BBA - Molecular Cell Research (v.1539, #1-2)
A novel and highly divergent Arabidopsis cyclin isolated by complementation in budding yeast by Sharon Abrahams; Guy Cavet; E.Ann Oakenfull; Jeremy P Carmichael; Zahid H Shah; Rajeev Soni; James A.H Murray (1-6).
A novel cyclin, CycJ18, was isolated by complementation of G1 cyclin-deficient budding yeast with an Arabidopsis cDNA library. CycJ18 shares only 20% identity in its conserved cyclin box domain with other cyclins, and is predominantly expressed in young seedlings. CycJ18 is a member of a potential new plant cyclin class.
Keywords: Cyclin; Yeast; Complementation; Cell cycle; Arabidopsis;
Modulation of the host immune system by phosphorylcholine-containing glycoproteins secreted by parasitic filarial nematodes by William Harnett; Margaret M. Harnett (7-15).
Phosphorylcholine (PC) is increasingly becoming recognised as a carbohydrate-associated component of a wide variety of procaryotic and eucaryotic pathogens. Studies employing nematode PC-containing molecules indicate that it possesses a plethora of immunomodulatory activities. ES-62 is a PC-containing glycoprotein, which is secreted by the rodent filarial nematode Acanthocheilonema viteae and which provides a model system for the dissection of the mechanisms of immune evasion induced by related PC-containing glycoproteins expressed by human filarial nematodes. At concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitised humans, ES-62 is able to inhibit antigen receptor-stimulated proliferation of B and T lymphocytes in vitro and in vivo. The active component of ES-62 appears to be PC, as PC conjugated to albumin or even PC alone broadly mimic the results obtained with ES-62. PC-induced impaired lymphocyte responsiveness appears to reflect uncoupling of the antigen receptors from key intracellular proliferative signalling events such as the phosphoinositide 3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways. Although PC-ES-62 can desensitise B and T cells, not all cells are affected, and in fact it is still possible to generate an antibody response to the molecule. Dissection of this response indicates that it is of the TH-2 type. This appears to reflect the ability of ES-62 to direct the polarity of the T cell response by suppressing the production of proinflammatory cytokines, inducing the induction of anti-inflammatory cytokines and by driving the maturation of dendritic cells that direct TH-2 T cell responses.
Keywords: Cytokine; Immunomodulation; Lymphocyte; Nematode; Phosphorylcholine; Signal transduction;
Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain by Daniela Giordano; Maria Egle De Stefano; Gennaro Citro; Anna Modica; Mauro Giorgi (16-27).
We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDE5). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDE5 activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDE5 activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC50=0.6 μM) and sildenafil (IC50=3.5 nM). Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDE5 mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment.
Keywords: Phosphodiesterase; Cyclic guanosine monophosphate; Phosphodiesterase-5 antibody; Glutathione S-transferase fusion protein; Murine neuroblastoma;
Multipathways for transdifferentiation of human prostate cancer cells into neuroendocrine-like phenotype by Stanislav Zelivianski; Michael Verni; Carissa Moore; Dmitriy Kondrikov; Rodney Taylor; Ming-Fong Lin (28-43).
The neuroendocrine (NE) cell is a minor cell population in normal human prostate glands. The number of NE cells is increased in advanced hormone-refractory prostate carcinomas (PCA). The mechanism of increased NE cell population in these advanced tumors is poorly understood. We examined molecular mechanisms which may be involved in the regulation of the transdifferentiation process of human PCA cells leading to a NE phenotype. We compared PCA cell lines LNCaP and PC-3 in the following medium conditions: steroid-reduced (SR), interleukin-6 (IL-6)-supplemented, or dibutyrate cAMP (db-cAMP)-supplemented. We found that androgen-responsive C-33 LNCaP cells responded to all treatments, having a neuronal-like morphology. In contrast, C-81 LNCaP cells, having a decreased androgen responsiveness, had a less pronounced effect although followed a similar trend. Androgen-unresponsive PC-3 cells showed little change in their morphology. Grown in the SR condition, the level of neuron-specific enolase (NSE), a marker of neuronal cells, was upregulated in C-33 LNCaP cells, while to a lesser degree in the presence of IL-6. In the presence of db-cAMP, the NSE level in C-33 cells was decreased, lower than that in control cells. An opposite effect was observed for C-81 LNCaP cells. Nevertheless, the NSE level was only elevated in db-cAMP-treated PC-3 cells, but no change was found in PC-3 cells grown in the SR- or IL-6-supplemented medium. Thus, a similar gross phenotypic change may correlate with differential molecular expressions. We also analyzed the expression of protein tyrosine phosphatase α (RPTPα) since it plays a critical role in normal neuronal differentiation and signaling. Our results showed that the expression of RPTPα correlates with the NE phenotypic change of LNCaP cells in the SR condition. In summary, our data clearly show that the molecular process by which cultured human prostate cancer cells undergo a transdifferentiation process to a NE cell-like phenotype is accompanied by differential expressions of different markers, and a gross NE cell-like phenotype can occur by exposing PCA cells to different pharmacological agents.
