Dendritic cell ontogeny: A human dendritic cell lineage of myeloid���origin

Dendritic cells (DC) have been thought to represent a family of closely related cells with similar functions and developmental pathways. The best-characterized precursors are the epidermal Langerhans cells, which migrate to lymphoid organs and become activated DC in response to inflammatory stimuli. Here, we demonstrate that a large subset of DC in the T cell-dependent areas of human lymphoid organs are nonactivated cells and belong to a separate lineage that can be identified by high levels of the interleukin 3 receptor �� chain (IL-3R��hi). The CD34+IL-3R��hi DC progenitors are of myeloid origin and are distinct from those that give rise to Langerhans cells in vitro. The IL-3R��hi DC furthermore appear to migrate to lymphoid organs independently of inflammatory stimuli or foreign antigens. Thus, DC are heterogeneous with regard to function and ontogeny.

Tyrosine Phosphorylation Regulates Cell Cycle-dependent Nuclear Localization of Cdc48p

Cdc48p from Saccharomyces cerevisiae and its highly conserved mammalian homologue VCP (valosin-containing protein) are ATPases with essential functions in cell division and homotypic fusion of endoplasmic reticulum vesicles. Both are mainly attached to the endoplasmic reticulum, but relocalize in a cell cycle-dependent manner: Cdc48p enters the nucleus during late G1; VCP aggregates at the centrosome during mitosis. The nuclear import signal sequence of Cdc48p was localized near the amino terminus and its function demonstrated by mutagenesis. The nuclear import is regulated by a cell cycle-dependent phosphorylation of a tyrosine residue near the carboxy terminus. Two-hybrid studies indicate that the phosphorylation results in a conformational change of the protein, exposing the nuclear import signal sequence previously masked by a stretch of acidic residues.

The PDZ Domain of the LIM Protein Enigma Binds to ��-Tropomyosin

PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein ��-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal ��-TM). The interaction between Enigma and skeletal ��-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal ��-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal ��-TM in transfected cells. The association of Enigma with skeletal ��-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells.

Gene content phylogeny of herpesviruses

Clusters of orthologous groups [COGs; Tatusov, R. L., Koonin, E. V. & Lipman, D. J. (1997) Science 278, 631���637] were identified for a set of 13 completely sequenced herpesviruses. Each COG represented a family of gene products conserved across several herpes genomes. These families were defined without using an arbitrary threshold criterion based on sequence similarity. The COG technique was modified so that variable stringency in COG construction was possible. High stringencies identify a core set of highly conserved genes. Varying COG stringency reveals differences in the degree of conservation between functional classes of genes. The COG data were used to construct whole-genome phylogenetic trees based on gene content. These trees agree well with trees based on other methods and are robust when tested by bootstrap analysis. The COG data also were used to construct a reciprocal tree that clustered genes with similar phylogenetic profiles. This clustering may give clues to genes with related functions or with related histories of acquisition and loss during herpesvirus evolution.

Increased expression of preprotachykinin-I and neurokinin receptors in human breast cancer cells: Implications for bone marrow metastasis

Neuropeptides are implicated in many tumors, breast cancer (BC) included. Preprotachykinin-I (PPT-I) encodes multiple neuropeptides with pleiotropic functions such as neurotransmission, immune/hematopoietic modulation, angiogenesis, and mitogenesis. PPT-I is constitutively expressed in some tumors. In this study, we investigated a role for PPT-I and its receptors, neurokinin-1 (NK-1) and NK-2, in BC by using quantitative reverse transcription���PCR, ELISA, and in situ hybridization. Compared with normal mammary epithelial cells (n = 2) and benign breast biopsies (n = 21), BC cell lines (n = 7) and malignant breast biopsies (n = 25) showed increased expression of PPT-I and NK-1. NK-2 levels were high in normal and malignant cells. Specific NK-1 and NK-2 antagonists inhibited BC cell proliferation, suggesting autocrine and/or intercrine stimulation of BC cells by PPT-I peptides. NK-2 showed no effect on the proliferation of normal cells but mediated the proliferation of BC cells. Cytosolic extracts from malignant BC cells enhanced PPT-I translation whereas extracts from normal mammary epithelial cells caused no change. These enhancing effects may be protein-specific because a similar increase was observed for IL-6 translation and no effect was observed for IL-1�� and stem cell factor. The data suggest that PPT-I peptides and their receptors may be important in BC development. Considering that PPT-I peptides are hematopoietic modulators, these results could be extended to understand early integration of BC cells in the bone marrow, a preferred site of metastasis. Molecular signaling transduced by PPT-I peptides and the mechanism that enhances translation of PPT-I mRNA could lead to innovative strategies for BC treatments and metastasis.

