Glutathione improves early somatic embryogenesis in Araucaria angustifolia (Bert) O. Kuntze by alteration in nitric oxide emission

In this work, it was observed a straight relationship between the manipulation of the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, nitric oxide emission and quality and number of early somatic embryos in Araucaria angustifolia, a Brazilian endangered native conifer. In low concentrations GSH (0.01 and 0.1 mM) is a potential NO scavenger in the culture medium. Furthermore, it can increase the number of early SE formed in cell suspension culture media in a few days. However, the maintenance in this low redox state lead to a loss of early somatic embryos polarization. In gelled culture medium, high levels of GSH (5 mM) allows the development of globular embryos presenting a high NO emission on embryo apex, stressing its importance in the differentiation and cell division. Taken together these results indicate that the modification of the embryogenic cultures redox state might be an effective strategy to develop more efficient embryogenic systems in A. angustifolia. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

WBC count and functional changes induced by co-administration of clofazimine and clarithromycin, in single and multiple doses, in Wistar rats

Clofazimine and clarithromycin are used to treat leprosy and infections caused by Mycobacterium avium complex. Little data on the toxicity of co-administration of these two drugs are available. Here we evaluated the potential adverse effects of polytherapy with these two drugs in male Wistar rats by determining WBCs counts and other blood cell counts, neutrophilic phagocytosis, and burst oxidative, by flow cytometry. We observed an increase in WBCs, in multiple-dose regimens, and in polymorphonuclear cells, in both single- clarithromycin only and multiple dose regimens. We also observed a reduction in mononuclear cell counts in single and multiple doses. The drugs seem to reverse the mononuclear and polymorphonuclear cell ratio. An increase in oxidative burst was observed in animals treated with the drugs administered either individually or combined. In conclusion, clofazimine and clarithromycin change WBCs counts. Our results may contribute for a better understanding of the mechanisms related to the effects of co-administrating the two drugs.

Squamous Cell Carcinoma of Mammary Gland in Domestic Cat

Background: In the feline species, 80% to 93% of neoplasias in the mammary gland are malignant, being the majority carcinomas. Among them, there is the mammary squamous cell carcinoma, which amounts to a very rare neoplasm in the domestic cat, with considerable potential for malignancy. This study aimed to report a case of squamous cell mammary carcinoma in the feline species. Case: A female cat, mixed breed, ten years old, presented history of skin lesion. The cat had been spayed two years before, but with previous administration of contraceptives. At the physical examination, it was observed ulcer between the caudal abdominal mammary glands. The occurrence of skin or mammary neoplasia was conceived. The following complementary tests were requested: complete blood count, serum biochemical profile (renal and hepatic), chest radiographs, abdominal ultrasound, and incisional biopsy of the ulcerated region periphery, followed by classic histopathology. The lesion histopathology was compatible with squamous cell carcinoma of the mammary gland. Due to such a diagnosis, bilateral mastectomy was recommended. The material obtained during the surgical procedure was sent for anatomopathological analysis. Microscopically, surgical margins infiltration and a regional lymph node were verified. The owner was advised of the need for complementary therapies and medical monitoring of the cat. However, there was no return. It is noteworthy that the animal's physical and laboratory examinations showed no neoplasia in other regions, being the squamous cell carcinoma of the mammary gland considered primary. Discussion: The malignant mammary neoplasia genesis in feline species, in general, seems to be related to steroid hormones. The ovariectomized females are less likely to develop the disease when compared to intact cats, but there is no protective effect of surgery on those spayed after two years of age regarding the appearance of the neoplasia. Thus, at the time the reported patient was ovariectomized, this effect no longer occurred. The synthetic progestins regularly used to prevent estrus increase by three times the risk of breast carcinomas onset. In humans, there is no clear definition of the etiology and pathogenesis of mammary squamous cell carcinoma. However, it has been suggested its association with extreme forms of squamous metaplasia present in pre-existing mammary adenocarcinoma, besides cysts, chronic inflammations, abscesses and mammary gland adenofibromas. In a hypothetical way, this etiology could also be related to the feline mammary carcinoma, although, for the case at issue, the exogenous and endogenous hormonal influence should not be excluded. It has been reported that mammary squamous cell carcinomas in cats are classified in grades II and III (ie, moderately and poorly differentiated, respectively). Thus, they are considered tumors with more unfavorable prognosis. However, the monitoring of the clinical course, in order to evaluate possible recurrence of the neoplasia and metastases to distant sites, was not possible as the animal under discussion did not return. The squamous cell carcinoma is the most common skin tumor in feline species, despite the primary location in the mammary gland. It is, therefore, important to differentiate squamous cell carcinoma originated in the breast from histological types derived from skin. The description of this special and rare feline mammary carcinoma is important due to its particular characteristics and potential for malignancy.

