B82 Cells Research Articles

Identification of NRAS Downstream Genes with CRISPR Activation Screening.

Mutations in NRAS constitutively activate cell proliferation signaling in malignant neoplasms, such as leukemia and melanoma, and the clarification of comprehensive downstream genes of NRAS might lead to the control of cell-proliferative signals of NRAS-driven cancers. We previously established that NRAS expression and proliferative activity can be controlled with doxycycline and named as THP-1 B11. Using a CRISPR activation library on THP-1 B11 cells with the NRAS-off state, survival clones were harvested, and 21 candidate genes were identified. By inducting each candidate guide RNA with the CRISPR activation system, DOHH, HIST1H2AC, KRT32, and TAF6 showed higher cell-proliferative activity. The expression of DOHH, HIST1H2AC, and TAF6 was definitely upregulated with NRAS expression. Furthermore, MEK inhibitors resulted in the decreased expression of DOHH, HIST1H2AC, and TAF6 proteins in parental THP-1 cells. The knockdown of DOHH, HIST1H2AC, and TAF6 was found to reduce proliferation in THP-1 cells, indicating that they are involved in the downstream proliferation of NRAS. These molecules are expected to be new therapeutic targets for NRAS-mutant leukemia cells.

Germline development during embryogenesis of the larvacean, Oikopleura dioica

Germ cells develop into eggs and sperms and represent a lineage that survives through multiple generations. Germ cell specification during embryogenesis proceeds through one of two basic modes: either the cell-autonomous mode or the inductive mode. In the cell-autonomous mode, specification of germ cell fate involves asymmetric partitioning of the specialized maternal cytoplasm, known as the germplasm. Oikopleura dioica is a larvacean (class Appendicularia) and a chordate. It is regarded as a promising animal model for studying chordate development because of its short life cycle (5 days) and small genome size (∼60 ​Mb). We show that their embryos possess germplasm, as observed in ascidians (class Ascidiacea). The vegetal cytoplasm shifted towards the future posterior pole before the first cleavage occurred. A bilateral pair of primordial germ cells (PGC, B11 ​cells) was formed at the posterior pole at the 32-cell stage through two rounds of unequal cleavage. These B11 ​cells did not undergo further division before hatching of the tadpole-shaped larvae. The centrosome-attracting body (CAB) is a subcellular structure that contains the germplasm and plays crucial roles in germ cell development in ascidians. The presence of CAB with germplasm was observed in the germline lineage cells of larvaceans via electron microscopy and using extracted embryos. The CAB appeared at the 8-cell stage and persisted until the middle stage of embryogenesis. The antigen for the phosphorylated histone 3 antibody was localized to the CAB and persisted in the PGC until hatching after the CAB disappeared. Maternal snail mRNA, which encodes a transcription factor, was co-localized with the antigen for the H3S28p antibody. Furthermore, we found a novel PGC-specific subcellular structure that we call the germ body (GB). This study thus highlights the conserved and non-conserved features of germline development between ascidians and larvaceans. The rapid development and short life cycle (five days) of O. dioica would open the way to genetically analyze germ cell development in the future.

IL-2 Plus IL-15 Leads to Enhanced Ex Vivo Expansion of Human Invariant Natural Killer T Cells