Nonlinear indicial response of complex nonstationary oscillations as pulmonary hypertension responding to step hypoxia

This paper is devoted to the quantization of the degree of nonlinearity of the relationship between two biological variables when one of the variables is a complex nonstationary oscillatory signal. An example of the situation is the indicial responses of pulmonary blood pressure (P) to step changes of oxygen tension (��pO2) in the breathing gas. For a step change of ��pO2 beginning at time t1, the pulmonary blood pressure is a nonlinear function of time and ��pO2, which can be written as P(t-t1 | ��pO2). An effective method does not exist to examine the nonlinear function P(t-t1 | ��pO2). A systematic approach is proposed here. The definitions of mean trends and oscillations about the means are the keys. With these keys a practical method of calculation is devised. We fit the mean trends of blood pressure with analytic functions of time, whose nonlinearity with respect to the oxygen level is clarified here. The associated oscillations about the mean can be transformed into Hilbert spectrum. An integration of the square of the Hilbert spectrum over frequency yields a measure of oscillatory energy, which is also a function of time, whose mean trends can be expressed by analytic functions. The degree of nonlinearity of the oscillatory energy with respect to the oxygen level also is clarified here. Theoretical extension of the experimental nonlinear indicial functions to arbitrary history of hypoxia is proposed. Application of the results to tissue remodeling and tissue engineering of blood vessels is discussed.

Glutathione biosynthesis in Arabidopsis trichome cells

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode ��-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238 �� 25, 80 �� 2, and 144 �� 19 ��M were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH]cyt is reduced to 47 �� 5 ��M. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.

Regulation of ribonuclease III processing by double-helical sequence���antideterminants

The double helix is a ubiquitous feature of RNA molecules and provides a target for nucleases involved in RNA maturation and decay. Escherichia coli ribonuclease III participates in maturation and decay pathways by site-specifically cleaving double-helical structures in cellular and viral RNAs. The site of cleavage can determine RNA functional activity and half-life and is specified in part by local tertiary structure elements such as internal loops. The involvement of base pair sequence in determining cleavage sites is unclear, because RNase III can efficiently degrade polymeric double-stranded RNAs of low sequence complexity. An alignment of RNase III substrates revealed an exclusion of specific Watson���Crick bp sequences at defined positions relative to the cleavage site. Inclusion of these ���disfavored��� sequences in a model substrate strongly inhibited cleavage in vitro by interfering with RNase III binding. Substrate cleavage also was inhibited by a 3-bp sequence from the selenocysteine-accepting tRNASec, which acts as an antideterminant of EF-Tu binding to tRNASec. The inhibitory bp sequences, together with local tertiary structure, can confer site specificity to cleavage of cellular and viral substrates without constraining the degradative action of RNase III on polymeric double-stranded RNA. Base pair antideterminants also may protect double-helical elements in other RNA molecules with essential functions.

The major murine systemic lupus erythematosus susceptibility locus, Sle1, is a cluster of functionally related genes

The major murine systemic lupus erythematosus (SLE) susceptibility locus Sle1 is syntenic to a chromosomal region linked with SLE susceptibility in multiple human studies. Congenic analyses have shown that Sle1 breaks tolerance to chromatin, a necessary step for full disease induction that can be suppressed by specific modifier loci. In the present study, our fine mapping analysis of the location of Sle1 has determined that three loci within this congenic interval, termed Sle1a, Sle1b, and Sle1c, can independently cause a loss of tolerance to chromatin. Each displays a distinctive profile of serological and cellular characteristics, with T and B cell functions being more affected by Sle1a and Sle1b, respectively. The epistatic interactions of Sle1 with other susceptibility loci to cause severe nephritis cannot be accounted, however, by these three loci alone, suggesting the existence of an additional locus, termed Sle1d. These findings indicate that the potent autoimmune phenotype caused by the Sle1 genomic interval reflects the combined impact of four, separate, susceptibility genes. This level of genetic complexity, combined with similar findings in other systems, supports the possibility that many complex trait loci reflect the impact of polymorphisms in linked clusters of genes with related functions.