Keywords: Cellular transdifferentiation; Prostate cancer; Neuroendocrine cell; Neuron-specific enolase; Receptor protein tyrosine phosphatase α;
3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces caspase-dependent apoptosis in mononuclear cells by Takashi Hashimoto; Hitoshi Ashida; Takashi Sano; Takashi Furuyashiki; Yutaka Hatanaka; Ken-ichiro Minato; Masashi Mizuno; Keiichi Nomura; Atsushi Kumatori; Kazuki Kanazawa; Gen-ichi Danno (44-57).
3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the tryptophan pyrolysates, is a dietary carcinogen and is formed in cooked meat and fish in our daily diet. Trp-P-1 will affect the cells in the blood circulation system before it causes carcinogenicity in target organs such as the liver. In this study, the cytotoxicity of Trp-P-1 was investigated in mononuclear cells (MNCs) from blood. Trp-P-1 (10–15 μM) decreased cell viability and induced apoptosis characterized both by morphological changes and by DNA fragmentation 4 h after treatment. DNA fragmentation was also observed following treatment at 1 nM after 24 h in culture. This result suggested that apoptosis would occur in the body following unexpected intake of foods containing Trp-P-1. To determine the mechanism of apoptosis, we investigated the activation of the caspase cascade in MNCs. Trp-P-1 (10–15 μM) activated the caspase cascade, i.e. the activity of caspase-3, -6, -7, -8 and -9 increased dose-dependently using peptide substrates, the active forms of caspase-3, -8 and -9 were detected by immunoblotting, and cleavage of poly(ADP-ribose) polymerase and protein kinase C-δ as the intracellular substrates for caspases was observed. A peptide inhibitor of caspase-8 completely suppressed activation of all other caspases, while an inhibitor of caspase-9 did not. These results indicated that caspase-8 may act as an apical caspase in the Trp-P-1-activated cascade.
Keywords: Heterocyclic amine; 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; Apoptosis; Caspase; Mononuclear cell; Rat;
Micro-Raman characterisation of the R to T state transition of haemoglobin within a single living erythrocyte by Bayden R Wood; Brian Tait; Donald McNaughton (58-70).
We present the first recorded Raman spectra of haemoglobin in both the R and T states from within a single living erythrocyte using 632.8 nm excitation. Bands characteristic of low spin haems are observed in oxygenated and carboxylated erythrocytes at approx. 1636 (ν10), 1562–1565 (ν2), 1250–1245 cm−1 (ν13) and 1226–1224 cm−1 (ν5+ν8). The spectra of deoxygenated and methaemoglobin erythrocytes have characteristic high spin bands at approx. 1610–1606 cm−1 (ν10), 1582–1580 (ν37), 1547–1544 (ν11), 1230–1220 cm−1 (ν13) and 1215–1210 cm−1 (ν5+ν8). Bands at 1172 (ν30), 976 (ν45) and 672 (ν7) cm−1 appear to be enhanced at 632.8 nm in low spin haems. The oxidation state marker band (ν4) at 1364–1366 cm−1 appeared invariant within this domain in all single cells and conditions investigated contrary to other resonance Raman studies on haem isolates. The information gained by in vivo single erythrocyte molecular analysis has important ramifications to the understanding of fundamental physiological processes and may have applications in the diagnosis and treatment of red blood cell disorders.
