Photocyclization by a triplet-triplet annihilation upconversion pair in water - avoiding UV-light and oxygen removal.

We present a formal [2 + 2]-cycloaddition of unsaturated ketones enabled by a green-to-ultraviolet triplet-triplet annihilation upconversion (TTA-UC) pair, using commercially available Ru(bpy)32+ and pyrene as sensitizer and annihilator, respectively. In the developed protocol, visible light irradiation at λmax = 520 nm allows for the reaction to proceed without the need for UV-light and the aqueous medium eliminates the need for oxygen removing protocols. Through this study, the application of the readily available upconversion pair is broadened to include cyclization reactions. We showcase the utility of the system by generating bicyclo[2.1.1]hexanes that are valuable bioisosteres of ortho-substituted benzenes, a promising motif for pharmaceuticals.

Role of PRL-3, a human muscle-specific tyrosine phosphatase, in angiotensin-II signaling.

Action of protein kinases and phosphatases contributes to myocardial hypertrophy. PRL-3, a protein tyrosine phosphatase, was identified in a cDNA library from an explanted human heart obtained from a patient with idiopathic cardiomyopathy. PRL-3 is expressed in heart and skeletal muscle, exhibiting approximately 76% identity to the ubiquitous tyrosine phosphatase PRL-1, which was reported to increase cell proliferation. PRL-3 was cloned into E. coli and purified using affinity chromatography. PRL-3 activity was determined using the substrate 6,8-difluoro-4-methylumbelliferyl phosphate, and was inhibited by vanadate and analogs. HEK293 cells expressing PRL-3 demonstrated increased growth rates versus nontransfected cells or cells transfected with the catalytically inactive C104S PRL-3 mutant. The tyrosine phosphatase inhibitor, potassium bisperoxo (bipyridine) oxovanadate V, normalizes the growth rate of PRL-3 expressing cells to that of parental HEK293 cells in a concentration-dependent manner. Using FLIPR analysis, parental HEK293 cells mobilize calcium when stimulated with angiotensin-II (AngII). However, calcium mobilization is inhibited in cells expressing wild-type PRL-3 when stimulated with AngII, while cells expressing the inactive mutant of PRL-3 mobilize calcium to the same extent as parental HEK293 cells. Western blots comparing PRL-3 transfected cells to parental HEK293 cells showed dephosphorylation of p130(cas) in response to AngII. These data suggest a role for PRL-3 in the modulation of intracellular calcium transients induced by AngII.

A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9.

ACE2, the first known human homologue of angiotensin-converting enzyme (ACE), was identified from 5' sequencing of a human heart failure ventricle cDNA library. ACE2 has an apparent signal peptide, a single metalloprotease active site, and a transmembrane domain. The metalloprotease catalytic domains of ACE2 and ACE are 42% identical, and comparison of the genomic structures indicates that the two genes arose through duplication. In contrast to the more ubiquitous ACE, ACE2 transcripts are found only in heart, kidney, and testis of 23 human tissues examined. Immunohistochemistry shows ACE2 protein predominantly in the endothelium of coronary and intrarenal vessels and in renal tubular epithelium. Active ACE2 enzyme is secreted from transfected cells by cleavage N-terminal to the transmembrane domain. Recombinant ACE2 hydrolyzes the carboxy terminal leucine from angiotensin I to generate angiotensin 1-9, which is converted to smaller angiotensin peptides by ACE in vitro and by cardiomyocytes in culture. ACE2 can also cleave des-Arg bradykinin and neurotensin but not bradykinin or 15 other vasoactive and hormonal peptides tested. ACE2 is not inhibited by lisinopril or captopril. The organ- and cell-specific expression of ACE2 and its unique cleavage of key vasoactive peptides suggest an essential role for ACE2 in the local renin-angiotensin system of the heart and kidney. The full text of this article is available at http://www. circresaha.org.

Endothelin-1 induces production of the neutrophil chemotactic factor interleukin-8 by human brain-derived endothelial cells.

