RESUMO
AIM: Using mice deficient in the CaV 3.1 T-type Ca(2+) channel, the aim of the present study was to elucidate the molecular identity of non-L-type channels involved in vascular tone regulation in mesenteric arteries and arterioles. METHODS: We used immunofluorescence microscopy to localize CaV 3.1 channels, patch clamp electrophysiology to test the effects of a putative T-type channel blocker NNC 55-0396 on whole-cell Ca(2+) currents, pressure myography and Ca(2+) imaging to test diameter and Ca(2+) responses of the applied vasoconstrictors, and Q-PCR to check mRNA expression levels of several Ca(2+) handling proteins in wild-type and CaV 3.1(-/-) mice. RESULTS: Our data indicated that CaV 3.1 channels are important for the maintenance of myogenic tone at low pressures (40-80 mm Hg), whereas they are not involved in high-voltage-activated Ca(2+) currents, Ca(2+) entry or vasoconstriction to high KCl in mesenteric arteries and arterioles. Furthermore, we show that NNC 55-0396 is not a specific T-type channel inhibitor, as it potently blocks L-type and non-L-type high-voltage-activated Ca(2+) currents in mouse mesenteric vascular smooth muscle cell. CONCLUSION: Our data using mice deficient in the CaV 3.1 T-type channel represent new evidence for the involvement of non-L-type channels in arteriolar tone regulation. We showed that CaV 3.1 channels are important for the myogenic tone at low arterial pressure, which is potentially relevant under resting conditions in vivo. Moreover, CaV 3.1 channels are not involved in Ca(2+) entry and vasoconstriction to large depolarization with, for example, high KCl. Finally, we caution against using NNC 55-0396 as a specific T-type channel blocker in native cells expressing high-voltage-activated Ca(2+) channels.
Assuntos
Canais de Cálcio Tipo T/deficiência , Hipotensão/fisiopatologia , Artérias Mesentéricas/fisiopatologia , Tono Muscular/fisiologia , Músculo Liso Vascular/fisiopatologia , Animais , Benzimidazóis/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/genética , Ciclopropanos/farmacologia , Modelos Animais de Doenças , Hipotensão/metabolismo , Hipotensão/patologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tono Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Naftalenos/farmacologia , Técnicas de Patch-Clamp , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
AIM: In rat afferent arterioles we investigated the role of Na(+) entry in noradrenaline (NA)-induced depolarization and voltage-dependent Ca(2+) entry together with the importance of the transient receptor potential channel (TRPC) subfamily for non-voltage-dependent Ca(2+) entry. METHODS: R (340/380) Fura-2 fluorescence was used as an index for intracellular free Ca(2+) concentration ([Ca(2+)](i)). Immunofluorescence detected the expression of TRPC channels. RESULTS: TRPC 1, 3 and 6 were expressed in afferent arteriolar vascular smooth muscle cells. Under extracellular Na(+)-free (0 Na) conditions, the plateau response to NA was 115% of the baseline R(340/380) (control response 123%). However, as the R(340/380) baseline increased (7%) after 0 Na the plateau reached the same level as during control conditions. Similar responses were obtained after blockade of the Na(+)/Ca(2+) exchanger. The L-type blocker nifedipine reduced the plateau response to NA both under control (from 134% to 116% of baseline) and 0 Na conditions (from 112% to 103% of baseline). In the presence of nifedipine, the putative TRPC channel blockers SKF 96365 (30 µm) and Gd(3+) (100 µm) further reduced the plateau Ca(2+) responses to NA (from 117% to 102% and from 117% to 110% respectively). CONCLUSION: We found that Na(+) is not crucial for the NA-induced depolarization that mediates Ca(2+) entry via L-type channels. In addition, the results are consistent with the idea that TRPC1/3/6 Ca(2+) -permeable cation channels expressed in afferent arteriolar smooth muscle cells mediate Ca(2+) entry during NA stimulation.