Keywords: Erythrocyte; Haemoglobin; Raman spectroscopy; R-T transition;
Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype by Valérie Gouyer; Antje Wiede; Marie-Pierre Buisine; Sophie Dekeyser; Odile Moreau; Thécla Lesuffleur; Werner Hoffmann; Guillemette Huet (71-84).
Trefoil factor family (TFF) peptides are typical secretory products of mucin-producing cells, e.g. of the gastrointestinal tract. Here, the expression and secretion of mucins and TFF peptides was studied in the HT-29 cell line throughout cellular growth and differentiation in relation to a mucin-secreting (HT-29 MTX) or an enterocyte-like (HT-29 G−) phenotype. mRNAs of several MUC and TFF genes were expressed in both cell subpopulations. However, for most MUC and TFF genes, the expression appeared strongly induced with the differentiation into the mucin-secreting phenotype. On the other hand, TFF2 was specifically expressed in the mucin-secreting HT-29 MTX cells. The differentiation of HT-29 MTX cells into the mucin-secreting phenotype was characterised by secretion of the gel-forming mucins MUC2, MUC5AC, and MUC5B, however, according to a different pattern in the course of differentiation. A significant amount of TFF1 and TFF3 was secreted after differentiation, also according to a different pattern, whereas TFF2 was only faintly detected. Secretagogues, known to induce the secretion of mucus, increased the secretion of all three TFF peptides. In contrast, neither a secretory mucin nor a TFF peptide was found in the culture medium of HT-29 G− cells. Overlay assays indicated that HT-29 MTX mucins bound to secretory peptides of HT-29 MTX cells with relative molecular mass similar to TFF peptides. TFF1 and TFF3 were specifically localised in the mucus layer of HT-29 MTX cells by confocal microscopy. Finally, the secretion of TFF peptides and mucins appears as a co-ordinated process which only occurs after differentiation into goblet cell-like phenotype.
Keywords: Trefoil factor family peptide; Mucin; HT-29 cell;
Effect of the β3-adrenergic agonist Cl316,243 on functional differentiation of white and brown adipocytes in primary cell culture by Susanne Klaus; Anita Seivert; Stéphane Boeuf (85-92).
We investigated the effect of the specific β3-adrenergic receptor agonist CL 316,243 (CL) on proliferation and functional differentiation of the Siberian hamster (Phodopus sungorus) white and brown preadipocytes in primary cell culture. Proliferation of both white and brown preadipocytes was stimulated by a general β-adrenergic agonist (isoproterenol) but not by CL. Lipolysis of differentiated white and brown adipocytes was stimulated similarly by CL with maximum effect at 10 nM. Thermogenic properties of cells were assessed by immunodetection of UCP-1, the brown adipocyte specific uncoupling protein, and measurement of cytochrome c oxidase (COx) activity as an index of mitochondrial capacity. UCP-1 content was largely increased by CL in BAT but not in WAT cultures. Basal UCP-2 mRNA levels were similar in WAT and BAT cultures and increased by both CL and isoproterenol. COx activity of BAT cultures was twice as high as that of WAT cultures but in neither cell culture system could it be increased by β-adrenergic stimulation. We suggest (i) that white and brown preadipocyte proliferation is increased in vitro via β1 or β2, but not β3-adrenergic pathways, (ii) that white and brown preadipocytes represent different cell types, and (iii) that in vitro β-adrenergic stimulation it is not sufficient to induce complete thermogenic adaptation of brown adipocytes.
Keywords: Thermogenesis; Mitochondria; Uncoupling protein; Obesity; Adipose; Adrenergic receptor;
Confocal microscopic study of GABAA receptors in Xenopus oocytes after rat brain mRNA injection: modulation by tyrosine kinase activity by Raffaella Balduzzi; Aroldo Cupello; Alberto Diaspro; Paola Ramoino; Mauro Robello (93-100).
The expression of GABAA receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The β2/3 subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 μM, resulted in a lower presence of GABAA receptors on the membrane. The inactive genistein analogue daidzein, 200 μM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABAA receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABAA receptors.
