Thymosin beta4 and AcSDKP inhibit the proliferation of HL-60 cells and induce their differentiation and apoptosis

Our previous works have shown that bone marrow stromal cells secrete thymosin beta4 (Tbeta4) and AcSDKP. Tbeta4 and AcSDKP are existed in the conditioned medium of bone marrow endothelial cells. They exerted inhibitory effects on hematopoietic cells and then had protective effect on the early hematopoietic cells, which were cultured in the presence of hematopoietic stimulators. Thymosin beta4 consists of 43 peptides with a molecular weight of 4963. It contains at its N-terminal end the sequence of the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP). This study was performed to evaluate the effect of Tbeta4 and AcSDKP on the growth of HL-60 cells. It was showed that Tbeta4 (10(-11)-10(-7)mol/L) and AcSDKP (10(-11)-10(-7)mol/L) had the dose-dependent inhibitory effect on the proliferation of HL-60 cells. Based on cell morphology and NBT reduction, Tbeta4 and AcSDKP induced differentiation of HL-60 cells. Morphologic and DNA fragment analysis proved that Tbeta4 and AcSDKP induced apoptosis of HL-60 cells. In order to analyze the mechanism of the effects of Tbeta4 and AcSDKP, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of HL-60 leukemic cells was tested and Atlas cDNA Expression Array was performed. The results showed that Tbeta4 and AcSDKP could increased [Ca(2+)](i) by stimulating the release of Ca(2+) from intracellular Ca(2+) pool. Moreover, AcSDKP could also elicit a potent extracelluar calcium influx in HL-60 cells. Tbeta4 could also change apoptotic-related gene expression in leukemic cells, and resulted in the inhibition of proliferation and induction of differentiation and apoptosis of leukemic cells.     

Ku80 as a novel receptor for thymosin beta4 that mediates its intracellular activity different from G-actin sequestering

Our data demonstrate that increased intracellular expression of thymosin beta4(Tbeta4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tbeta4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tbeta4((AcSDKPT/4A)), Tbeta4((KLKKTET/7A)), and Tbeta4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tbeta4. Overexpression of Tbeta4, Tbeta4((AcSDKPT/4A)), and Tbeta4((K16A)) affected intracellular formation of actin filaments. As expected, Tbeta4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of Tbeta4((KLKKTET/7A)) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tbeta4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tbeta4 and Tbeta4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants Tbeta4((KLKKTET/7A)) and Tbeta4((K16A)) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tbeta4. Ku80 and Tbeta4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tbeta4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tbeta4 and mediates its intracellular activity.     

Complement- and IgE-mediated release of histamine from basophils in vitro. V. Differential effects of drugs modulating arachidonic acid metabolism

Basophil degranulation probably plays a significant role in the pathogenesis of different hypersensitivity reactions. These cells can be stimulated to secrete lysosomal histamine in vitro in response to various secretagogues. We compared the effects of drugs, modulating arachidonic acid (AA) metabolism, on histamine release (HR) from human basophils stimulated by anti-IgE antibody or C5a anaphylatoxin. Leukocytes from normal donors were preincubated with drug for 15 min at 22 degrees C, followed by the addition of C5a or anti-IgE for 30 min at 37 degrees C. Bromophenacyl bromide, an inhibitor of AA formation by phospholipase, blocked the effects of C5a and anti-IgE (greater than or equal to 3.3 X 10(-6) M, p less than 0.05 and p less than 0.01, respectively). 3-Amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C, greater than or equal to 3.3 X 10(-5) M) and 5, 8, 11, 14-eicosatetraynoic acid (greater than or equal to 3.3 X 10(-4) M), known inhibitors of both cyclooxygenase (COX) and lipoxygenase (LPX) pathways of AA metabolism, blocked both C5a- and anti-IgE-induced HR (p less than 0.01). Nordihydroguaiaretic acid, an inhibitor of LPX, decreased HR induced by anti-IgE (greater than or equal to 3.3 X 10(-6) M, p less than 0.01) and allergens, but reduced C5a-initiated HR only at a higher concentration (greater than or equal to 7 X 10(-5) M, p less than 0.01). Indomethacin (INDO), an inhibitor of COX, significantly reduced HR caused by C5a (greater than or equal to 3.3 X 10(-8) M, p less than 0.01) and its degradation product C5adesArg, but had no effect or caused slight enhancement of HR initiated by anti-IgE. We confirmed that INDO augments allergen-induced HR. Our findings suggest that there are basic differences in the regulation of C5a- and IgE-mediated basophil degranulation.     

Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhbition of cell proliferation

GH(4)C(1) cells are a clonal strain of rat pituitary cells that synthesize and secrete prolactin and growth hormone. Chronic treatment (longer than 24 h) of GH(4)C(1) cells with epidermal growth factor (EGF) (10(-8) M) decreased by 30-40 percent both the rate of cell proliferation and the plateau density reached by cultures. Inhibition of cell proliferation was accompanied by a change in cellular morphology from a spherical appearance to an elongated flattened shape and by a 40-60 percent increase in cell volume. These actions of EGF were qualitatively similar to those of the hypothalamic tripeptide thyrotropin-releasing hormone (TRH) (10(-7) M) which decreased the rate of cell proliferation by 10-20 percent and caused a 15 percent increase in cell volume. The presence of supramaximal concentrations of both EGF (10(-8)M) and TRH (10(-7)M) resulted in greater effects on cell volume and cell multiplication than either peptide alone. EGF also altered hormone production by GH(4)C(1) cells in the same manner as TRH. Treatment of cultures with 10(-8) M EGF for 2-6 d increased prolactin synthesis five- to ninefold compared to a two- to threefold stimulation by 10(-7) M TRH. Growth hormone production by the same cultures was inhibited 40 percent by EGF and 15 percent by TRH. The half- maximal effect of EGF to increase prolactin synthesis, decrease growth hormone production, and inhibit cell proliferation occurred at a concentration of 5 x 10 (-11) M. Insulin and multiplication stimulating activity, two other growth factors tested, did not alter cell proliferation, cell morphology, or hormone production by GH(4)C(1) cells, indicating the specificity of the EGF effect. Fibroblast growth factor, however, had effects similar to those of EGF and TRH. Of five pituitary cell strains tested, all but one responded to chronic EGF treatment with specifically altered hormone production. Acute chronic EGF treatment with specifically altered hormone production. Acute treatment (30 min) of GH(4)C(1) cells with 10(-8) M EGF caused a 30 percent enhancement of prolactin release compared to a greater than twofold increase caused by 10(-7) M TRH. Therefore, although EGF and TRH have qualitatively similar effects on GH(4)C(1) cells, their powers to affect hormone release acutely or hormone synthesis and cell proliferation chronically are distinct.     

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.     

Mast-cell degranulation induced by physical stimuli involves the activation of transient-receptor-potential channel TRPV2

A characteristic of mast cells is the degranulation in response to various stimuli. Here we have investigated the effects of various physical stimuli in the human mast-cell line HMC-1. We have shown that HMC-1 express the transient receptor potential channels TRPV1, TRPV2 and TRPV4. In the whole-cell patch-clamp configuration, increasing mechanical stress applied to the mast cell by hydrostatic pressure (-30 to -90 cm H(2)O applied via the patch pipette) induced a current that could be inhibited by 10 microM of ruthenium red. This current was also inhibited by 20 microM SKF96365, an inhibitor that is among TRPV channels specific for the TRPV2. A characteristic of TRPV2 is its activation by high noxious temperature; temperatures exceeding 50 °C induced a similar ruthenium-red-sensitive current. As another physical stimulus, we applied laser light of 640 nm. Here we have shown for the first time that the application of light (at 48 mW for 20 min) induced an SKF96365-sensitive current. All three physical stimuli that led to activation of SKF96365-sensitive current also induced pronounced degranulation in the mast cells, which could be blocked by ruthenium red or SKF96365. The results suggest that TRPV2 is activated by the three different types of physical stimuli. Activation of TRPV2 allows Ca(2+) ions to enter the cell, which in turn will induce degranulation. We, therefore, suggest that TRPV2 plays a key role in mast-cell degranulation in response to mechanical, heat and red laser-light stimulation.     

Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model.

The effect of alpha- and beta-thymosin peptides, namely prothymosin alpha (ProT(alpha)), thymosin alpha(1) (T(alpha)1), parathymosin alpha (ParaT(alpha)), thymosin beta(4) (Tbeta4), thymosin beta(10) (Tbeta10), and thymosin beta(9) (Tbeta9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 microl aliquots containing 0.01-4 nmoles of Tbeta4, Tbeta10 or Tbeta9, 0.016-6.66 nmoles of T(alpha)1, 4.1 pmoles-1.66 nmoles of ProT(alpha), and 4.4 pmoles-1.76 nmoles of ParaT(alpha). Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tbeta4, ProT(alpha) and T(alpha)1 were found to enhance angiogenesis, while Tbeta10, Tbeta9 and ParaT(alpha) exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tbeta4 and Tbeta10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tbeta4 on angiogenesis was reversed in the presence of increasing concentrations of Tbeta10 and vice versa. The effect of Tbeta10, Tbeta9, ProT(alpha) and ParaT(alpha), in parallel with Tbeta4 and T(alpha)1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.     

Substance P augmentation of CSF-1-stimulated in vitro myelopoiesis. A two-signal progenitor restricted, tuftsin-like effect

The inflammatory neuropeptide substance P acted as a costimulant for macrophage CSF-1-induced clonal proliferation of murine marrow-derived two signal-dependent mononuclear phagocyte progenitors. Substance P had no effect on clonal proliferation by progenitors responding solely to CSF-1. Substance P fragment 2-11 had no costimulatory activity; however, SP fragment 1-4 retained the full activity of the parent undecapeptide. Fragment 1-4 (ARG-PRO-LYS-PRO), a peptide containing a PRO residue between two positive charges, is a tuftsin-like (THR-LYS-PRO-ARG) tetrapeptide, and tuftsin exerted an identical costimulatory effect. Substance P, SP:1-4, and tuftsin were optimally effective as costimulants at 10(-7) to 10(-6) M. (ALA1)-tuftsin, an inhibitory analog of tuftsin, was a potent negative regulator of two signal-dependent colony formation. (ALA1)-tuftsin at concentrations less than or equal to 10(-9) M exerted dose-dependent inhibition of the positive effects of optimal concentrations of all of the co-stimulants tested, including bacterial LPS. The inhibitory tetrapeptide was equivalent in activity to ferritin, an established inhibitor of two signal-dependent colony formation. The results indicated that SP may influence myelopoiesis in addition to its other inflammatory and immunopotentiating properties. In addition, a potentially valuable modulator of SP and LPS responses in this system, (ALA1)-tuftsin, was identified.     

The effects of A-ring and D-ring metabolites of estradiol on the proliferation of vascular endothelial cells

