate synthesis of ribosomal precursors are activated in the early G1 phase. The G1 synthesis of ribosomal RNA is accelerated by phosphorylation of the hypophosphorylated UBF pool. As blastic transformation develops UBF translation is triggered in the S phase and neosynthesized UBF, activated by phosphorylation, pushes the synthesis of ribosomal precursors to maximal efficiency. The process of blastic transformation interferes throughout the entire prolonged G1 phase of the first cell division cycle.

[Long-term culture of plasma cells from patients with myeloma and establishment of a permanent cell line]. / Dlouhodobá kultivace plazmatických bunek nemocných s myelomem a moznosti získání permanentních bunecných linií.

UNLABELLED: Multiple myeloma plasma cells are actively dividing cells with the long surviving ability in the ex-vivo culture. In the effort for better understanding of the proliferative potential of malignant myeloma cells and establishment of permanent myeloma cell lines we performed long term cultures of human myeloma cells ex-vivo. During the last two years we cultured 41 bone marrow samples from 39 patients with multiple myeloma. Cells were cultured in the RPMI 1640 culture medium with 15% fetal calf serum at 37 degrees C in 5% CO2 and approximately one third of the culture medium was changed regularly twice a week. Most of the marrows cultures died by apoptosis within 30 days. Four bone marrow samples were cultured for more than 11 months, however, no culture can be qualified as an established cell line. In three cases permanent B-lymphoblastoid cell lines were established (UHKT-55, UHKT-56 a UHKT-57) but secondary immortalization by Epstein-Barr virus was suggested. CONCLUSIONS: Presented results suggest that myeloma plasma cells can survive and are able to proliferate in the ex-vivo culture for several months up to one year independently on the addition of any external growth factor without spontaneous apoptosis or necrosis. The probability of the establishment of a permanent cell line of plasma cell origin is, however, low. Presence of accessory bone marrow cells was the most important factor for the long-term survival of myeloma cells.