Keywords: GABAA receptor; Xenopus oocyte; Immunocytochemistry; Protein tyrosine kinase; Genistein; Phosphatase;
Nitric oxide regulates actin reorganization through cGMP and Ca2+/calmodulin in RAW 264.7 cells by XinChen Ke; Masaharu Terashima; Yuko Nariai; Yukie Nakashima; Toumei Nabika; Yoshinori Tanigawa (101-113).
Nitric oxide (NO) has been reported to be involved in the regulation of pseudopodia formation, phagocytosis and adhesion in macrophages through the reorganization of actin. In the present study, we directly separated the globular (G) and filamentous (F) actin from quiescent or NO-stimulated macrophage-like cell line RAW 264.7 cells in order to investigate the dynamic redistribution of actin pools. We also focused on the regulatory mechanisms of actin assembly, induced by NO and its possible subsequent signaling pathway. We showed that predominant G-actin coexisted with Triton X-100-insoluble filamentous (TIF) and Triton X-100-soluble filamentous actin in resting RAW 264.7 cells. The exogenous NO produced by (±)-(E)-2-[(E)-hydroxyimino]-6-methoxy-4-methyl-5-nitro-3-hexenamide (NOR1), the endogenous NO induced by lipopolysaccharide (LPS) plus interferon-γ (IFNγ), and dibutyryl-cGMP increased the contents of TIF-actin in dose- and time-dependent manners and altered its morphology. The increase in the TIF-actin contents induced by NOR1 or LPS plus IFNγ was efficiently blocked by the radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or the arginine analogue N G-monomethyl-l-arginine acetate, respectively. Preincubation with the calmodulin antagonist W-7 almost completely blocked the NO-induced TIF-actin increase and morphological change. On the other hand, preincubation with C3 transferase, an inhibitor of Rho protein, efficiently prevented the change in cell morphology, but had no effect on the TIF-actin increase. We postulate that cGMP and subsequent Ca2+/calmodulin may be key regulators of actin reorganization in NO-stimulated RAW 264.7 cells.
Keywords: Nitric oxide; Actin; Macrophage; (±)-(E)-2-[(E)-hydroxyimino]-6-methoxy-4-methyl-5-nitro-3-hexenamide; Guanosine 3′:5′-cyclic monophosphate;
Possible mechanisms regulating ATP- and thimerosal-induced Ca2+ oscillations in the HSY salivary duct cell line by Yosuke Tojyo; Akihiko Tanimura; Akihiro Nezu; Takao Morita (114-121).
The ATP-induced oscillatory changes in cytosolic Ca2+ concentration ([Ca2+]i) were analysed in HSY cells, a salivary ductal cell line from human parotid, using a fluorescence ratio imaging system. At concentrations higher than 1 μM, ATP caused sinusoidal [Ca2+]i oscillations due to the periodic release and reuptake of Ca2+ by intracellular Ca2+ stores. The phorbol ester 4β-phorbol 12,13-dibutyrate (PDBu) changed the [Ca2+]i oscillations to a single spike. The inhibitory effect of PDBu on the [Ca2+]i signals was reversed by protein kinase C (PKC) inhibitors such as staurosporine and chelerythrine chloride. However, preincubation of the cells with the PKC inhibitors did not affect the pattern of the ATP-induced [Ca2+]i oscillations. The desensitization of the [Ca2+]i response observed during prolonged stimulation with ATP was also not prevented by the PKC inhibitors. Incubation of HSY cells with the sulphydryl reagent thimerosal, which enhances the sensitivity of inositol 1,4,5-trisphosphate (IP3) receptors, caused repetitive Ca2+ release from intracellular Ca2+ stores resulting in baseline spikes of [Ca2+]i. The thimerosal-induced [Ca2+]i oscillations did not change in the presence of PDBu and the phospholipase C inhibitor U73122. Thus, we could not provide evidence that negative feedback by PKC plays a central role in the regulation of ATP-induced [Ca2+]i oscillations. These results suggest that the [Ca2+]i oscillations, at least the baseline spikes, in HSY cells can be generated without stimulating the formation of IP3.
Keywords: Ca2+ oscillation; Protein kinase C; IP3 receptor; Thimerosal; HSY cell;
Vitamin D3 enhances the expression of I-mfa, an inhibitor of the MyoD family, in osteoblasts by Kunikazu Tsuji; Norbert Kraut; Mark Groudine; Masaki Noda (122-130).
