Associations of functional candidate genes derived from gene-expression profiles of prenatal porcine muscle tissue with meat quality and muscle deposition.

Ten genes (ANK1, bR10D1, CA3, EPOR, HMGA2, MYPN, NME1, PDGFRA, ERC1, TTN), whose candidacy for meat-quality and carcass traits arises from their differential expression in prenatal muscle development, were examined for association in 1700 performance-tested fattening pigs of commercial purebred and crossbred herds of Duroc, Pietrain, Pietrain x (Landrace x Large White), Duroc x (Landrace x Large White) as well as in an experimental F(2) population based on a reciprocal cross of Duroc and Pietrain. Comparative sequencing revealed polymorphic sites segregating across commercial breeds. Genetic mapping results corresponded to pre-existing assignments to porcine chromosomes or current human-porcine comparative maps. Nine of these genes showed association with meat-quality and carcass traits at a nominal P-value of < or = 0.05; PDGFRA revealed no association reaching the P < or = 0.05 threshold. In particular, HMGA2, CA3, EPOR, NME1 and TTN were associated with meat colour, pH and conductivity of loin 24 h postmortem; CA3 and MYPN exhibited association with ham weight and lean content (FOM) respectively at P-values of < 0.003 that correspond to false discovery rates of < 0.05. However, none of the genes showed significant associations for a particular trait across all populations. The study revealed statistical-genetic evidence for association of the functional candidate genes with traits related to meat quality and muscle deposition. The polymorphisms detected are not likely causal, but markers were identified that are in linkage disequilibrium with causal genetic variation within particular populations.

Documentation of normal and leukemic myelopoietic progenitor cells with high-resolution phase-contrast time-lapse cinematography.

The high-resolution phase-contrast, time-lapse cinematography using oil immersion lenses and 16-mm film demonstrates the kinetic cell events as maturation, locomotion, mitosis, and apoptosis of cells cultivated at 37 degrees C for up to 10 days. 0.5 v/v frozen-thawed sera with presumably high cytokine concentrations were added to the plasma or agar clot. Vital progenitor cells from human bone marrow and blood have a large, bright, unstructured nucleus with a large nucleolus and a narrow rim of cytoplasm (nuclear/cytoplasmic volume ratio = 0.7). Their nuclei are 6-14 micrometer in diameter and double their volume within 8 h. Many (70%) move at a mean speed of 2 micrometer/min, and many (30%) multiply with alpha-2alpha mitoses, generating progenitor cell families. Various disturbances during the course of mitosis lead to the formation of polyploid cells, thereby yielding the megakaryocytic cell line. Some of the progenitor cells undergo asymmetric alpha-alphan mitoses: One of the two initially identical daughter cells remains a progenitor cell in the morphological sense, whereas the other daughter cell - depending on the size of its mother cell - matures in the same culture medium to form a granulocytopoietic, monocytopoietic or erythrocytopoietic cell line. - In acute myeloid leukemias (AML), the blasts and their nuclei are slightly larger than the corresponding progenitor cells and move faster (5 micrometer/min). Symmetric alpha-2alpha mitoses permit unlimited multiplication of the leukemic blasts if contact with cytotoxic lymphocytes does not render them apoptotic. This results in more stromal cells than normal. Granulocytopenia, monocytopenia, and anemia occur due to the genetic impairment of signaling control for asymmetric alpha-alphan mitoses, and thrombocytopenia occurs due to the reduction in polyploidization.

Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor. Importance of a glutamate residue in the transmembrane region.

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological changes, expression of alpha(4)- and beta(1)-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.

Human megakaryoblastic proliferation and differentiation events observed by phase-contrast cinematography.

Microcinematographic documentation of mitoses, amitoses, endomitoses, or cytoplasmic fusion shortly after completion of mitoses was done in bone marrow specimens of patients with quantitative platelet disorders and controls. In patients with platelet disorders, most mitoses with cell duplication occurred in large promegakaryocytes after 4-fold nuclear and cytoplasmic enhancement. Normal specimens showed polyploidization happening in small megakaryoblasts, while mitoses with cell duplication were seen only after cultivation in freeze-thawed sera of patients with platelet disorders. Frequently, lymphocytes were observed to contact megakaryoblastic cells undergoing mitoses, amitoses and endomitoses and to enter the cytoplasm of megakaryocytes (emperipolesis), leaving it again after several hours.

A long-term clinical trial of interferon alpha-therapy in essential thrombocythemia.

