TURNOVER RATE OF THE NEURONAL CONNEXIN CX36 IN HELA CELLS

Electrical synapses formed of the gap junction protein Cx36 show a great deal of functional plasticity, much dependent on changes in phosphorylation state of the connexin. However, gap junction turnover may also be important for regulating cell-cell communication, and turnover rates of Cx36 have not been studied. Connexins have relatively fast turnover rates, with short half-lives measured to be 1.5 to 3.5 hours in pulse-chase analyses of connexins (Cx26 and Cx43) in tissue culture cells and whole organs. We utilized HaloTag technology to study the turnover rate of Cx36 in transiently transfected HeLa cells. The HaloTag protein forms irreversible covalent bonds with chloroalkane ligands, allowing pulse-chase experiments to be performed very specifically. The HaloTag open reading frame was inserted into an internal site in the C-terminus of Cx36 designed not to disrupt the regulatory phosphorylation sites and not to block the C-terminal PDZ interaction motif. Functional properties of Cx36-Halo were assessed by Neurobiotin tracer coupling, live cell imaging, and immunostaining. For the pulse-chase study, transiently transfected HeLa cells were pulse labeled with Oregon Green (OG) HaloTag ligand and chase labeled at various times with tetramethylrhodamine (TMR) HaloTag ligand. Cx36-Halo formed large junctional plaques at sites of contact between transfected HeLa cells and was also contained in a large number of intracellular vesicles. The Cx36-Halo transfected HeLa cells supported Neurobiotin tracer coupling that was regulated by activation and inhibition of PKA in the same manner as wild-type Cx36 transfected cells. In the pulse-chase study, junctional protein labeled with the pulse ligand (OG) was gradually replaced by newly synthesized Cx36 labeled with the chase ligand (TMR). The half-life for turnover of protein in junctional plaques was 2.8 hours. Treatment of the pulse-labeled cells with Brefeldin A (BFA) prevented the addition of new connexins to junctional plaques, suggesting that the assembly of Cx36 into gap junctions involves the traditional ER-Golgi-TGN-plasma membrane pathway. In conclusion, Cx36-Halo is functional and has a turnover rate in HeLa cells similar to that of other connexins that have been studied. This turnover rate is likely too slow to contribute substantially to short-term changes in coupling of neurons driven by transmitters such as dopamine, which take minutes to achieve. However, turnover may contribute to longer-term changes in coupling.

Cell survival in early embryogenesis requires proper dosage of GCN5 and p300

Histone acetylation is a central event in transcriptional activation. The importance of this modification in mammalian development is highlighted by knockout studies that revealed loss of the histone acetyltransferases GCN5, p300, or CBP results in embryonic lethality. Furthermore, early embryogenesis is sensitive to the dosage of p300 and CBP since double p300 +/−CBP+/− heterozygotes die in utero, although either single heterozygote survives. PCAF and GCN5 physically interact with p300 and CBP in vitro. To determine whether these two groups of HATs interact functionally in vivo, we created mice lacking one or more allele of p300, GCN5 or PCAF. As expected, we found that mice heterozygous for any one of these null alleles are viable. The majority of GCN5 p300 double heterozygotes also survive to adulthood with no apparent abnormalities. However, a portion of these mice die prior to birth. These embryos are developmentally stunted and exhibit increased apoptosis compared to wild type or single GCN5 or p300 heterozygous littermates at E8.5. Tissue specification is unaffected in these embryos but organ formation is compromised. In contrast, no abnormalities were observed in mice harboring mutations in both PCAF and p300 , emphasizing the specificity of HAT functions in mammalian development. ^ Since GCN5 null embryos die early in embryogenesis because of a marked increase in apoptosis, studies of its function and mechanism in late development and in tissue specific differentiation are precluded. Here, we also report the establishment of a GCN5 null embryonic stem cell line and a conditional floxGCN5 mouse line, which will serve as powerful genetic tools to examine in depth the function of GCN5 in mammalian development and in adult tissues. ^

Micropropagación de la enredadera nativa Mutisia subspinosa Cav.

La especie nativa Mutisia subspinosa es una enredadera perenne, resistente a heladas, con hermosas flores anaranjadas que presenta gran interés como ornamental. En trabajos previos de propagación a través de semillas o estacas se obtuvieron escasos resultados, lo que justificó el uso de la micropropagación. Para el establecimiento in vitro se utilizó el medio de Murashige Skoog (MS) diluido a la mitad. Se evaluó la adición de 6-bencilaminopurina (BAP) sola o en combinación con thidiazurón (TDZ) y un testigo sin hormonas. En la etapa de multiplicación se utilizó el mismo medio basal y se probaron dos auxinas: los ácidos indol butírico (AIB) y naftalén acético (ANA), además del uso de carbón activado. La composición del medio de cultivo más adecuada para el establecimiento fue la del testigo, mientras que para la multiplicación los mejores resultados se obtuvieron con la adición de 4 μmoles.L-1 de ANA. El agregado de 1 g.L-1 de carbón activado al medio de cultivo con 2,7 μmoles.L-1 mejoró la tasa de multiplicación y el enraizamiento.

In vitro propagation of Glandularia peruviana (L.) Small, an ornamental native plant from South America

The flower market is characterized by being both eager for novelties and highly competitive. The exploration of native species with ornamental potential represents a remarkable area of research, since it entails the introduction and development of novel promising ornamental crops. The genus Glandularia, widely distributed in Argentina, holds an enormous ornamental potential, due to the variety of colors of its inflorescences (red, violet, white, rose and lily), and extended flowering period. There is little information on tissue culture of Glandularia, thus highlighting the relevance of this research. In this work, the conditions for in vitro multiplication of G. peruviana were optimized. It was concluded that WPM supplemented with TDZ, in concentrations ranging from 1.1 to 9.0 μM, was the most adequate treatment, rendering a multiplication rate of approximately 10 de novo shoots per explant. This paper presents a protocol for the in vitro propagation of this species and introduces interesting prospects in the application of biotechnological tools to breed Glandularia.

Micropropagación de Glandularia, género nativo con potencial ornamental

En este trabajo se estableció un protocolo de propagación in vitro de tres especies nativas del género Glandularia: G. peruviana, G. sp. y G. laciniata. Para el establecimiento in vitro se evaluó el medio de Murashige Skoog (MS) con macro y micronutrientes diluidos a la mitad adicionado con 0,05 μM de bencilaminopurina (BAP) sola o en combinación con 0,1 μM thiadiazuron (TDZ) y un testigo sin reguladores del crecimiento. En la etapa de multiplicación se evaluó el medio de cultivo MS diluido a la ½ ó ¼ y adicionado de 20 ó 40 gr.L-1 de sacarosa. Es posible establecer y micropropagar estas especies en medios de cultivo sencillos. El medio más eficiente para el establecimiento fue aquel sin reguladores, mientras que el más adecuado para la multiplicación fue MS ½ adicionado de 20 gr.L-1 de sacarosa, en el cual la tasa de multiplicación cada 30 días fue de 6 en G. sp. y G. peruviana y 4 para G. laciniata.

Growth data from a field trial of Quercus suber plants regenerated from selected trees and their half-sib progenies by somatic embryogenesis

The development of reliable clonal propagation technologies is a requisite for performing Multi-Varietal Forestry (MVF). Somatic embryogenesis is considered the tissue culture based method more suitable for operational breeding of forest trees. Vegetative propagation is very difficult when tissues are taken from mature donors, making clonal propagation of selected trees almost impossible. We have been able to induce somatic embryogenesis in leaves taken from mature oak trees, including cork oak (Quercus suber). This important species of the Mediterranean ecosystem produces cork regularly, conferring to this species a significant economic value. In a previous paper we reported the establishment of a field trial to compare the growth of plants of somatic origin vs zygotic origin, and somatic plants from mature trees vs somatic plants from juvenile seedlings. For that purpose somatic seedlings were regenerated from five selected cork oak trees and from young plants of their half-sib progenies by somatic embryogenesis. They were planted in the field together with acorn-derived plants of the same families. After the first growth period, seedlings of zygotic origin doubled the height of somatic seedlings, showing somatic plants of adult and juvenile origin similar growth. Here we provide data on height and diameter increases after two additional growth periods. In the second one, growth parameters of zygotic seedlings were also significantly higher than those of somatic ones, but there were not significant differences in height increase between seedlings and somatic plants of mature origin. In the third growth period, height and diameter increases of somatic seedlings cloned from the selected trees did not differ from those of zygotic seedlings, which were still higher than data from plants obtained from somatic embryos from the sexual progeny. Therefore, somatic seedlings from mature origin seem not to be influenced by a possible ageing effect, and plants from somatic embryos tend to minimize the initial advantage of plants from acorns

Plant regeneration from an endangered valuable cork oak tree by somatic embryogenesis.