I-mfa (inhibitor of the MyoD family) is a transcription modulator that binds to and suppresses the transcriptional activity of MyoD family members. I-mfa transcripts are expressed in sclerotome, suggesting a role of I-mfa in skeletogenesis. The aim of this study was to examine the expression and regulation of I-mfa in osteoblasts. We found that I-mfa is expressed at a low level in an osteoblast-like cell line, MC3T3E1, and a pluripotent differentiation modulator, 1,25-dihydroxyvitamin D3, specifically enhanced I-mfa mRNA expression. This effect was completely blocked by the presence of an RNA polymerase inhibitor, but not by a protein synthesis inhibitor, suggesting that 1,25-dihydroxyvitamin D3 upregulates transcription of the I-mfa gene without requirement for new protein synthesis. Western blot analysis indicated that 1,25-dihydroxyvitamin D3 increased the I-mfa protein levels severalfold in MC3T3E1 cells. I-mfa expression was also observed in primary mouse calvaria cells and ROS17/2.8 cells and 1,25-dihydroxyvitamin D3 enhanced I-mfa expression in these cells. These data indicate that I-mfa is a novel transcriptional regulator gene expressed in osteoblasts and that its level is under the control of 1,25-dihydroxyvitamin D3.
Keywords: Inhibitor of MyoD family A; Basic helix-loop-helix; 1,25-Dihydroxyvitamin D3; Osteoblast;
Glycosylation of human CRLR at Asn123 is required for ligand binding and signaling by Shigeki Kamitani; Tsuneaki Sakata (131-139).
Calcitonin receptor-like receptor (CRLR) constitutes either a CGRP receptor when complexed with receptor activity-modifying protein 1 (RAMP1) or an adrenomedullin receptor when complexed with RAMP2 or RAMP3. RAMP proteins modify the glycosylation status of CRLR and determine their receptor specificity; when treated with tunicamycin, a glycosylation inhibitor, CHO-K1 cells constitutively expressing both RAMP2 and CRLR lost the capacity to bind adrenomedullin. Similarly, in HEK293 EBNA cells constitutively expressing RAMP1/CRLR receptor complex CGRP binding was remarkably inhibited. Whichever RAMP protein was co-expressing with CRLR, the ligand binding was sensitive to tunicamycin. There are three putative Asn-linked glycosylation sites in the extracellular, amino terminal domain of CRLR at positions 66, 118 and 123. Analysis of CRLR mutants in which Gln was substituted for selected Asn residues showed that glycosylation of Asn123 is required for both the binding of adrenomedullin and the transduction of its signal. Substituting Asn66 or Asn118 had no effect. FACS analysis of cells expressing FLAG-tagged CRLRs showed that disrupting Asn-linked glycosylation severely affected the transport of the CRLR protein to the cell surface on N66/118/123Q mutant, and slightly reduced the level of the cell surface expression of N123Q mutant compared with wild-type CRLR. But other single mutants (N66Q, N118Q) had no effect for other single mutants. Our data shows that glycosylation of Asn66 and Asn118 is not essential for ligand binding, signal transduction and cell surface expression, and Asn123 is important for ligand binding and signal transduction rather than cell surface expression. It thus appears that glycosylation of Asn123 is required for CRLR to assume the appropriate conformation on the cell surface through its interaction with RAMPs.
Keywords: Adrenomedullin; Calcitonin receptor like receptor; Receptor activity modifying protein; N-Glycosylation;
Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction by P.K Vadiveloo; E Keramidaris; W.A Morrison; A.G Stewart (140-146).
Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII). We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM). The addition of the NO donor DETA NONOate (200 μM) inhibited BMM proliferation by approx. 80%. However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII−/− mice as from wild-type mice. Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea. Moreover, in both normal and NOSII−/− BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types. Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression. Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.
Keywords: Lipopolysaccharide; Cell cycle; Nitric oxide; Macrophage;
Thromboxane A2 receptor mediated activation of the mitogen activated protein kinase cascades in human uterine smooth muscle cells by Sinead M Miggin; B.Therese Kinsella (147-162).