Increased levels of endothelin-1 (Et-1), a potent vasoconstrictor, have been correlated with hypertension and neuronal damage in ischemic/reperfusion injury. The presence of polymorphonuclear cells (PMNs) in the brain has been shown to be directly responsible for this observed pathology. To address the question of whether Et-1 plays a role in this process, human brain-derived endothelial cells (CNS-ECs) were cultured with Et-1. The results demonstrate that Et-1 induces production of the neutrophil chemoattractant interleukin-8 (IL-8) twofold to threefold after 72 hours; mRNA was maximal after 1 hour of stimulation. Conditioned culture medium derived from Et-1-stimulated CNS-ECs induced a chemotactic response in the PMN migration assay. The inflammatory cytokines tumor necrosis factor-alpha (TNF) and IL-1beta functioned additively with Et-1 in increasing IL-8 production. In contrast, transforming growth factor-beta (TGF-beta), but not IL-10, completely abolished the effect of Et-1 on IL-8 production. However, Et-1 did not modulate intercellular adhesion molecule-1 (ICAM-1) expression. These data demonstrate that Et-1 may be a risk factor in ischemic/reperfusion injury by inducing increased levels of the neutrophil chemoattractant IL-8.

Human immunodeficiency virus Tat protein induces interleukin 6 mRNA expression in human brain endothelial cells via protein kinase C- and cAMP-dependent protein kinase pathways.

The intracellular signal transduction pathways utilized by the HIV-1-derived protein, Tat, in the activation of human central nervous system-derived endothelial cells (CNS-ECs) were examined using specific enzymatic assays. Tat induced an increase in interleukin 6 (IL-6) mRNA within 1 hr of treatment. This biological effect of Tat involved activation of both protein kinase C (PK-C) and cAMP-dependent protein kinase (PK-A) in CNS-ECs. Tat at 10 ng/ml induced a sharp, transient increase in membrane PK-C activity within 30 sec of incubation, and reached maximum levels at 2 min, declining to control values within 10 min. Tat also induced a sharp increase in intracellular cAMP levels and PK-A activity in these cells, with the PK-A activity reaching a maximum at 10 min and slowly declining to control values in 4 hr of incubation. Activation of PK-A was dependent on a Tat-induced increase in membrane PK-C activity as demonstrated by calphostin C (a PK-C inhibitor) abolishing this effect. Incubation of cells with the cyclooxygenase inhibitor indomethacin did not affect Tat-induced activation of PK-A, indicating that prostacyclins are not involved in this process. Tat-induced increase in IL-6 mRNA was abolished in the presence on PK-A inhibitor H-89, demonstrating that activation of PK-A is necessary and sufficient for the increase in IL-6 production by these cells. Both the Tat-induced increase in intracellular cAMP and IL-6 mRNA levels in CNS-ECs may play a role in altering the blood-brain barrier and thereby inducing pathology often observed in AIDS dementia.

A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.