Invariant natural killer T (iNKT) cells have been shown to play an important role in immune regulation, defense against pathogens, and cancer immunity, despite representing a minority population of lymphocytes. In particular, emerging murine and human data demonstrate that within this immunoregulatory capacity, iNKT cells function to suppress graft-versus-host disease without loss of the graft-versus-tumor effect, making them ideal to exploit in hematopoietic cell transplantation. Given the key functions of this relatively understudied population, the ability to ex vivo expand human iNKT cells has vital scientific and clinical applications. Therefore, we sought to determine the optimal conditions for iNKT cell expansion and the effects of expansion method on the iNKT cell cytokine production profile. Several expansion culture conditions were explored following initial magnetic bead-based iNKT enrichment and stimulation with α-galactosylceramide (α-GalCer)-loaded irradiated autologous peripheral blood mononuclear cells (PBMCs) on day 0: inclusion of either IL-2 or IL-2 plus IL-15 and day 10 restimulation with either α-GalCer-loaded irradiated autologous PBMCs or α-GalCer-loaded irradiated K562 cells engineered to express CD1d, CD86, 4-1BBL, and OX40L (“B82” cells; Tian G et al. J Clin Invest. 2016 Jun 1;126(6):2341-55). We found significant heterogeneity in the numbers of circulating iNKT cells in healthy donors, ranging from 0.05-0.3% of CD3+ cells, as well as in the fold expansion obtained following two weeks of in vitro culture. Ex vivo expansion using B82 cells for day 10 stimulation and IL-2 alone routinely yielded viability >90% and purity >95% (Figure 1A), and we obtained a median of 3.5x107 iNKT cells starting from a median of 7.2x108 PBMCs following two weeks of expansion. However, we found that when iNKT cells were expanded with autologous PBMCs used on day 10 for restimulation, fold expansion was higher than when B82 cells were used on day 10 and the inclusion of IL-15 along with IL-2 in the culture media led to substantially enhanced expansion (mean fold expansion for PBMC with IL-2: 458; B82 with IL-2: 314; PBMC with IL-2 plus IL-15: 634; B82 with IL-2 plus IL-15: 375; Figure 1B). Following PMA/ionomycin stimulation, cytokine production profiles were similar regardless of expansion conditions, with nearly all iNKT cells producing IFNγ (ranging 94.4-99.9%), and a smaller fraction of iNKT cells producing IL-4 (ranging 21.0-36.1%). These data describe the ex vivo expansion capacity of iNKT cells and culture conditions which lead to optimal expansion. These results contribute to our understanding of human iNKT cells and could inform the design of future clinical trials utilizing iNKT cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Carotenoid biosynthetic gene expression, pigment and n-3 fatty acid contents in carotenoid-rich Tetraselmis striata CTP4 strains under heat stress combined with high light

In this study, two carotenoid-rich strains of the euryhaline microalga Tetraselmis striata CTP4 were isolated by random mutagenesis combined with selection via fluorescence activated cell sorting and growth on norflurazon. Both strains, ED5 and B11, showed an up to 1.5-fold increase in carotenoid contents as compared with the wildtype, independent of the growth conditions. More specifically, violaxanthin, β-carotene and lutein contents reached as high as 1.63, 4.20 and 3.81 mg g−1 DW, respectively. Genes coding for phytoene synthase, phytoene desaturase, lycopene-β-cyclase and ε-ring hydroxylase involved in carotenoid biosynthesis were found to be upregulated in ED5 and B11 cells as compared to the wildtype. Both strains showed higher contents of eicosapentaenoic acid as compared with those of the wildtype, reaching up to 4.41 and 2.88 mg g−1 DW, respectively. Overall, these results highlight the complexity of changes in carotenoid biosynthesis regulation that are required to improve pigment contents in microalgae.

Distal control of mitochondrial biogenesis and respiratory activity by extracellular lactate caused by large-scale deletion of mitochondrial DNA

Lactate is highly produced under conditions of respiratory dysfunction such as anaerobic respiration and various types of mitochondrial diseases, and it was also known as an active molecule that plays various roles both within and between cells. High levels of extracellular lactate may lead to lactic acidosis, which has been related to pathology of the mitochondrial diseases with mutated mitochondrial DNA (mtDNA). In this study, to elucidate the poorly understood molecular roles of extracellular lactate in mitochondrial regulation, we analyzed mouse B82 cells and their cybrid cells carrying mutated mtDNA with a large-scale deletion (ΔmtDNA). Inhibition of lactate production by sodium dichloroacetate (DCA) treatment improved mitochondrial respiration in cells carrying ΔmtDNA through the activation of mitochondrial biogenesis. Chronic exposure to extracellular lactate (more than 3 days) repressed mitochondrial respiration in healthy cells via calcium and CaMK signaling, leading to a decrease in PGC1α-mediated mitochondrial biogenesis. These mitochondrial dysfunctions induced by the lactate treatment were repressed by pH buffering of the medium. These results suggest that lactate, produced in respiration-deficient cells, acts not only as an intracellular source of energy through the TCA cycle, but also as an extracellular messenger molecule regulating the respiratory activity of both cells carrying ΔmtDNA and the surrounding cells, which could cause whole-body repression of respiratory activity.

