Somatic embryogenesis and in vitro plant regeneration from pejibaye adult plant leaf primordia

The aim of this work was to evaluate a protocol for plant regeneration by means of somatic embryos obtained from isolated adult pejibaye leaf primordia, and to describe histological origin of embryos and morphogenetic response. Explants were cultivated in modified MS medium. Mesophyll parenchymatous cells originated meristemoids (preembryonic complex formation) induced with 7.1 µM BAP in the first two subculture periods. After polarized structures with 12.9 µM NAA and 3.55 µM BAP were formed in the third subculture, somatic embryos developed and regenerated normal plants. The mesophyll parenchymatous cells display high capacity of direct response to the auxin and cytokinin.

Manual reaction times and brain dynamics after 'awake surgery' of slow-growing tumors invading the parietal area. A case report

Primary objectives: Awake surgeries of slow-growing tumours invading the brain and guided by direct electrical stimulation induce major brain reorganizations accompanied with slight impairments post-operatively. In most cases, these deficits are so slight after a few days that they are often not detectable on classical neuropsychological evaluations. Consequently, this study investigated whether simple visuo-manual reaction time paradigms would sign some level of functional asymmetries between both hemispheres. Importantly, the visual stimulus was located in the saggital plane in order to limit attentional biases and to focus mainly on the inter-hemispheric asymmetry. Methods and procedures: Three patients (aged 41, 59 and 59 years) after resections in parietal regions and a control group (age¼44, SD¼6.9) were compared during simple uni- and bimanual reaction times (RTs). Main outcomes and results: Longer RTs were observed for the contralesional compared to the ipsilesional hand in the unimanual condition. This asymmetry was reversed for the bimanual condition despite longer RTs. Conclusion and clinical implications: Reaction time paradigms are useful in these patients to monitor more precisely their functional deficits, especially their level of functional asymmetry, and to understand brain (re)organization following slowgrowing lesions.

Evidence of energy sparing in Gambian women during pregnancy: a longitudinal study using whole-body calorimetry.

Components of daily energy expenditure were measured serially by whole-body calorimetry in Gambian women before pregnancy and at 6, 12, 18, 24, 30, and 36 wk gestation. Weight gain was (mean +/- SD) 6.8 +/- 2.8 kg, fat deposition was 2.0 +/- 2.5 kg and lean tissue deposition was 5.0 +/- 2.5 kg. Basal metabolic rate (BMR) was depressed during the first 18 wk of gestation, causing total cumulative maintenance costs by week 36 to be 8.4 MJ. Individual responses to pregnancy correlated with changes in body mass (36 wk: delta BMR vs delta weight; r = 0.60, P < 0.01 delta BMR vs delta LBM; r = 0.62, P < 0.01). There was no significant increase in the cost of treadmill exercise (0% slope: F = 0.71, P = 0.64; 5% slope: F = 1.97, P = 0.10), 24-h energy expenditure (F = 0.72, P = 0.64), activity or diet-induced thermogenesis (F = 1.02, P = 0.43), during pregnancy in spite of body weight gain. Total metabolic costs over 36 wk were 144 MJ (fetus 43 MJ, fat deposition 92 MJ, cumulative maintenance costs 8.4 MJ). These were far lower than reported for well-nourished Western populations.

ERK1/2 controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell of the cortical collecting duct of the mouse kidney.