Assuntos
Agonistas Adrenérgicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Cálcio/metabolismo , Rim/irrigação sanguínea , Músculo Liso Vascular/efeitos dos fármacos , Nifedipino/farmacologia , Norepinefrina/farmacologia , Sódio/metabolismo , Canais de Cátion TRPC/efeitos dos fármacos , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/metabolismo , Compostos de Benzil/farmacologia , Canais de Cálcio Tipo L/metabolismo , Imunofluorescência , Gadolínio/metabolismo , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Potenciais da Membrana , Microscopia de Fluorescência , Músculo Liso Vascular/metabolismo , Ratos , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo , Canais de Cátion TRPC/metabolismo , Tiazolidinas/farmacologia , Fatores de TempoRESUMO
The identification of orthologous relationships forms the basis for most comparative genomics studies. Here, we present the second version of the eggNOG database, which contains orthologous groups (OGs) constructed through identification of reciprocal best BLAST matches and triangular linkage clustering. We applied this procedure to 630 complete genomes (529 bacteria, 46 archaea and 55 eukaryotes), which is a 2-fold increase relative to the previous version. The pipeline yielded 224,847 OGs, including 9724 extended versions of the original COG and KOG. We computed OGs for different levels of the tree of life; in addition to the species groups included in our first release (i.e. fungi, metazoa, insects, vertebrates and mammals), we have now constructed OGs for archaea, fishes, rodents and primates. We automatically annotate the non-supervised orthologous groups (NOGs) with functional descriptions, protein domains, and functional categories as defined initially for the COG/KOG database. In-depth analysis is facilitated by precomputed high-quality multiple sequence alignments and maximum-likelihood trees for each of the available OGs. Altogether, eggNOG covers 2,242 035 proteins (built from 2,590,259 proteins) and provides a broad functional description for at least 1,966,709 (88%) of them. Users can access the complete set of orthologous groups via a web interface at: http://eggnog.embl.de.
Assuntos
Motivos de Aminoácidos/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Animais , Archaea , Biologia Computacional/tendências , Bases de Dados de Proteínas , Peixes , Genoma Bacteriano , Humanos , Armazenamento e Recuperação da Informação/métodos , Internet , Primatas , Estrutura Terciária de Proteína , Ratos , SoftwareRESUMO
To assess the potential of protein function prediction in environmental genomics data, we analyzed shotgun sequences from four diverse and complex habitats. Using homology searches as well as customized gene neighborhood methods that incorporate intergenic and evolutionary distances, we inferred specific functions for 76% of the 1.4 million predicted ORFs in these samples (83% when nonspecific functions are considered). Surprisingly, these fractions are only slightly smaller than the corresponding ones in completely sequenced genomes (83% and 86%, respectively, by using the same methodology) and considerably higher than previously thought. For as many as 75,448 ORFs (5% of the total), only neighborhood methods can assign functions, illustrated here by a previously undescribed gene associated with the well characterized heme biosynthesis operon and a potential transcription factor that might regulate a coupling between fatty acid biosynthesis and degradation. Our results further suggest that, although functions can be inferred for most proteins on earth, many functions remain to be discovered in numerous small, rare protein families.
Assuntos
Genoma Bacteriano , Genoma , Biblioteca Genômica , Proteínas/genética , Animais , Biofilmes , Bases de Dados Factuais , Variação Genética , Modelos Genéticos , Fases de Leitura Aberta , Proteínas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The taxonomic composition of environmental communities is an important indicator of their ecology and function. We used a set of protein-coding marker genes, extracted from large-scale environmental shotgun sequencing data, to provide a more direct, quantitative, and accurate picture of community composition than that provided by traditional ribosomal RNA-based approaches depending on the polymerase chain reaction. Mapping marker genes from four diverse environmental data sets onto a reference species phylogeny shows that certain communities evolve faster than others. The method also enables determination of preferred habitats for entire microbial clades and provides evidence that such habitat preferences are often remarkably stable over time.
Assuntos
Bactérias/classificação , Ecossistema , Microbiologia Ambiental , Genômica , Filogenia , Animais , Bactérias/genética , Evolução Biológica , Osso e Ossos/microbiologia , Genes Bacterianos , Genes de RNAr , Marcadores Genéticos , Funções Verossimilhança , Mineração , Água do Mar/microbiologia , Microbiologia do Solo , Microbiologia da Água , Baleias/microbiologiaRESUMO
At present, diabetic kidney disease affects about 15 - 25% of all type 1 diabetic patients and 20 - 40% of all patients with type 2 diabetes. The mechanisms underlying the development of diabetic kidney disease are extremely complex and not yet fully understood. Among the many potential pathogenic mechanisms responsible for the progression in diabetic kidney disease, the involvement of metabolic factors beyond blood glucose (such as advanced glycation end products (AGEs)) has been suggested. This review will present the emerging evidence in support of a significant role of AGE formation in the development of diabetic kidney disease. AGEs mediate their effects through two main pathways - through a receptor-independent AGE cross-link formation pathway and through a receptor-dependent pathway where AGEs bind to specific cell surface associated receptors, the receptor for AGE (RAGE) being the most well-characterised so far. First, we will describe the AGE-RAGE system, including its localisation in the normal kidney, and then move on to discuss in vitro and in vivo studies (that is, experimental and clinical data) in support of a pathogenic role of AGE-RAGE and AGE cross-link interaction in the development of diabetic kidney disease. Finally, the effects of known and potential inhibitors of AGE-RAGE and AGE cross-link systems in diabetic kidney disease will be examined.
