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Protein phosphatase 2A (PP2A) is one kind of serine/ threonine protein phosphatase regulating mainly cell growth and division. It comprises three subunits, A, B, and C. The B-subunit is involved in enzyme activity and substrate specificity. The B-subunit family is of great diversity and is divided into three classes, the B1, B2, and B3 subfamilies. Until now, two members of the B1 subfamily, B1alpha and B1beta, have been identified in human. In this report, the third member of the sub-family, B1gamma, was identified, and its cDNA was isolated from a human brain cDNA library. This novel cDNA is 4,120 bp in length and contains an open reading frame (nt 55-1,398) encoding 447 amino acid residues. The putative protein shares 81 and 85% identity with B1alpha (PPP2R2A) and B1beta (PPP2R2B), respectively, and was named PPP2R2C for its high level of homology to the other two isoforms. One remarkable characteristic of this novel gene is that it is highly expressed in brain with a 4.7-kb transcript while it is nearly undetectable in other tissues. In addition, the PPP2R2C gene was localized to human chromosome 4p16 between markers D4S2925 and D4S3007 with 5.45 cR (LOD > 14) and 2.63 cR (LOD > 15) RH distance, respectively, by radiation hybrid panel mapping.
NAE:I is transformed from DNA endonuclease to DNA topoisomerase and recombinase by a single amino acid substitution. The crystal structure of NAE:I was solved at 2.3 A resolution and shows that NAE:I is a dimeric molecule with two domains per monomer. Each domain contains one potential DNA recognition motif corresponding to either endonuclease or topoisomerase activity. The N-terminal domain core folds like the other type II restriction endonucleases as well as lambda-exonuclease and the DNA repair enzymes MutH and Vsr, implying a common evolutionary origin and catalytic mechanism. The C-terminal domain contains a catabolite activator protein (CAP) motif present in many DNA-binding proteins, including the type IA and type II topoisomerases. Thus, the NAE:I structure implies that DNA processing enzymes evolved from a few common ancestors. NAE:I may be an evolutionary bridge between endonuclease and DNA processing enzymes.
Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.
Discrepancies in the levels of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) following hemorrhagic shock (HS) may be due to the inconsistent rates of bleeding. The purpose of this study was to investigate the effects of rapid versus slow bleeding rates on TNF-alpha levels and if inhibition of TNF-alpha convertase by a matrix metalloproteinase inhibitor (MMPI) affects hepatic integrity in animals exposed to 35% HS. Sprague-Dawley male rats (n = 24, 300-350 g) were divided into four groups: HS 15 (produced over 15 min), HS 30 (produced over 30 min), and HS with MMPI (2.5 mg/kg British Biotech 1101: HS15 + MMPI, HS30 + MMPI). Mean arterial blood pressure (MAP), serum TNF-alpha,levels, and hepatic resting membrane potentials (E(m)) were obtained. A Student t test was performed. TNF-alpha levels for HS 15, HS15 + MMPI, HS 30, and HS 30 + MMPI were 474, 40, 32, and 50 pg/ml, respectively. The hepatic resting membrane potentials for HS 15, HS15 + MMPI, HS 30, and HS 30 + MMPI were -26, -30, -23, and -31 mV, respectively. In conclusion, circulating TNF-alpha levels are affected by the rate of bleeding in hemorrhagic shock. However, despite the differences in the magnitude of TNF-alpha in untreated animals, hepatic integrity was compromised. Interestingly, MMPI, an inhibitor of TNF-alpha convertase, stabilizes the membrane potential in both types of hemorrhagic shock.
Cu(2+) and Zn(2+) inhibit all of the NADPH-dependent reactions catalyzed by neuronal nitric-oxide synthase (nNOS) including ferricytochrome c reduction, NADPH oxidation, and citrulline formation. Cu(2+) and Zn(2+) also inhibit ferricytochrome c reduction by the independent reductase domain. Zn(2+) affects all activities of the full-length nNOS and the reductase domain to the same extent (estimated IC(50) values from 9 to 31 microm), suggesting Zn(2+) occupation of a single site in the reductase domain. Citrulline formation and NADPH oxidation by the full-length nNOS and ferricytochrome c reduction by the reductase domain are affected similarly by Cu(2+), with estimated IC(50) values ranging from 6 to 33 microm. However, Cu(2+) inhibits ferricytochrome c reduction by the full-length nNOS 2 orders of magnitude more potently, with an estimated IC(50) value of 0.12 microm. These data suggest the possibility that Cu(2+) may interact with nNOS at two sites, one composed exclusively of the reductase domain (which is perhaps also involved in Zn(2+)-mediated inhibition), and another that includes components of both domains. Occupation of the second (higher affinity) site could then promote the selective inhibition of ferricytochrome c reduction in full-length nNOS. Neither the inhibition by Cu(2+) nor that by Zn(2+) is dependent on calmodulin.
