Role of Chk1 in the differentiation program of hematopoietic stem cells

Hematopoietic stem cells (HSC) isolated from umbilical cord blood (UCB) were treated with ionizing radiation (IR) and sensitivity and IR induced checkpoints activation were investigated. No difference in the sensitivity and in the activation of DNA damage pathways was observed between CD133+ HSC and cells derived from them after ex vivo expansion. Chk1 protein was very low in freshly isolated CD133+ cells, and undetectable in ex vivo expanded UCB CD133+ cells. Chk1 was expressed only on day 3 of the ex vivo expansion. This pattern of Chk1 expression was corroborated in CD133+ cells isolated from peripheral blood apheresis collected from an healthy donor. Treatment with a specific Chk1 inhibitor resulted in a strong reduction in the percentage of myeloid precursors (CD33+) and an increase in the percentage of lymphoid precursors (CD38+) compared to untreated cells, suggesting a possible role for Chk1 in the differentiation program of UCB CD133+ HSC.     

Rhythms of enzymatic activity in maternal and umbilical cord blood

The 24-h activity patterns of variouns enzymes were determined in human serum, red blood cells and white blood cells of maternal and umbilical cord blood. Blood was drawn from the brachial vein of mothers and from the umbilical cord within ten minutes after delivery. Corresponding blood specimens were obtained from 83 spontaneous labors, occurring at different hours over a period of 60 days. For each variable (variable=activity of a specific enzyme in one of the blood components) the results were grouped according to delivery hour, forming a 24-h pattern which was analyzed to elucidate time dependency. Five out of six corresponding maternal and fetal variables were similar with regard to pattern and peak time. The activity rhythms of glyceraldehyde-3-phosphate dehydrogenase and glucose phosphate isomerase in red blood cells of mothers and fetuses possessed a significant bimodal pattern. The activity rhythms of the latter enzyme in white blood cells and sera exhibited a significant 24-h period. Hexosaminidase activity exhibited a distinct 24-h rhythm in maternal white blood cells, but no significant rhythm could be detected in the fetal white blood cells. The activity of hexosaminidase showed, identical 24-h patterns in maternal and cord serum when analyzed by best fit cosine, and no significant time-dependency when analyzed by ANOVA.     

Rhythms of enzymatic activity in maternal and umbilical cord blood.

The 24-h activity patterns of various enzymes were determined in human serum, red blood cells and white blood cells of maternal and umbilical cord blood. Blood was drawn from the brachial vein of mothers and from the umbilical cord within ten minutes after delivery. Corresponding blood specimens were obtained from 83 spontaneous labors, occurring at different hours over a period of 60 days. For each variable (variable = activity of a specific enzyme in one of the blood components) the results were grouped according to delivery hour, forming a 24-h pattern which was analyzed to elucidate time dependency. Five out of six corresponding maternal and fetal variables were similar with regard to pattern and peak time. The activity rhythms of glyceraldehyde-3-phosphate dehydrogenase and glucose phosphate isomerase in red blood cells of mothers and fetuses possessed a significant bimodal pattern. The activity rhythms of the latter enzyme in white blood cells and sera exhibited a significant 24-h period. Hexosaminidase activity exhibited a distinct 24-h rhythm in maternal white blood cells, but no significant rhythm could be detected in the fetal white blood cells. The activity of hexosaminidase showed, identical 24-h patterns in maternal and cord serum when analyzed by best fit cosine, and no significant time-dependency when analyzed by ANOVA.     

Ultrastructural identification of human tonsil T-lymphocytes by peroxidase-conjugated anti-HTLA serum.

A horse anti-serum rendered specific for human T-lymphocytes was conjugated with peroxidase and used for ultrastructural identification of human tonsil T-lymphocytes. With T- and B-enriched suspensions, virtually all T-lymphocytes were labelled with Po-anti-HTLA, whereas no B-cells were stained with this conjugate. The labelling HTLA conjugate confirm the ultrastructural characteristics of thymus-dependant cells.     

Immunoglobulin synthesis by cord blood lymphocytes

Summary The IgG, IgA and IgM synthesis by adult peripheral blood and cord blood lymphocytes incubated alone and with pokeweed mitogen was quantitated. The cord blood lymphocytes produced no immunoglobulin even with mitogen stimulation while the adult peripheral blood lymphocytes responded to the mitogen with a significant (p<0.04) increase in immunoglobulin production.     