In a prospective clinical trial involving six patients suffering from essential thrombocythemia (ET), recombinant human interferon alpha 2b significantly and consistently lowered highly elevated peripheral platelet numbers over long time periods. One patient has now been on continuous treatment for 4 years. During the treatment phase none of the patients suffered from bleeding episodes, thrombosis or disturbances of the microcirculation. The interferon maintenance dose varies considerably from patient to patient, but it is usually much lower than the induction dose. One of the patients had to be withdrawn from the study due to interferon-specific chronic toxicity concomitant with the development of non-neutralizing interferon antibodies. With the exception of one patient, stopping interferon treatment led to an increase in peripheral platelet numbers of up to one million cells per microliter of blood within 4 to 12 weeks. We conclude that interferon alpha can correct peripheral thrombocytosis in selected patients with essential thrombocythemia over a period of years and prevent morbidity attributable to this disease.

Locomotion of human bone marrow and peripheral blood leukocytes is associated with maturational stage and altered in malignancy.

Using high-resolution phase contrast time-lapse microcinematography, slow movements (0.8-2.0 microns/minute) of human myeloblasts, monoblasts, and megakaryocytes can be recorded. Upon maturation to promyelocytes, motility is lost until cells have reached the stage of metamyelocytes (0.4 microns/minute). Motility increases sharply following maturation into segmented neutrophils (20.4 microns/minute). Monocytes and promonocytes display a mean track velocity of 7.1 microns/minute. The distribution of lymphocyte velocities is not bell-shaped but shows three maxima of 2.1 microns/minute, 7.8 microns/minute, and 18.4 microns/minute. Atypical lymphocytes from patients with infectious mononucleosis belong to the fast group, whereas lymphocytes activated in vitro by mitogens belong to the slow group. Red blood cell precursors from normal human bone marrow do not move actively. In contrast, erythroleukemic blasts show a motility comparable to normal myeloblasts. Similarly, acute promyelocytic leukemia cells move at 6.7 microns/minute, while their normal counterparts are sessile. Increased motility is also observed in blast cells from a variety of acute myelogenous and lymphoblastic leukemias.

Erythroblastopenia in two patients after splenectomy and polychemotherapy.

Acquired erythroblastopenia is a rare disorder of the hematopoietic system associated with viral infections, autoimmune diseases, and drugs. We report on two patients who became anemic due to maturation-arrest at the proerythroblast level, without alterations of white blood cell or platelet counts. Both patients had been splenectomized and had undergone chemotherapy for nephroblastoma or Hodgkin's disease, respectively, at the same pediatric oncology unit. Erythropoietin levels were elevated in both patients. Antibodies against specific viruses, particularly parvovirus B 19, could not be detected in patient sera. Both patients responded to infusions of 7 S immunoglobulin with a rapid increase of the reticulocyte counts. In both cases, complete clinical remission was observed after a duration of 5 months. Heat-inactivated serum obtained during the acute phase and after remission as well was found to be inhibitory for normal bone marrow granulocyte and erythrocyte progenitor growth in vitro. The simultaneous appearance of this rare disorder in two otherwise unrelated patients treated at the same unit prompts speculations about a viral etiology.

Nocodazole sensitivity, age-related aneuploidy, and alterations in the cell cycle during maturation of mouse oocytes.

To detect age-related alterations in the formation and function of the spindle apparatus, we examined in vitro maturing oocytes obtained from young (2-4 mo) and aged (greater than 9 mo) diestrous CBA/Ca mice. Observation of cells processed for antitubulin immunofluorescence revealed that oocytes from aged females progress faster through first maturation division than those from young animals. They are also more prone to nondisjunction, as shown by a significantly higher level of aneuploidy in C-banded cells arrested at metaphase II. The ability of oocytes to recover from treatment with a microtubule inhibitor, nocodazole, and the effect of the drug on spindle integrity and chromosome segregation were also studied. In both age groups, treatment of metaphase I oocytes with 10 microM nocodazole caused rapid and complete microtubule depolymerization and chromosome scattering. Upon recovery, oocytes from both age groups were able to reestablish a spindle apparatus, proceed through anaphase, and extrude a first polar body. However, nocodazole treatment led to a dramatic increase of aneuploidy. Unexpectedly, the relative rise in hyperploids was greater in oocytes from young mice than in those from aged mice, so that the absolute percentage of hyperploid metaphase II cells was similar in both age groups after drug treatment. Concomitantly, nocodazole exposure abolished or, at least, diminished intrinsic differences in the cell cycle and anaphase trigger present in the controls (e.g., the earlier onset of chromosome separation in oocytes from aged females). It shortened the period available for spindle formation before chromosome segregation in all oocytes. Therefore, our study implies that temporal differences in the progression of oocytes through maturation, in particular, the shortening of the time available for alignment of bivalents before chromosome separation occurs in oocytes of old females, are mainly responsible for age-related rises in aneuploidy. There is no indication that (1) the spindle apparatus of oocytes from aged mammals is more labile or susceptible to disturbances than the spindle apparatus of oocytes from young individuals or that (2) an increase in the number of univalents makes oocytes from aged mammals particularly prone to nondisjunction.