Among the several applications of in vitro tissue culture techniques, the conservation of plant germplasm is one of the most widely used. The cork oak is one of the principal tree species in the Western Mediterranean región. Within this área, the Balearic Islands are considered to be a glacial refuge, and therefore a reservoir of genetic resources. A singular tree has been found in the small Minorca Island population. The haplotype of this tree is of Tyrrhenian origin, showing a past link between Minorca and Sardinia. Moreover, this tree do not bear a deletion within an ITS from ribosimic nuclear DNA, which is fairly common in many populations of this species, and indicates that ir may be the descendant of a very ancient population. This tree is currently in a precarious condition, and it has not produced acorns in the last years. Hence there is a clear need of vegetative propagation to conserve this genotype. We have previously developed methods to clone adult cork oak tres by somatic embryogenesis, and therefore the aim of the present work was to clone this singular tree. There braches from the corwn were collected in November 2004, and methods previously described were carried out. By February 2005 somatic embryogenesis was obtained from leaves of the tree with percentages on induction ranging from 17 to 54% depending on the branch, which may show a novel source of variation that requires further study. Spontaneously matured somatic embryos germinated at 46% in average, and the first somatic seedlings from the Alfavaret's cork oak tree were obtained. Therefore, this study shows one of the most relevant applications of somatic embryogenesis: the plant regeneration of valuable genotypes for the in situ and ex situ conservation of forest genetic resources.

Effective inhibition of influenza virus production in cultured cells by external guide sequences and ribonuclease P

The polymerase (PB2) and nucleocapsid (NP) genes encoded by the genome of influenza virus are essential for replication of the virus. When synthetic genes that express RNAs for external guide sequences targeted to the mRNAs of the PB2 and NP genes are stably incorporated into mouse cells in tissue culture, infection of these cells with influenza virus is nonproductive. Endogenous RNase P cleaves the targeted influenza virus mRNAs when they are in a complex with the external guide sequences. Targeting two different mRNAs simultaneously inhibits viral particle production more efficiently than does targeting only one mRNA.

Stimulation of neurite outgrowth using an electrically conducting polymer

Damage to peripheral nerves often cannot be repaired by the juxtaposition of the severed nerve ends. Surgeons have typically used autologous nerve grafts, which have several drawbacks including the need for multiple surgical procedures and loss of function at the donor site. As an alternative, the use of nerve guidance channels to bridge the gap between severed nerve ends is being explored. In this paper, the electrically conductive polymer—oxidized polypyrrole (PP)—has been evaluated for use as a substrate to enhance nerve cell interactions in culture as a first step toward potentially using such polymers to stimulate in vivo nerve regeneration. Image analysis demonstrates that PC-12 cells and primary chicken sciatic nerve explants attached and extended neurites equally well on both PP films and tissue culture polystyrene in the absence of electrical stimulation. In contrast, PC-12 cells interacted poorly with indium tin oxide (ITO), poly(l-lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) surfaces. However, PC-12 cells cultured on PP films and subjected to an electrical stimulus through the film showed a significant increase in neurite lengths compared with ones that were not subjected to electrical stimulation through the film and tissue culture polystyrene controls. The median neurite length for PC-12 cells grown on PP and subjected to an electrical stimulus was 18.14 μm (n = 5643) compared with 9.5 μm (n = 4440) for controls. Furthermore, animal implantation studies reveal that PP invokes little adverse tissue response compared with poly(lactic acid-co-glycolic acid).