Assuntos
Nefropatias Diabéticas/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Rim/metabolismo , Receptores Imunológicos/metabolismo , Animais , Nefropatias Diabéticas/patologia , Humanos , Rim/patologia , Receptor para Produtos Finais de Glicação AvançadaRESUMO
MOTIVATION: The human genome project has led to the discovery of many human protein coding genes which were previously unknown. As a large fraction of these are functionally uncharacterized, it is of interest to develop methods for predicting their molecular function from sequence. RESULTS: We have developed a method for prediction of protein function for a subset of classes from the Gene Ontology classification scheme. This subset includes several pharmaceutically interesting categories-transcription factors, receptors, ion channels, stress and immune response proteins, hormones and growth factors can all be predicted. Although the method relies on protein sequences as the sole input, it does not rely on sequence similarity, but instead on sequence derived protein features such as predicted post translational modifications (PTMs), protein sorting signals and physical/chemical properties calculated from the amino acid composition. This allows for prediction of the function for orphan proteins where no homologs can be found. Using this method we propose two novel receptors in the human genome, and further demonstrate chromosomal clustering of related proteins.
Assuntos
Algoritmos , Sistemas de Gerenciamento de Base de Dados , Redes Neurais de Computação , Proteínas/química , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Bases de Dados de Proteínas , Perfilação da Expressão Gênica/métodos , Humanos , Armazenamento e Recuperação da Informação/métodos , Reconhecimento Automatizado de Padrão , Proteínas/classificação , Proteínas/genética , Homologia de Sequência , Relação Estrutura-AtividadeRESUMO
Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads ( Bufo bufo) and frogs ( Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl(-)) on the external side. Active proton secretion was 34.2+/-2.0 pmol.cm(-2).s(-1) ( n=18) in frog skin, and 16.7+/-1.7 pmol.cm(-2).s(-1) ( n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential ( V(T)), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO(2)/HCO(3)(-), indicating the presence of a rheogenic proton pump. Cl(-) influx was 37.4+/-7.5 pmol.cm(-2).s(-1) ( n=14) in frog skin and 19.5+/-3.5 pmol.cm(-2).s(-1) ( n=11) in toad skin. In toad skin, the mean Cl(-) flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl(-). Cl(-) influx in toad skin was unaffected by large perturbations (100-150 mV) of V(T), which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl(-) efflux seemed to be dependent on V(T). 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl(-) influx and, surprisingly, increased Cl(-) efflux in toad skin. Influx and efflux of Cl(-) in toad skin were highly dependent on the external [Cl(-)] in the freshwater range (0.1-4 mM). (36)Cl(-) influx decreased whereas the total Cl(-) efflux increased as a function of external [Cl(-)]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl(-). Ethoxzolamide (100 micro M), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl(-) influx in frog skin. Concanamycin A (0.1-10 micro M), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 micro M) significantly reduced Cl(-) influx in frog skin. We suggest that the active proton secretion and Cl(-) influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl(-) uptake in fresh water by creating a favourable gradient for an apical HCO(3)(-) exit in exchange for external Cl(-). The data also suggest that a carbonic anhydrase activity provides H(+) and HCO(3)(-) for apically co-expressed proton pumps and Cl(-)/HCO(3)(-) exchangers.
Assuntos
Cloretos/farmacocinética , Água Doce , Macrolídeos , Bombas de Próton/metabolismo , Pele/metabolismo , Animais , Antibacterianos/farmacologia , Bufo bufo , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Inibidores da Bomba de Prótons , Prótons , Rana esculentaRESUMO
We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects such as the length, isoelectric point and composition of the polypeptide chain.