The effects of 14 estradiol metabolites on the proliferation of cultured endothelial cells of human umbilical cord veins were examined and compared with that of their parent substance estradiol. The relationship between dosage and effect was tested over the pharmacological concentration range of 10(-8) to 10(-5) M. Estradiol showed a biphasic behaviour, in the form of stimulation at low concentrations and inhibition at the highest concentration. All 10 A-ring metabolites tested stimulated the growth of the endothelial cells at the lower concentrations. At the highest concentration, the 5 A-ring metabolites: 2-hydroxyestrone, 2-hydroxyestradiol, 2-hydroxyestriol, 4-hydroxyestrone and 4-hydroxyestradiol caused significant inhibitions. Except for the 2-hydroxyestradiol, methylation of these metabolites resulted in the loss of the proliferation inhibiting effect. The D-ring metabolites showed no marked effects compared to the A-ring metabolites except for 16alpha-hydroxyestrone which had an inhibiting effect from 10(-7) to 10(-5) M. Our results show that estradiol metabolites can influence the growth of vascular endothelial cells in the concentration range tested. While the antiproliferative action of 2-methoxyestradiol has been known for some time this study is the first to show the potential capacity of non-methylated metabolites of the A-ring metabolism in inhibiting endothelial proliferation. This may open up new clinical pharmacological aspects in the anti-angiogenetic treatment of tumors.     

The comparative analysis of the effect of oligopeptides and amino acids on the development of rat different tissue culture

The effect the synthetic tetra-, tripeptides and amino acids, composed them, in concentration 10(-12) M was investigated in organotypic tissue culture on the cell proliferation and apoptosis development in six tissue explants (ecto-, meso- and entodermal genesis) in rats at age 3 months old. The tetrapeptide demonstrated the greater tissue-specificity, as compared to the tripeptides and amino acids. Each tetrapeptide stimulated proliferation in only one corresponded tissue type. Less tissue-specificity was observed in the tripeptides, which stimulated proliferation and apoptosis in one (one case)--four tissues. The amino acids stimulated proliferation and apoptosis in three--five tissues. It can be suggested, that tissue-specificity in these biologically active substances is depended on the structure complexity. It is discussed, that the tetrapeptide tissue-specificity is related to the complementar interaction to the DNA site-specific blocks.     

The effect of insulin on amino acid metabolism and glycogen content in isolated liver cells of juvenile coho salmon, Oncorhynchus kisutch

Amino acid transport and [14C]leucine incorporation into liver proteins as well as the secretion of proteins into incubation medium were studied in liver cells isolated from coho salmon (Oncorhynchus kisutch) parr. Pink salmon (Oncorhynchus gorbuscha) or mammalian (bovine) insulin caused a significant increase in TCA-precipitable radioactivity from both cells and incubation medium. The effects appeared at insulin concentration of 10(-8) M with a maximal response at 5 X 10(-8) M. The radioactivity of the TCA-soluble fraction was not changed by insulin. Insulin increased the amount of the non-metabolized amino acid [14C]cycloleucine, in the TCA-soluble fraction of hepatocytes. The glycogen content of hepatocytes was increased in the presence of insulin at 10(-9) M but was not changed from the control value in the presence of insulin at 10(-8) M.     

Pertussis toxin pretreatment reveals differential effects of adenosine analogs on IgE-dependent histamine and peptidoleukotriene release from RBL-2H3 cells

The effects of adenosine (A) and the nonmetabolizable adenosine analogs, N-ethylcarboxamidoadenosine (NECA), L-phenylisopropyladenosine (L-PIA), D-PIA and 2-chloroadenosine (2CHA) were examined on the IgE-dependent mediator release from RBL-2H3 cells, a model for mast-cell function. Adenosine and the adenosine analogs failed to influence mediator release from cells, previously sensitized with monoclonal anti-TNP mouse immunoglobulin E (anti-TNP IgE), when added alone. When added prior to conjugated trinitrophenol-ovalbumin (TNP-OVA), adenosine and the adenosine analogs (10(-8)-10(-4) M) significantly potentiated the release of both histamine (marker for degranulation) and peptidoleukotrienes (LT) (marker for de novo synthesized mediators). The effects were concentration-dependent with the potency order being L-PIA greater than NECA greater than A greater than D-PIA, 2CHA. The stimulatory effect on both histamine and LT release were reversed by prior treatment of the cells with pertussis toxin but not by the purinoceptor antagonists, theophylline and 8-phenyltheophylline, nor adenosine uptake blockers. At higher concentrations (above 10(-5) M), adenosine and adenosine analogs were also inhibitory on LT but not on histamine release. This inhibition was more evident on pertussis-toxin-treated cells in which there was no effect of adenosine or adenosine analogs on histamine release, but a concentration-dependent inhibition of IgE-dependent LT release. These findings demonstrate that adenosine analogs have two distinct mechanisms on mediator release from RBL-2H3 cells; a stimulatory effect on both histamine and LT release, mediated via a pertussis-toxin-sensitive G protein and an inhibitory effect on LT release via a pertussis-toxin-insensitive pathway. An abstract of this work has been published.     

Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125I-labeled human FSH to testis receptor.     

Thymosin beta4 and thymosin beta4-derived peptides induce mast cell exocytosis

The peptide thymosin beta4 (Tbeta4) promotes angiogenesis and wound healing. Mast cells are involved in these processes as well and therefore we investigated the effect of Tbeta4 on mast cells. Exposure to 0.2-2000nM Tbeta4 induced mediator release (up to 23%) in murine peritoneal and human HMC-1 mast cells in a concentration-dependent manner. While the peptide AcSDKP, matching the 4 N-terminal amino acid residues of Tbeta4, mediated low but detectable mediator release, peptides corresponding to the Tbeta4 amino acid sequences 16-38 and 17-23 stimulated mast cells mediator release on a level equal to or higher than that observed with native Tbeta4. These observations and certain characteristics of Tbeta4-mediated mast cell activation suggest that the actin-binding motif LKKTET present in Tbeta4 (amino acid 17-22) might be implicated in this process. Thus, Tbeta4 activates mediator release in mast cells by a process that possibly involves an actin-binding motif and this could be important for understanding the mechanisms of Tbeta4-mediated effects in vivo.     

Mitogenic, melanogenic, and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens.

The following studies have been undertaken to compare and correlate the effects of 12-O-tetradecanoylphorbol acetate (TPA), basic fibroblast growth factor (bFGF), cholera toxin (CT), and isobutyl methylxanthine (IBMX) on neonatal human melanocyte (NHM) proliferation, tyrosinase activity, and cyclic adenosine monophosphate (cAMP) concentration. NHM proliferated at a maximal rate in medium containing 8 nM TPA, 200 ng/ml CT, and 10(-4) M IBMX. TPA alone did not result in optimal melanocyte proliferation, and, as previously shown, its mitogenic effect was greatly enhanced by the addition of CT and IBMX individually or concomitantly. Human recombinant (hr) bFGF could replace TPA in the NHM growth medium. Maximal proliferation was achieved using 3 ng/ml hrbFGF, 20 ng/ml CT, and 10(-4) M IBMX. The mitogenic effect of 1.2 ng/ml hrbFGF was potentiated in the concomitant but not individual presence of CT and IBMX. TPA alone in the absence of CT and IBMX caused a dose-dependent stimulation of tyrosinase activity. Maximal tyrosinase activity was obtained in the presence of 0.8 nM TPA, 20 ng/ml CT, and 10(-4) M IBMX. Unlike TPA, hrbFGF alone resulted in inhibition of tyrosinase activity. In the presence of hrbFGF, tyrosinase activity was potentiated by CT and IBMX, but not by CT alone. Neither TPA nor hrbFGF alone could increase intracellular cAMP levels. The effects of CT and IBMX on intracellular cAMP concentration were enhanced to a greater extent by TPA than by hrbFGF. Under our experimental conditions, in the presence of hrbFGF, CT but not IBMX resulted in a dose-dependent increase in cAMP concentration. Further studies on NHM will be aimed at determining the exact role of protein kinase C (PKC) in regulating proliferation and melanogenesis and the mechanism(s) activated by hrbFGF.     

In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.     