Myocyte nuclear factor (MNF) is a winged helix transcription factor that is expressed selectively in myogenic stem cells (satellite cells) of adult animals. Using a gene knockout strategy to generate a functional null allele at the Mnf locus, we observed that mice lacking MNF are viable, but severely runted. Skeletal muscles of Mnf-/- animals are atrophic, and satellite cell function is impaired. Muscle regeneration after injury is delayed and incomplete, and the normal timing of expression of cell cycle regulators and myogenic determination genes is dysregulated. Mnf mutant mice were intercrossed with mdx mice that lack dystrophin and exhibit only a subtle myopathic phenotype. In contrast, mdx mice that also lack MNF die in the first few weeks of life with a severe myopathy. Haploinsufficiency at the Mnf locus (Mnf+/-) also exacerbates the mdx phenotype to more closely resemble Duchenne's muscular dystrophy in humans. We conclude that MNF acts to regulate genes that coordinate the proliferation and differentiation of myogenic stem cells after muscle injury. Animals deficient in MNF may prove useful for evaluation of potential therapeutic interventions to promote muscle regeneration for patients having Duchenne's muscular dystrophy.
Soluble gamma-globin chains were expressed in bacteria and purified to assess the mechanism of gamma- and alpha-chain assembly to form Hb F. Formation of Hb F in vitro following incubation of equimolar mixtures of gamma and alpha chains was about 4 x 10(5)-fold slower than assembly of alpha and beta chains to form Hb A in vitro. Results of assembly for gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains were similar to that of beta chains, whereas assembly of gamma(112Thr-->Cys) and alpha chains was similar to wild type gamma chains, indicating that amino acid differences at alpha1beta1 and alpha1gamma1 interaction sites between gamma116 Ile and beta116 His are responsible for the different assembly rates in vitro in the formation of Hb F and Hb A. Homoassembly in vitro of individual gamma chains as assessed by size-exclusion chromatography shows that gamma and gamma(112Thr-->Cys) chains form stable dimers like alphabeta and alphagamma that do not dissociate readily into monomers like beta chains. In contrast, gamma(116Ile-->His) chains form monomers and dimers upon dilution. These results are consistent with the slower assembly rate in vitro of gamma and gamma(112Thr-->Cys) with alpha chains, whereas the faster rate of assembly of gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains, like beta chains, may be caused by dissociation to monomers. These results suggest that dissociation of gamma(2) dimers to monomers limits formation of Hb F in vitro. However, yields of soluble Hb F expressed in bacteria were similar to Hb A, and no unassembled alpha and gamma chains were detected. These results indicate that gamma chains assemble in vivo with alpha chains prior to forming stable gamma(2) dimers, possibly binding to alpha chains as partially folded nascent gamma-globin chains prior to release from polyribosomes.
BACKGROUND: Xenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymus and liver tissue (FP THY/LIV) to thymectomized (ATX), T/NK cell-depleted mice. By using neonatal pig tissue, we hoped to overcome two obstacles that arise with the use of fetal pig tissue: (1) the inability to keep fetal pigs alive after harvesting their thymic tissue, resulting in unavailability of their skin or other organs for grafting; and (2) the limited fetal thymic tissue yield, making application to large animals and humans more difficult. METHODS: Neonatal pig thymus tissue (NP THY) was grafted into ATX, T/NK cell-depleted, 3Gy whole body-irradiated, originally immunocompetent B6 mice to evaluate the ability of NP THY to reconstitute mouse CD4+ T cells and to induce xenogeneic tolerance to donor pig skin grafts. RESULTS: Repopulation of mouse CD4+ T cells in the peripheral tissues was observed in T/NK cell-depleted, ATX B6 mice that received NP THY with or without neonatal pig spleen (NP SPL), but not in those receiving NP SPL alone, indicating that pig thymus grafting was necessary and sufficient for mouse T cell recovery. Seven of nine NP THY/SPL-grafted ATX mice and two of six NP THY-grafted ATX mice that reconstituted >5% CD4+ cells in PBL accepted donor pig skin long-term without lymphocyte infiltration, whereas they rejected allogeneic BALB/c skin and third party pig skin grafts as rapidly as euthymic mice. CONCLUSIONS: NP THY can support the development of mouse CD4+ T cells that are functional and specifically tolerant to donor pig antigens in ATX, T/NK cell-depleted, 3 Gy whole body-irradiated, originally immunocompetent B6 mice. Additional grafting of NP SPL with NP THY improves the efficiency of tolerance induction in this model.