A database and tools for 3-D protein structure comparison and alignment using the Combinatorial Extension (CE) algorithm

The database reported here is derived using the Combinatorial Extension (CE) algorithm which compares pairs of protein polypeptide chains and provides a list of structurally similar proteins along with their structure alignments. Using CE, structure���structure alignments can provide insights into biological function. When a protein of known function is shown to be structurally similar to a protein of unknown function, a relationship might be inferred; a relationship not necessarily detectable from sequence comparison alone. Establishing structure���structure relationships in this way is of great importance as we enter an era of structural genomics where there is a likelihood of an increasing number of structures with unknown functions being determined. Thus the CE database is an example of a useful tool in the annotation of protein structures of unknown function. Comparisons can be performed on the complete PDB or on a structurally representative subset of proteins. The source protein(s) can be from the PDB (updated monthly) or uploaded by the user. CE provides sequence alignments resulting from structural alignments and Cartesian coordinates for the aligned structures, which may be analyzed using the supplied Compare3D Java applet, or downloaded for further local analysis. Searches can be run from the CE web site, http://cl.sdsc.edu/ce.html, or the database and software downloaded from the site for local use.

A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus.

Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant revealed defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. The Hk polypeptide, determined from analysis of cDNA clones, is a homologue of mammalian K+ channel beta subunits (Kv beta). Coexpression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence alignments show that Hk, together with mammalian Kv beta, represents an additional branch of the aldo-keto reductase superfamily. These results are relevant to understanding the function and evolutionary origin of Kv beta.

Sphingosylphosphocholine, a signaling molecule which accumulates in Niemann-Pick disease type A, stimulates DNA-binding activity of the transcription activator protein AP-1.

Sphingosylphosphocholine (SPC) is the deacylated derivative of sphingomyelin known to accumulate in neuropathic Niemann-Pick disease type A. SPC is a potent mitogen that increases intracellular free Ca2+ and free arachidonate through pathways that are only partly protein kinase C-dependent. Here we show that SPC increased specific DNA-binding activity of transcription activator AP-1 in electrophoretic mobility-shift assays. Increased DNA-binding activity of AP-1 was detected after only 1-3 min, was maximal after 6 hr, and remained elevated at 12-24 hr. c-Fos was found to be a component of the AP-1 complex. Northern hybridization revealed an increase in c-fos transcripts after 30 min. Since the increase in AP-1 binding activity preceded the increase in c-fos mRNA, posttranslational modifications may be important in mediating the early SPC-induced increases in AP-1 DNA-binding activity. Western analysis detected increases in nuclear c-Jun and c-Fos proteins following SPC treatment. SPC also transactivated a reporter gene construct through the AP-1 recognition site, indicating that SPC can regulate the expression of target genes. Thus, SPC-induced cell proliferation may result from activation of AP-1, linking signal transduction by SPC to gene expression. Since the expression of many proteins with diverse functions is known to be regulated by AP-1, SPC-induced activation of AP-1 may contribute to the pathophysiology of Niemann-Pick disease.

Expression cloning of a cDNA encoding a fish prolactin receptor.

By using an expression cloning strategy, we isolated a single positive clone encoding a tilapia prolactin (PRL) receptor. Tilapia PRL188 was used to screen a freshwater tilapia kidney expression library transfected in COS cells. The tilapia PRL receptor is a mature protein of 606 amino acids. The extracellular domain is devoid of the tandem repeat units present in birds and has two pairs of cysteine residues, a Trp-Ser-Xaa-Trp-Ser motif, and two potential N-glycosylation sites. The cytoplasmic domain contains 372 amino acids, including box 1, a sequence previously shown to be important for signal transduction in mammalian species. Thus, the general structure is similar to the long form of mammalian PRL receptors; however, amino acid comparisons reveal a rather low identity (approximately 37%). Northern blot analysis shows the existence of a single transcript in osmoregulatory tissues and reproductive organs. This localization is in agreement with known functions of PRL in teleosts.