Kinetic and morphological changes induced in human blood leucocytes by cytochalasin D and E.

The effect of increasing concentrations of cytochalasin D and E, up to toxicity, on the velocity of blood leucocytes from normal subjects was measured in vitro using a high-resolution objective and phase-contrast time-lapse photography. The dose-response effect for the two different cytochalasins differed in accordance with the different cell specificity of their membrane binding. The average velocity of granulocytes was reduced at cytochalasin D concentrations above 5 x 10(-7)M and cytochalasin E concentrations above 5 x 10(-5)M. The effect on monocytes and eosinophils was similar. In contrast the velocity of lymphocytes was not affected until cytotoxic concentrations were reached. The concentration ranges which inhibited locomotion corresponded well with the concentration ranges of the cytochalasins which have an in vitro effect on microfilaments. The concentrations which induced additional morphological changes in lymphocytes also correlate well with the concentrations found to inhibit cross-linking in vitro, as well as those known to induce morphological changes in, for example, fibroblasts in vivo. Cytotoxic effects were first observed with ten-fold higher concentrations of cytochalasin E than of cytochalasin D.

[Effect of different glucocorticoids on the in vitro cell kinetics of human hematopoiesis represented by a bone marrow culture and by phytohemagglutinin- and pokeweed mitogen-stimulated blood cultures]. / Die Wirkung verschiedener Glukokortikoide auf die Zellkinetik der menschlichen Hämatopoese in vitro, dargestellt an der Knochenmarkkultur und an der mit Phytohämagglutinin und Pokeweed Mitogen stimulierten Blutkultur.

The effect of nine glucocorticoids, DHEA, cyclohexanol and dextran sulphate on the hematopoietic proliferation was investigated. Three glucocorticoids inhibited the proliferation of normal bone marrow cells, the others enhanced it. The T-cell stimulation was not inhibited by fluorcortolone contrary to prednisolone. Specially the proliferation of acute myeloid leukemic blasts was inhibited by fluorcortolone hemisulfate, which significantly stimulated the proliferation of normal bone cells.

Interactions between lymphocytes and hematopoietic progenitor cells in normal and leukemic human bone marrow (phase contrast observations).

Single cell observations of normal and of leukemic human bone marrow cells demonstrated cell-cell interactions of lymphocytes with hematopoietic progenitor cells. In all cases lymphocytes and target cells were from the same individual. Lymphocyte-target cell interactions occurred more frequently with normal committed progenitor cells and leukemic blast cells from acute myeloid leukemia than with precursor cells of the proliferative cell pool, including myeloblasts, promonocytes, erythroblasts and megakaryocytes. Both induction of mitosis and degeneration of the progenitor cells occurred after cell-cell interaction with almost the same frequency. Acute myeloid leukemic blast cells degenerated after contact with lymphocytes with the same frequency as normal progenitor cells (i. e. in 16% of cell contacts), but especially during mitosis. In contrast, normal and regenerating bone marrow progenitor cells from myeloproliferative diseases demonstrated no degeneration after cell-cell interaction with lymphocytes during mitosis. Otherwise the induction of mitoses by lymphocyte-target cell interactions was more frequently observed in normal progenitor cells than in leukemic blasts.

Measurements of nuclear sizes of committed hematopoietic progenitor cells compared to leukemic blasts from human bone marrow smears.

By measurements of the nuclear sizes of normal and of leukemic myeloblasts and monoblasts in colored smears, the differences estimated in surviving cells with phase contrast were confirmed. The median nuclear size of normal myeloblasts is the smallest one and differs significantly from that of the normal monoblasts and AML myeloblasts. The median nuclear size of normal monoblasts significantly exceeds values of normal myeloblasts and of AMoL monoblasts. Both leukemic blasts cover a wide range of nuclear diameters, but do not differ from one another, while normal myeloblasts have a smaller dispersion than normal monoblasts. The difference of leukemic blasts and normal progenitor cells is to be emphasized.