Doxorubicin (Dox), a cardiotoxic antineoplastic drug, disrupts the cardiac-specific program of gene expression (Kurabayashi, M., Dutta, S., Jeyaseelan, R., and Kedes, L. (1995) Mol. Cell. Biol. 15, 6386-6397; Jeyaseelan, R., Poizat, C., Wu, H. Y., and Kedes, L. (1997) J. Biol. Chem. 272, 5828-5832). To determine whether this drug might interfere with the function of cardiac-specific regulatory pathways, we used a differential display strategy to clone from neonatal rat cardiomyocyte candidate mRNAs that were rapidly sensitive to Dox. We report here the identification of a constitutively expressed, cardiac-restricted, nuclear protein whose mRNA level is exquisitely sensitive to Dox. Hence we have named this protein cardiac adriamycin-responsive protein (CARP). CARP mRNA is present at the earliest stages of cardiac morphogenesis. It was detected by in situ hybridization within the cardiogenic plate of 7. 5-day post coitum (p.c.) embryos, and in 8.5-day p.c. embryos CARP transcripts are present in uniformly high levels in the myocardium. Throughout cardiac development, CARP expression is specific for the myocardium; endocardial cushions and valves exhibit only background levels of signal. Transcript levels persist but gradually decrease in neonatal, 2-week-old, and adult hearts. There were no stages when CARP mRNA could not be detected. The pattern and timing of CARP mRNA expression, including transient expression in the tongue at 14.5 days p.c., coincides with that of Nkx2.5/Csx (a putative homolog of tinman, the Drosophila melanogaster gene responsible for cardiac development). The cloned full-length 1749 nucleotide CARP cDNA encodes a 319-amino acid 40-kDa polypeptide containing five tandem ankyrin repeats. CARP appears to be the rat homolog of a previously reported human single-copy gene (C-193; Chu, W., Burns, D. K., Swerlick, R. A., and Presky, D. H. (1995) J. Biol. Chem. 270, 10236-10245), whose mRNA is inducible by cytokines only in human endothelial cells. CARP appears to function as a negative regulator of cardiac-specific gene expression. Overexpression of CARP in cardiomyocytes suppresses cardiac troponin C and atrial natriuretic factor transcription. Cotransfection experiments in HeLa cells indicate that CARP inhibits Nkx2.5 transactivation of atrial natriuretic factor promoter. When fused to a GAL4 DNA-binding domain, CARP has transcriptional inhibitory properties in noncardiac cells. CARP thus represents the first example of a cardiac-restricted transcriptional regulatory protein that is sensitive to Dox.

Molecular mechanisms of doxorubicin-induced cardiomyopathy. Selective suppression of Reiske iron-sulfur protein, ADP/ATP translocase, and phosphofructokinase genes is associated with ATP depletion in rat cardiomyocytes.

Doxorubicin, a cardiotoxic antineoplastic, disrupts the cardiac-specific program of gene expression (Kurabayashi, M., Dutta, S., Jeyaseelan, R., and Kedes, L. (1995) Mol. Cell. Biol. 15, 6386-6397). We have now identified neonatal rat cardiomyocyte mRNAs rapidly sensitive to doxorubicin, or its congener daunomycin, including transcripts of nuclear genes encoding enzymes critical in production of energy in cardiomyocytes: ADP/ATP translocase, a heart- and muscle-specific isoform; Reiske iron-sulfur protein (RISP), a ubiquitously expressed electron transport chain component; and a muscle isozyme of phosphofructokinase. Loss of these mRNAs following doxorubicin or daunomycin is evident as early as 2 h and precedes significant reduction of intracellular ATP. ATP levels in control cardiomyocytes (17.9 +/- 2.9 nM/mg of protein) fall only after 14 h and reach residual levels of 10.4 +/- 0.9 nM (doxorubicin; p = <0.006) and 6.7 +/- 1.9 nM (daunomycin; p = <0. 001) by 24 h. Loss of mRNAs generating ATP was highly selective since mRNAs for other energy production enzymes, (cytochrome c, cytochrome b, and malate dehydrogenase), and genes important in glycolysis (pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase) were unaffected even at 24 and 48 h. The drugs had no effect on levels of ubiquitously expressed RISP mRNA in fibroblasts. These findings could link doxorubicin-induced damage to membranes and signaling pathways with 1) suppression of transcripts encoding myofibrillar proteins and proteins of energy production pathways and 2) depletion of intracellular ATP stores, myofibrillar degeneration, and related cardiotoxic effects.

Doxorubicin inhibits Tat-dependent transactivation of HIV type 1 LTR.