Modula����o da diferencia����o neural de c��lulas troco embrion��rias por transientes de c��lcio intracelulares: pap��is dos receptores purin��rgicos e de canais de c��lcio voltagem-dependentes

Receptores purin��rgicos e canais de c��lcio voltagem-dependentes est��o envolvidos em diversos processos biol��gicos como na gastrula����o, durante o desenvolvimento embrion��rio, e na diferencia����o neural. Quando ativados, canais de c��lcio voltagem-dependentes e receptores purin��rgicos do tipo P2, ativados por nucleot��deos, desencadeiam transientes de c��lcio intracelulares controlando diversos processos biol��gicos. Neste trabalho, n��s estudamos a participa����o de canais de c��lcio voltagem-dependentes e receptores do tipo P2 na gera����o de transientes de c��lcio espont��neos e sua regula����o na express��o de fatores de transcri����o relacionados com a neurog��nese utilizando como modelo c��lulas tronco (CTE) induzidas �� diferencia����o em c��lulas tronco neurais (NSC) com ��cido retin��ico. Descrevemos que CTE indiferenciadas podem ter a prolifera����o acelerada pela ativa����o de receptores P2X7, enquanto que a express��o e a atividade desse receptor precisam ser inibidas para o progresso da diferencia����o em neuroblasto. Al��m disso, ao longo da diferencia����o neural, por an��lise em tempo real dos n��veis de c��lcio intracelular livre identificamos 3 padr��es de oscila����es espont��neas de c��lcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequ��ncia e amplitude aumentadas conforme o andamento da diferencia����o. C��lulas tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas n��o ondas, indicando que ondas dependem exclusivamente de c��lcio oriundo do ret��culo endoplasm��tico pela ativa����o de IP3R. NSC de telenc��falo de embri��o de camundongos transg��nicos ou pr��-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequ��ncia e a amplitude das oscila����es espont��neas de c��lcio do tipo pico. Dados, obtidos por microscopia de luminesc��ncia, da express��o em tempo real de gene rep��rter luciferase fusionado �� Mash1 e Ngn2 revelou que a ativa����o dos receptores P2Y2/P2Y4 aumentou a express��o est��vel de Mash1 enquanto que ativa����o do receptor P2X7 levou ao aumento de Ngn2. Al��m disso, c��lulas na presen��a do quelante de c��lcio extracelular (EGTA) ou do depletor dos estoques intracelulares de c��lcio do ret��culo endoplasm��tico (thapsigargin) apresentaram redu����o na express��o de Mash1 e Ngn2, indicando que ambos s��o regulados pela sinaliza����o de c��lcio. A investiga����o dos canais de c��lcio voltagem-dependentes demonstrou que o influxo de c��lcio gerado por despolariza����o da membrana de NSC diferenciadas de CTE �� decorrente da ativa����o de canais de c��lcio voltagem-dependentes do tipo L. Al��m disso, esse influxo pode controlar o destino celular por estabilizar express��o de Mash1 e induzir a diferencia����o neuronal por fosforila����o e transloca����o do fator de transcri����o CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de c��lcio voltagem-dependentes do tipo L podem modular as oscila����es espont��neas de c��lcio durante a diferencia����o neural e consequentemente alteram o padr��o de express��o de Mash1 e Ngn2 favorecendo a decis��o do destino celular neuronal.