The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 pathway as shown in vivo by immunostaining of phosphorylated active ERK1/2 (pERK1/2). The MEK1/2-ERK1/2 pathway controls many different ion transports both in proximal and distal nephron, raising the question of whether this pathway is involved in the basal and/or hormone-dependent transepithelial sodium reabsorption in the principal cell of the cortical collecting duct (CCD), a process mediated by the apical epithelial sodium channel and the basolateral sodium pump (Na,K-ATPase). To answer this question we used ex vivo microdissected CCDs from normal mouse kidney or in vitro cultured mpkCCDcl4 principal cells. Significant basal levels of pERK1/2 were observed ex vivo and in vitro. Aldosterone and vasopressin, known to up-regulate sodium reabsorption in CCDs, did not change ERK1/2 activity either ex vivo or in vitro. Basal and aldosterone- or vasopressin-stimulated sodium transport was down-regulated by the MEK1/2 inhibitor PD98059, in parallel with a decrease in pERK1/2 in vitro. The activity of Na,K-ATPase but not that of epithelial sodium channel was inhibited by MEK1/2 inhibitors in both unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface biotinylation showed that intrinsic activity rather than cell surface expression of Na,K-ATPase was controlled by pERK1/2. PD98059 also significantly inhibited the activity of Na,K-ATPase ex vivo. Our data demonstrate that the ERK1/2 pathway controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell and indicate that basal constitutive activity of the ERK1/2 pathway is a critical component of this control.

Lysine and arginine requirements of Salminus brasiliensis

The objective of this work was to determine the dietary lysine (DL) and dietary arginine (DA) requirements of dourado (Salminus brasiliensis), through dose-response trials using the amino acid profiles of whole carcasses as a reference. Two experiments were carried out in a completely randomized design (n=4). In the first experiment, groups of 12 feed-conditioned dourado juveniles (11.4±0.2 g) were stocked in 60 L cages placed in 300 L plastic indoor tanks in a closed circulation system. Fish were fed for 60 days on diets containing 1.0, 1.5, 2.0, 2.5, 3.0, or 3.5 % dietary lysine. In the second experiment, dourado juveniles (27.0±0.8 g) were fed for 60 days on semipurified diets containing arginine at 1.0, 1.5, 2.0, 2.5 or 3.0%, in similar conditions to those of the first experiment. Optimal DL requirements, as determined by broken-line analysis method for final weight, weight gain and specific growth rate, were 2.15% DL or 5% lysine in dietary protein, and 1.48% DA or 3.43% arginine in dietary protein. The best feed conversion ratio is attained with 2.5% DL or 5.8% lysine in dietary protein and 1.4% DA or 3.25% arginine in dietary protein.

Regulatory Mechanisms involved in Th2 Cell Differentiation. A Proteomics Approach.

Th2-solujen erilaistumista ohjaavat säätelyverkostot ja niiden tutkiminen proteomiikan avulla Astma ja allergiat ovat laajalle levinneitä ja vakavia sairauksia, joista kärsivät miljoonat ihmiset ympäri maailmaa. Koe-eläimillä tehdyt tutkimukset osoittavat, että interleukiini-4 (IL-4) on tärkeä allergisen astman ja allergioiden kehittymiselle ja kroonistumiselle. Se ohjaa T-auttajasolujen (Th-solujen) kehittymistä Th2-tyypin soluiksi, joilla on merkittävä rooli näiden tautien puhkeamisessa. Th2-solut tuottavat myös itse IL-4:ä, joka edesauttaa taudin seuraavien vaiheiden kehittymistä. Erityisesti STAT6-proteiini, joka aktivoituu IL-4-stimulaation seurauksena, on tarpeen Th2- vasteen syntymiselle ja kroonistumiselle antigeenin aiheuttamassa keuhkoputkien astmaattisessa tulehduksessa. Väitöskirjatyöni tarkoituksena oli käyttää kaksidimensionaaliseen elektroforeesiin (2- DE) perustuvaa proteomiikkaa ja massaspektrometriaa uusien Th2-solujen erilaistumista säätelevien proteiinien tunnistamiseksi. Erilaistumattomat Th-solut eristettiin vastasyntyneen napaverestä tai hiiren pernasta. Solut aktivoitiin Tsolureseptorin ja ns. ko-stimulatoristen reseptorien kautta ja erilaistettiin joko Th1- tai Th2-suuntaan vastaavasti erilaistavien IL-12- ja IL-4-sytokiinien avulla. Ensimmäisessä tutkimuksessa in vitro -erilaistettujen Th1- ja Th2-solujen proteomeja verrattiin keskenään proteiinien ilmenemisessä tai proteiinimodifikaatioissa olevien erojen tunnistamiseksi. Kaksi muuta päätutkimusta keskittyivät IL-4:n aiheuttamaan proteiinitason säätelyyn ensimmäisen vuorokauden aikana T-soluaktivaation jälkeen. Näistä ensimmäisessä IL-4:n aiheuttamia eroja tunnistettiin aktivoiduista ihmisen Thsoluista. IL-4:n todettiin säätelevän useita proteiineja kaspaasien välittämissä signalointiteissä sekä lisäävän T-solujen elävyyttä ja aktivoitumista. Toisessa tutkimuksessa STAT6-poistogeenisten hiirien lymfosyyttien proteomia verrattiin villityypin kontrollisoluihin T-soluaktivaation ja IL-4-stimulaation jälkeen. Näissä tutkimuksissa karakterisoitiin useita uusia IL-4:n ja STAT6:n kohdeproteiineja ja löydettiin uusia säätelyverkostoja. Tutkimustulokset ovat johtaneet uusiin Th2-erilaistumismekanismeja koskeviin hypoteeseihin.

