Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells.

Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

Cell cycle control in Alphaproteobacteria.

NlmCategory="UNASSIGNED">Alphaproteobacteria include many medically and environmentally important organisms. Despite the diversity of their niches and lifestyles, from free-living to host-associated, they usually rely on very similar mechanisms to control their cell cycles. Studies on Caulobacter crescentus still lay the foundation for understanding the molecular details of pathways regulating DNA replication and cell division and coordinating these two processes with other events of the cell cycle. This review highlights recent discoveries on the regulation and the mode of action of conserved global regulators and small molecules like c-di-GMP and (p)ppGpp, which play key roles in cell cycle control. It also describes several newly identified mechanisms that modulate cell cycle progression in response to stresses or environmental conditions.

Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

CCTα and CCTδ chaperonin subunits are essential and required for cilia assembly and maintenance in Tetrahymena

Background - The eukaryotic cytosolic chaperonin CCT is a hetero-oligomeric complex formed by two rings connected back-to-back, each composed of eight distinct subunits (CCTalpha to CCTzeta). CCT complex mediates the folding, of a wide range of newly synthesised proteins including tubulin (alpha, beta and gamma) and actin, as quantitatively major substrates. Methodology/Principal findings - We disrupted the genes encoding CCTalpha and CCTdelta subunits in the ciliate Tetrahymena. Cells lacking the zygotic expression of either CCTalpha or CCTdelta showed a loss of cell body microtubules, failed to assemble new cilia and died within 2 cell cycles. We also show that loss of CCT subunit activity leads to axoneme shortening and splaying of tips of axonemal microtubules. An epitope-tagged CCTalpha rescued the gene knockout phenotype and localized primarily to the tips of cilia. A mutation in CCTalpha, G346E, at a residue also present in the related protein implicated in the Bardet Biedel Syndrome, BBS6, also caused defects in cilia and impaired CCTalpha localization in cilia. Conclusions/Significance - Our results demonstrate that the CCT subunits are essential and required for ciliary assembly and maintenance of axoneme structure, especially at the tips of cilia.

Islet-brain1/JNK-interacting protein-1 is required for early embryogenesis in mice.

Islet-brain1/JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein that organizes the JNK, MKK7, and MLK1 to allow signaling specificity. Targeted disruption of the gene MAPK8IP1 encoding IB1/JIP-1 in mice led to embryonic death prior to blastocyst implantation. In culture, no IB1/JIP-1(-/-) embryos were identified indicating that accelerated cell death occurred during the first cell cycles. IB1/JIP-1 expression was detected in unfertilized oocytes, in spermatozoa, and in different stages of embryo development. Thus, despite the maternal and paternal transmission of the IB1/JIP-1 protein, early transcription of the MAPK8IP1 gene is required for the survival of the fertilized oocytes.

Relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the corpuscles of stannius and the ultimobranchial gland in the goldfish, Carassius auratus L. /

The relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the Corpuscles of Stannius and the Ultimobranchial gland were investigated. Neither the plasma concentration of ionic calcium nor histologically apparent prolactin cell activity could be correlated with photoperiod. Some evidence of a photoperiodic effect on both the Corpuscles of Stannius and the Ultimobranchial gland was obtained. The expected reciprocal relationship between the activity of these glands was not obvious at the histological level . Quantitative and qualitative analysis at the light microscope level revealed, however, that the hormone prolactin-secreting eta cells of the rostral pars distalis and the hypocalcin-secreting cells of the Corpuscles of Stannius may be arranged in a lamellar pattern comprized of synchronous bands of cells in like-phase of a secretory cycle consisting of four stages - synthesis, storage, release and reorganization. Such synchronized cell cycles in these glands have not heretofore been described in literature. It is suggested that the maintenance of at least 255? of the cells in any one phase of the cycle ensures a supply of the required hormone at all times.