Mouse somatic mutation orthologous to MELAS A3302G mutation in the mitochondrial tRNALeu(UUR) gene confers respiration defects

We searched for mtDNA harboring somatic mutations in mouse B82 cells, and found an A2748G mutation orthologous to the A3302G mutation in tRNALeu(UUR) gene reported in a patient with MELAS, the most prevalent mitochondrial disease. We isolated subclones of B82 cells until we obtained one subclone harboring >95% A2748G mtDNA. Cytoplasmic transfer of A2748G mtDNA resulted in cotransfer of A2748G mtDNA and respiration defects into mouse ES cells. Thus, A2748G mtDNA is responsible for respiration defects, and the ES cells harboring A2748G mtDNA may be useful for generation of transmitochondrial mice harboring A2748G mtDNA as potential disease models of MELAS.

Effect of TGF-β/Smad signaling on sertoli cell and possible mechanism related to complete sertoli cell-only syndrome

The roles of TGF-beta and the interaction between TGF-beta and EGFR signaling are critical in Sertoli cell, though the knowledge about them is limited. RT-PCR was used to characterize the status of TGF-beta signaling in clinical testicular specimens with complete Sertoli cell-only syndrome (SCOS). The mouse Sertoli cell TM4 was used to investigate the interaction between TGF-beta and EGFR signaling by using mitogenic assay, luciferase assay, and western blot, while TM3 (mouse leydig cell), 3T3 (mouse embryo fibroblasts), and B82 (mouse lung fibroblasts) were selected as control. The RT-PCR assay indicated that the expression levels of TbetaRII and Smad2 in SCOS testes were upregulated compared to that in the normal controls. In the in vitro experiment, the TGF-beta1 downregulated cellular proliferation of TM3 and B82 cell (P < 0.05), but it did not changed the proliferation of TM4 and 3T3 cells (P > 0.05). On contrast, TGF-beta1 only increased the TGF response elements p3TP-lux activity significantly (P < 0.05) in Sertoli cell TM4. Also, the Western blot assay shows an obvious increase of Smad2 in TM4, 3T3, and TM3 cells after TGF-beta1 treatment while the EGFR expression level was significantly increased in TM4 cells only. In conclusion, the TGF-beta pathway and the cross-link between TGF-beta and EGFR signaling may play an important role on the dysfunction of Sertoli cells which induce germ stem cells' disappearance in SCOS.

Epidermal Growth Factor Receptor Is a Critical Mediator of Ultraviolet B Irradiation-Induced Signal Transduction in Immortalized Human Keratinocyte HaCaT Cells

Epidermal growth factor receptor (EGFR) is a critical mediator of several types of epithelial cancers. Skin cancer arising from exposure to ultraviolet B irradiation (UVB) from the sun is a prominent form of human cancer. Recent data indicate that in addition to cognate ligands, EGFR is activated by UVB irradiation. We used pharmacological and genetic approaches to investigate the function of EGFR in mediating UVB-induced signal transduction in human skin keratinocyte HaCaT cells. Pharmacological inhibition of EGFR tyrosine kinase significantly inhibited UVB-mediated induction of ERK, p38, and JNK MAP kinases, and their effectors, transcription factors c-Fos and c-Jun. Inhibition of UVB activation of EGFR also suppressed activation of AKT-, PKC-, and PKA-dependent signal transduction pathways. B82 mouse L cells devoid of EGFR were used to further investigate EGFR dependence of UVB-induced signal transduction. UVB failed to induce ERK, and JNK activation was reduced 60% in B82 cells compared to B82K+ cells, which express EGFR. In addition, UVB induced both c-Fos and c-Jun proteins in B82K+ cells, whereas neither were induced in B82 cells. Taken together, these data demonstrate that EGFR is required for UVB-mediated induction of multiple signaling pathways that are known to mediate tumor formation in skin.

Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn2+

Epidemiological studies have implicated zinc (Zn2+) in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn2+-induced EGFR activation in HAEC, we treated HAEC with 500 microM ZnSO4 for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn2+ results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn2+-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn2+ treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn2+. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn2+ or V4+ was significantly diminished. Moreover, exposure of HAEC to Zn2+ also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn2+-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn2+ exposure.