Marrow mesenchymal stem cells transduced with TPO/FL genes as support for ex vivo expansion of hematopoietic stem/progenitor cells

A new marrow-derived mesenchymal stem cell (hMSC) line that could support expansion of hematopoietic stem/progenitor cells (HSPCs) was developed. Primary hMSCs were infected with retrovirus containing Flt-3 ligand and thrombopoietin genes. CD34+ cells from cord blood were expanded with primary hMSCs or transduced hMSCs. The expansion of total nucleated cells, CD34+ cells and mixed colonies containing erythroid and myeloid cells and megakaryocytes for 2 weeks coculture with transduced hMSCs was remarkably increased. The outputs of long-term culture-initiating cells for 2 and 4 weeks coculture with transduced hMSCs were also largely increased. The expansion rates of HSPCs with transduced hMSCs were unchanged for 6 weeks. In contrast, the expansion rates of HSPCs with primary hMSCs declined drastically through 6 weeks. SCID-repopulating cell expansion with transduced hMSCs for 4 weeks was significantly higher than that of uncultured CD34+ cells and HSPCs expanded with primary hMSCs. Received 21 June 2005; received after revision 30 July 2005; accepted 24 August 2005     

Immunoglobulin synthesis by cord blood lymphocytes.

The IgG, IgA and IgM synthesis by adult peripheral blood and cord blood lymphocytes incubated alone and with pokeweed mitogen was quantitated. The cord blood lymphocytes produced no immunoglobulin even with mitogen stimulation while the adult peripheral blood lymphocytes responded to the mitogen with a significant (p less than 0.04) increase in immunoglobulin production.     

Augmentation of natural antiganglioside IgM antibodies in lower motor neuron disease (LMND) and role of CD5+ B cells

IgM antibodies directed against neuronal gangliosides GM₁ , GM₂ , GD_1a , GD_1b and GT_1b occur in normal individuals and their level significantly decreases with age. Patients with lower motor neuron disease (LMND) produce high levels of these autoantibodies. AntiGM₁ IgM is selectively augmented. In these patients, the CD5+ (B1) and CD5− (B2) subsets of B cells are not distinct entities but range from those without detectable CD5 marker to those with high CD5+ expression. B1 B cells were sorted to homogeneity, but B2 B cell cannot be isolated to homogeneity because of the presence of B1 cells with low CD5 expression. In short term cultures both the subsets produced IgM antibodies, but the antibodies reacted better with desialylated GM₁ than with GM₁ . Cycloheximide (Cx) (0.35 mM) largely blocked IgM synthesis of the B1 B cells but inhibition of the B2 B cells was incomplete, possibly due to shedding of cytophilic antibodies as well as to the presence of B1 phenotype with loss of CD5 expression. CD5+ B cells may be involved in the production of antiglycolipid IgM. Received 9 June 1997; received after revision 21 July 1997; accepted 28 July 1997     

The influence of sodium-8-chlorotheophyllinate (S8CT) on immune processes

Summary S8CT injected at the time of immunization significantly enhances specific IgM-production but has no effect on IgG-formation. Mitogen (PHA-P) induced macrophage migration inhibition of cells of S8CT pretreated animals is reduced. The same effect is observed, when normal cells are tested in the presence of S8CT in vitro. Blast transformation of B-lymphocytes but not of thymocytes is significantly stimulated by S8CT. Acid phosphatase activity is also stimulated in B-cells and- to a lesser degree-in cortisone-resistant T-lymphocytes whereas the activity of the total thymocyte population is reduced. No effect was seen on phagocytosis and intracellular bactericidal activity. A stimulatory effect of S8CT for B-cells is postulated.Presented in part at the Xth International Congress of Allergology and Clinical Immunology, Jerusalem/Israel 1979, Nov. 4–11.     

Ultrastructural identification of human tonsil T-lymphocytes by peroxidase-conjugated anti-HTLA serum

Summary A horse anti-serum rendered specific for human T-lymphocytes was conjugated with peroxidase and used for ultrastructural identification of human tonsil T-lymhocytes. With T- and B-enriched suspensions, virtually all T-lymphocytes were labelled with Po-anti-HTLA, whereas no B-cells were stained with this conjugate. The labelling was found to be irregularly distributed on the plasma membrane of T-cells. Direct identification with specific Po-anti-HTLA conjugate confirm the ultrastructural characteristics of thymus-dependant cells.This work was supported by I.N.S.E.R.M. grants No. 74-5-027-01 and A.T.P. No. 73-16.Acknowlegments. We are indebted toMartine Blanc who prepared the cell suspensions and toDanielle Germain who did the preparations for electron microscopic examination.     