An��lise da microbiota intestinal em adultos�� com h��bitos alimentares distintos e de associa����es com a inflama����o e resist��ncia �� insulina

Introdu����o: A microbiota intestinal possui grande diversidade de bact��rias, predominantemente dos filos Bacteroidetes e Firmicutes, com m��ltiplas fun����es. A alimenta����o pode alterar sua composi����o e fun����o. Alto teor de gordura saturada altera a permeabilidade intestinal, eleva os lipopolissacar��deos e predisp��e �� inflama����o subcl��nica cr��nica. Dieta rica em fibras, como a vegetariana, induz eleva����o de ��cidos graxos de cadeia curta e benef��cios metab��licos. Objetivos: Para analisar a composi����o da microbiota intestinal de adventistas com diferentes h��bitos alimentares e associ��-los �� inflama����o subcl��nica e resist��ncia �� insulina, esta tese incluiu: 1) revis��o dos mecanismos que associam a alimenta����o �� microbiota intestinal e ao risco cardiometab��lico; 2) verifica����o da composi����o da microbiota intestinal segundo diferentes h��bitos alimentares e de associa����es com biomarcadores de doen��as cardiometab��licas; 3) avalia����o da associa����o entre a abund��ncia de Akkermansia muciniphila e o metabolismo da glicose; 4) an��lise da presen��a de enter��tipos e de associa����es com caracter��sticas cl��nicas. M��todos: Este estudo transversal incluiu 295 adventistas estratificados segundo h��bitos alimentares (vegetariano estrito, ovo-lacto-vegetariano e on��voro). Foram avaliadas associa����es com dados cl��nicos, bioqu��micos e inflamat��rios. O perfil da microbiota foi obtido por sequenciamento do gene 16S rRNA (Illumina�� Miseq). Resultados: 1) H�� evid��ncias de que as rela����es entre dieta, inflama����o, resist��ncia �� insulina e risco cardiometab��lico s��o em parte mediadas pela composi����o da microbiota intestinal. 2) Vegetarianos apresentaram melhor perfil cl��nico quando comparados aos on��voros. Confirmou-se maior abund��ncia de Firmicutes e Bacteroidetes, que n��o diferiram segundo a adiposidade corporal. Entretanto, vegetarianos estritos apresentaram mais Bacteroidetes, menos Firmicutes e maior abund��ncia do g��nero Prevotella quando comparados aos outros dois grupos de h��bitos alimentares. Entre os ovo-lactovegetarianos verificou-se maior propor����o de Firmicutes especialmente do g��nero Faecalibacterium. Nos on��voros, houve super-representa����o do filo Proteobacteria (Succinivibrio e Halomonas) comparados aos vegetarianos. 3) Indiv��duos normoglic��micos apresentaram maior abund��ncia de Akkermansia muciniphila que aqueles com glicemia alterada. A abund��ncia desta bact��ria correlacionou-se inversamente �� glicemia e hemoglobina glicosilada. 4) Foram identificados tr��s enter��tipos (Bacteroides, Prevotella e Ruminococcaceae), similares ��queles previamente descritos. As concentra����es de LDL-C foram menores no enter��tipo 2, no qual houve maior frequ��ncia de vegetarianos estritos. Discuss��o: 1) Conhecimentos sobre participa����o da microbiota na fisiopatologia de doen��as poder��o reverter em estrat��gias para manipul��-la para promover sa��de. 2) Apoia-se a hip��tese de que h��bitos alimentares se associam favor��vel ou desfavoravelmente a caracter��sticas metab��licas e inflamat��rias do hospedeiro via altera����es na composi����o da microbiota intestinal. Sugerimos que a exposi����o a alimentos de origem animal possa impactar negativamente nas propor����es de comunidades bacterianas. 3) Sugerimos que a abund��ncia da Akkermansia muciniphila possa participar do metabolismo da glicose. 4) Refor��amos que a exist��ncia de tr��s enter��tipos n��o deva ser espec��fica de certas popula����es/continentes. Apesar de desconhecido o significado biol��gico destes agrupamentos, as correla����es com o perfil lip��dico podem sugerir sua utilidade na avalia����o do risco cardiometab��lico. Conclus��es: Nossos achados fortalecem a ideia de que a composi����o da microbiota intestinal se altera mediante diferentes h��bitos alimentares, que, por sua vez, est��o associados a altera����es nos perfis metab��licos e inflamat��rios. Estudos prospectivos dever��o investigar o potencial da dieta na preven����o de dist��rbios cardiometab��licos mediados pela microbiota.