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1.
ISME Commun ; 3(1): 3, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36690784

RESUMO

The meltwater streams of the McMurdo Dry Valleys are hot spots of biological diversity in the climate-sensitive polar desert landscape. Microbial mats, largely comprised of cyanobacteria, dominate the streams which flow for a brief window of time (~10 weeks) over the austral summer. These communities, critical to nutrient and carbon cycling, display previously uncharacterized patterns of rapid destabilization and recovery upon exposure to variable and physiologically detrimental conditions. Here, we characterize changes in biodiversity, transcriptional responses and activity of microbial mats in response to hydrological disturbance over spatiotemporal gradients. While diverse metabolic strategies persist between marginal mats and main channel mats, data collected from 4 time points during the austral summer revealed a homogenization of the mat communities during the mid-season peak meltwater flow, directly influencing the biogeochemical roles of this stream ecosystem. Gene expression pattern analyses identified strong functional sensitivities of nitrogen-fixing marginal mats to changes in hydrological activities. Stress response markers detailed the environmental challenges of each microhabitat and the molecular mechanisms underpinning survival in a polar desert ecosystem at the forefront of climate change. At mid and end points in the flow cycle, mobile genetic elements were upregulated across all mat types indicating high degrees of genome evolvability and transcriptional synchronies. Additionally, we identified novel antifreeze activity in the stream microbial mats indicating the presence of ice-binding proteins (IBPs). Cumulatively, these data provide a new view of active intra-stream diversity, biotic interactions and alterations in ecosystem function over a high-flow hydrological regime.

2.
Mol Cell Biol ; 19(1): 515-25, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9858575

RESUMO

The regulatory elements that restrict transcription of genes encoding contractile proteins specifically to either slow- or fast-twitch skeletal muscles are unknown. As an initial step towards understanding the mechanisms that generate muscle diversity during development, we have identified a 128-bp troponin I slow upstream element (SURE) and a 144-bp troponin I fast intronic element (FIRE) that confer fiber type specificity in transgenic mice (M. Nakayama et al., Mol. Cell. Biol. 16:2408-2417, 1996). SURE and FIRE have maintained the spatial organization of four conserved motifs (3' to 5'): an E box, an AT-rich site (A/T2) that binds MEF-2, a CACC site, and a novel CAGG motif. Troponin I slow (TnIs) constructs harboring mutations in these motifs were analyzed in transiently and stably transfected Sol8 myocytes and in transgenic mice to assess their function. Mutations of the E-box, A/T2, and CAGG motifs completely abolish transcription from the TnI SURE. In contrast, mutation of the CACC motif had no significant effect in transfected myocytes or on the slow-specific transcription of the TnI SURE in transgenic mice. To assess the role of E boxes in fiber type specificity, a chimeric enhancer was constructed in which the E box of SURE was replaced with the E box from FIRE. This TnI E box chimera, which lacks the SURE NFAT site, confers essentially the same levels of transcription in transgenic mice as those conferred by wild-type SURE and is specifically expressed in slow-twitch muscles, indicating that the E box on its own cannot determine the fiber-type-specific expression of the TnI promoter. The importance of the 5' half of SURE, which bears little homology to the TnI FIRE, in muscle-specific expression was analyzed by deletion and linker scanning analyses. Removal of the 5' half of SURE (-846 to -811) results in the loss of expression in stably transfected but not in transiently expressing myocytes. Linker scanning mutations identified sequences in this region that are necessary for the function of SURE when integrated into chromatin. One of these sites (GTTAATCCG), which is highly homologous to a bicoid consensus site, binds to nuclear proteins from several mesodermal cells. These results show that multiple elements are involved in the muscle-specific activity of the TnIs promoter and that interactions between upstream and downstream regions of SURE are important for transcription in the context of native chromatin.


