Automated maintenance of embryonic stem cell cultures

Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.     

A region encompassing the FERM domain of Jak1 is necessary for binding to the cytokine receptor gp130

Cardiac L-type Ca(2+) channel is facilitated by protein kinase A (PKA)-mediated phosphorylation. Here, we investigated the role of Ser(1901), a putative phosphorylation site in the carboxy-terminal of rat brain type-II alpha(1C) subunit (rbCII), in the PKA-mediated regulation. Forskolin (3 microM) enhanced Ca(2+) channel currents (I(Ca)) and shifted the activation curve to negative voltages, which were abolished by protein kinase inhibitor. Replacement of Ser(1901) of rbCII by Ala abolished the enhancement of I(Ca) by forskolin but not the shift of the activation curve. These results indicate that Ser(1901) is required for the PKA-mediated enhancement of I(Ca), and that the voltage-dependence of the activation of I(Ca) appears to be modulated via another PKA phosphorylation site.     

Phosphoinositide-specific phospholipase C is involved in cytokinin and gravity responses in the moss Physcomitrella patens

The phosphoinositide signalling pathway is important in plant responses to extracellular and intracellular signals. To elucidate the physiological functions of phosphoinositide-specific phopspholipase C, PI-PLC, targeted knockout mutants of PpPLC1, a gene encoding a PI-PLC from the moss Physcomitrella patens, were generated via homologous recombination. Protonemal filaments of the plc1 lines show a dramatic reduction in gametophore formation relative to wild type: this was accompanied by a loss of sensitivity to cytokinin. Moreover, plc1 appeared paler than the wild type, the result of an altered differentiation of chloroplasts and reduced chlorophyll levels compared with wild type filaments. In addition, the protonemal filaments of plc1 have a strongly reduced ability to grow negatively gravitropically in the dark. These effects imply a significant role for PpPLC1 in cytokinin signalling and gravitropism.     

Correcting mass isotopomer distributions for naturally occurring isotopes

In one method of metabolic flux analysis, simulated mass spectrometry data is fitted to measured mass distributions of metabolites that are isolated from cultures with defined feeding of (13)C-labeled substrates. Doing so, simulated mass distributions must be corrected for the presence of naturally occurring isotopes. A method that was recently introduced for this purpose consists of consecutive correction steps for each isotope of each element in the considered compound. Here we show that all isotopes of each individual element must, however, be corrected in one single step. Furthermore, it is shown that the source of information with respect to isotopic compositions of the elements needs to be chosen with care.     

Recherches dans le massif d’Ardines : nouvelles galeries ornées de la grotte de Tito Bustillo

During the year 2002 we have continued the works in the massive of Ardines at Ribadesella, Asturias, and especially in its fundamental cave, Tito Bustillo. Here the prospection has permitted us to find several new elements of great cultural and graphic value. A consistent deposit in four cutted contours in the form of head of hind and also two new galleries, called gallery of the Bisons and gallery of the Anthropomorphes, communicated with the Principal Gallery of the cave and mutually. In this last one exist remains of adaptation of the space, bony remains that we are yet digging and two figures of painted anthropomorphes with an exceptional interest. Finally, in the ensemble N XI, place where it is found the habitation deposit, and where we have found large masses of red colourings prepared for its use, exists a small final cave, where also it has been painted in its interior, and where the mouth is opened among the remains of a great deposit in surface that occupies all its northern part.     

The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.     

Java editor for biological pathways

SUMMARY: A visual Java-based tool for drawing and annotating biological pathways was developed. This tool integrates the possibilities of charting elements with different attributes (size, color, labels), drawing connections between elements in distinct characteristics (color, structure, width, arrows), as well as adding links to molecular biology databases, promoter sequences, information on the function of the genes or gene products, and references. It is easy to use and system independent. The result of the editing process is a PNG (portable network graphics) file for the images and XML (extended markup language) file for the appropriate links.     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Hepatocytes of Wistar and Sprague Dawley rats differ significantly in their central metabolism

Wistar and Sprague‐Dawley (SD) rats are most commonly used experimental rats. They have similar genetic background and are therefore, not discriminated in practical research. In this study, we compared metabolic profiles of Wistar and SD rat hepatocytes from middle (6 months) and old (23 months) age groups. Principle component analysis (PCA) on the specific uptake and production rates of amino acids, glucose, lactate and urea indicated clear differences between Wistar and SD rat hepatocytes. SD rat hepatocytes showed higher uptake rates of various essential and non‐essential amino acids, particularly in early culture phases (0‐12 h) compared to later phases (12‐24 h). SD hepatocytes seem to be more sensitive to isolation procedure and in vitro culture requiring more amino acids for cellular maintenance and repair. Major differences between Wistar and SD rat hepatocytes were observed for glucose and branched chain amino acid metabolism. We conclude that the observed differences in the central carbon metabolism of isolated hepatocytes from these two rats should be considered when using one or the other rat type in studies on metabolic effects or diseases such as diabetes or obesity.     

Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition

Disruption of the functional protein balance in living cells activates protective quality control systems to repair damaged proteins or sequester potentially cytotoxic misfolded proteins into aggregates. The established model based on Saccharomyces cerevisiae indicates that aggregating proteins in the cytosol of eukaryotic cells partition between cytosolic juxtanuclear (JUNQ) and peripheral deposits. Substrate ubiquitination acts as the sorting principle determining JUNQ deposition and subsequent degradation. Here, we show that JUNQ unexpectedly resides inside the nucleus, defining a new intranuclear quality control compartment, INQ, for the deposition of both nuclear and cytosolic misfolded proteins, irrespective of ubiquitination. Deposition of misfolded cytosolic proteins at INQ involves chaperone‐assisted nuclear import via nuclear pores. The compartment‐specific aggregases, Btn2 (nuclear) and Hsp42 (cytosolic), direct protein deposition to nuclear INQ and cytosolic (CytoQ) sites, respectively. Intriguingly, Btn2 is transiently induced by both protein folding stress and DNA replication stress, with DNA surveillance proteins accumulating at INQ. Our data therefore reveal a bipartite, inter‐compartmental protein quality control system linked to DNA surveillance via INQ and Btn2.