Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways

Extraembryonic ectoderm-derived factors instruct the pluripotent epiblast cells to develop toward a restricted primordial germ cell (PGC) fate during murine gastrulation. Genes encoding Bmp4 of the Dpp class and Bmp8b of the 60A class are expressed in the extraembryonic ectoderm and targeted mutation of either results in severe defects in PGC formation. It has been shown that heterodimers of DPP and 60A classes of bone morphogenetic proteins (BMPs) are more potent than each homodimers in bone and mesoderm induction in vitro, suggesting that BMP4 and BMP8B may form heterodimers to induce PGCs. To investigate how BMP4 and BMP8B interact and signal for PGC induction, we cocultured epiblasts of embryonic day 6.0–6.25 embryos with BMP4 and BMP8B proteins produced by COS cells. Our data show that BMP4 or BMP8B homodimers alone cannot induce PGCs whereas they can in combination, providing evidence that two BMP pathways are simultaneously required for the generation of a given cell type in mammals and also providing a prototype method for PGC induction in vitro. Furthermore, the PGC defects of Bmp8b mutants can be rescued by BMP8B homodimers whereas BMP4 homodimers cannot mitigate the PGC defects of Bmp4 null mutants, suggesting that BMP4 proteins are also required for epiblast cells to gain germ-line competency before the synergistic action of BMP4 and BMP8B.

Retinal engineering: engrafted neural cell lines locate in appropriate layers.

A major question in central nervous system development, including the neuroretina, is whether migrating cells express cues to find their way and settle at specific locations. We have transplanted quail neuroretinal cell lines QNR/D, a putative amacrine or ganglion cell, and QNR/K2, a putative Müller cell into chicken embryo eyes. Implanted QNR/D cells migrate only to the retinal ganglion and amacrine cell layers and project neurites in the plane of retina; in contrast, QNR/K2 cells migrate through the ganglion and amacrine layers, locate in the inner nuclear layer, and project processes across the retina. These data show that QNR/D and QNR/K2 cell lines represent distinct neural cell types, suggesting that migrating neural cells express distinct address cues. Furthermore, our results raise the possibility that immortalized cell lines can be used for replacement of specific cell types and for the transport of genes to given locations in neuroretina.

The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein-1.

Bone morphogenic protein-1 (BMP-1) was originally identified as one of several BMPs that induced new bone formation when implanted into ectopic sites in rodents. BMP-1, however, differed from other BMPs in that it its structure was not similar to transforming growth factor beta. Instead, it had a large domain homologous to a metalloendopeptidase isolated from crayfish, an epidermal growth-factor-like domain, and three regions of internal sequence homology referred to as CUB domains. Therefore, BMP-1 was a member of the "astacin families" of zinc-requiring endopeptidases. Many astacins have been shown to play critical roles in embryonic hatching, dorsal/ventral patterning, and early developmental decisions. Here, we have obtained amino acid sequences and isolated cDNA clones for procollagen C-proteinase (EC 3.4.24.19), an enzyme that is essential for the processing of procollagens to fibrillar collagens. The results demonstrate that procollagen C-proteinase is identical to BMP-1.

Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development.

Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4 RNA reduces goosecoid and forkhead-1 transcription in whole embryos and in activin-treated animal cap explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of goosecoid mRNA are detected in the organizer. In addition, the organizer loses the ability to dorsalize neighboring ventral marginal zone to muscle. Overexpression of BMP-4 in ventral mesoderm inhibits its response to dorsalization signals. Ventral marginal zone explants ectopically expressing BMP-4 form less muscle when treated with soluble noggin protein or when juxtaposed to a normal Spemann organizer in comparison to control explants. Endogenous BMP-4 transcripts are downregulated in ventral marginal zone explants dorsalized by noggin, in contrast to untreated explants. Thus, while BMP-4 inhibits noggin protein activity, noggin downregulates BMP-4 expression by dorsalizing ventral marginal zone to muscle. Noggin and BMP-4 activities may control the lateral extent of dorsalization within the marginal zone. Competition between these two molecules may determine the final degree of muscle formation in the marginal zone, thus defining the border between dorsolateral and ventral mesoderm.

Cloning and characterization of a human type II receptor for bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily. Several members of this family have been shown to transduce their signals through binding to type I and type II serine-(threonine) kinase receptors. Here we report the cDNA cloning and characterization of a human type II receptor for BMPs (BMPR-II), which is distantly related to DAF-4, a BMP type II receptor from Caenorhabditis elegans. In transfected COS-1 cells, osteogenic protein (OP)-1/BMP-7, and less efficiently BMP-4, bound to BMPR-II. BMPR-II bound ligands only weakly alone, but the binding was facilitated by the presence of previously identified type I receptors for BMPs. Binding of OP-1/BMP-7 to BMPR-II was also observed in nontransfected cell lines. Moreover, a transcriptional activation signal was transduced by BMPR-II in the presence of type I receptors after stimulation by OP-1/BMP-7.

Degradação enzimática de clorofenol em microrreator.