Assuntos
Fibras Musculares Esqueléticas , Transcrição Gênica , Troponina I/genética , Animais , Sequência Conservada , Regulação da Expressão Gênica , Genes Reporter , Camundongos , Camundongos Transgênicos , Mutagênese , Relação Estrutura-Atividade , Transfecção
3.
Mol Cell Biol ; 21(24): 8490-503, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11713284

RESUMO

Transcription is a major regulatory mechanism for the generation of slow- and fast-twitch myofibers. We previously identified an upstream region of the slow TnI gene (slow upstream regulatory element [SURE]) and an intronic region of the fast TnI gene (fast intronic regulatory element [FIRE]) that are sufficient to direct fiber type-specific transcription in transgenic mice. Here we demonstrate that the downstream half of TnI SURE, containing E box, NFAT, MEF-2, and CACC motifs, is sufficient to confer pan-skeletal muscle-specific expression in transgenic mice. However, upstream regions of SURE and FIRE are required for slow and fast fiber type specificity, respectively. By adding back upstream SURE sequences to the pan-muscle-specific enhancer, we delineated a 15-bp region necessary for slow muscle specificity. Using this sequence in a yeast one-hybrid screen, we isolated cDNAs for general transcription factor 3 (GTF3)/muscle TFII-I repeat domain-containing protein 1 (MusTRD1). GTF3 is a multidomain nuclear protein related to initiator element-binding transcription factor TF II-I; the genes for both proteins are deleted in persons with Williams-Beuren syndrome, who often manifest muscle weakness. Gel retardation assays revealed that full-length GTF3, as well as its carboxy-terminal half, specifically bind the bicoid-like motif of SURE (GTTAATCCG). GTF3 expression is neither muscle nor fiber type specific. Its levels are highest during a period of fetal development that coincides with the emergence of specific fiber types and transiently increases in regenerating muscles damaged by bupivacaine. We further show that transcription from TnI SURE is repressed by GTF3 when overexpressed in electroporated adult soleus muscles. These results suggest a role for GTF3 as a regulator of slow TnI expression during early stages of muscle development and suggest how it could contribute to Williams-Beuren syndrome.


Assuntos
Proteínas Musculares , Proteínas Nucleares , Análise de Sequência de DNA , Transativadores , Fatores de Transcrição/química , Transcrição Gênica , Animais , Sequência de Bases , Northern Blotting , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Homeodomínio/metabolismo , Humanos , Hibridização In Situ , Íntrons , Luciferases/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Músculos/patologia , Fator de Transcrição PAX7 , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Distribuição Tecidual , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Síndrome de Williams
4.
J Med Chem ; 38(13): 2302-10, 1995 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-7608895

RESUMO

We recently demonstrated in animal models that a new conformationally defined RA isomer (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) was as effective as RA in the prevention of skin papillomas but was less toxic. In order to provide more details concerning this improved action, we report here the preparation of a homologous conformationally defined 6-s-trans-retinoid (1) and investigate its ability to interact with proteins and to activate gene expression. Four configurational isomers of 1 were evaluated in binding assays for cellular retinoic acid binding protein, CRABP (isolated from chick skin); CRABP-I and CRABP-II (cloned from mouse); nuclear retinoic acid receptors (RARs); and nuclear retinoid X receptors (RXRs). In each assay the all-E-isomer of this retinoid had an activity that was comparable to that of (all-E)-RA. However, the 9Z-isomer was at least 200-fold less active than (all-E)-RA in binding to different RARs, while it was only 6-20 times less active than (9Z)-RA in binding to different RXRs. In an in vivo transient transfection assay, the all-E-isomer activated a reporter gene containing a retinoic acid response element (RARE) with efficiency similar to (all-E)-RA when expression vectors for either RAR alpha, RAR beta, RAR gamma alone or RAR alpha together with RXR alpha were cotransfected. In contrast, the 9Z-isomer was much less active than (9Z)-RA in the same assay systems. However, (9Z)-1 efficiently enhanced the DNA binding and transactivational activity of RXR alpha homodimers. Taken together, these studies demonstrate that the all-E- and 9Z-isomers of this retinoid are selective and potent agonists of RAR and RXR binding and activation.


Assuntos
Receptores do Ácido Retinoico/agonistas , Fatores de Transcrição/agonistas , Transcrição Gênica/efeitos dos fármacos , Tretinoína/análogos & derivados , Animais , Linhagem Celular , Galinhas , Camundongos , Conformação Molecular , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Espectrofotometria Ultravioleta , Estereoisomerismo , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia
5.
J Med Chem ; 39(19): 3625-35, 1996 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-8809153