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.

Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.

Gene expression during early somatic embryogenesis in Brazilian pine (Araucaria angustifolia (Bert) O. Ktze)

Brazilian pine (Araucaria angustifolia (Bert) O. Ktze) is the only native conifer species with economic importance in Brazil. Recently, due to intensive exploitation Brazilian pine was included in the official list of endangered Brazilian plants, under the "vulnerable" category. Biotechnology tools like somatic embryogenesis (SE) are potentially useful for mass clonal propagation and ex situ conservation strategies of commercial and endangered plant species. In spite of that, numerous obstacles still hamper the full application of SE technology for a wider range of species, including Brazilian pine. To enhance somatic embryogenesis in Brazilian pine and to gain a better understanding of the molecular events associated with somatic embryo development, we analyzed the steady-state transcript levels of genes known to regulate somatic embryogenesis using semiquantitative reverse transcription polymerase chain reaction (sqRT-PCR). These genes included Argonaute (AaAGO), Cup-shaped cotyledon1 (AaCUC), wushel-related WOX (AaWOX), a S-locus lectin protein kinase (AaLecK), Scarecrow- like (AaSCR), Vicilin 7S (AaVIC), Leafy Cotyledon 1 (AaLEC), and a Reversible glycosylated polypeptide (AaRGP). Expression patterns of these selected genes were investigated in embryogenic cultures undergoing different stages of embryogenesis, and all the way to maturation. Up-regulation of AaAGO, AaCUC, AaWOX, AaLecK, and AaVIC was observed during transition of somatic embryos from stage I to stage II. During the maintenance phase of somatic embryogenesis, expression of AaAGO and AaSCR, but not AaRPG and AaLEC genes was influenced by presence/ absence of plant growth regulators, both auxins and cytokinins. The results presented here provide new insights on the molecular mechanisms responsible for somatic embryo formation, and how selected genes may be used as molecular markers for Brazilian pine embryogenesis.

WUSCHEL-related genes are expressed during somatic embryogenesis of the basal angiosperm Ocotea catharinensis Mez. (Lauraceae)

Ocotea catharinensis is a basal angiosperm and an endangered tree species from the Brazilian Atlantic Rain Forest. Despite its economical and ecological importance, mass-propagation of this species is hampered by seldom-produced short-lived seeds, and in vitro propagation is challenged by frequently malformed somatic embryos. Therefore, O. catharinensis somatic embryos are also a good experimental material to study the physiological and molecular mechanisms underlying in vitro morphogenesis. In an ongoing effort to characterize genes expressed during somatic embryogenesis of O. catharinensis we have cloned two Ocotea WUSCHEL-related genes. According to our RT-PCR data, both genes were preferentially expressed in embryogenic cell aggregates. One of them, OcWUS, is a possible ortholog of the Arabidopsis WUSCHEL (WUS) gene, which codes for a homeodomain-containing protein involved in the specification and maintenance of the shoot apical meristem. We analyzed the expression patterns of OcWUS and OcWOX4 by RT-PCR, and OcWUS expression was also assessed by in situ hybridization. The expression patterns of OcWUS were very similar to those described for the Arabidopsis WUS. OcWUS transcripts were generally restricted to a small group of cells in the center of the putative shoot apical meristem of O. catharinensis somatic embryos. Perturbed expression of OcWUS might be related to abnormally formed somatic embryos of O. catharinensis obtained through tissue culture.