No elevation of the frequencies of chromosomal alterations as a consequence of handling cytostatic drugs. Analyses with peripheral blood and urine of hospital personnel.

Peripheral blood lymphocytes of hospital personnel handling cytostatic drugs showed no elevation of structural chromosomal aberrations or sister-chromatid exchanges (SCE) over the baseline. The urine of the probands did not induce SCE in peripheral lymphocytes in vitro.

[Different effects of cytostatics on cell kinetics of normal and leukemic human bone marrow in vitro]. / Unterschiedliche Wirkungen von Zytostatika auf die Zellkinetik des normalen und leukämischen menschlichen Knochenmarkes in vitro.

The kinetics of the erythropoietic and of the granulocytopoietic pool of normal und leukemic human bone marrow cells were investigated in vitro under the influence of 23 cytostatic drugs. Clot cultures were evaluated up to 72 hours by differential and mitotic counting in smears stained according to Pappenheim. Based on mitotic index, caryological curve and percentage of proliferative cells different patterns of proliferation were observed: spindle poisons enhanced the mitotic index, all other investigated cytostatic drugs diminished it, but in a different degree the one of the erythroblasts, of the granuloblasts and of the leukemic blasts. The decrease of the percentage of proliferative precursors did not always correlate with the decrease of the mitotic index, because sometimes an ineffective granulocytopoiesis arised or leukemic blasts entered the Go-phase of the cell cycle. 1-asparaginase was the only drug which increased erythroblasts. Chloramphenicol was the only out of 18 drugs tested on leukemic bone marrow that decreased only the leukemic proliferation and had no influence on the normal one.

Lymphocytes as inducers of mitoses of human morphologically identifiable progenitor cells. Phase contrast observations.

Phase contrast time lapse observations of human bone marrow cells demonstrate the induction of progenitor cell mitoses or amitoses by the touch of locomotive lymphocytes on the cell surface. Interkinetic progenitor cells as well as precursor cells before mitosis were very rarely touched by lymphocytes. We suppose this cell-cell interaction is essential in the T-cell participation in hematopoietic stem cell proliferation.

Clinical and prognostic heterogeneity of non-Hodgkin lymphomas of high-grade malignancy.

Comparison of clinical data of 64 patients with centroblastic lymphoma, 55 patients with immunoblastic lymphoma and 31 patients with lymphoblastic lymphoma not only confirmed the original assumption of high-grade malignancy as proposed by the concept of the Kiel classification but also demonstrated distinct clinical differences, particularly between lymphoblastic lymphoma and the two other entities. Rapid lymph node enlargement as well as steep fall of survival curves within the first year after diagnosis were common characteristics. Bimodal age distribution, predominance of males and early generalization of disease were typical features of lymphoblastic lymphoma; elderly patients and patients with the unclassified subtypes of lymphoblastic lymphoma exhibited the worst prognosis. Whereas patients with centroblastic and immunoblastic lymphomas showed similar distribution of age, sex and initial stage of disease, patients with immunoblastic lymphoma presented more frequently with a reduced performance status and showed a poorer response to radio- and chemotherapy resulting in a worse prognosis discernible after the first year of follow-up. Generalization during course of the disease was significantly more frequent in immunoblastic than in centroblastic lymphoma.

Origin of the early megakaryopoietic progenitor cell and further polyploidization in the thrombopoietic cell series. Phase-contrast observations of human bone marrow.

Continuous observation of living cells by phase-contrast time lapse microcinematography revealed different origins of multinuclear cells and cells with lobed nuclei in the hematopoietic progenitor cell series, which must be classified as megakaryoblasts, the determined progenitor cells of the megakaryopoietic series. Hematopoietic progenitor cells with single nuclei of nuclear diameter 5-13 microns, the promegakaryoblasts, may become polyploid by various means: by postmitotic cytoplasmic fusion, by consecutive nuclear fusion, by endomitosis, or by nuclear amitosis or lobe formation presumably after endoreduplication and nuclear growth. Including amitotic nuclear division in the mitotic rate of normal progenitor cells nearly the half of all these cells in G2 phase (0.4) produce in vitro octoploid megakaryoblasts, respectively osteoclasts. Thrombopoietin-rich sera in the medium enhance the mitotic index of the hematopoietic progenitor cell line from 0.1 to 0.25, but do not change the proportion of polyploid mitoses.