Evidence for an adaptive DNA repair pathway in CHO and human skin fibroblast cell lines

When Escherichia coli is chronically exposed to very low, nontoxic doses of a monofunctional alkylating agent (notably N-methyl-N'-nitro-nitrosoguanidine, MNNG), the adaptive DNA repair pathway is induced which enables the bacteria to resist the killing and mutagenic effects of further alkylation damage. Mutation resistance in adapted bacteria is achieved, at least partly, by a greatly increased capacity of the cells to eliminate the minor DNA alkylation product O6-methyl-guanine, which has been strongly implicated as premutagenic and precarcinogenic. We now show that the chronic treatment of a Chinese hamster ovary (CHO) and a SV40-transformed human skin fibroblast (GM637) cell line with non-toxic levels of MNNG renders the cells resistant to the induction of sister chromatid exchange (SCE) by further alkylation damage. CHO cells also become resistant to killing (GM637 cells have not yet been tested). Having ruled out explanations such as changes in cell cycle distribution, mutagen permeability and mutagen detoxification, we conclude that resistance is probably achieved by the cells becoming more efficient at repairing alkylation damage, analogous to the adaptive response of E. coli.     

Strand- and sequence-specific attenuation of N-methyl-N'-nitro-N-nitrosoguanidine-induced G.C to A.T transitions by expression of human 6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells

The effect of human O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxicity, the mutagenicity, and the specific kinds of base substitutions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were examined in non-MGMT transfected Chinese hamster ovary cells (CHOM cells) and in those cells which had been transfected with human MGMT complementary DNA (AGT cells). AGT cells containing a high level of human MGMT activity were markedly more resistant to the cytotoxic and mutagenic effects of MNNG than CHOM cells which had no detectable MGMT activity. The dosages of MNNG which reduced to 50% of colony forming ability were estimated to be 0.8 microM for CHOM and 10 microM for AGT cells. The induction frequency of 6-thioguanine-resistant cells was significantly declined in AGT cells. At 4 microM MNNG, this frequency was declined from 273 mutants/10(6) viable CHOM cells to 13 mutants/10(6) viable AGT cells. The entire coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene in 37 AGT and 22 CHOM mutants was characterized by direct sequencing of the mRNA-polymerase chain reaction-amplified complementary DNA. Base changes at the intron-exon boundaries of the hprt DNA in the splicing mutants were further examined. Those results indicated that G to A transitions were significantly reduced in MNNG-treated AGT cells (chi 2 test, P < 0.001), suggesting that O6-methylguanine was repaired error free by human MGMT. In contrast, no difference arose in the frequencies of T to C transitions induced by MNNG in these two populations. All of the G to A transitions induced in AGT cells were located on the nontranscribed strand, assuming that the causative lesion was O6-methylguanine (P < 0.05). Such a strand specificity was not observed in CHOM mutants. Most of the G to A transitions observed in CHOM mutants were located at the middle guanine of 5'-GGPu sequences. Transitions observed at these sites, particularly 5'-GGG, were significantly reduced in AGT mutants (P < 0.05). Our results have suggested that human MGMT specifically repairs O6-methylguanine with a preference to remove those located on the transcribed strand and middle guanine of 5'-GGG.     

Changes in urinary tissue kallikrein excretion in black African women with hypertensive disorders of pregnancy

The induction of micronuclei by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and their reduction by the cardioprotective synthetic antioxidant, stobadine were studied in hamster V79 cells cultured in vitro. The micronuclei derived from acentric fragments or from whole chromosomes were evaluated with the help of an immunofluorescent staining using antikinetochore antibodies from the serum of scleroderma (CREST syndrome) patients. Our results showed that MNNG (0.5 microgram/ml) induced mainly kinetochore-negative micronuclei. At 6, 24 and 48 h after MNNG treatment, we measured a 2.7-, 4.3-, and 7.0-fold increase, respectively, of kinetochore-negative micronuclei over the controls. The increase of kinetochore-positive micronuclei was rather low and represented at 6, 24 and 48 h, respectively 0.9-, 1.8- and 2.6-fold increases over the controls. Stobadine decreased the level of kinetochore-negative micronuclei at 6, 24 and 48 h to approximately one-half; the frequency of kinetochore-positive micronuclei was reduced only at 6 h. We suppose that the antioxidant stobadine reduces the induction of micronuclei by MNNG by scavenging of MNNG-induced highly reactive OH radicals which cause chromosomal damage.     