Assuntos
Biologia Computacional/métodos , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Proteínas/química , Proteínas/classificação , Bases de Dados de Proteínas , Enzimas/química , Enzimas/classificação , Enzimas/metabolismo , Genoma Humano , Glicosilação , Humanos , Ponto Isoelétrico , Linguística , Redes Neurais de Computação , Fosforilação , Mapeamento Físico do Cromossomo , Ligação Proteica , Transporte Proteico , Proteínas/metabolismo , SoftwareRESUMO
In sequenced microbial genomes, some of the annotated genes are actually not protein-coding genes, but rather open reading frames that occur by chance. Therefore, the number of annotated genes is higher than the actual number of genes for most of these microbes. Comparison of the length distribution of the annotated genes with the length distribution of those matching a known protein reveals that too many short genes are annotated in many genomes. Here we estimate the true number of protein-coding genes for sequenced genomes. Although it is often claimed that Escherichia coli has about 4300 genes, we show that it probably has only approximately 3800 genes, and that a similar discrepancy exists for almost all published genomes.
Assuntos
Escherichia coli/genética , Genoma Bacteriano , Genoma , Bases de Dados Factuais , Modelos Estatísticos , Fases de Leitura Aberta , Saccharomyces cerevisiae/genéticaRESUMO
MOTIVATION: The whole genomes submitted to GenBank contain valuable information about the function of genes as well as the upstream sequences and whole cell expression provides valuable information on gene regulation. To utilize these large amounts of data for a biological understanding of the regulation of gene expression, new automatic methods for pattern finding are needed. RESULTS: Two word-analysis algorithms for automatic discovery of regulatory sequence elements have been developed. We show that sequence patterns correlated to whole cell expression data can be found using Kolmogorov-Smirnov tests on the raw data, thereby eliminating the need for clustering co-regulated genes. Regulatory elements have also been identified by systematic calculations of the significance of correlations between words found in the functional annotation of genes and DNA words occurring in their promoter regions. Application of these algorithms to the Saccharomyces cerevisiae genome and publicly available DNA array data sets revealed a highly conserved 9-mer occurring in the upstream regions of genes coding for proteasomal subunits. Several other putative and known regulatory elements were also found. AVAILABILITY: Upon request.
Assuntos
Algoritmos , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas , Automação , Sítios de Ligação , Cisteína Endopeptidases , Perfilação da Expressão Gênica/métodos , Complexos Multienzimáticos , Complexo de Endopeptidases do Proteassoma , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismoRESUMO
We have performed a computational analysis of DNA structural features in 18 fully sequenced prokaryotic genomes using models for DNA curvature, DNA flexibility, and DNA stability. The structural values that are computed for the Escherichia coli chromosome are significantly different from (and generally more extreme than) that expected from the nucleotide composition. To aid this analysis, we have constructed tools that plot structural measures for all positions in a long DNA sequence (e.g. an entire chromosome) in the form of color-coded wheels (http://www.cbs.dtu. dk/services/GenomeAtlas/). We find that these "structural atlases" are useful for the discovery of interesting features that may then be investigated in more depth using statistical methods. From investigation of the E. coli structural atlas, we discovered a genome-wide trend, where an extended region encompassing the terminus displays a high of level curvature, a low level of flexibility, and a low degree of helix stability. The same situation is found in the distantly related Gram-positive bacterium Bacillus subtilis, suggesting that the phenomenon is biologically relevant. Based on a search for long DNA segments where all the independent structural measures agree, we have found a set of 20 regions with identical and very extreme structural properties. Due to their strong inherent curvature, we suggest that these may function as topological domain boundaries by efficiently organizing plectonemically supercoiled DNA. Interestingly, we find that in practically all the investigated eubacterial and archaeal genomes, there is a trend for promoter DNA being more curved, less flexible, and less stable than DNA in coding regions and in intergenic DNA without promoters. This trend is present regardless of the absolute levels of the structural parameters, and we suggest that this may be related to the requirement for helix unwinding during initiation of transcription, or perhaps to the previously observed location of promoters at the apex of plectonemically supercoiled DNA. We have also analyzed the structural similarities between groups of genes by clustering all RNA and protein-encoding genes in E. coli, based on the average structural parameters. We find that most ribosomal genes (protein-encoding as well as rRNA genes) cluster together, and we suggest that DNA structure may play a role in the transcription of these highly expressed genes.