Tat, the human immunodeficiency virus (HIV)-encoded transcription factor, is vital for HIV replication and transcription. Any drug that inhibits Tat's activity is a valuable candidate for chemotherapeutic applications. We show here that doxorubicin (Dox), a well-known anticancer drug and its derivative, daunomycin, inhibit the ability of Tat to activate the HIV-1 LTR. We contransfected HeLa cells with pSV40TAT and a chloramphenicol acetyltransferase gene driven by an HIV LTR promoter. CAT transcription was vigorously stimulated many fold by Tat production but the effect of Tat was inhibited by Dox in a dose-dependent manner. The transcriptional activation domain of Tat, located in its 67 amino terminal residues, remains Dox sensitive. A TAR-deleted reporter gene with a Gal binding domain is transactivated by a Gal-Tat fusion protein. This transcription complex retains a high level of activity in the presence of Dox, suggesting that Dox primarily affects RNA-Tat, rather than DNA-Tat, mediated transactivation. RNA gel mobility analysis reveals that Dox does not affect the binding of Tat to TAR-RNA in vitro but does increase the binding activity of cellular nuclear proteins with TAR-RNA. Induction or activation of such TAR-binding proteins in cells that might interfere with the activity of Tat could explain the observed inhibitory effects of Dox on Tat-activated transcription. These results suggest that Dox may have chemotherapeutic effects on HIV expression mediated through TAR RNA.

Attenuation of interleukin 2-induced pulmonary vascular leak syndrome by low doses of oral methotrexate.

Pulmonary vascular leak induced in mice by interleukin 2 (IL-2) was attenuated by pretreatment with single or multiple doses of oral methotrexate. Methotrexate also attenuated pulmonary vascular leak when either larger doses of IL-2 or when lymphokine-activated killer (LAK) cells or LAK cells plus IL-2 were administered. Lymphoid infiltrates in the lungs of mice treated with IL-2 and methotrexate were significantly lower. The number of mice surviving treatment with high doses of IL-2 was also significantly increased when these mice were treated with methotrexate. Methotrexate prevented the IL-2-induced increase in the number of splenocytes that were asialo GM1+ but had no effect on Lyt 2+ or L3T4+ cell content. A marginal but significant inhibition in the generation of effector splenocytes that were cytolytic to either YAC or MCA-205 tumor targets was observed in mice treated with methotrexate and IL-2. In vivo studies indicated that methotrexate did not compromise the anti-tumor efficacy of treatment regimens that contained IL-2, LAK cells, or IL-2 and LAK cells. These results demonstrate the potential clinical utility of methotrexate in attenuating pulmonary vascular leak induced by IL-2 without compromising its efficacy. One potential mechanism of action of methotrexate is related to its ability to stimulate the release of adenosine followed by the inhibition of the adhesion of leukocytes to the IL-2-activated endothelium.

Doxorubicin-induced Id2A gene transcription is targeted at an activating transcription factor/cyclic AMP response element motif through novel mechanisms involving protein kinases distinct from protein kinase C and protein kinase A.

We have recently shown that doxorubicin (Dox), an antineoplastic drug and an inhibitor of terminal differentiation of myogenic and adipogenic cells, induces expression of Id, a gene encoding a helix-loop-helix transcriptional inhibitor. In this study we have investigated the molecular mechanisms underlying Dox-induced Id2A expression. We have also attempted to determine whether the genetic responses to Dox are related to the UV response, a well-characterized set of reactions to UV and DNA-damaging compounds that is partly mediated by AP-1. Transient transfection of a series of deletions and point mutation derivatives of the human Id2A promoter sequence shows that two closely spaced and inverted short elements similar to an activating transcription factor (ATF) binding site or a cyclic AMP response element (CRE) are necessary and sufficient for a full response to Dox. We refer to this element as the IdATF site. Sequences containing an IdATF site conferred Dox inducibility on a minimal heterologous promoter. An electrophoretic mobility shift assay showed nuclear proteins specifically interacting with the IdATF sequence. While oligonucleotides containing either legitimate ATF/CRE or AP-1 binding sequences competed for binding, antibody supershift experiments suggested that neither CREB/ATF-1 nor AP-1 are major factors binding to IdATF. Several independent criteria suggest that Dox inducibility was independent of Ca2+/phospholipid-dependent protein kinase (protein kinase C), cyclic AMP-dependent protein kinase (protein kinase A), and tyrosine kinase. Moreover, we found that Dox also induces transcription from promoters of immediate-early genes through an AP-1-independent pathway. Taken together, our results suggest that Dox elicits a novel genetic response distinct from the classical UV response.

Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2.

Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin-15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2.

Sequences of the 5'-flanking region of the human helix-loop-helix protein-encoding Id2A gene, and promoter activity regulated by serum and c-Jun/AP-1.

The 5'-flanking region of the human Id2A gene between nt -1213 and +36 from the putative transcription start point was sequenced. Transient transfection studies showed that the region between nt -834 and +30 directs the expression of a reporter gene and contains regulatory elements responsive to serum and c-Jun/AP-1 over production.

Doxorubicin represses the function of the myogenic helix-loop-helix transcription factor MyoD. Involvement of Id gene induction.

We have shown recently that Doxorubicin (Dox, Adriamycin), a potent broad spectrum chemotherapeutic agent with a major side effect of cardiomyopathy, completely prevents myoblast fusion and accumulation of muscle-specific transcripts in the mouse C2 skeletal muscle cell line. Here we use mouse embryonic fibroblast 10T1/2 cells to demonstrate that Dox represses muscle-specific gene expression by interfering with MyoD activity. As assayed by transient cotransfection, Dox inhibits the ability of MyoD to trans-activate muscle-specific reporter genes. A stable cell system was developed in which MyoD is constitutively expressed in 10T1/2 cells (M10 cells). Dox-treated M10 cells express MyoD from a long terminal repeat-driven vector but fail to activate endogenous MyoD and myogenin loci. Dox did not effect E2A gene transcript levels, but Id mRNA levels are significantly increased in Dox-treated M10 cells. Interestingly, overexpression of E2-5, which forms inactive heterodimers with Id, can overcome the Dox-induced suppression of the trans-activation function of MyoD in 10T1/2 cells. Furthermore, we demonstrate that the 5'-flanking region of the Id2 gene mediates its Dox-inducible transcriptional expression. These findings support a model in which Dox inhibits muscle-specific gene expression by interfering with the function of MyoD protein through, at least in part, induction of Id gene expression. The implications of our results for the molecular mechanisms underlying the myofibrillar loss observed in Dox-induced cardiomyopathy are discussed.

Effect of CL 184,005, a platelet-activating factor antagonist in a murine model of Staphylococcus aureus-induced gram-positive sepsis.

Experiments using a murine model of heat-killed Staphylococcus aureus-induced gram-positive bacterial sepsis indicate that the lethal bacterial effects can be prevented if mice are pretreated with CL 184,005, a platelet-activating factor (PAF) antagonist. CL 184,005 was ineffective when administered after bacterial challenge. Plasma of mice pretreated with CL 184,005 contained significantly less tumor necrosis factor (TNF), suggesting that CL 184,005 interferes with TNF synthesis induced by S. aureus. Spleen-associated TNF protein was also decreased by pretreatment with CL 184,005. Although TNF levels were significantly decreased in mice treated with CL 184,005, interleukin-6 levels in serum were significantly increased. Athymic mice were also susceptible to the lethal effects of S. aureus, suggesting that T cells were not involved. When rats rendered hypotensive with S. aureus were treated with CL 184,005, their blood pressure was normalized. Mice treated with enterotoxin B were not protected if they were pretreated with CL 184,005; however, TNF levels in these mice were significantly lower, suggesting that mediators other than PAF and TNF may contribute to the lethal effects of enterotoxin.

Two distinct cDNA sequences encoding the human helix-loop-helix protein Id2.

Two distinct Id2 cDNA clones, Id2A and Id2B (inhibitor of differentiation), have been isolated from a human heart cDNA library and sequenced. The deduced amino acid sequences show nearly complete identity, except for the nonsense mutation at codon 37 in Id2B. Characterization of the genomic clones revealed that Id2B is encoded by an intronless gene which probably arose after the evolutionary divergence of rats and humans.