Antinociceptive effects of SC-39566, an opioid dipeptide arylalkylamide, in the Rhesus monkey

We stably expressed the rat D1A dopamine receptor in mouse fibroblast LTK- cells and obtained specific ligand binding and functional activity characteristic of the D1A dopamine receptor coupled to stimulation of adenylyl cyclase. In the transfected cells, the selective D1 agonist fenoldopam caused a concentration-dependent inhibition of Na+/K(+)-ATPase activity, achieving maximum inhibition of approximately 30%. The latter was abolished by the selective D1 antagonist (+)-SCH 23390 and by the specific protein kinase A inhibitor protein kinase inhibitor-(6-22) amide. In the nontransfected cells, fenoldopam did not affect Na+/K(+)-ATPase activity. 8-Chlorophenylthio-cAMP inhibited Na+/K(+)-ATPase activity in both transfected and nontransfected cells; this effect was blocked by protein kinase inhibitor-(6-22). These results indicate that the inhibition of Na+/K(+)-ATPase activity induced by agonist occupancy of D1A receptors is mediated by protein kinase A.     

Increase in telomere sequence-binding activity in normal human fibroblasts in senescence or in cells treated with phorbol ester or N-methyl-N'-nitro-N-nitrosoguanidine

The telomere is a specialized chromatin structure composed of unique repetitive DNA sequences and specific nuclear proteins. Telomere sequence-binding activity was measured by a mobility shift assay using nuclear extract from normal human fibroblasts. The specific binding activity to the telomere sequence increased in cells that were in a senescence state compared to that in cells at early population doublings. Treatment of cells with tumor promoting phorbol ester TPA induced an increase in the telomere sequence binding activity of nuclear extract in young cells, but the increase was marginal in senescent cells. DNA-damaging N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also increased the telomere sequence binding activity in young cells, but not in senescent cells. As a reference, we measured the binding activity to NFkB sequence. It was activated by TPA or okadaic acid, but was not affected by MNNG or in senescence. The increase in telomere sequence-binding activity seemed to depend on activation of tyrosine phosphorylation, since an inhibitor of Tyr-kinase abolished the increase in telomere-binding activity. The molecular weight of the major binding factor in the normal human fibroblasts was approximately 32 kDa which is different from that of the telomere-associated protein, TRF-1.     

A role for glutamate and its receptors in the regulation of oligodendrocyte development in cerebellar tissue slices

We tested the hypothesis that the neurotransmitter glutamate would influence glial proliferation and differentiation in a cytoarchitecturally intact system. Postnatal day 6 cerebellar slices were maintained in organotypic culture and treated with glutamate receptor agonists or antagonists. After dissociation, cells were stained with antibodies for different oligodendrocyte developmentally regulated antigens. Treatment of the slices with the glutamate receptor agonists kainate or alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid significantly decreased the percentage of LB1(+), NG2(+) and O4(+) cells, and their bromodeoxyuridine labeling index. The non-N-methyl-D-aspartate glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione increased the percentage and bromodeoxyuridine labeling of LB1(+), NG2(+) and O4(+) cells. In intact slices, RNA levels of the oligodendrocyte gene for 2',3'-cyclic nucleotide 3'-phosphodiesterase were decreased by kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and increased by 6,7-dinitroquinoxaline-2,3-dione. The percentage of astrocytes was not modified by kainate, alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or 6, 7-dinitroquinoxaline-2,3-dione. Treatment with the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid did not alter the percentage of O4(+) cells, nor their proliferation. Incubation with the gamma-aminobutyric acid receptor antagonist bicuculline did not modify the percentage of LB1(+), A2B5(+) and O4(+) cells. In purified cerebellar oligodendrocyte progenitor cells, glutamate receptor agonists blocked K+ currents, and inhibited cell proliferation and lineage progression. The K+ channel blocker tetraethylammonium also inhibited oligodendrocyte progenitor cell proliferation. These findings indicate that in rat cerebellar tissue slices: (i) glutamate specifically modulates oligodendrocyte but not astrocyte development through selective activation of alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and (ii) cell depolarization and blockage of voltage-dependent K+ channels is likely to be the triggering mechanism.     

Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples

To assess genotoxic burdens from chemicals, it is necessary to relate observations in experimental animals to humans. The success of this extrapolation would be increased by including data on chemical activities in human tissues. Therefore, we have developed techniques to assess DNA damage in human gastric and nasal mucosa (GM, NM) cells. Biopsy samples were obtained during gastroscopy from macroscopically healthy tissue of the stomach or from healthy nasal epithelia during surgery. The specimens were incubated for 30-45 min at 37 degrees C with a digestive solution. We obtained 1.5-8 x 10(6) GM cells and 5-10 x 10(5) NM cells per donor, both with viabilities of 80-95%. The cells were incubated in vitro for 1 hr at 37 degrees C with the test compounds added in their appropriate solvents. In GM cells, we studied N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium dichromate (Na2Cr2O7), nickel sulphate (NiSO4), cadmium sulphate (CdSO4), and lindane. In NM cells, lindane was investigated. Each compound was assessed for DNA damaging activity in cells of at least three different human donor samples using the microgel single cell assay. Similar studies were performed with GM and NM cells obtained from Sprague-Dawley rats. We have found human GM cells to be more sensitive to the genotoxic activity of MNNG than rat GM cells (low effective concentration [LEC] = 0.16 and 0.625 micrograms/ml for human and rat, respectively). Human cells were also more sensitive to the cytotoxic/genotoxic activity of NiSO4 (LEC = 5 and 19 mumoles/ml for human and rat, respectively). CdSO4 was genotoxic in human GM cells (LEC = 0.03-0.125 mumoles/ml), whereas no dose-related genotoxicity was observed in rat GM at concentrations up to 0.5 mumoles/ml. In contrast, approximately equal responses regarding genotoxicity and cytotoxicity were observed in rat and human GM for Na2Cr2O7 (0.25-1 mumoles/ml). Lindane, however, was genotoxic in three out of four rat GM but not in human GM cells (0.5-1 mumoles/ml), whereas it was active in both rat and human NM cells. Together with other recently published in vivo findings, our results with lindane can be interpreted according to a parallelogram approach. In view of possible human exposure situations and the sensitivities of the two target tissues from both species, the data imply that lindane will pose a health risk to humans by inhalation but not by ingestion.     

Diacylglycerol molecular species in plasma membrane and microsomes change transiently with endothelin-1 treatment of glioma cells

Agonist-induced intracellular signal transduction often involves activation of protein kinase C by diacylglycerol (DAG) released from membrane phospholipids by phospholipases. Using either DAG kinase or HPLC assays to quantitatively determine DAG mass, we observed a time-dependent increase in DAG accumulation upon incubation of rat C6 glioma cells with 200 nM endothelin-1 (ET-1). Total cell DAG rapidly increased by 25-35% from a basal level of 4.5 +/- 0.3 nmol/mg protein during one min of ET-1 treatment and remained constant or slightly decreased between 1 and 2 min. Thereafter, DAG increased to a maximum (1.6-fold above basal) by 5-10 min. and remained elevated to 30 min. Resolution of DAG molecular species by HPLC after incubation of cells with ET-1 revealed that accumulation of DAG species differed in total cell lysate and subcellular compartments. In plasma membrane, major DAG species increased at 1 min. followed by a decrease at 10 min. whereas in microsomes DAG species did not change at 1 min. and decreased at 10 min. Although phospholipid sources of DAG species were not identified specifically, there was preferential hydrolysis of molecular species of phospholipid for DAG production. We propose that molecular species of DAG produced at the plasma membrane may be transferred to the endoplasmic reticulum so that phospholipid resynthesis can replenish molecular species initially utilized in signal transduction.     