RESUMO

We recently demonstrated that conformationally defined 6-s-trans-retinoic acid (RA) analogs were effective in the prevention of skin papillomas (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) and selective agonists for nuclear receptor binding and activation (Alam et al. J. Med. Chem. 1995, 38, 2302-2310). In order to probe important structure-activity relationships, we evaluated a homologous series of four 6-s-trans-retinoids that are 8-(2'-cyclohexen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acids with different substituents at 2' (R2) and 3' (R1) positions on the cyclohexene ring. UAB1 (R1 = R2 = H), UAB4 (R1 = R2 = Me), UAB7 (R1 = Me, R2 = iPr), and UAB8 (R1 = Et, R2 = iPr) contain alkyl R groups that mimic, to different extents, portions of the trimethylcyclohexenyl ring of RA. Both 9Z- and all-E-isomers of these retinoids were evaluated in binding assays for cellular retinoic acid-binding proteins (CRABP-I and CRABP-II), a nuclear retinoic acid receptor (RAR alpha), and a nuclear retinoid X receptor (RXR alpha). The all-E-isomers of UAB retinoids bound tightly to CRABPs and RAR alpha, the binding affinity of the all-E-isomer increased systematically from UAB1 to UAB8, and binding for the latter was comparable to that of all-E-RA. In contrast to RA, the (9Z)-UAB retinoids were at least 200-fold less active than the all-E-isomers in binding to RAR alpha. The (9Z)-UAB isomers exhibited increasingly stronger binding to RXR alpha, and (9Z)-UAB8 was nearly as effective as (9Z)-RA in binding affinity. The retinoids were also evaluated in gene expression assays mediated by RAR alpha and RXR alpha homodimers or RAR alpha/RXR alpha heterodimers. Consistent with the binding affinities, the (all-E)-UAB retinoids activated gene transciption mediated by RAR alpha homodimers or RAR alpha/RXR alpha heterodimers, while the (9Z)-UAB isomers activated only the RXR alpha homodimer-mediated transcription. The all-E- and 9Z-isomers of the UAB retinoids were further evaluated for their capacity to prevent the induction of mouse skin papillomas. When compared to RA, only the (all-E)-UAB retinoids containing bulky R1 and R2 groups were effective in this chemoprevention assay. (9Z)-RA displayed equal capacity as RA to prevent papillomas, while the 9Z-isomers of the UAB retinoids were much less effective. Taken together, these studies demonstrate that the cyclohexenyl ring substituents of 6-s-trans-UAB retinoids are important for their biological activities and that the chemopreventive effect of the all-E-isomers of these retinoids correlates well with their capacity to bind to RARs and activate RAR/RXR-mediated transcription.


Assuntos
Anticarcinógenos , Núcleo Celular/metabolismo , Receptores do Ácido Retinoico/metabolismo , Retinoides/química , Transcrição Gênica/efeitos dos fármacos , Animais , Camundongos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Papiloma/prevenção & controle , Receptores X de Retinoides , Retinoides/metabolismo , Retinoides/uso terapêutico , Neoplasias Cutâneas/prevenção & controle , Estereoisomerismo , Relação Estrutura-Atividade , Termodinâmica , Fatores de Transcrição/metabolismo
6.
J Med Chem ; 41(10): 1679-87, 1998 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-9572893

RESUMO

We recently synthesized several conformationally constrained retinoic acid (RA) analogues [8-(2'-cyclohexen-1'-ylidene)-3, 7-dimethyl-2,4,6-octatrienoic acids with different alkyl substituents at 2' (R1) and 3' (R2) positions on the cyclohexene ring] (Muccio et al. J. Med. Chem. 1996, 39, 3625) as cancer chemopreventive agents. UAB8 (R1 = Et; R2 = iPr), which contains sufficient steric bulk at the terminal end of the polyene chain to mimic the trimethylcyclohexenyl ring of RA, displayed biological properties similar to those of RA. To explore the efficacy of this retinoid in acute promyelocytic leukemia (APL) and juvenile myelomonocytic leukemia (JMML), we evaluated UAB8 isomers in in vitro assays which measure the capacity of retinoids to inhibit aberrant myeloid colony growth from blood or bone marrow cells obtained from human JMML patients and in assays measuring the potential of retinoids to differentiate NB4 cells (an APL cell line). Both (all-E)- and (13Z)-UAB8 were 2-fold more active than RA in the NB4 cell differentiation assay; however, only (all-E)-UAB8 had comparable activity to the natural retinoids in the JMML cell assays. These results were compared to the biological effectiveness of a new retinoid, UAB30 [8-(3', 4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4, 6-octatrienoic acid], which had different nuclear receptor binding and transactivational properties than UAB8. Relative to (all-E)-RA and (all-E)-UAB8, (all-E)-UAB30 bound well to RARalpha but did not activate transcription-mediated RARalpha homodimers, even though it was effective in RARbeta- and RARgamma-mediated transactivational assays. In APL assays, this retinoid had much reduced activity and was only moderately effective in JMML assays and in cancer chemoprevention assays.