Loss of huntingtin-associated protein 1 impairs insulin secretion from pancreatic β-cells

Hap1 was originally identified as a neuronal protein that interacts with huntingtin, the Huntington’s disease (HD) protein. Later studies revealed that Hap1 participates in intracellular trafficking in neuronal cells and that this trafficking function can be adversely affected by mutant huntingtin. Hap1 is also present in pancreatic β-cells and other endocrine cells; however, the role of Hap1 in these endocrine cells remains unknown. Using the Cre-loxP system, we generated conditional Hap1 knockout mice to selectively deplete the expression of Hap1 in mouse pancreatic β-cells. Mutant mice with Hap1 deficiency in pancreatic β-cells had impaired glucose tolerance and decreased insulin release in response to intraperitoneally injected glucose. Using cultured pancreatic β-cell lines and isolated mouse pancreatic islets, we confirmed that decreasing Hap1 could reduce glucose-mediated insulin release. Electron microscopy suggested that there was a reduced number of insulin-containing vesicles docked at the plasma membrane of pancreatic islets in Hap1 mutant mice following intraperitoneal glucose injection. Glucose treatment decreased the phosphorylation of Hap1A in cultured β-cells and in mouse pancreatic tissues. Moreover, this glucose treatment increased Hap1’s association with kinesin light chain and dynactin p150, both of which are involved in microtubule-dependent trafficking. These studies suggest that Hap1 is important for insulin release from β-cells via dephosphorylation that can regulate its intracellular trafficking function.     

Erythrocyte catechol-0-methyltransferase and plasma dopamine-beta-hydroxylase in human umbilical cord blood

Plasma dopamine-beta-hydroxylase enzymatic activity and immunoreactive protein levels in human umbilical cord blood are only about 2% as great as values in the blood of older subjects. Erythrocyte catechol-0-methyltransferase activity levels in umbilical cord blood are very similar to those in the blood of adult subjects.     

Shape change of blood platelets induced by myelin basic protein.

Myelin basic protein (MBP) isolated from bovine spinal cord caused a marked shape change reaction of human blood platelets which was not accompanied by the release reaction and not inhibited by methysergide and spiroperidol. Only those basic proteins, including MBP, which had previously shown to exert neuronal depolarisation also induced the shape change reactions. Therefore, these findings may extend the use of platelets as neuronal models.     

Shape change of blood platelets induced by myelin basic protein

Summary Myelin basic protein (MBP) isolated from bovine spinal cord caused a marked shape change reaction of human blood platelets which was not accompanied by the release reaction and not inhibited by methysergide and spiroperidol. Only those basic proteins, including MBP, which had previously shown to exert neuronal depolarisation also induced the shape change reaction. Therefore, these findings may extend the use of platelets as neuronal models.Acknowledgment. We thank Miss B. Gieux and Miss M. Handschin for skilful technical assistance.     

Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63 Ag8 cells

Summary Using the suspension cell line P3X63 Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (M_r 40 kD) and Semliki Forest virus core protein (M_r 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0°C. Subsequent resealing of the pores was completed after a 5-min incubation at 37°C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37°C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K⁺, Mg²⁺, and Ca²⁺) and resealing (in the presence of K⁺, Mg²⁺, and Ca²⁺), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.     

Zur zellulären Aminosäure-Inkorporation im in- und exkretorischen Pankreas unter experimentellen Bedingungen

Summary After glucagon and tolbutamide administration, the protein synthesis in the nucleus increases, especially in the B- but to a lesser extent also in the A-cells. Similar effects are noted in the cytoplasma of B-cells. Already 2 h following alloxan premedication, there is no activity in the nucleus and cytoplasm of B-cells, whereas in the A-cells they are markedly labelled.     

HAb18G/CD147-mediated calcium mobilization and hepatoma metastasis require both C-terminal and N-terminal domains

HAb18G/CD147 is a heavily glycosylated protein containing two immunoglobulin superfamily domains. Our previous studies have indicated that overexpression of HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca²⁺ entry by nitric oxide (NO)/cGMP. In the present study, we investigated the structure-function of HAb18G/CD147 by transfecting truncated HAb18G/CD147 fragments into human 7721 hepatoma cells. The inhibitory effect of HAb18G/CD147 on 8-bromo-cGMP-regulated thapsigargin-induced Ca²⁺ entry was reversed by the expression of either C or N terminus truncated HAb18G/CD147 in T7721C and T7721N cells, respectively. The potential effect of HAb18G/CD147 on metastatic potentials, both adhesion and invasion capacities, of hepatoma cells was abolished in T7721C cells, but not affected in T7721N cells. Release and activation of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were found to be enhanced by the expression of HAb18G/CD147, and this effect was abolished by both truncations. Thapsigargin significantly enhanced release and activation of MMPs (MMP-2 and MMP-9) in non-transfected 7721 cells, and this effect was negatively regulated by SNAP. However, no effects of thapsigargin or SNAP were observed in T7721 cells, and expression of HAb18G/CD147 enhanced secretion and activation of MMPs at a stable and high level. Taken together, these results suggest that both ectodomain and intracellular domains of HAb18G/CD147 are required to mediate the effect of HAb18G/CD147 on the secretion and activation of MMPs and metastasis-related processes in human hepatoma cells by disrupting the regulation of NO/cGMP-sensitive intracellular Ca²⁺ mobilization although each domain may play different roles.Received 1 April 2004; received after revision 15 June 2004; accepted 22 June 2004