Pre-procambial cells are niches for pluripotent and totipotent stem-like cells for organogenesis and somatic embryogenesis in the peach palm: a histological study

The direct induction of adventitious buds and somatic embryos from explants is a morphogenetic process that is under the influence of exogenous plant growth regulators and its interactions with endogenous phytohormones. We performed an in vitro histological analysis in peach palm (Bactris gasipaes Kunth) shoot apexes and determined that the positioning of competent cells and their interaction with neighboring cells, under the influence of combinations of exogenously applied growth regulators (NAA/BAP and NAA/TDZ), allows the pre-procambial cells (PPCs) to act in different morphogenic pathways to establish niche competent cells. It is likely that there has been a habituation phenomenon during the regeneration and development of the microplants. This includes promoting the tillering of primary or secondary buds due to culturing in the absence of NAA/BAP or NAA/TDZ after a period in the presence of these growth regulators. Histological analyses determined that the adventitious roots were derived from the dedifferentiation of the parenchymal cells located in the basal region of the adventitious buds, with the establishment of rooting pole, due to an auxin gradient. Furthermore, histological and histochemical analyses allowed us to characterize how the PPCs provide niches for multipotent, pluripotent and totipotent stem-like cells for vascular differentiation, organogenesis and somatic embryogenesis in the peach palm. The histological and histochemical analyses also allowed us to detect the unicellular or multicellular origin of somatic embryogenesis. Therefore, our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells. Key message Our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells.

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.

Hematological differences between patients with different subtypes of sickle cell disease on hydroxyurea treatment

OBJECTIVE: Sickle cell anemia and the interaction S/Beta thalassemia differ in hematological values due to microcytosis and hypochromia caused by the thalassemic mutation. The clinical benefit of long-term hydroxyurea treatment is undeniable in sickle cell disease with monitoring of the biological action of the drug being by the complete blood count. The objective of this work is to compare changes in some of the erythrocytic indexes between S/Beta thalassemia and sickle cell anemia patients on long-term hydroxyurea treatment. METHODS: The values of erythrocyte indexes (mean corpuscular volume and mean corpuscular hemoglobin) were compared in a retrospective study of two groups of patients (Sickle cell anemia and S/Beta thalassemia) on hydroxyurea treatment over a mean of six years. RESULTS: The quantitative values of the two parameters differed between the groups. Increases in mean corpuscular volume and reductions in mean corpuscular hemoglobin delay longer in S/Beta thalassemia patients (p-value = 0.018). CONCLUSION: Hematological changes are some of the beneficial effects of hydroxyurea in sickle cell disease as cellular hydration increases and the hemoglobin S concentration is reduced. The complete blood count is the best test to monitor changes, but the interpretation of the results in S/Beta thalassemia should be different.

Axenic cell culture of the seagrass Cymodocea nodosa from cotyledonary tissue

[EN] Plant Tissue Culture, also called “micropropagation”, is the propagation of plants from different tissues (or explants) in a shorter time than conventional propagation, making use of the ability that many plant cells have to regenerate a whole plant (totipotency).There are two alternative mechanisms by which an explant can regenerate an entire plant, namely organogenesis and somatic embryogenesis. Since the last decades, the number of higher terrestrial plants species from which these techniques have been successfully applied has continually increased. However, few attempts have been carried out in marine plants. Previous seagrasses authors have focused their studies on i) vegetative propagation of rhizome fragments as explants in Ruppia maritima, Halophila engelmannii, Cymodocea nodosa and Posidonia oceanica; ii) culture of meristems in Heterozostera tasmanica, C. nodosa or P. oceanica; and iii) culture of germinated seeds on aseptic conditions, in Thalassia testudinum, H. ovalis, P. coriacea, P. oceanica, and H. decipiens. All these studies determine the most adequate culture medium for each species (seawater, nutrients, vitamins, carbon sources, etc...), often supplemented with different plant growth regulators and the necessary conditions for the culture maintenance, such as light and temperature. On the other hand, several studies have previously established protocols for cell or protoplast isolation in the species Zostera marina, Z. muelleri, P. oceanica, and C. nodosa, using shoots collected from natural meadows as original vegetal source, but further cell growth was never accomplished. Due to the absence of somatic embryogenesis or organogenetic studies in seagrasses we wonder: IS THE SUCCESSFUL APPLICATION OF TISSUE CULTURE TECHNIQUES POSSIBLE IN SEAGRASSES?