Isolation of human and mouse genes based on homology to REC2, a recombinational repair gene from the fungus Ustilago maydis

A human and a mouse gene have been isolated based on homology to a recombinational repair gene from the corn smut Ustilago maydis. The new human (h) gene, termed hREC2, bears striking resemblance to several others, including hRAD51 and hLIM15. hREC2 is located on human chromosome 14 at q23–24. The overall amino acid sequence reveals characteristic elements of a RECA-like gene yet harbors an src-like phosphorylation site curiously absent from hRAD51 and hLIM15. Unlike these two relatives, hREC2 is expressed in a wide range of tissues including lung, liver, placenta, pancreas, leukocytes, colon, small intestine, brain, and heart, as well as thymus, prostate, spleen, and uterus. Of greatest interest is that hREC2 is undetectable by reverse transcription-coupled PCR in tissue culture unless the cells are treated by ionizing radiation.

Emergence of a highly pathogenic simian/human immunodeficiency virus in a rhesus macaque treated with anti-CD8 mAb during a primary infection with a nonpathogenic virus

Although simian/human immunodeficiency virus (SHIV) strain DH12 replicates to high titers and causes immunodeficiency in pig-tailed macaques, virus loads measured in SHIVDH12-infected rhesus monkeys are consistently 100-fold lower and none of 22 inoculated animals have developed disease. We previously reported that the administration of anti-human CD8 mAb to rhesus macaques at the time of primary SHIVDH12 infection resulted in marked elevations of virus loads. One of the treated animals experienced rapid and profound depletions of circulating CD4+ T lymphocytes. Although the CD4+ T cell number partially recovered, this monkey subsequently suffered significant weight loss and was euthanized. A tissue culture virus stock derived from this animal, designated SHIVDH12R, induced marked and rapid CD4+ cell loss after i.v. inoculation of rhesus monkeys. Retrospective analyses of clinical specimens, collected during the emergence of SHIVDH12R indicated: (i) the input cloned SHIV remained the predominant virus during the first 5–7 months of infection; (ii) variants bearing only a few of the SHIVDH12R consensus changes first appeared 7 months after the administration of anti-CD8 mAb; (iii) high titers of neutralizing antibody directed against the input SHIV were detected by week 10 and persisted throughout the infection; and (iv) no neutralizing antibody against SHIVDH12R ever developed.

Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow

We have identified a rare (≈0.05–0.1%) population of cells (Thy-1hiCD16+CD44hiCD2−TCRαβ−B220−Mac-1−NK1.1−) in the adult mouse bone marrow that generates CD4+ and CD8+ TCRαβ+ T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2−CD16+CD44hiTCRαβ− to CD2+CD16int/−CD44int/−TCRαβ− cells, and a later transition to CD4+CD8+TCRαβ+ double-positive T cells that rapidly generate the CD4+ and CD8+ single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2+TCRαβ− stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.

Transgenic Drosophila expressing human amyloid precursor protein show γ-secretase activity and a blistered-wing phenotype

The importance of the amyloid precursor protein (APP) in the pathogenesis of Alzheimer’s disease (AD) became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment (βA4) in amyloid plaques and vascular deposits. However, the physiological role of APP is still unclear. In this work, Drosophila melanogaster is used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in fly tissue culture cells as well as in transgenic fly lines. After expression of full-length APP forms, secretion of APP but not of βA4 was observed in both systems. By using SPA4CT, a short APP form in which the signal peptide was fused directly to the βA4 region, transmembrane domain, and cytoplasmic tail, we observed βA4 release in flies and fly-tissue culture cells. Consequently, we showed a γ-secretase activity in flies. Interestingly, transgenic flies expressing full-length forms of APP have a blistered-wing phenotype. As the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion/signaling pathways in Drosophila, independently of βA4 generation.

The mechanism of Golgi segregation during mitosis is cell type-specific

Golgi membranes in Drosophila embryos and tissue culture cells are found as discrete units dispersed in the cytoplasm. We provide evidence that Golgi membranes do not undergo any dramatic change in their organization during the rapid mitotic divisions of the nuclei in the syncitial embryo or during cell division postcellularization. By contrast, in Drosophila tissue culture cells, the Golgi membranes undergo complete fragmentation during mitosis. Our studies show that the mechanism of Golgi partitioning during cell division is cell type-specific.