Effects of H2O2 Treatment of Mouse B82 Cells on mtDNA Amounts and Respiratory Function

Effects of H2O2 Treatment of Mouse B82 Cells on mtDNA Amounts and Respiratory Function

The role of epidermal growth factor receptor in ethanol-mediated inhibition of activator protein-1 transactivation

A potential mechanism underlying ethanol-induced alterations in gene expression is the disruption of transcription factor activity. Growth factor receptors, particularly receptor tyrosine kinases, play an important role in modulating many biological effects of ethanol. We demonstrated here that the expression of epidermal growth factor receptor (EGFR) mediated the effect of ethanol on the activity of transcription factor activator protein-1 (AP-1). Ethanol had little effect on AP-1 activity in the fibroblast cells devoid of EGFR (B82); however, it significantly suppressed AP-1 activity in B82 cells that were stably transfected with either a wild-type EGFR (B82L) or a kinase-deficient receptor (B82M721) in a concentration-dependent manner. EGF activated AP-1 only in B82L cells; the activation was mediated primarily by Akt and ERK. Ethanol inhibited EGF-induced EGFR autophosphorylation, phosphorylation of ERK as well as Akt and its substrate GSK-3β, and subsequently blocked EGF-stimulated AP-1 activation in B82L cells. On the other hand, ethanol had little effect on EGF-stimulated JNK activation. Phorbol ester 12- O-teradecanoyl-phorbol-13-acetate (TPA) activated AP-1 in B82L and B82M721 cells, but not B82 cells. TPA-induced activation of ERK and PKCδ was dependent on the expression of EGFR although the intrinsic kinase activity of EGFR was not required. In contrast, TPA-induced phosphorylation of p38 MAPK, JNKs and other PKC isoforms was independent of EGFR. Ethanol selectively inhibited TPA-induced phosphorylation of ERK and PKCδ, and modestly suppressed TPA-stimulated AP-1 activation in B82L and B82M721 cells. Thus, EGFR plays a critical role in the interaction between ethanol and AP-1.

Evidence for Domain-specific Recognition of SK and Kv Channels by MTX and HsTx1 Scorpion Toxins

Maurotoxin (MTX) and HsTx1 are two scorpion toxins belonging to the alpha-KTx6 structural family. These 34-residue toxins, cross-linked by four disulfide bridges, share 59% sequence identity and fold along the classical alpha/beta scaffold. Despite these structural similarities, they fully differ in their pharmacological profiles. MTX is highly active on small (SK) and intermediate (IK) conductance Ca(2+)-activated (K(+)) channels and on voltage-gated Kv1.2 channel, whereas HsTx1 potently blocks voltage-gated Kv1.1 and Kv1.3 channels only. Here, we designed and chemically produced MTX-HsTx1, a chimera of both toxins that contains the N-terminal helical region of MTX (sequence 1-16) and the C-terminal beta-sheet region of HsTx1 (sequence 17-34). The three-dimensional structure of the peptide in solution was solved by (1)H NMR. MTX-HsTx1 displays the activity of MTX on SK channel, whereas it exhibits the pharmacological profile of HsTx1 on Kv1.1, Kv1.2, Kv1.3, and IK channels. These data demonstrate that the helical region of MTX exerts a key role in SK channel recognition, whereas the beta-sheet region of HsTx1 is crucial for activity on all other channel types tested.

A Novel Fluorescent Toxin to Detect and Investigate Kv1.3 Channel Up-regulation in Chronically Activated T Lymphocytes

T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3(high) cells by flow cytometry. ShK-F6CA blocked Kv1.3 at picomolar concentrations with a Hill coefficient of 1 and exhibited >80-fold specificity for Kv1.3 over Kv1.1 and other K(V) channels. In flow cytometry experiments, ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of approximately 600 channels per cell. Rat and human T cells that had been repeatedly stimulated 7-10 times with antigen were readily distinguished on the basis of their high levels of Kv1.3 channels (>600 channels/cell) and ShK-F6CA staining from resting T cells or cells that had undergone 1-3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelin-specific rat T cell line following myelin antigen stimulation, peaking at 15-20 h and then declining to baseline over the next 7 days, in parallel with the acquisition and loss of encephalitogenicity. Both calcium- and protein kinase C-dependent pathways were required for the antigen-induced Kv1.3 up-regulation. ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3(high) expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.

Differential requirement of EGF receptor and its tyrosine kinase for AP-1 transactivation induced by EGF and TPA.

The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including cell differentiation, proliferation, apoptosis and oncogenic transformation. It is thought that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activity is because of the activation of the PKC/MAPK/AP-1 pathway, although the detailed molecular mechanism has not been fully characterized. The tyrosine kinases of epidermal growth factor receptor (EGFR) lie at the head of a complex of signal transduction cascade that modulates cell proliferation, survival, adhesion, migration and differentiation. Currently, little is known about whether EGFR or its tyrosine kinase is necessary for TPA-induced AP-1 activation. In the present study, we investigated this issue using a well-characterized mouse fibroblast B82 cell line, which is devoid of the EGFR, and its stable transfectants with either wild-type EGFR (B82L) or tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721). We demonstrated that the TPA or epidermal growth factor (EGF) induced AP-1 activation in the B82L cells that express wild-type EGFR, but not in the B82 cell, whereas autophosphorylation at tyrosine(1173) of EGFR in B82L cells was only induced by EGF, but not TPA. The expression of tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721) resulted in deficiency of AP-1 induction in cellular response to EGF, while TPA treatment led to fully AP-1 activation. Furthermore, the mutation at Lys-721 of EGFR resulted in impairing of EGFR autophosphorylation at tyrosine(1173) induced by EGF. Based on these results, we conclude that TPA-induced AP-1 activation requires the basal level-EGFR protein, but not EGFR tyrosine kinase and EGFR autophosphorylation at tyrosine(1173), whereas both EGFR tyrosine kinase and EGFR autophosphorylation at Y(1173) play a critical role in EGF-induced AP-1 activation.

An electrophysiological study on the effects of Pa-1G (a phospholipase A 2) from the venom of king brown snake, Pseudechis australis, on neuromuscular function

The effects of Pa-1G, a phospholipase A 2 (PLA 2) from the venom of the Australian king brown snake ( Pseudechis australis) were determined on the release of acetylcholine, muscle resting membrane potential and motor nerve terminal action potential at mouse neuromuscular junction. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations revealed that Pa-1G (800 nM) significantly reduced the amplitude of endplate potentials within 10 min exposure. The quantal content of endplate potentials was decreased to 58±6% of control after 30 min exposure to 800 nM Pa-1G. The toxin also caused a partial depolarisation of mouse muscle fibres within 60 min exposure. Extracellular recording of action potentials at motor nerve terminals showed that Pa-1G reduced the waveforms associated with both sodium and potassium conductances. To investigate whether this was a direct or indirect effect of the toxin on these ionic currents, whole cell patch clamp experiments were performed using human neuroblastoma (SK-N-SH) cells and B82 mouse fibroblasts stably transfected with rKv1.2. Patch clamp recording experiments confirmed that potassium currents sensitive to α-dendrotoxin recorded from B82 cells and sodium currents in SK-N-SH cells were not affected by the toxin. Since neither facilitation of acetylcholine release at mouse neuromuscular junction nor depression of potassium currents in B82 cells has been observed, the apparent blockade of potassium currents at mouse motor nerve endings induced by the toxin is unlikely to be due to a selective block of potassium channels.

Extinction of expression of the genes encoding haematopoietic cell-restricted transcription factors in T-lymphoma x fibroblast cell hybrids.

We previously reported that expression of the T-cell receptor (TCR) alpha and lck genes is extinguished in hybrids between mouse T-lymphoma EL4 cells and mouse fibroblast B82 cells. In the present study, we found that the activities of the TCRalpha minimum enhancer and the lck promoter monitored by the luciferase or chloramphenicol acetyltransferase (CAT) assays were markedly inhibited in the hybrids. Expression of the TCF-1, LEF-1, GATA-3, Ikaros, c-myb and Fli-1 genes, which encode the haematopoietic cell-restricted transcription factors that appear to be responsible for the activities of the enhancer and the promoter, was fully extinguished or markedly suppressed in the hybrids. On the other hand, expression of the transcription factor genes observed in both parental cells, such as the AML1 and c-ets-1 genes, and that of the genes encoding ubiquitously expressed transcription factors, such as the E2A, CREB and c-ets-2 genes, was not significantly suppressed in the hybrids. These results suggest that the genes encoding haematopoietic cell-restricted transcription factors are targets for negative regulation in fibroblastic background and that the repression of these genes may consequently lead to suppression of the promoter and/or enhancer activities of several T-cell-specific structural genes in T-lymphoma x fibroblast cell hybrids.