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Genoma Bacteriano , Conformação de Ácido Nucleico , Proteínas de Bactérias/genética , Pareamento de Bases/genética , Cor , Biologia Computacional , Simulação por Computador , Cristalografia por Raios X , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Desoxirribonuclease I/metabolismo , Genes Bacterianos/genética , Modelos Moleculares , Nucleossomos/química , Nucleossomos/genética , Reconhecimento Automatizado de Padrão , Filogenia , Maleabilidade , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , Software , Estatística como Assunto , TermodinâmicaRESUMO
The recently published complete DNA sequence of the bacterium Thermotoga maritima provides evidence, based on protein sequence conservation, for lateral gene transfer between Archaea and Bacteria. We introduce a new method of periodicity analysis of DNA sequences, based on structural parameters, which brings independent evidence for the lateral gene transfer in the genome of T.maritima. The structural analysis relates the Archaea-like DNA sequences to the genome of Pyrococcus horikoshii. Analysis of 24 complete genomic DNA sequences shows different periodicity patterns for organisms of different origin. The typical genomic periodicity for Bacteria is 11 bp whilst it is 10 bp for Archaea. Eukaryotes have more complex spectra but the dominant period in the yeast Saccharomyces cerevisiae is 10.2 bp. These periodicities are most likely reflective of differences in chromatin structure.
Assuntos
Biologia Computacional , DNA/genética , Genoma Bacteriano , Modelos Genéticos , Recombinação Genética , Thermotoga maritima/genética , Cromatina/química , Cromatina/genética , DNA/química , Análise de Fourier , Genoma Arqueal , Genoma Fúngico , Conformação de Ácido Nucleico , Filogenia , Pyrococcus/genética , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise Espectral , TermodinâmicaRESUMO
We show here how pathogenicity islands can be analysed using GenomeAtlases, which is a method for visualising repeats, DNA structural characteristics, and base composition of chromosomes and plasmids. We have applied this method to the E. coli plasmid pO157, and the Y. pestis plasmid pPCP1. In both cases pathogenic genes were shown to differ in A + T content and structural properties. Furthermore, examination of an antibiotic resistance gene cluster from S. typhimurium showed that the same was true for genes encoding antibiotic resistance.
Assuntos
Resistência Microbiana a Medicamentos/genética , Escherichia coli O157/patogenicidade , Salmonella typhimurium/patogenicidade , Yersinia pestis/patogenicidade , Composição de Bases , DNA Bacteriano/análise , DNA Bacteriano/genética , Escherichia coli O157/genética , Genes Bacterianos/genética , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico , Salmonella typhimurium/genética , Virulência/genética , Yersinia pestis/genéticaRESUMO
A low-bicarbonate concentration and an acidic pH in the luminal fluid of the epididymis and vas deferens are important for sperm maturation. These factors help maintain mature sperm in an immotile but viable state during storage in the cauda epididymidis and vas deferens. Two proton extrusion mechanisms, an Na(+)/H(+) exchanger and an H(+)ATPase, have been proposed to be involved in this luminal acidification process. The Na(+)/H(+) exchanger has not yet been localized in situ, but we have reported that H(+)ATPase is expressed on the apical membrane of apical (or narrow) and clear cells of the epididymis. These cells are enriched in carbonic anhydrase II, indicating the involvement of bicarbonate in the acidification process and suggesting that the epididymis is a site of bicarbonate reabsorption. Previous unsuccessful attempts to localize the Cl/HCO(3) anion exchanger AE1 in rat epididymis did not investigate other anion exchanger (AE) isoforms. In this report, we used a recently described SDS antigen unmasking treatment to localize the Cl/HCO(3) exchanger AE2 in rat and mouse epididymis. AE2 is highly expressed in the initial segment, intermediate zone, and caput epididymidis, where it is located on the basolateral membrane of epithelial cells. The cauda epididymidis and vas deferens also contain basolateral AE2, but in lower amounts. The identity of the AE2 protein was further confirmed by the observation that basolateral AE2 expression was unaltered in the epididymis of AE1-knockout mice. Basolateral AE2 may participate in bicarbonate reabsorption and luminal acidification, and/or may be involved in intracellular pH homeostasis of epithelial cells of the male reproductive tract.