Assuntos
Antineoplásicos , Ácidos Graxos Insaturados , Leucemia Mielomonocítica Crônica/prevenção & controle , Leucemia Promielocítica Aguda/prevenção & controle , Naftalenos , Tretinoína/análogos & derivados , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular , Galinhas , Criança , Ácidos Graxos Insaturados/síntese química , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Células HL-60 , Humanos , Técnicas In Vitro , Camundongos , Conformação Molecular , Naftalenos/síntese química , Naftalenos/química , Naftalenos/metabolismo , Naftalenos/farmacologia , Papiloma/prevenção & controle , Ensaio Radioligante , Receptores do Ácido Retinoico/metabolismo , Pele/metabolismo , Neoplasias Cutâneas/prevenção & controle , Estereoisomerismo , Transcrição Gênica/efeitos dos fármacos , Tretinoína/química , Tretinoína/metabolismo , Tretinoína/farmacologia , Ensaio Tumoral de Célula-Tronco
7.
Biochem Pharmacol ; 53(7): 1049-53, 1997 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-9174119

RESUMO

All-trans-3,4-Didehydroretinoic acid (vitamin A2 acid; DDRA) is one of the retinoids present in human skin, the most responsive tissue to retinoid treatment. To understand the mechanism of action of DDRA in the control of differentiation and tumorigenesis, we studied its interaction with cellular retinoic acid-binding proteins (CRABPs) and nuclear all-trans-retinoic acid (RA) receptors (RARs), and 9-cis-retinoic acid receptors (RXRs). The IC50 plots of DDRA for inhibition of [3H]RA binding to CRABP I and II and to RAR alpha, beta and gamma illustrate that this retinoid binds with the same affinity as RA to these proteins. DDRA, however, showed higher affinity than RA for RXR alpha. Evaluation of the transcriptional activation potential of DDRA in CV-1 cells showed that this retinoid induced RAR alpha-mediated transcription to the same magnitude as RA in the 10(-9) to 10(-6) M concentration range. However, in comparison to RA, DDRA produced a 2- to 3-fold higher activation of the transcription mediated by RXR alpha homodimers, as well as RAR beta-RXR alpha heterodimers. These results suggest that the biological activity of retinoids in the skin may be attained through the joint potential of both RA and DDRA.


Assuntos
Receptores do Ácido Retinoico/efeitos dos fármacos , Vitamina A/análogos & derivados , Animais , Ligação Competitiva , Células COS , Humanos , Camundongos , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Tretinoína/farmacologia , Vitamina A/farmacologia
8.
Anticancer Res ; 16(3A): 1177-81, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8702232

RESUMO

3-Methyl-4-oxoretinoic acid and 3-cinnamyl-4-oxoretinoic acid bind to a cellular retinoic acid-binding protein (CRABP-II) and to a retinoic acid-receptor protein (RARa). These analogs of 4-oxoretinoic acid, as well as the parent compound, have less binding affinity than retinoic acid. Cotransfection assays in CV-1 cells with plasmids containing cDNAs for RAR alpha, RAR beta and RAR gamma (homodimers) and RAR alpha-RXR alpha and RAR beta-RXR alpha (heterodimers), indicate that 3-cinnamyl-4-oxoretinoic acid induces relatively less transcriptional activity than 4-oxoretinoic acid and its 3-methyl analog, both of which are less effective than retinoic acid. In differentiating mouse F9 embryocarcarcinoma cells, the order of effectiveness is retinoic acid > 4-oxoretinoic acid = 3-methyl-4-oxoretinoic acid > 3-cinnamyl-4-oxoretinoic acid. This order of potency is similar to that for inhibition of induction of ornithine decarboxylase (ODC) activity and for prevention of papillomas on the skin of mice. Binding to CRABP-II and activation of RARs appear to be important factors for expression of differentiating activity, inhibition of induction of ODC activity and prevention of papillomas on the skin of mice.