The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm(-1). Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.
Estrogen biosynthesis is catalyzed by aromatase cytochrome P-450 (the product of the CYP19 gene). Adipose tissue is the major site of estrogen biosynthesis in postmenopausal women, with the local production of estrogen in breast adipose tissue implicated in the development of breast cancer. In human adipose tissue, aromatase is primarily expressed in the mesenchymal stromal cells and is a marker of the undifferentiated preadipocyte phenotype. Aromatase expression in adipose tissue is regulated via the distal promoter I.4, under the control of glucocorticoids and class I cytokines such as oncostatin M, interleukin 6, and interleukin 11, as well as tumor necrosis factor alpha. These cytokines, which are expressed in adipose, also inhibit adipocyte differentiation. Therefore, we hypothesized that factors which stimulate adipocyte differentiation should inhibit aromatase expression. These factors include synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligands such as thiazolidinediones, e.g., troglitazone and rosiglitazone (BRL49653) and the endogenous PPARgamma ligand 15-deoxy-delta12,14-prostaglandin J2. We have demonstrated by measurement of aromatase activity and by reverse transcription-PCR/Southern blotting that these PPARgamma ligands inhibit aromatase expression in cultured breast adipose stromal cells stimulated with oncostatin M or tumor necrosis factor alpha plus dexamethasone in a concentration-dependent manner, whereas a metabolite of troglitazone that does not activate PPARgamma has no effect. We have also shown that troglitazone inhibits luciferase activity of reporter constructs containing various lengths of the upstream region of promoter I.4 transfected into mouse 3T3-L1 preadipocyte mesenchymal cells, whereas the troglitazone metabolite does not. Because local estrogen production in breast fat is implicated in breast cancer development in postmenopausal women, the actions of PPARgamma ligands suggest that they may have potential therapeutic benefit in the treatment and management of breast cancer.
Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the Rsk-2 gene was disrupted by homologous recombinant gene targeting. Rsk-2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-Fos. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF- and IGF-1-stimulated Elk-1 phosphorylation. By contrast, PDGF- and IGF-1-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.
Two diastereomeric saponins, julibrosides J1 (1) and J9 (2), both of which show cytotoxic activity, were obtained from the stem bark of Albizia julibrissin Durazz. On the basis of chemical and spectral evidence [L.B. Ma et al., Carbohydr. Res., 281 (1996) 35-46], the structure of 1 was revised as 3-O-[beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6) -beta-D-glucopyranosyl]-21-O-[(6S)-2-trans-2-hydroxymethyl-6-methyl-6-O- [4-O-((6R)-2-trans-2,6-dimethyl-6-O-(beta-D-quinovopyranosyl)-2,7- octadienoyl)-beta-D-quinovopyranosyl]-2,7-octadienoyl] acacic acid-28-O-beta-D-glucopyranosyl-(1-->3)-[alpha-L-arabinofuranosyl-(1-->4 )]-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl ester. The diastereoisomer 2 of 1 was identified as 3-O-[beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6) -beta-D-glucopyranosyl]-21-O-[(6S)-2-trans-2-hydroxymethyl-6-methyl-6-O- [4-O-((6S)-2-trans-2,6-dimethyl-6-O-(beta-D-quinovopyranosyl)-2,7- octadienoyl)-beta-D-quinovopyranosyl]-2,7-octadienoyl] acacic acid-28-O-beta-D-glucopyranosyl-(1-->3)-[alpha-L-arabinofuranosyl-(1-->4 )]-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl ester. Saponin 2 is a new saponin named julibroside J9. Both julibrosides J1 and J9 show good inhibitory action against the KB cancer cell line in vitro.
Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-beta1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta1 increased IL-1beta+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-beta1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-beta1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta1. Increase in P450arom expression by TGF-beta1 was attributable to expression driven by promoter I.4. TGF-beta1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (-410/+14 and-340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-beta1. Deletion and mutation of the GRE in P450-I.4/Luc (-340/+14) abolished the TGF-beta1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta1. These results indicate that TGF-beta1 increases the expression of P450arom at the level of transcription through promoter I.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-beta1 is suggested to be one of the physiological up-regulatory factors of bone aromatase.