T lymphocytes and their CD4 subset are direct targets for the inhibitory effect of calcitriol

We studied the direct effects of the hormone calcitriol on the activation and proliferation of pure T lymphocytes and their subsets. Calcitriol inhibited the proliferation of T lymphocytes stimulated in the absence of monocytes with phytohemagglutinin (PHA) and either a monocytic culture supernatant or a combination of monocyte-derived interleukin 1 and interleukin 6. This inhibition was not influenced by the concentration of the stimulating agents. The minimal effective concentration of calcitriol was 10(-10) M. In contrast, the interleukin 2 (10 U/ml)-driven growth of PHA-stimulated T lymphocytes was not significantly altered by calcitriol at 10(-8) M. The hormone had also no influence on the T lymphocyte proliferation induced by a combination of PHA and the anti-CD28 monoclonal antibody 9.3. Pure T lymphocytes, after incubation for 5 days with PHA and monocytic factors, expressed a high level of transferrin receptors. This phenomenon was strongly suppressed on both CD4 and CD8 subsets when 10(-8) M calcitriol had been present during the culture. Moreover, the proliferation of pure CD4 cells was directly inhibited by calcitriol in similar conditions as for unseparated T lymphocytes. We conclude that T lymphocytes and their CD4 subset are direct targets for the inhibitory effect of calcitriol.     

Mouse fetal kidneys in serum-free organ culture: effects of epidermal growth factor and hydrocortisone.

1. The present study was undertaken to determine whether epidermal growth factor (EGF, 100 ng/ml) or hydrocortisone (HC, 10(-8)-10(-5) M) directly influence proliferation and differentiation of mouse fetal kidney maturing in serum-free organ culture. 2. Addition of EGF to the medium significantly stimulated DNA synthesis after 2 and 5 days of culture. Labelled nuclei were mainly localized in the mesenchymal tissue. Protein synthesis remained unchanged. Activities of three hydrolases, markers of brush border differentiation, were reduced. 3. Hydrocortisone (HC), at all concentrations used, significantly inhibited DNA synthesis. Labelled nuclei were distributed in various cell populations of both control and treated explants. Protein synthesis was stimulated by 10(-7) M after 5 days of culture. Hydrolase activities were slightly modified by HC treatment. 4. The present results indicate that EGF stimulates whereas HC decreases proliferation. Both factors have regulatory effects on brush border maturation. 5. Thus, this culture model is a valuable tool for the study of nephrogenesis.     

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.     

Effect of apoptosis-related compounds on Ca2+ transport system in isolated rat liver nuclei

The effect of various inhibitors of DNA topoisomerase II, which has been shown to induce apoptotic cell death, on Ca2+ transport in isolated rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. The presence of aurintricarboxylic acid (ATA; 10-6 to 10-4 M), etoposide (10-4 M), genistein (10-5 and 10-4 M) or amsacrine (10-4 M) in the reaction mixture caused a significant increase in Ca2+ release from the nuclei. Also, these compounds (10-4 M) significantly inhibited Ca2+ uptake by the nuclei. However, the presence of ATA (10-5 and 10-4 M) in the enzyme reaction mixture did not significantly inhibit Ca2+-ATPase activity, which is involved in the nuclear Ca2+ uptake, in the liver nuclei, while etoposide (10-4 M), genistein (10-4 M) and amsacrine (10-4 M) appreciably decreased the enzyme activity. Meanwhile, addition of Ca2+ clearly activated DNA fragmentation in the liver nuclei. The Ca2+ activated DNA fragmentation was significantly prevented by the presence of etoposide, genistein and amsacrine with the concentrations of 10-5 and 10-4 M in the reaction mixture, although ATA (10-5 and 10-4 M) had no effect. The present study demonstrates that some apoptosis inducible compounds used can influence on Ca2+ transport system in isolated rat liver nuclei, suggesting a decrease of nuclear Ca2+ level involved in nuclear functions. (Mol Cell Biochem 166: 183-189, 1997)