Carga viral de seis tipos de Virus del Papiloma Humano de alto riesgo y su asociacion con lesiones cervicales

Introducción: La infección por un tipo de Virus del Papiloma Humano de alto riesgo (VPH-AR), es el factor principal en el desarrollo de Cáncer de Cérvix (CC). La carga viral puede modular esta asociación, por lo que resulta importante su cuantificación y el establecimiento de su relación con lesiones precursoras de CC. Metodología: 60 mujeres con lesiones escamosas intraepiteliales (LEI) y 120 mujeres sin LEI, confirmadas por colposcopia, fueron incluidas en el estudio. Se determinó la carga viral de 6 tipos de VPH-AR, mediante PCR en tiempo real. Se estimaron OR crudos y ajustados para evaluar la asociación entre la carga viral de cada tipo y las lesiones cervicales. Resultados: 93.22% de mujeres con LEI y 91.23% de mujeres negativas, fueron positivas para al menos un tipo de VPH. VPH-18 y VPH-16 fueron los tipos más prevalentes, junto con VPH-31 en mujeres sin LEI. No se encontraron diferencias estadísticamente significativas de las cargas virales entre éstos dos grupos, aunque se observó un mayor carga viral en lesiones para algunos tipos virales. Una mayor frecuencia de lesiones se asoció a infecciones con carga baja de VPH-16 (ORa: 3.53; IC95%: 1.16 – 10.74), en comparación a mujeres con carga alta de VPH-16, (ORa: 2.63; IC95%: 1.09 – 6.36). En infecciones por VPH-31, la presencia de carga viral alta, se asoció con una menor frecuencia de lesiones (ORa: 0.34; IC95%: 0.15 – 0.78). Conclusiones: La prevalencia tipo-específica de VPH se corresponde con las reportadas a nivel mundial. La asociación entre la carga viral del VPH y la frecuencia de LEI es tipo específica y podría depender de la duración de la infección, altas cargas relacionadas con infecciones transitorias, y bajas cargas con persistentes. Este trabajo contribuye al entendimiento del efecto de la carga viral en la historia natural del CC; sin embargo, estudios prospectivos son necesarios para confirmar estos resultados.

CDK9 a potential target for drug development

The family of Cyclin-Dependent Kinases (CDKs) can be subdivided into two major functional groups based on their roles in cell cycle and/or transcriptional control. CDK9 is the catalytic subunit of positive transcription elongation factor b (P-TEFb). CDK9 is the kinase of the TAK complex (Tat-associated kinase complex), and binds to Tat protein of HIV, suggesting a possible role for CDK9 in AIDS progression. CDK9 complexed with its regulatory partner cyclin T1, serves as a cellular mediator of the transactivation function of the HIV Tat protein. P-TEFb is responsible for the phosphorylation of the carboxyl-terminal domain of RNA Pol II, resulting in stimulation of transcription. Furthermore, the complexes containing CDK9 induce the differentiation in distinct tissue. The CDK9/cyclin T1 complex is expressed at higher level in more differentiated primary neuroectodermal and neuroblastoma tumors, showing a correlation between the kinase expression and tumor differentiation grade. This may have clinical and therapeutical implications for these tumor types. Among the CDK inhibitors two have shown to be effective against CDK9: Roscovitine and Flavopiridol. These two inhibitors prevented the replication of human immunodeficiency virus (HIV) type 1 by blocking Tat transactivation of the HIV type 1 promoter. These compounds inhibit CDKs by binding to the catalytic domain in place of ATP, preventing transfer of a phosphate group to the substrate. More sensitive therapeutic agents of CDK9 can be designed, and structural studies can add information in the understanding of this kinase. The major features related to CDK9 inhibition will be reviewed in this article.

Histochemistry and Protein Profile of The Venom Glands of Workers of Neoponera villosa Ants (Hymenoptera: Ponerinae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of the nucleolar cycle in the seminiferous epithelium of rodents