Identification of potential HIV restriction factors by combining evolutionary genomic signatures with functional analyses.

BACKGROUND: Known antiretroviral restriction factors are encoded by genes that are under positive selection pressure, induced during HIV-1 infection, up-regulated by interferons, and/or interact with viral proteins. To identify potential novel restriction factors, we performed genome-wide scans for human genes sharing molecular and evolutionary signatures of known restriction factors and tested the anti-HIV-1 activity of the most promising candidates. RESULTS: Our analyses identified 30 human genes that share characteristics of known restriction factors. Functional analyses of 27 of these candidates showed that over-expression of a strikingly high proportion of them significantly inhibited HIV-1 without causing cytotoxic effects. Five factors (APOL1, APOL6, CD164, TNFRSF10A, TNFRSF10D) suppressed infectious HIV-1 production in transfected 293T cells by >90% and six additional candidates (FCGR3A, CD3E, OAS1, GBP5, SPN, IFI16) achieved this when the virus was lacking intact accessory vpr, vpu and nef genes. Unexpectedly, over-expression of two factors (IL1A, SP110) significantly increased infectious HIV-1 production. Mechanistic studies suggest that the newly identified potential restriction factors act at different steps of the viral replication cycle, including proviral transcription and production of viral proteins. Finally, we confirmed that mRNA expression of most of these candidate restriction factors in primary CD4+ T cells is significantly increased by type I interferons. CONCLUSIONS: A limited number of human genes share multiple characteristics of genes encoding for known restriction factors. Most of them display anti-retroviral activity in transient transfection assays and are expressed in primary CD4+ T cells.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

Utilização de resíduos agroindustriais para a produção de proteases pelo termofílico Bacillus sp em fermentação submersa: otimização do meio de cultura usando a técnica de planejamento experimental

Thermophilic Bacillus sp. SMIA-2, produced protease when grown on apple pectic, whey protein and corn step liquor medium, whose concentration was varied from 3 to 10 gL-1, according to the central composite design 2³. The experiments were conducted in shaker, at 50 °C, 150 rpm and initial pH 6.5. The results revealed that the culture medium affected both, cell growth and enzyme production. After graphical and numerical optimization procedure, the enzyme production reached its maximum value at 30 h fermentation, reaching, approximately, 70 U protein mg-1, suggesting that this process was partially associated to the growth.