O microrreator faz parte de conjunto de dispositivos de uma nova e promissora tecnologia, que podem ser chamados de micro fabricados, atuante em campos como a da química, biológica, farmacêutica, engenharia química e biotecnologia. Trata-se de um dispositivo que possibilita reação química, tais como os reatores convencionais, mas com dimensões menores, com canais na escala micrométrica. A tecnologia de miniaturização de dispositivos para reações químicas vem se expandindo promovendo uma importante evolução, com microssistemas que abrange dispositivos mais eficazes, com configuração e geometrias específicas e menor consumo de energia, onde reações com elevadas taxas de transporte podem ser usadas para muitas finalidades diferentes, tais como, reações rápidas, mistura, reações sensíveis à temperatura, temperatura de homogeneização, ou até mesmo precipitação de nano partículas. Devido sua escala ser extremamente reduzida em relação à escala macro, oferecem um sistema que permite uma investigação do processo em um curto espaço de tempo, sendo muito útil para o rastreio de substratos, enzimas, condições de reação, bem como a determinação de parâmetros cinéticos. O presente trabalho teve por objetivo estudar a biodegradação enzimática de 2,4,6-Triclorofenol, com a utilização das enzimas Lacase e Soybean Peroxidase em microrreator da Syrris com volume de 250 ?l, que permite o estudo de cinéticas muito rápidas. Para as análises de degradação utilizou-se duas enzimas, a Lacase em concentrações de 0,05; 0,1 e 0,2 mg/ml; e a Soybean Peroxidase em concentrações de 0,0005; 0,001 e 0,002 mg/ml com a adição de Peróxido de Hidrogênio. Através dos ensaios realizados obteve-se dados experimentais da reação enzimática, possibilitando a verificação da taxa inicial de reação e sua cinética. Posteriormente, realizou-se as análises em simulação utilizando os dados experimentais, que através de um sistema de EDOs estimando inicialmente as constantes cinéticas k1, k2 e k3 usando a ferramenta ESTIMA, onde apresentaram duas respostas, uma resposta típica de mínimos quadrados, e a outra resposta que a velocidade inicial, que foi melhor representada pelos parâmetros obtidos. O método empregado na degradação do substrato, o microrreator mostrou-se eficiente, permitindo a detecção de baixo consumo de substrato para a determinação da taxa inicial, em curto tempo de residência. Perante os ensaios realizados com Lacase e Soybean Peroxidase, o microrreator é também um equipamento eficaz na repetitividade e na reprodutibilidade dos dados obtidos em diferentes concentrações.

La leyenda de la antigua Kanisia

Recreación divulgativa de la transformación del complejo episcopal visigodo de Eio/Madinat Iyyuh (El Tolmo de Minateda, Hellín, Albacete) en un barrio islámico.

La casa de Aixa

Recreación divulgativa del espacio social doméstico islámico de El Tolmo de Minateda (S. IX) a la luz de las excavaciones arqueológicas.

Madinat iyyuh: El Tolmo de Minateda en el siglo IX

Explicación de los datos arqueológicos de El Tolmo de Minateda (Hellín, Albacete) que permiten construir los relatos novelados de La casa de Aixa y La leyenda de la Kanisia.

Non-Viral Gene Therapy to Cells of the bovine Intervertebral Disc

Background. Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and degeneration. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM).1 Further studies showed that growth factors from transforming growth factor β (TGFβ) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC).2 Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods. Bovine nucleus pulposus (bNPC) and annulus fibrosus cells (bAFC) were harvested from bovine coccygeal IVD. Primary cells were then electroporized with plasmid GDF6 (Origene, vector RG211366) by optimizing parameters using the Neon Transfection system (Life Technologies, Basel). After transfection, cells were cultured in 2D monolayer or 3D alginate beads for 7, 14 or 21 days. Transfection efficiency of pGDF6 was analyzed by immunohistochemistry and fluorescent microscopy. Cell phenotype was quantified by real-time RT-PCR. To test a non-viral gene therapy applied directly to 3D whole organ culture, coccygeal bovine IVDs were harvested as previously described. Bovine IVDs were transfected by injection of plasmid GDF6 into the center. Electroporation was performed with ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) using 2-needle array electrode or tweezertrodes. 72 h after tranfection discs were fixed and cryosectioned and analyzed by immunofluorescence against GDF6. Results. RT-PCR and immunohistochemistry confirmed up-regulation of GFP and GDF6 in the primary bNPC/bAFC culture. The GFP-tagged GDF6 protein, however, was not visible, possibly due to failure of dimer formation as a result of fusion structure. Organ IVD culture transfection revealed GDF6 positive staining in the center of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GDF6 positive cells. Conclusion. Non-viral transfection is an appealing approach for gene therapy as it fulfills the translational safety aspects of transiency and lacks the toxic effects of viral transduction. We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgements. This project was funded by the Lindenhof Foundation (Funds “Research & Teaching”) Project no. 13-02-F. The imaging part of this study was performed with the facility of the Microscopy Imaging Center (MIC), University of Bern. References. Roughly PJ (2004): Spine (Phila), 29:2691-2699 Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014), Arthritis Research & Therapy, 16:R67