Assuntos
Proteínas de Transporte de Ânions , Epididimo/química , Proteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Animais , Antiporters/genética , Antiporters/fisiologia , Antiportadores de Cloreto-Bicarbonato , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Coelhos , Ratos , Ratos Sprague-Dawley , Proteínas SLC4ARESUMO
An acidic environment is important for sperm maturation in the epididymis and also helps to maintain mature sperm in an immotile state during storage in this organ. Both an Na+/H+ exchanger and an H+ATPase have been implicated in this process. The H+ATPase is concentrated in specialized apical (and/or narrow) and clear cells of the epididymis, while the Na+/H+ exchanger has not yet been localized in situ. As in other proton-secreting epithelia, bicarbonate transport occurs in the epididymis, where it is implicated in luminal acidification. In this study we used an antibody raised against a fusion protein (maltose-binding protein: MBP-NBC-5) from the C-terminus of the recently cloned rat kidney Na+/HCO3- cotransporter (NBC) to localize this protein in the epididymis and vas deferens of the rat. The distribution of the respective mRNA was mapped by in situ hybridization. NBC message was strongly expressed in the initial segment and the intermediate zone of the epididymis, and the NBC-5 antibody gave a strong basolateral staining in both principal cells and apical/narrow cells in this region. Western blotting revealed a single band at about 160 kDa in the epididymis. The intensity of staining as well as mRNA levels decreased in the cauda epididymidis and in the vas deferens, where only weak staining was seen. Basolateral NBC may function in parallel with apical proton secretion to regulate luminal acidification and/or bicarbonate reabsorption in the excurrent duct system.
Assuntos
Proteínas de Transporte/análise , Proteínas de Transporte/genética , Epididimo/química , RNA Mensageiro/análise , Animais , Western Blotting , Imunofluorescência , Técnicas Imunoenzimáticas , Hibridização In Situ , Masculino , Microscopia Eletrônica , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Simportadores de Sódio-BicarbonatoRESUMO
We describe here GenomeAtlases as a method for visualising three different aspects of complete microbial chromosomes: repeats, DNA structural characteristics, and base composition. We have applied this method to all publicly available genomes, and find a general strand preference of global repeats. The atlas for the Mycoplasma genitalium genome is presented as an example, and results from all three views are consistent with known characteristics of the genome.
Assuntos
Genoma Bacteriano , Composição de Bases/genética , Cromossomos/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Mycoplasma/genética , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
Protein engineering by inserting stretches of random DNA sequences into target genes in combination with adequate screening or selection methods is a versatile technique to elucidate and improve protein functions. Established compounds for generating semi-random DNA sequences are spiked oligonucleotides which are synthesised by interspersing wild type (wt) nucleotides of the target sequence with certain amounts of other nucleotides. Directed spiking strategies reduce the complexity of a library to a manageable format compared with completely random libraries. Computational algorithms render feasible the calculation of appropriate nucleotide mixtures to encode specified amino acid subpopulations. The crucial element in the ranking of spiked codons generated during an iterative algorithm is the scoring function. In this report three scoring functions are analysed: the sum-of-square-differences function s, a modified cubic function c, and a scoring function m derived from maximum likelihood considerations. The impact of these scoring functions on calculated amino acid distributions is demonstrated by an example of mutagenising a domain surrounding the active site serine of subtilisin-like proteases. At default weight settings of one for each amino acid, the new scoring function m is superior to functions s and c in finding matches to a given amino acid population.
Assuntos
Algoritmos , Desoxirribonucleotídeos/genética , Modelos Genéticos , Mutagênese Insercional/estatística & dados numéricos , Aminoácidos/genética , Composição de Bases , Códon , Desoxirribonucleotídeos/síntese química , Biblioteca Gênica , Subtilisinas/genéticaRESUMO
We have hypothesized that a major role of the apical H(+)-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl- via an apical Cl-/HCO3(-)-exchanger. The activity of the H+ pump was studied by monitoring mucosal [H+]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 +/- 0.07 (mean +/- SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 +/- 18 microns, corresponding to an estimated "unstirred layer" thickness of 329 +/- 29 microns. Mucosal acidification was dependent on serosal pCO2, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H+. The [H+] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H+]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H+ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace's equation. Model calculations predicted that with a physiological density of MR cells, the [H+] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H+]-gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H+]-gradient depended on the mucosal anion such as to indicate that base (HCO3-) is excreted in exchange not only for Cl-, but also for Br- and I-, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.