Tyrosine Phosphorylation of Syndecan-1 and -4 Cytoplasmic Domains in Adherent B82 Fibroblasts

The syndecans, a family of cell surface proteoglycans, have highly conserved cytoplasmic domains that bind proteins containing PDZ domains and co-localize with the actin cytoskeleton. The syndecan cytoplasmic domains contain four conserved tyrosine residues, two of which are located within favorable sequences for phosphorylation. Endogenous tyrosine phosphorylation of syndecans-1 and -4 is detected in adherent B82 fibroblasts. Approximately 1.5% of total syndecan is endogenously phosphorylated, while most, if not all, cell surface syndecan is phosphorylated following treatment with the tyrosine phosphatase inhibitor pervanadate. Syndecan phosphorylation is also detected in Raji-S1 and NMuMG cells, but only following treatment with vanadate or pervanadate, suggesting that endogenous phosphorylation is maintained in an "off" state in these cells. Endogenous syndecan phosphorylation in B82 cells is rapidly blocked by genistein (IC50 < 10 microM) confirming the presence of a constitutively active kinase and a corresponding tyrosine phosphatase. Phosphorylation is also inhibited by herbimycin A (IC50 < 1.0 microM) and staurosporine (IC50 < 1.0 nM), suggesting a role for Src family kinases in regulating syndecan phosphorylation. Together, these data suggest an important role for tyrosine phosphorylation of the syndecan cytoplasmic domains in regulating downstream signaling events in response to cell adhesion and/or growth factor activity.

Identification of adenylyl cyclase isoenzymes in CHO and B82 cells.

The identification of adenylyl cyclase isoenzymes in mammalian host cells is important for the interpretation of data obtained from cell lines heterologously expressing G-protein coupled receptors. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify adenylyl cyclase cDNAs from Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. The isolated fragments were identified by restriction analyses and by sequencing. We found mRNAs for adenylyl cyclases VI and VII in CHO and adenylyl cyclases IX and VII in B82 cells.

Signal transduction through atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in immortal murine cells.

The exposure of mammalian cells to ultraviolet (u.v.) irradiation leads to activation of transcription factors, such as AP-1 and NFkappaB. It is postulated that the EGF receptor but not protein kinase C (PKC) is the major membrane mediator in UVC-induced signal transduction. We demonstrate here that the antisense oligonucleotides of PKC zeta and the dominant negative mutant of PKC lambda/iota as well as dominant negative PKC zeta markedly blocked UVC-induced AP-1 activity. In contrast, UVC-induced AP-1 activity in cells devoid of the EGF receptor (B82), is not significantly different from that of the stable transfectants with a kinase-deficient EGF receptor (B82M721), or wild-type EGF receptor (B82L). This was found at all UVC irradiation doses and time courses studied, while high levels of EGF-induced AP-1 activity were observed in B82L cells but not in B82 cells. This evidence strongly suggests that atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in JB6 and B82 cells.

The origin of the most common mutation of adenine phosphoribosyltransferase deficiency among Japanese goes back to a prehistoric era

Cells with and without hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity were used to examine the transfer of purine metabolites through the medium and via cell contacts. HGPRT− Chinese hamster and human fibroblasts were able to incoporate 3H-labeled purine metabolite(s) from medium in which mouse HGPRT+ B82 cells had been grown for 24 h with [3H]hypoxanthine, but mouse A9 fibroblasts that were deficient in HGPRT, adenine phosphoribosyltransferase (APRT), and methylthioadenosine phosphorylase (MTAP) were unable to incorporate these metabolites. This suggests that in recipient cells incorporation is due to [3H]MTA, which has been shown previously to be the major 3H-labeled purine metabolite to accumulate in B82 medium, being cleaved by MTAP to [3H]adenine, which is phosphoribosylated by APRT to [3H]AMP. Incorporation by recipient cells of metabolites from the medium is referred to as contact-independent metabolite transfer (CIMT). In autoradiograms of B82/A9 cocultures that were labeled with [3H]hypoxanthine, grains were found over A9 that were not in contact with B82, although A9 did not act as recipients of CIMT. This is termed proximity-dependent metabolite transfer (PDMT). Both CIMT and PDMT interfered with the assessment of nucleotide exchange between HGPRT+ and HGPRT− cells through cell contacts, which is referred to as contact-dependent metabolite transfer (CDMT). These problems were unique to HGPRT+ mouse L cells. However, HGPRT− mouse L cells, A9, could be used as potential recipients. A9 were positive recipients of CDMT with only one of five cell lines tested, which suggested that these cells were selective communicators. CDMT could not be studied with [3H]guanine because the nuclei of HGPRT− cells became labeled.