Anisotropy-magnetic susceptibility from the Krems Wachtberg archaeological Site (Austria) and high temperature susceptibility

The loess sediment embedding the main Gravettian layer at the Krems-Wachtberg archaeological site facilitates exceptional preservation. To gain insight in the sedimentation process before and after the Paleolithic settlement, the magnetic fabric (preferential orientation of magnetic particles) of loess of the Krems-Wachtberg site is investigated. Magnetic fabric properties clearly show an eolian origin of the loess, but may indicate some relocation in the meter above the cultural layer. The magnetic fabric properties can be divided into three intervals, the top interval shows lowest foliation and inconsistent magnetic fabric directions. The middle interval around the main cultural layer shows low foliation, but a clear preferential NW - SE direction of the lineation. This lineation is interpreted as preferential direction of the eolian loess accumulation from the South-East. The interval below ca. 0.5 m underneath the main find horizon shows a northeast-southwest lineation, but an imbrication suggesting that sediment accumulation occurred perpendicular to this direction, similar to the interval around the find horizon.

Data on mineral composition of volcanic glass and radiocarbon age of Upper Quaternary sediments from the Sea of Okhotsk

Lithological horizons have been distinguished in sediments cores from different parts of the Sea of Okhotsk based on primary descriptions of sediments and smear slides, and analyses of contents of both calcium carbonate and organic carbon, and opal. Sediment lithology has been correlated with oxygen isotope records and the standard isotope scale and radiocarbon data by AMS method for three cores studied in detail. This allowed to determine in detail periods of carbonaceous and diatomaceous ooze accumulation in the Sea of Okhotsk. Changes in magnetic susceptibility and grain size composition of sediments have been also compared with oxygen-isotope curves and radiocarbon datings. Obtained results confirm that variations in magnetic susceptibility are related with oxygen-isotope stages and influenced by climatic changes. Tephra interlayers K0, TR, K2, K3 have been identified by mineralogical analyses in all studied cores. Stratigraphic location of these tephra interlayers in detailed studied cores and their radiocarbon ages (8.1, 8.05, 26.8, and about 60 ka, respectively) provided base correlation between the interlayers and volcanic eruptions on the Kamchatka Peninsula and the Kuril Islands. This allows to use the former ones as time markers for deep-sea sediments of the Sea of Okhotsk. New lithostratigraphic and tephrochronologic data obtained allowed to correlate Upper Quaternary sediments from the Sea of Okhotsk.

Magnetic properties of ODP Site 191-1179 sediments

As reported by Shipboard Scientific Party (2001b, doi:10.2973/odp.proc.ir.191.104.2001) in the Site 1179 chapter of the Initial Reports volume, Leg 191 Site 1179 is located on abyssal seafloor northwest of Shatsky Rise, ~1650 km east of Japan. This part of the Pacific plate was formed during the Early Cretaceous, as shown by northeast-trending M-series magnetic lineations that become younger toward the northwest (Larson and Chase, 1972, doi:10.1130/0016-7606(1972)83[3627:LMEOTW]2.0.CO;2; Sager et al., 1988, doi:10.1029/JB093iB10p11753; Nakanishi et al., 1989, doi:10.1029/1999JB900002). The site is situated on magnetic Anomaly M8 (Nakanishi et al., 1999, doi:10.1029/1999JB900002), corresponding to an age of ~129 Ma and the Hauterivian stage of the Early Cretaceous (Gradstein et al., 1994, doi:10.1029/94JB01889; 1995). The sediments recovered at Site 1179 are split into four lithostratigraphic units based on composition and color (Shipboard Scientific Party, 2001b, doi:10.2973/odp.proc.ir.191.104.2001). Unit I (0-221.52 meters below seafloor [mbsf]) is a dominantly olive-gray clay- and radiolarian-bearing diatom ooze. Unit II (221.52-246.0 mbsf) is a yellowish brown to light brown clay-rich and diatom-bearing radiolarian ooze. Unit III (246.0-283.53 mbsf) is composed of brown pelagic clay. Unit IV (283.53-377.15 mbsf) is composed of chert and some porcellanite; any softer sediments present were washed out of the core barrel by the fluid circulating during the coring process.

Calcium carbonate content and anisotropy of magnetic susceptibility of ODP Leg 172 sites

Sediments from Sites 1057 and 1061 of Ocean Drilling Program Leg 172 on the Blake Outer Ridge exhibit nearly isotropic magnetic susceptibility. Resolving the degree of anisotropy of magnetic susceptibility proved difficult in many samples because of the generally weak magnetic susceptibility of the sediments relative to the noise level of the susceptibility meters used. Lineation varies from 1.0 to 1.013 and foliation varies from 1.0 to 1.08 in the samples that pass rejection criteria. In general the foliation is better resolved than the lineation, particularly at Site 1061, where the foliation exhibits long-term trends that mimic the mean susceptibility. The changes in the foliation at this site are likely the result of changes in the magnetic mineralogy of the sediment. The poorly developed or absent magnetic fabric in the sediments overall can be attributed to high carbonate concentrations and to a circulation regime that was diffuse or with currents too weak to effectively align magnetic particles.