Assuntos
Tretinoína/análogos & derivados , Tretinoína/farmacologia , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Embrião de Galinha , Indução Enzimática/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Papiloma/prevenção & controle , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Neoplasias Cutâneas/prevenção & controle , Ativação Transcricional/efeitos dos fármacos , Tretinoína/metabolismo
9.
J Biol Chem ; 270(43): 25402-10, 1995 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-7592707

RESUMO

Two sequence elements located at -111 to -100 base pairs and -70 to -50 base pairs in the 5'-flanking region of the bovine CYP11A gene and in closely related positions in CYP11A of other species contain G-rich regions that are similar to the consensus Sp1-binding site. These sequences bind the purified transcription factor Sp1 as well as nuclear proteins from mouse Y1 adrenal cells that interact with an antibody specific for Sp1. Both of these CYP11A sequences support basal and cAMP-dependent transcription of reporter gene plasmids transfected into Y1 cells, and mutations within the G-rich -111/-100-base pair sequence that reduce or eliminate the binding of Sp1-related Y1 nuclear proteins also markedly reduce cAMP-induced transcription. cAMP-dependent transcription supported by both CYP11A sequence elements is mediated by protein kinase A at levels comparable to that promoted by different cAMP-response sequences and transcription factors in other genes involved in steroidogenesis. These results indicate that ACTH-dependent regulation of cholesterol side chain cleavage cytochrome P450 levels in the adrenal cortex which is mediated through cAMP involves the ubiquitous transcription factor Sp1.


Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Regiões Promotoras Genéticas/genética , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Córtex Suprarrenal/enzimologia , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Análise Mutacional de DNA , Regulação da Expressão Gênica , Genes Reporter , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Esteroides/biossíntese
10.
J Biol Chem ; 267(24): 17333-8, 1992 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-1324931

RESUMO

To analyze the transcriptional regulatory elements in rabbit cytochrome P450IIC genes, varying lengths of the 5'-flanking regions of CYP2C1 and CYP2C2 were fused to a luciferase reporter gene. Promoter activity was assayed by transfection into HepG2 cells, a hepatic cell line, and monkey kidney COS-1 cells, a nonhepatic cell line. Activity of the CYP2C1 promoter in HepG2 cells increased slightly with progressive 5' deletions of the 5'-flanking region from nucleotide -3600 to -1500 relative to the transcription start site. Additional deletions to -900, -358, and -116 each reduced activity by about 50%, and deletion of the sequence from -116 to -67 reduced activity by a factor of 12. Activity of the CYP2C2 promoter increased about 3-fold with progressive 5' deletions of sequence from nucleotide -3500 to -410. In contrast, deletions of sequences from -251 to -193 and from -133 to -64 reduced promoter activity by factors of 2 and 8, respectively. In COS-1 cells, the maximum activities of the CYP2C1 and CYP2C2 promoters normalized to a Rous sarcoma viral promoter were about 10-20% of that in the HepG2 cells. The changes in activity between different constructions in COS-1 cells largely paralleled those in the HepG2 cells except for deletions of the sequences -133 to -64 and -116 to -67 for CYP2C1 and CYP2C2, respectively, which produced the largest reduction of promoter activity in HepG2 cells but had little effect in COS-1 cells. These results show that HepG2-specific regulatory elements are present in the regions between -120 and -65 in both genes. Nuclear proteins from HepG2 cells, but not from COS-1 cells, bound to sequences within these regions, and the binding was inhibited by an oligonucleotide containing a sequence conserved in rabbit P450IIC genes which has been designated the HepG2-specific P450 2C factor-1 (HPF1) motif. Mutation of this sequence eliminated the binding of nuclear proteins and reduced transcriptional activity 25-fold. The HPF1 binding sequence is conserved in CYP2A, CYP2C, and CYP2D genes and resembles the binding motif for hepatic nuclear factor-4. These results demonstrate that CYP2C1 and CYP2C2 contain several potential regulatory elements for basal expression, including one HepG2-specific sequence that may be important for liver expression.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Animais , Vírus do Sarcoma Aviário/genética , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Deleção Cromossômica , Sistema Enzimático do Citocromo P-450/biossíntese , Humanos , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transfecção
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