The nucleolus is a subcompartment of the nucleus and the site of ribosome biogenesis. During the mitotic and meiotic cell cycles, a disorganization and later reorganization of the nucleolar material occur, an event called nucleologenesis. In the spermatogenesis of mammals and other vertebrates, there is evidence of the disorganization of the nucleolus at the end of meiosis I, which supplies material for the cytoplasmic formation of an organelle called the chromatoid body (CB). The CB is a structure characteristic of spermatogenic cells and seems to be responsible for RNA metabolism in these cells and for some events of spermiogenesis, such as the formation of the acrosome, cellular communication between spermatids, and the formation of the spermatozoon middle piece and tail. The aim of this paper was to obtain information about the cytochemical and ultrastructural nature of the nucleolar cycle and the distribution of cytoplasmic RNAs in the seminiferous tubule cells of Rattus novergiucus, Mus musculus and Meriones unguiculatus. The testis was fixed in Bouin and Karnovsky solutions for conventional histological analysis and for cytochemical study that included: periodic acid-Schiff, hematoxylin-eosin, Feulgen reaction, silver-ion impregnation, Gomori's reticulin stain, toluidine blue, modified method of critical electrolyte concentration, and basic and acid fast green. The blocks of testis fixed in glutaraldehyde were used for ultrastructural analysis by transmission electron microscopy. Ultrathin sections were double-stained with uranyl acetate and lead citrate. All the techniques used provided information on the origin and function of the CB in the spermatogenic cells. Therefore, considering the persistence of the RNA and nucleolar ribonucleoproteins during spermatogenesis of Rattus novergicus, Mus musculus and Meriones unguiculatus, our findings corroborate the statement that these molecular complexes are very important in the spermiogenesis phases. It can be suggested that these ribonucleoprotein corpuscles (chromatoid bodies) are of nuclear origin and have a role in the successive series of events that occur in the formation of the spermatozoon. Furthermore, these results reinforce the conservation of the mechanisms involved in preserving necessary levels of protein stocks in different stages of cell differentiation, from spermatid to spermatozoon, in these rodent species. ©FUNPEC-RP.

Avaliação dos efeitos citotóxicos, genotóxicos e mutagênicos de 2 classes de agrotóxicos utilizados em cultura de cana-de-açúcar no Estado de São Paulo-Brasil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks, alkylations, or x-ray damage.

The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plus-Giemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions.

Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide.

To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN-promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6-20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles.

Identificazione di miRNA correlati ad EGFR: nuovi potenziali biomarcatori di risposta alla terapia di 2a-3a linea in pazienti con NSCLC metastatico