New mechanisms regulating human Th1 and Th2 cell differentiation

Selective development of human T helper (Th) cells into functionally distinct Th1 and Th2 subtypes plays an essential role in the host immune response towards pathogens. However, abnormal function or differentiation of these cells can lead to development of various autoimmune diseases as well as asthma and allergy. Therefore, identification of key factors and the molecular mechanisms mediating Th1 and Th2 cell differentiation is important for understanding the molecular mechanisms of these diseases. The goal of this study was to identify novel factors involved in the regulation of Th1 and Th2 differentiation processes. A new method was optimized for enrichment of transiently transfected resting human primary T lymphocytes, that allowed the study of the influence of genes of interest in human Th1/Th2 cell differentiation and other primary Th cell functions. Functional characterization of PRELI, a novel activation-induced protein in human Th cells, identified it as a mitochondrial protein involved in the regulation of Th cell differentiation and apoptosis. By influencing the intracellular redox state, PRELI induces mitochondrial apoptosis pathway and downregulates STAT6 and Th2 differentiation. The data suggested that Calpain, an oxidative stress induced cysteine protease, is involved as a mediator in PRELI-induced downregulation of STAT6. PIM serine/threonine-specific kinases were identified as new regulators of human Th1 cell differentiation. PIM1 and PIM2 kinases were shown to be preferentially expressed in Th1 cells as compared to Th2 cells. RNA interference studies showed that PIM kinases enhance the production of IFN, the hallmark cytokine produced by Th1 cells. They also induce the expression of the key Th1-driving factor T-bet and the IL-12 signaling pathway during early phases of Th1 cell differentiation. Taken together, new regulators of human T helper cell differentiation were identified in this study, which provides new insights into the signaling mechanisms controlling the selective activation of human Th cell subsets.

The Integrin Tail: A Tale of Cell Motility and Division

Integrin transmembrane receptor functions are regulated by adaptor molecules binding to their alpha and beta subunit intracellular domains, or tails, thus affecting integrin traffic and adhesion during e.g. cell motility. Interestingly, many cellular proteins function in both cell motility and cell division, thus raising the possibility that integrins might be involved in regulating the cell cycle. A thorough understanding of cell division is essential in cell biology and in human malignancies. It is well established that failures to complete cell cycle can give rise to genetically unstable cells with tumorigenic properties. Transformed cells promote the disruption of intercellular adhesions such as tight junctions, and this correlates with the onset of cell motility, invasion and unfavorable prognosis in cancer. In this study, we analyzed integrin regulation, mediated by adaptor binding to the  subunit tail, during cell motility and cell division. We revealed a novel molecular mechanism by which Rab21, through association with the integrin alpha subunits, drives integrin endosomal traffic during mitotic phases. In addition, we found indications for this finding in vivo, as RAB21 gene deletions were mapped in ovarian and prostate cancer samples. Importantly, the multinucleated phenotype of cultured ovarian cancer cells could be reverted by Rab21 overexpression. In this thesis work, we also show how the tight junction protein ZO-1 unexpectedly interacts with the 5 integrin cytoplasmic domain in the lamellipodia to promote cell motility and at the cleavage furrow to support separation of the daughter cells. The alpha5-ZO-1 complex formation was dependent on PKC which regulates ZO-1 phosphorylation and its subcellular localization. In addition, by an in situ detection method, we showed that a subset of metastatic human lung cancers expressed the alpha5beta-ZO-1 complex. Taken together, we were able to identify new molecular pathways that regulate integrin functions in an alpha tail-mediated fashion. These findings firmly suggest that genetic alterations in integrin traffic may lead to progression of tumorigenesis as a result of failed cell division. Also, the interplay of integrins and ZO-1 in forming spatially regulated adhesive structures broadens our view of crosstalk between pathways and distinct adhesive structures that can be involved in cancer cell biology.