Molecular cloning, heterologous expression and characterization of Strictosidine Glucosidase from Rauvolfia serpentina cell suspension cultures

Die vorliegende Arbeit hatte zum Ziel, die enzymatische Deglucosylierung von Strictosidin in Zellsuspensionskulturen von Rauvolfia serpentina zu charakterisieren.Ein Verfahren zur Isolierung und Reinigung von Strictosidin aus pflanzlicher Zellkulturen wurde entwickelt. Zwei somatische Hybridzellkulturen zwischen R. serpentina und Rhazya stricta wurden als potenzielle Quelle dieses Glucoalkaloides untersucht. Der Sekundärstoffwechsel der pflanzlichen Zellen wurde mit Methyljasmonat induziert und 15 Stoffe wurden identifiziert, u. a. das neue Indolalkaloid 3-Oxo-rhazinilam. Die Gehaltsänderung von 7 Indolalkaloiden nach Behandlung mit Methyljasmonat wurde untersucht.Deglucosylierung von Strictisidin bei in E. coli exprimierter Raucaffricin Glucosidase wurde detektiert.Die Strictosidin Glucosidase kodierende cDNA wurde aus R. serpentina Zellsuspensionskulturen cloniert und in E. coli exprimiert. Das Enzyme wurde mit Hilfe des Inteintages gereinigt und seine Eigenschaften wurden untersucht, u. a. optimale Temperatur und pH Wert und Substratspezifität.Die Produkte von der enzymatischen Strictosidinhydrolyse wurden als Cathenamin (unter normalen Bedingungen) und Sitsirikin und Isositsirikin (im Gegenwart von Reduktoren) identifiziert. Das neue Indolalkaloid 3-Isocorreantin A wurde nach der enzymatischen Deglucosylierung von Dolichantosid (Nß-Methylstrictosidin) gebildet.

Computation of direction selectivity in retinal starburst amacrine cell dendrites – studied using electrophysiological recordings and two-photon imaging

Neuronal circuits in the retina analyze images according to qualitative aspects such as color or motion, before the information is transmitted to higher visual areas of the brain. One example, studied for over the last four decades, is the detection of motion direction in ‘direction selective’ neurons. Recently, the starburst amacrine cell, one type of retinal interneuron, has emerged as an essential player in the computation of direction selectivity. In this study the mechanisms underlying the computation of direction selective calcium signals in starburst cell dendrites were investigated using whole-cell electrical recordings and two-photon calcium imaging. Analysis of the somatic electrical responses to visual stimulation and pharmacological agents indicated that the directional signal (i) is not computed presynaptically to starburst cells or by inhibitory network interactions. It is thus computed via a cell-intrinsic mechanism, which (ii) depends upon the differential, i.e. direction selective, activation of voltage-gated channels. Optically measuring dendritic calcium signals as a function of somatic voltage suggests (iii) a difference in resting membrane potential between the starburst cell’s soma and its distal dendrites. In conclusion, it is proposed that the mechanism underlying direction selectivity in starburst cell dendrites relies on intrinsic properties of the cell, particularly on the interaction of spatio-temporally structured synaptic inputs with voltage-gated channels, and their differential activation due to a somato-dendritic difference in membrane potential.

Atypical expression and distribution of embryonic stem cell marker, OCT4, in human lung adenocarcinoma

Lung cancer is one of the leading causes of cancer-related deaths in the world. Although the origin still remains to be resolved, a prevailing hypothesis implies the involvement of cancer stem cells (CSCs) responsible for tumor initiation, maintenance, and progression. Embryonic stem cell marker, OCT4, encoding the spliced variants OCT4A and OCT4B, has recently been shown to have a dual role; as a potential adult stem cell marker and as a CSC marker in germline and somatic tumors.