Identification of Gas6 as a ligand for Mer, a neural cell adhesion molecule related receptor tyrosine kinase implicated in cellular transformation

Mer/Nyk/Eyk is an orphan receptor tyrosine kinase expressed at high levels in monocytes and cells derived from epithelial and reproductive tissues. Overexpression of Mer has been associated with lymphoid malignancies. Here we identify Gas6, the product of a growth arrest specific gene, as a ligand for Mer. Gas6 has previously been shown to activate both Axl and Rse/Tyro3, two other receptor tyrosine kinases in the same family as Mer. The apparent relative association and dissociation rate constants of Gas6 for soluble Axl, Rse/Tyro3 and Mer were compared using surface plasmon resonance. Gas6 was shown to induce rapid phosphorylation of Mer expressed in several different types of cells. We also observed a transient activation of p42 MAP kinase following activation of Mer by Gas6. Thus, Gas6 exerts its biological effects through multiple receptor tyrosine kinases.     

Conjugative mobilization of a vancomycin resistance plasmid by a putative Enterococcus faecium sex pheromone response plasmid

The purpose of this study was to develop a model of gastrointestinal carcinogenesis using C57BL/6 mice. Treatment regimens consisted of one control group and 2 groups which received N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in drinking water: 50 micrograms/ml x 52 weeks and 100 micrograms/ml x 27 weeks. In addition, 2 protocols using adjuvant agents intended to increase tumor formation were used: MNNG (100 micrograms/ml) x 27 weeks + 0.2% taurocholic acid added to the diet from weeks 13-52, and MNNG (50 micrograms/ml) x 33 weeks+caerulein (10 micrograms/kg) subcutaneously 3 times/week from weeks 21-52. High-grade dysplasia was observed in the duodenum of 1/13 mice treated with MNNG (50 micrograms/ml). The combination of the latter and caerulein did not augment tumorigenesis. Mice treated with MNNG (100 micrograms/ml) frequently developed neoplasia in the duodenum and upper jejunum. Foci of low-grade and high-grade dysplasia alone were found in 3/12 (25%) mice; and intramucosal and invasive adenocarcinoma were found in 7/12 (58.3%) mice. The addition of taurocholic acid significantly increased the number and histological stages of the tumors (adenocarcinoma occurred in 100%, P = 0.03) and decreased the time for tumor formation.     

Chemically induced mutations in mitochondrial DNA of human cells: mutational spectrum of N-methyl-N'-nitro-N-nitrosoguanidine

We have observed a reproducible mitochondrial mutational spectrum in the MT1 human lymphoblastoid line treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The MNNG spectrum was distinct from the spontaneous mutational spectrum. However, our ability to observe MNNG-induced mitochondrial mutations above the high level of accumulated spontaneous mutations was dependent on the MT1 phenotype. MT1 cells are markedly resistant to the cytotoxicity but not the mutagenicity of MNNG, presumably as a result of inactivation of both copies of the hMSH6 (GTBP) mismatch repair gene. Thus, we were able to use conditions of treatment that yielded induced mitochondrial mutant fractions beyond the practical limits for human cell experiments in mismatch-proficient human cell lines. In contradistinction, when MT1 cells were treated repeatedly with maximum tolerated concentrations of (+/-) anti-benzo(a)pyrene diol-epoxide, no induced mitochondrial mutations above the spontaneous background were observed. A single dose of 4 microM MNNG (survival, 0.85) induced a mutant fraction of 8 x 10(-3) in the nuclear hypoxanthine-guanine phosphoribosyltrans. ferase gene, and a clear and reproducible pattern of seven MNNG-induced hotspot mutations was observed within the mitochondrial DNA target sequence studied (mitochondrial bp 10,030-10,130). All of the MNNG-induced hotspot mutations were G:C to A:T transitions present at frequencies between 6 x 10(-5) and 30 x 10(-5). Additional experiments supported the conclusion that MNNG-induced hotspot mutations observed were generated in living cells as a result of MNNG treatment and not from mismatch intermediates or DNA adducts converted into mutations during the PCR process.     

Nitrogenase activity and respiration of cultures of Rhizobium spp. with special reference to concentrations of dissolved oxygen

Studies of nitrogenase in cultures of the cowpea rhizobia (Rhizobium spp.) strains 32H1 and CB756 are reported. Preliminary experiments established that, even when agar cultures were grown in air, suspensions of bacteria prepared anaerobically from them were most active at low concentrations of free dissolved O2. Consequently, assays for activity used low concentrations of O2, stabilized by adding the nodule pigment leghaemoglobin. In continuous, glutamine-limited cultures of 32H1, nitrogenase activity appeared only when the concentration of dissolved O2 in the cultures approached 1 muM. Lowering the glutamine concentration in the medium supplied to the culture from 2 to 1 mM halved the cell yield and nitrogenase activity was also diminished. Omitting succinate from the medium caused the concentration of dissolved O2 to rise and nitrogenase activity was lost. Upon restoration of the succinate supply, the O2 concentration immediately fell and nitrogenase was restored. The activity doubled in about 8 h, whereas the doubling time of this culture was 14 h. Sonic extracts of 32H1 cells from continuous cultures with active nitrogenase contained components reacting with antiserum against nitrogenase Mo-Fe protein from soybean bacteroids. Continuous cultures grown at higher O2 concentration, with only a trace of active nitrogenase, contained less of these antigens and they were not detected in highly aerobic cultures. Nitrogenase activity of a continuous culture was repressed by NH+4; the apparent half-life was about 90 min. Cells of 32H1 from a continuous culture growing at between 30 and 100 muM dissolved O2 possessed a protective mechanism which permitted respiration to increase following exposure to a rapid increase in O2 concentration from low levels (O2 shock). This effect disappeared as the O2 concentration for growth was reduced towards 1 muM.     

Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts

NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.     

K+ fluxes mediated by Na(+)-K(+)-Cl- cotransport and Na(+)-K(+)-ATPase pumps in renal tubule cell lines transformed by wild-type and temperature-sensitive strains of Simian virus 40

The relative contributions of Na(+)-K(+)-ATPase pumps and Na(+)-K(+)-Cl- cotransport to total rubidium (Rb+) influx into primary cultures of renal tubule cells (PC.RC) and cells transformed either with the wild-type or a temperature-sensitive mutant of the simian virus 40 (SV40), were measured under various growth conditions. The Na(+)-K(+)-ATPase-mediated component represented 74% and 44-48% of total Rb+ influx into PC.RC and SV40-transformed cells, respectively. Proliferating transformed cells showed substantial ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx (41-45% of total) which indicated the presence of a Na(+)-K(+)-Cl- cotransport. The Or-Bs component of Rb+ influx was greatly reduced when temperature-sensitive transformed renal cells (RC.SVtsA58) grown in Petri dishes or on permeable filters were shifted from the permissive (33 degrees C) to the restrictive temperature (39.5 degrees C) to arrest cell growth. The ouabain-sensitive Rb+ influx mediated by the Na(+)-K(+)-ATPase, the total and amiloride-sensitive Na+ uptakes were not modified following inhibition of cell proliferation. A similar fall in the Or-Bs influx was obtained when renal tubule cells transformed by the wild-type SV40 (RC.SV) were incubated with the K+ channel blocker, tetraethylammonium (TEA) ion, which we had previously shown to arrest cell growth without affecting cell viability (Teulon et al.: J. Cell. Physiol., 151:113-125, 1992). Reinitiation of cell growth by removal of TEA or return to 33 degrees C of the temperature-sensitive cells restored the Or-Bs component of Rb influx. Taken together, these results indicate that the Na(+)-K(+)-Cl- cotransport activity is critically dependent on cell growth conditions.     

Identification of the dopamine D3 receptor in oligodendrocyte precursors: potential role in regulating differentiation and myelin formation

Expression of the dopamine D3 receptor (D3r) was found in primary mixed glial cultures from newborn brain and in the corpus callosum in vivo during the peak of myelination. Expression of the D3r mRNA, but not D2r mRNA, was detected as early as 5 d in vitro (DIV) by RT-PCR. Immunoblot studies revealed D3r protein was also expressed in the cultures. Double immunofluorescence analysis for the D3r and for surface markers of specific stages of oligodendrocyte development indicated that D3r expression occurred in precursors and in immature oligodendrocytes but not in mature oligodendrocytes (i.e. , A2B5(+) 007(-) 01(-) and A2B5(+) 007(+) 01(-) cells but not A2B5(-) 007(+) 01(+) cells). Confocal microscopic analysis indicated that D3r was associated with cell bodies and cell membranes but not with the processes emanating from cell somas. Immunohistochemistry of brain sections revealed the presence of D3r in some oligodendrocytes located mainly within the genu and radiato of the corpus callosum during the active period of myelination. Treatment of cultures with 20 microM quinpirole led to decreased numbers of O1(+) oligodendrocytes possessing myelin-like membranes as well as an increase in the number of precursors in 14 DIV cultures. This effect was prevented by the dopamine antagonist haloperidol. These results show that the D3r expression is not restricted to neurons but it is also expressed in differentiating oligodendrocytes before terminal maturation. It also suggests that dopamine or some other D3r ligand may play a role in oligodendrocyte differentiation and/or the formation of myelin by mature oligodendrocytes.     

Monoacyl-sn-glycerol 3-phosphate acyltransferase specificity in bovine mammary microsomes

The acyl-CoA:acyl-sn-glycerol 3-phosphate acyltransferases located in the microsomal fraction of lactating bovine mammary tissue show a preference for palmityl-CoA particularly above the apparent Km values of the acyl acceptors. Using saturating levels of monopalmityl-sn-glycerol 3-phosphate, the order of acylation was palmityl- greater than myristyl- greater than oleyl- greater than stearyl- greater than linoleyl-CoA. Apparent Km values for monopalmityl- and mono-oleyl-sn-glycerol 3-phosphate with palmityl-CoA as donor were 16 and 13muM, respectively while the Km values for palmityl-CoA with these two acyl acceptors were 5 and 5.2muM, RESPECTIVELY. The apparent Vmax values for the palmityl acceptor and donor were 25 and 30 nmol/min/mg protein. Phosphatidic acid was the principal product. The inclusion of magnesium in the assay depressed activity while the addition of ethylenediaminetetraacetate doubled the rate of acylation.     

Thrombopoietin requires additional megakaryocyte-active cytokines for optimal ex vivo expansion of megakaryocyte precursor cells

Little is known concerning the interaction of thrombopoietin (TPO) with other megakaryocyte-active cytokines in directing the early events of megakaryocyte development. Culture of CD34(+) cells in interleukins (IL) -1, -6, -11, plus stem cell factor (SCF; S) results in a 10- to 12-fold expansion in total cell numbers, whereas total CD41(+) megakaryocytes are expanded approximately 120-fold over input levels. Addition of TPO to IL-1, -6, -11, S generates a biphasic proliferation of CD41(+) cells, accelerates their rate of production, and results in an ex vivo expansion of more than 200-fold. The addition of Flt-3 ligand (FL) increases CD41+ cell expansion to approximately 380-fold over input levels. In the absence of TPO, approximately 95% of the expanded cells show the phenotype of promegakaryoblasts; TPO and/or FL addition increases CD41 antigen density and ploidy in a subpopulation of promegakaryoblasts. A moderate (approximately sevenfold) expansion of megakaryocyte progenitor cells (colony-forming unit-megakaryocyte) occurs in the presence of IL-1, -6, -11, S, and the addition of TPO to this cocktail yields an approximately 17-fold expansion. We conclude that early proliferative events in megakaryocyte development in vitro are regulated by multiple cytokines, and that TPO markedly affects these early developmental steps. However, by itself, TPO is neither necessary nor sufficient to generate a full proliferative/maturational in vitro response within the megakaryocyte compartment. TPO clearly affects terminal differentiation and the development of (some) high-ploidy human megakaryocytes. However, its limited in vitro actions on human cell polyploidization suggest that additional megakaryocyte-active cytokines or other signals are essential for the maximal development of human megakaryocytes.     