High Risk HPV E6/E7 Oncoprotein Expression in Women with High Grade Squamous Intraepithelial Lesion

Purpose To correlate the expression of high-risk HPV E6 mRNA with pap smear, colposcopy, and biopsy results in women with high grade squamous intraepithelial lesion (HSIL). Methods A cross-sectional study was performed on women referred for primary care services after cytological diagnosis of HSIL. We evaluated the expression of E6/E7 mRNA of HPV types 16,18,31,33, and 45 and correlated the results with those of Pap smear, colposcopy, and biopsy. For amplification/detection of mRNA E6 / E7 we used NucliSENSEasyQ kit to detect HPV mRNA by polymerase chain reaction with primers/ probes for HPV types 16, 18, 31, 33, and 45. Results Out of 128 valid tests, the results of 30 (23.4%) tests were negative and 98 (70%) tests were positive. Only one type of HPV was detected in 87.7% of the E6/E7 mRNA positive cases. HPV16 was detected in 61.2% of the cases, followed by HPV33 (26.5%), HPV31 (17.3%), HPV18 (10%), and HPV45 (4.08%). Pap smear tests revealed that the E6/E7 test was positive in 107 (83.8%) women with atypical squamous cells - high grade (ASC-H), HSIL, or higher. The E6/E7 test was positive in 69 (57.5%) specimens presenting negative cytology results. When analyzing the association with colposcopy results, the frequency of positive E6/E7 results increased with the severity of the injury, ranging from 57.1% in women without colposcopy-detected injury to 86.5% in those with higher levels of colposcopy findings. Of the 111 women who underwent biopsy and E6/E7 testing, the E6/E7 test was positive in 84.7% of the women who presented with lesions of cervical intraepithelial neoplasia (CIN) grade 2 or higher. Finally, 41.2% of women with a negative biopsy presented a positive E6/E7 test. Conclusions E6/E7mRNA expression was higher in women with HSIL and CIN grade 2 or higher.

Regulation of epidermal tight junctions by calcium ATPases and p38

The epidermis is the upper layer of the skin and keratinocytes are its most abundant cells. Tight junctions are cell junctions located in the granular layer of the epidermis. They maintain the polarity of the cells and regulate the movement of water-soluble molecules. Epidermal tight junctions may lose their integrity when there are defects in intercellular calcium regulation. Hailey-Hailey and Darier´s disease are dominantly inherited, blistering skin diseases. Hailey-Hailey disease is caused by mutations in the ATP2C1 gene encoding a calcium/manganese ATPase SPCA1 of the Golgi apparatus. Darier´s disease is caused by mutations in the ATP2A2 gene encoding a calcium ATPase SERCA2 of the endoplasmic reticulum. p38 regulates the differentiation of keratinocytes. The overall regulation of epidermal tight junctions is not well understood. The present study examined the regulation of tight junctions in the human epidermis with a focus on calcium ATPases and p38. Skin from Hailey-Hailey and Darier´s disease patients was studied by using immunofluorescence labeling which targeted intercellular junction proteins. Transepidermal water loss was also measured. ATP2C1 gene expression was silenced in cultured keratinocytes, by siRNA, which modeled Hailey-Hailey disease. Expression of intercellular junction proteins was studied at the mRNA and protein levels. Squamous cell carcinoma and normal human keratinocytes were used as a model for impaired and normal keratinocyte differentiation, and the role of p38 isoforms alpha and delta in the regulation of intercellular junction proteins was studied. Both p38 isoforms were silenced by adenovirus cell transduction, chemical inhibitors or siRNA and keratinocyte differentiation was assessed. The results of this thesis revealed that: i.) intercellular junction proteins are expressed normally in acantholytic skin areas of patients with Hailey-Hailey or Darier´s disease but the localization of ZO-1 expanded to the stratum spinosum; ii.) tight junction proteins, claudin-1 and -4, are regulated by ATP2C1 in non-differentiating keratinocytes; and iii.) p38 delta regulates the expression of tight junction protein ZO-1 in proliferating keratinocytes and in squamous cell carcinoma derived cells. ZO-1 silencing, however, did not affect the expression of other tight junction proteins, suggesting that they are differently regulated. This thesis introduces new mechanisms involved in the regulation of tight junctions revealing new interactions. It provides novel evidence linking intracellular calcium regulation and tight junctions.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained