A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.

Enhancement of the citrulline-nitric oxide cycle in astroglioma cells by the proline-rich peptide-10c from Bothrops jararaca venom

The biological activity of the proline rich decapeptde Bj PRO 10c a processing product of the C type natriuretic peptide precursor protein, expressed in the brain and the venom gland of the pit viper Bothrops jararaca, was originally attributed to the inhibition of the somatic angiotensm converting enzyme activity with subsequent ant hypertensive effect However recent results suggest broader biological activity may also be involved in the cardiovascular effects of this peptide Here we show that Bj PRO 10c enhances and sustains the generation of nitric made (NO) by regulating argininosuccinate synthase activity and thereby velocity of the citrulline NO cycle Bj PRO 10c-mediated effects not restricted to the cardiovascular system since NO production was also induced in cells of astroglial origin Bj PRO 10c was internalized by C6 astroglioma cells where it induces NO production and upregulation of the citrulline NO cycle cells in a dose dependent fashion In view of that, astroglial cells function as L arginine pool for NO production in neighboring neurons, we suggest a regulatory function for Bj PRO-10c on the metabolism of this gaseous neurotransmitter in the CNS Moreover, proliferation of astroglial cells was reduced in the presence of Bj PRO 10c however, cell death was not induced Since NO donors have been studied for the treatment of solid cancers Bj PRO 10c may serve as structural model for developing drugs to improve the effects of cancer therapy based on the peptide`s ability to augment NO production (C) 2010 Elsevier B V All rights reserved

Aberrant expression of E-cadherin and beta-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and beta-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n - 6) and control pregnancies (n - 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or beta-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total beta-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-beta-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/beta-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and beta-catenin proteins, along with defective beta-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

Somatic cell nuclear transfer is associated with altered expression of angiogenic factor systems in bovine placentomes at term

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

Ultrastructural changes in the ovary cells of engorged Rhipicephalus sanguineus female ticks treated with esters of ricinoleic acid from castor oil (Ricinus communis)

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

Follicular somatic cell factors and follicle development

Considerable attention is currently paid to oocyte-derived secreted factors that act upon cumulus and granulosa cells. Also important for follicle development are somatic cell-derived secreted factors. This is illustrated by the ability of granulosa cell-derived Kit ligand (KITL) to promote primordial follicle activation, and the loss of follicle development that accompanies KITL gene disruption. This review summarises our current understanding of somatic cell factors during both preantral and antral follicle growth, involving not only signalling from granulosa cells to the oocyte, but also signalling between granulosa and theca cells. Principal granulosa cell-derived factors include activin, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Theca cells also secrete BMPs and FGFs. The interplay between these factors is equally important for follicle growth as the activity of oocyte-derived factors.

Somatic meiosis and development of the juvenile gametophyte in members of the Batrachospermales sensu lato (Rhodophyta)

Seven populations (six in culture and one sampled directly from nature) of the freshwater red algal families Batrachospermaceae, Lemaneaceae and Thoreaceae were examined, involving three species of Batrachospermum, two of Paralemanea and one of Thorea. All 'Chantransia' stages ultimately produced juvenile gametophytes. The production of juvenile gametophytes in the three populations of Batrachospermum was generally most abundant at 15°C and low irradiances (47-68 μmol photons m-2 s-1). The most abundant gametophyte development in the Paralemanea species was observed at 10°C and low or high irradiances (47-142 μmol photons m-2 s-1). Gametophyte production in Thoreaceae occurred at higher temperatures (20°C) and also at low irradiances. In species of the Batrachospermaceae and Lemaneaceae, the 'elimination cells' can be situated on the basal or suprabasal cell of the juvenile gametophyte, but the position is usually fixed in individual species. The presence and position of the elimination cells remain to be established in Thoreaceae. Our results corroborate a previous study suggesting that the position of elimination cells is such a constant feature that it is of potential diagnostic value at the generic or infrageneric (sectional or specific) level. The characteristics observed in the development of the juvenile gametophytes in species of Batrachospermaceae and Lemaneaceae essentially agreed with general descriptions in the previous studies. The characteristics of the Thoreaceae, with a distinctive developmental pattern of the juvenile gametophyte and the occurrence of two morphological types in the 'Chantransia' stage, support the proposal to elevate it to the ordinal level. Two remarkable observations in Batrachospermum species were the production of numerous juvenile gametophytes from filaments of the same plant of the 'Chantransia' stage and the formation of a system of rhizoidal filaments or cell agglomeration of the juvenile gametophytes, which produced new gametophytes. These two characteristics potentially increase the formation of additional gametophytes under favourable conditions.

Prevalence and etiology of buffalo mastitis and milk somatic cell countin dry and rainy seasons in a buffalo herd from Analândia, São Paulo State,Brazil

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

Somatic Mosaic Activating Mutations in PIK3CA Cause CLOVES Syndrome

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.

A hormonal misunderstanding in Acca sellowiana embryogenesis: levels of zygotic embryogenesis do not match those of somatic embryogenesis

Endogenous levels of IAA, ABA and four types of CKs were analyzed in zygotic and indirect (ISE) and direct somatic embryogenesis of Acca sellowiana. Zygotic and somatic embryos at different developmental stages were sampled for morphological and hormonal analysis. Both embryo types showed substantial asymmetry in hormone levels. Zygotic embryos displayed a conspicuous peak of IAA in early developmental stages. The results outlined the hormonal variations occurring during zygotic and somatic embryogenesis regarding the timing, nature and hormonal status involved in both processes. The short transient pulse of IAA observed on the 3rd day in culture was suggested to be involved with the signaling for the induction of somatic embryogenesis. Fertilized ovule development was associated with increased IAA levels 21-24 days after pollination, followed by a sharp decrease in the cotyledonary stage, both in zygotic and somatic embryos. There was a prominent increase in ABA levels in cultures which generated ISE 24-30 days after pollination, a period that corresponds to the heart and torpedo stages. The levels of total CKs (Z, [9R]Z, iP and [9R]iP) were also always higher in zygotic than in somatic embryogenesis. While zygotic embryogenesis was dominated by the presence of zeatin, the somatic process, contrarily, was characterized by a large variation of the other cytokinin forms and amounts studied. The above results, when taken together, could be related to the previously observed high frequency formation of anomalous somatic embryos formed in A. sellowiana, as well as to their low germination ability.

Treatment of Nuclear-Donor Cells or Cloned Zygotes with Chromatin-Modifying Agents Increases Histone Acetylation But Does Not Improve Full-Term Development of Cloned Cattle

Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA + TSA or HH + VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH + VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.

Polymorphonuclear leukocytes CH138+apoptosis evaluation in milk with high and low somatic cell count - preliminary data

Polymorphonuclear leukocyte (PMNL) apoptosis is central to the successful resolution of inflammation. Since Somatic Cell Count (SCC) is an indicator of the mammary gland's immune status, this study sought to clarify the influence that these factors have on each other and on the evolution of the inflammatory process. Milk samples were stained with annexin-V, propidium iodide (PI), primary antibody anti-CH138A. Negative correlation between SCC and PMNL apoptosis was found, and a statistical difference between high SCC group and low SCC group was observed concerning the rate of viable PMNL, apoptotic PMNL, necrotic PMNL and necrotic and/or apoptotic PMNL. Overall, the high cellularity group presented lower proportions of CH138+ cells undergoing apoptosis and higher proportions of viable and necrotic CH138+ cells. Thus, it can be concluded that PMNL apoptosis and SCC are related factors, and that in high SCC, milk apoptosis is delayed. Although there is a greater amount of active phagocytes in this situation, apoptosis' anti-inflammatory effects are decreased, while necrosis' pro-inflammatory effects are increased, which can contribute to chronic inflammation.

A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis

The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.

Ultrastructural changes in the ovary cells of engorged Rhipicephalus sanguineus female ticks treated with esters of ricinoleic acid from castor oil (Ricinus communis)

Rhipicephalus sanguineus is a widely distributed tick species that has adapted to the urban environment, and the dog is its main host. This species is also known as a vector and reservoir of diseases caused by bacteria, protozoa, and viruses. Currently, acaricides of synthetic chemical origin have been widely and indiscriminately used, leading to the development of resistance to these products by ticks and causing damage to the environment. Thus, these issues have made it necessary to seek other forms of controlling these ectoparasites. R. sanguineus was artificially infested in host New Zealand White rabbits, which were divided into four treatment groups: control (CG1 and CG2) and treatment (TG1 and TG2) groups. TG1 and TG2 hosts were provided with feed supplemented with esters of ricinoleic acid from castor oil at a concentration of 5 g/kg of feed for 7 and 15 days. Afterward, the ovaries of the female ticks were removed for analysis by transmission electron microscopy. The results showed ultrastructural changes in the somatic and germ cells of ovaries from TG1 and TG2 females, particularly with respect to chorion deposition, a protective membrane of the oocyte, as well as in the transport process of vitellogenic materials via the hemolymph and pedicel cells. Moreover, the mitochondria were less electron-dense and had cristae that were more disorganized than the mitochondria from CG1 and CG2 individuals. Thus, this study demonstrated the action of esters on the ovaries of R. sanguineus, signaling the prospect of a way to control this ectoparasite without affecting nontarget organisms or the environment. Microsc. Res. Tech., 2012. (c) 2011 Wiley Periodicals, Inc.

Morphological Alterations in Newly Born Dentate Gyrus Granule Cells That Emerge after Status Epilepticus Contribute to Make Them Less Excitable

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.