During mammalian embryonic development, the ovaries and testes develop from somatic cells of the urogenital ridges as indifferent gonads, harbouring primordial germ cells that have migrated there. After sex determination of the gonads, the testes produce testosterone and anti-Mullerian hormone which mediate male sexual differentiation, and the female developmental pathway ensues in their absence. Here we show that transcripts of the LIM homeobox gene Lhx9 are present in urogenital ridges of mice at embryonic day 9.5; later they localize to the interstitial region as morphological differentiation occurs. In mice lacking Lhx9 function, germ cells migrate normally, but somatic cells of the genital ridge fail to proliferate and a discrete gonad fails to form. In the absence of testosterone and anti-Mullerian hormone, genetically male mice are phenotypically female. The expression of steroidogenic factor 1 (Sf1), a nuclear receptor essential for gonadogenesis, is reduced to minimal levels in the Lhx9-deficient genital ridge, indicating that Lhx9 may lie upstream of Sf1 in a developmental cascade. Unlike mice lacking other genes that mediate early stages of gonadogenesis, Lhx9 mutants do not exhibit additional major developmental defects. Thus, LHX9 mutations may underlie certain forms of isolated gonadal agenesis in humans.
An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.
We have previously established the presence of a pool of apoE sequestered on the macrophage cell surface by demonstrating its displacement from a cell monolayer at 4 degrees C. In this series of experiments, we use a cell surface biotinylation protocol to directly quantitate apoE on the macrophage cell surface and evaluate its transport to and from this cell surface pool. In human monocyte-derived macrophages labeled to equilibrium and in a mouse macrophage cell line transfected to constitutively express human apoE3, approximately 8% of total cellular apoE was present on the surface, but only a portion of this surface pool served as a direct precursor to secreted apoE. The half-life of apoE on the macrophage cell surface was calculated to be approximately 12 min. On SDS-polyacrylamide gel electrophoresis, the apoE isolated from the surface fraction of cells labeled to equilibrium migrated in an isoform pattern distinct from that observed from the intracellular fraction, with the surface fraction migrating predominantly in a higher molecular weight isoform. Pulse labeling experiments demonstrated that newly synthesized apoE reached the cell surface by 10 min but was predominantly in a low molecular weight isoform. There was also a lag between appearance of apoE on the cell surface and its appearance in the medium. Biotinylated apoE, which accumulated in the medium, even from pulse labeled cells, was predominantly in the high molecular weight isoform. Additional experiments demonstrated that low molecular weight apoE present on the cell surface was modified to higher molecular weight apoE by the addition of sialic acid residues prior to secretion and that this conversion was inhibited by brefeldin A. These results demonstrate an unexpected complexity in the transport and cellular processing of macrophage cell surface apoE. Factors that modulate the size and turnover of the cell surface pool of apoE in the macrophage remain to be identified and investigated.
We analyzed the frequency and relevance of mutations in the coding region of the androgen receptor (AR) in genomic DNA extracted from 137 specimens of prostate cancer. The specimens were obtained from the primary tumors of patients affected by stage B disease [15 nonmicrodissected (group 1A) and 84 microdissected (group 1B)] and from the metastatic deposits of individuals with stage D1 disease [8 nonmicrodissected (group 2A) and 30 microdissected (group 2B)] who had not undergone androgen ablation therapy. The study was conducted by PCR-single strand conformational polymorphism (SSCP) analysis of exons 2-8 in the four groups and direct sequence analysis of exon 1 in group 1B. As positive and negative controls, we used genomic DNA extracted from genital skin fibroblasts of patients affected by various forms of androgen resistance with known mutations in the AR. To control for genetic instability, PCR-SSCP analysis of exon 2 of the human progesterone receptor was carried out on each specimen. The overall number of mutations detected was 11 (8%). No mutations were detected in any of the 99 patients with stage B disease. Eleven mutations were detected in exons 2-8 in 8 of the 38 patients with stage D1 disease (all in group 2B). Simultaneous analysis of exon 2 of the progesterone receptor was carried out, and no SSCP changes were identified. These data suggest that AR mutations are rare and presumably do not play a role in the initial phase of prostatic carcinogenesis. The presence of a significant number of AR mutations in metastatic disease indicates that mutations of this molecule may play a role in the most advanced phases of the natural history of this disease, either by facilitating growth or acquisition of the metastatic phenotype.