Catecholamines and operant response rates in albino rats

Rabbit aortic strips (nerve-free, reserpine-pretreated or normal) whose noradrenaline-metabolizing enzymes were inhibited (by in vitro treatment with 0.5 mM pargyline for 30 min and by the presence of 0.1mM U-0521) were exposed to 1.18 muM labelled (-)- or (+)noradrenaline for 30 min. At the end of the incubation period some strips were used for analysis of radioactive (i.e., of noradrenaline and its metabolites), while for others the efflux of radioactivity was determined during 250 min of wash out with amine-free solution. An estimate of the original distribution of the amine into the various extraneuronal and neuronal compartments of the tissue was obtained by compartmental analysis of the efflux curves. 1. The mechanisms responsible for the accumulation of radioactivity in extraneuronal and axoplasmic compartments lack stereoselectivity; the rate constants for the efflux of radioactivity from these compartments are the same for (-)- and (+)noradrenaline. 2. The accumulation of radioactivity in storage vesicles is stereospecific with preference for the (-)isomer. 3. Despite the use of enzyme inhibitors, the "late neuronal efflux" of radioactivity (i.e., the efflux collected between the 200th and 250th min of wash out) contained a considerable proportion of metabolites of noradrenaline. The metabolism of noradrenaline was stereoselective: while dihydroxyphenylglycol (DOPEG) was the predominant metabolite in the efflux from strips incubated with (-)noradrenaline, a considerable part of the efflux from strips incubated with the (+) isomer consisted of dihydroxymandelic acid and "O-methylated and deaminated" metabolites (in addition to DOPEG).     

N-Methyl-D-aspartate attenuates opioid receptor-mediated G protein activation and this process involves protein kinase C

The effects of N-methyl-D-aspartate (NMDA) on opioid receptor-mediated G protein activation were explored in neuroblastoma X glioma hybrid (NG108-15) cells. Treatment of the cells with NMDA resulted in a remarkable attenuation of [35S]guanosine-5'-O-(3-thio)triphosphate binding stimulated by [D-Pen2,D-Pen5]-enkephalin (DPDPE), a delta-opioid receptor agonist. The effects of NMDA were dose and time dependent with an IC50 value of 5 nM and could be blocked by NMDA receptor antagonists. After NMDA treatment, the DPDPE dose-response curve shifted to the right (EC50 value increased approximately 7-fold, from 6 to 40 nM), and the maximal response induced by DPDPE was reduced by approximately 60%. The effects of NMDA were reversible, and the DPDPE response could recover within 60 min. The functional responses of delta-, mu-, and kappa-opioid receptors in primarily cultured neurons also were attenuated significantly by NMDA treatment. The inhibitory effects of NMDA on opioid receptor-mediated G protein activation could be blocked by coadministration of the protein kinase C (PKC) inhibitors or by elimination of the extracellular Ca2+. Correspondingly, NMDA treatment of NG108 cells significantly elevated cellular PKC activity and stimulated Gialpha2 phosphorylation. Transient transfection into NG108-15 cells of the wild-type Gialpha2 and a mutated Gialpha2 (Ser144Ala) resulted in a 2-fold increase in DPDPE-stimulated G protein activation. The DPDPE responses were greatly inhibited by NMDA treatment in the wild-type Gialpha2-transfected cells but much less affected in the mutant Gialpha2-transfected cells. In summary, NMDA attenuates opioid receptor/G protein coupling, and this process requires activation of PKC.     

Ischemic preconditioning in isolated perfused mouse heart: reduction in infarct size without improvement of post-ischemic ventricular function

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [3H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90 degrees C for at least 30 min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (delta ada, delta ogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.