Both thromboxane (TX) A2 and 8-epi prostaglandin (PG) F2α have been reported to stimulate mitogenesis of vascular smooth muscle (SM) in a number of species. However, TXA2 and 8-epiPGF2α mediated mitogenic signalling has not been studied in detail in human vascular SM. Thus, using the human uterine ULTR cell line as a model, we investigated TXA2 receptor (TP) mediated mitogenic signalling in cultured human vascular SMCs. Both the TP agonist U46619 and 8-epiPGF2α elicited time and concentration dependent activation of the extracellular signal regulated kinase (ERK)s and c-Jun N-terminal kinase (JNK)s in ULTR cells. Whereas the TP antagonist SQ29548 abolished U46619 mediated signalling, it only partially inhibited 8-epiPGF2α mediated ERK and JNK activation in ULTR cells. Both U46619 and 8-epiPGF2α induced ERK activations were inhibited by the protein kinase (PK) C, PKA and phosphoinositide 3-kinase inhibitors GF109203X, H-89 and wortmannin, respectively, but were unaffected by pertussis toxin. In addition, U46619 mediated ERK activation in ULTR cells involves transactivation of the epidermal growth factor (EGF) receptor. In humans, TXA2 signals through two distinct TP isoforms. In investigating the involvement of the TP isoforms in mitogenic signalling, both TPα and TPβ independently directed U46619 and 8-epiPGF2α mediated ERK and JNK activation in human embryonic kidney (HEK) 293 cells over-expressing the individual TP isoforms. However, in contrast to that which occurred in ULTR cells, SQ29548 abolished 8-epiPGF2α mediated ERK and JNK activation through both TPα and TPβ in HEK 293 cells providing further evidence that 8-epiPGF2α may signal through alternative receptors, in addition to the TPs, in human uterine ULTR cells.
Keywords: Thromboxane A2 receptor; TPα; TPβ; 8-EpiPGF2α; Smooth muscle; Human; MAPK; ERK; JNK; Mitogenesis;
Association of protein kinase Cλ with adducin in 3T3-L1 adipocytes by Palle G. Laustsen; William S. Lane; Vann Bennett; Gustav E. Lienhard (163-172).
There is evidence that the atypical protein kinases C (PKCλ, PKCζ) participate in signaling from the insulin receptor to cause the translocation of glucose transporters from an intracellular location to the plasma membrane in adipocytes. In order to search for downstream effectors of these PKCs, we identified the proteins that were immunoprecipitated by an antibody against PKCλ/ζ from lysates of 3T3-L1 adipocytes through peptide sequencing by mass spectrometry. The data show that PKCλ is the major atypical PKC in these cells. Moreover, an oligomeric complex consisting of α- and γ-adducin, which are cytoskeletal proteins, coimmunoprecipitated with PKCλ. Association of the adducins with PKCλ was further indicated by the finding that the adducins coimmunoprecipitated proportionally with PKCλ in repeated rounds of immunoprecipitation. Such an association is consistent with literature reports that the adducins contain a single major site for PKC phosphorylation in their carboxy termini. Using antibody against the phospho form of this site for immunoblotting, we found that insulin caused little or no increase in the phosphorylation of this site on the adducins in a whole cell lysate or on the small portion of the adducins that coimmunoprecipitated with PKCλ. PKCλ and the adducins were located in both the cytosol and subcellular membranous fractions. The binding of PKCλ to adducin may function to localize PKCλ in 3T3-L1 adipocytes.
Keywords: Protein kinase C; Adducin; 3T3-L1 adipocyte;
Identification and characterisation of PEX6 orthologues from plants by Claude P Kaplan; Josie E Thomas; Wayne L Charlton; Alison Baker (173-180).
The sunflower (Helianthus annuus) orthologue of PEX6, an AAA ATPase essential for the biogenesis of peroxisomes in yeasts and mammals, was isolated. HaPex6p is immunologically related to Pichia pastoris Pex6p. Like other genes involved in peroxisome biogenesis and function HaPEX6 mRNA and protein levels peak in early post-germinative growth and mRNA levels also increase in senescent tissue. HaPEX6 identifies probable orthologues in Arabidopsis and rice.
Keywords: Peroxin 6; PEX gene; Peroxisome biogenesis; Germination; Glyoxylate cycle;