Antineoplastic agent doxorubicin inhibits myogenic differentiation of C2 myoblasts.

Doxorubicin (Dox, adriamycin), an antineoplastic agent that can cause dilated cardiomyopathy, selectively inhibits muscle-specific gene expression in rodent cardiac muscle cells. This study shows that Dox treatment of proliferating C2 myoblasts, an established cell line from mouse skeletal muscle, completely prevents both fusion and accumulation of muscle-specific gene transcripts without significantly altering non-muscle gene transcripts. When added to high density cultures, Dox only blocked myotube formation but did not inhibit induction of muscle-specific genes. Transient transfection into C2 myoblasts showed that the transcriptional expression of chloramphenicol acetyltransferase reporter plasmids regulated by either the cardiac alpha-actin promoter or the muscle creatine kinase enhancer, but not with a viral or beta-actin promoter, was significantly diminished by Dox in a dose-dependent manner. Moreover, exposure of C2 myoblasts to Dox had a profound effect on the expression of regulatory genes critical to the myogenic differentiation program; mRNAs for MyoD and myogenin were dramatically reduced and Id mRNA was concomitantly increased. In addition, there was diminished DNA binding activity of the muscle-specific transcription factor, MEF-2. These results suggest that Dox inhibits myogenesis by preventing muscle-specific gene expression, possibly through affecting the myogenic programs controlled by muscle-specific transcription factors.

Studies on the effect of CL 306,293, a substituted quinoline carboxylic acid, on the clinical disease induced in mice with LP-BM5 virus.

CL 306,293, a substituted quinoline carboxylic acid, is a potent inhibitor of dihydroorotic acid dehydrogenase, an enzyme essential for the biosynthesis of pyrimidines. In mammalian cell culture, the agent exhibits antiproliferative properties that can be reversed by the addition of uridine. CL 306,293 inhibits the development of the clinical disease in a murine model of immunodeficiency induced by a mixture of LP-BM5 retroviruses. In infected mice, the agent prevents the development of hypergammaglobulinemia, lymphadenopathy, splenomegaly and induction of an IL-2 deficiency. The CD4/CD8 ratio and the number of B cells in the lymph nodes are decreased if the infected animals are treated with CL 306,293. CL 306,293 was more efficacious and potent than 3'-azido-3'-deoxythymidine. The beneficial effects of CL 306,293 observed in this model are most probably related to its antiproliferative properties.

Stimulation of protein synthesis by a 34 KD DNA-binding protein from human placenta.

A 34 KD DNA-binding protein fraction from human placenta stimulated endogenous protein synthesis in rabbit reticulocyte and wheat-germ cell-free systems. Though the synthesis of several proteins were stimulated by the 34 KD protein, a dose-dependent increase of two polypeptides of molecular weights 42,000 and 51,000 were distinctly observed in reticulocyte lysates. The synthesis of the major protein (beta-globin) was not affected by the 34 KD protein. In both hemin supplemented and unsupplemented lysates, the ability of 34 KD protein to stimulate the synthesis of high molecular weight (HMW) proteins was drastically reduced by Mg++ and not by dsRNA.

Human placental endonuclease cleaves Holliday junctions.

A partially purified endonuclease from human placenta cleaves cruciform structures. The placental enzyme is active both on extruded cruciform structures from negatively supercoiled covalently closed circular plasmid DNA and on synthetic X-junctions formed by reannealing short oligonucleotides. Plasmids containing natural or cloned palindromes such as pBR322 and pHD101-3 were used as substrates. The synthetic X-junction tetramer DNA formed by reannealing short oligonucleotides, was converted into dimer form by the enzyme. This is the first report of an enzyme activity involved in resolution of recombination intermediates in higher eukaryotes and second report of a cellular enzyme.