La diagnosi di neoplasia epiteliale maligna polmonare è legata tradizionalmente alla distinzione tra carcinoma a piccole cellule (small-cell lung cancer, SCLC) e carcinoma non-a piccole cellule del polmone (non-small-cell lung cancer, NSCLC). Nell’ambito del NSCLC attualmente è importante di-stinguere l’esatto istotipo (adenocarcinoma, carcinoma squamocellulare e carcinoma neuroendocrino) perchè l’approccio terapeutico cambia a seconda dell’istotipo del tumore e la chemioterapia si dimostra molto spesso inefficace. Attualmente alcuni nuovi farmaci a bersaglio molecolare per il gene EGFR, come Erlotinib e Gefitinib, sono utilizzati per i pazienti refrattari al trattamento chemioterapico tradizionale, che non hanno risposto a uno o più cicli di chemioterapia o che siano progrediti dopo questa. I test per la rilevazione di specifiche mutazioni nel gene EGFR permettono di utilizzare al meglio questi nuovi farmaci, applicandoli anche nella prima linea di trattamento sui pazienti che hanno una maggiore probabilità di risposta alla terapia. Sfortunatamente, non tutti i pazienti rispondono allo stesso modo quando trattati con farmaci anti-EGFR. Di conseguenza, l'individuazione di biomarcatori predittivi di risposta alla terapia sarebbe di notevole importanza per aumentare l'efficacia dei questi farmaci a target molecolare e trattare con farmaci diversi i pazienti che con elevata probabilità non risponderebbero ad essi. I miRNAs sono piccole molecole di RNA endogene, a singolo filamento di 20-22 nucleotidi che svolgono diverse funzioni, una delle più importanti è la regolazione dell’espressione genica. I miRNAs possono determinare una repressione dell'espressione genica in due modi: 1-legandosi a sequenze target di mRNA, causando così un silenziamento del gene (mancata traduzione in proteina), 2- causando la degradazione dello specifico mRNA. Lo scopo della ricerca era di individuare biomarcatori capaci di identificare precocemente i soggetti in grado di rispondere alla terapia con Erlotinib, aumentando così l'efficacia del farmaco ed evitan-do/riducendo possibili fenomeni di tossicità e il trattamento di pazienti che probabilmente non ri-sponderebbero alla terapia offrendo loro altre opzioni prima possibile. In particolare, il lavoro si è fo-calizzato sul determinare se esistesse una correlazione tra la risposta all'Erlotinib ed i livelli di espressione di miRNAs coinvolti nella via di segnalazione di EGFR in campioni di NSCLC prima dell’inizio della terapia. Sono stati identificati 7 microRNA coinvolti nel pathway di EGFR: miR-7, -21, 128b, 133a, -133b, 146a, 146b. Sono stati analizzati i livelli di espressione dei miRNA mediante Real-Time q-PCR in campioni di NSCLC in una coorte di pazienti con NSCLC metastatico trattati con Erlotinib dal 1° gennaio 2009 al 31 dicembre 2014 in 2°-3° linea dopo fallimento di almeno un ciclo di chemioterapia. I pazienti sottoposti a trattamento con erlotinib per almeno 6 mesi senza presentare progressione alla malattia sono stati definiti “responders” (n=8), gli altri “non-responders” (n=25). I risultati hanno mostrato che miR-7, -133b e -146a potrebbero essere coinvolti nella risposta al trat-tamento con Erlotinib. Le indagini funzionali sono state quindi concentrate su miR-133b, che ha mo-strato la maggiore espressione differenziale tra i due gruppi di pazienti. E 'stata quindi studiata la capacità di miR-133b di regolare l'espressione di EGFR in due linee di cellule del cancro del polmone (A549 e H1299). Sono stati determinati gli effetti di miR-133b sulla crescita cellulare. E’ stato anche analizzato il rapporto tra miR-133b e sensibilità a Erlotinib nelle cellule NSCLC. L'aumento di espressione di miR-133b ha portato ad una down-regolazione del recettore di EGF e del pathway di EGFR relativo alla linea cellulare A549. La linea cellulare H1299 era meno sensibili al miR-133b up-regulation, probabilmente a causa dell'esistenza di possibili meccanismi di resistenza e/o di com-pensazione. La combinazione di miR-133b ed Erlotinib ha aumentato l'efficacia del trattamento solo nella linea cellulare A549. Nel complesso, questi risultati indicano che miR-133b potrebbe aumentare / ripristinare la sensibilità di Erlotinib in una frazione di pazienti.

Inheritance of the CENP-A chromatin domain is spatially and temporally constrained at human centromeres.

BACKGROUND: Chromatin containing the histone variant CENP-A (CEN chromatin) exists as an essential domain at every centromere and heritably marks the location of kinetochore assembly. The size of the CEN chromatin domain on alpha satellite DNA in humans has been shown to vary according to underlying array size. However, the average amount of CENP-A reported at human centromeres is largely consistent, implying the genomic extent of CENP-A chromatin domains more likely reflects variations in the number of CENP-A subdomains and/or the density of CENP-A nucleosomes within individual subdomains. Defining the organizational and spatial properties of CEN chromatin would provide insight into centromere inheritance via CENP-A loading in G1 and the dynamics of its distribution between mother and daughter strands during replication. RESULTS: Using a multi-color protein strategy to detect distinct pools of CENP-A over several cell cycles, we show that nascent CENP-A is equally distributed to sister centromeres. CENP-A distribution is independent of previous or subsequent cell cycles in that centromeres showing disproportionately distributed CENP-A in one cycle can equally divide CENP-A nucleosomes in the next cycle. Furthermore, we show using extended chromatin fibers that maintenance of the CENP-A chromatin domain is achieved by a cycle-specific oscillating pattern of new CENP-A nucleosomes next to existing CENP-A nucleosomes over multiple cell cycles. Finally, we demonstrate that the size of the CENP-A domain does not change throughout the cell cycle and is spatially fixed to a similar location within a given alpha satellite DNA array. CONCLUSIONS: We demonstrate that most human chromosomes share similar patterns of CENP-A loading and distribution and that centromere inheritance is achieved through specific placement of new CENP-A near existing CENP-A as assembly occurs each cell cycle. The loading pattern fixes the location and size of the CENP-A domain on individual chromosomes. These results suggest that spatial and temporal dynamics of CENP-A are important for maintaining centromere identity and genome stability.