Characteristics of human chondrocyte cultures in completely defined medium

Summary Chondrocytes derived from normal human adult articular cartilage were established and maintained for over 5 months in a completely defined medium without the addition of serum or any other growth factors. At the end of 5 months, these cells were still metabolically active. The cells incorporated [³H]thymidine into DNA, incorporated [³⁵S]sulfate into proteoglycans, and exhibited lysosomal enzyme activities. The³⁵S-labeled proteoglycans isolated from the culture medium had elution profiles on high pressure liquid chromatography (HPCL) similar to those observed from proteoglycans from other mammalian sources. This self-contained growth competence may reflect a need produced by the unusual avascular and alymphatic character of articular cartilage. This research was supported, in part, by Grant AM22057 from the National Institutes of Health, Bethesda, MD.     

Effect of ageing on enzymes of phenylpropanoid metabolism in Solanum tuberosum discs

-Separation of cell fractions or cell organelles of potato tuber by differential centrifugation and by sucrose density gradient centrifugation showed that, in dormant tissue, nearly all the activity of shikimate and prephenate dehydrogenases, phenylalanine ammonia lyase, cinnamate-4-hydroxylase and an O-methyltransferase for caffeate was in the soluble fraction. All these enzymes increased in activity in slices aged in light for 18 hr. In contrast to the other enzymes, cinnamate hydroxylase becomes associated with the microsomal fraction in aged discs.     

Pitfalls in the Assay of Cyclic AMP-Dependent Protein Kinase Activity in Microsacs from Torpedo marmorata

Endogenous phosphorylation of the nicotinic acetylcholine receptor (nAChR) in microsacs from Torpedo marmorata was found to be affected by several reagents commonly used in the preparation of cyclic AMP (cAMP)-dependent protein kinases and in its activity determination. The presence of a Na+,K+-ATPase inhibitor is essential to avoid a rapid depletion of ATP, even when a membrane fraction highly enriched in the nAChR is used. The presence of the thiol reducing agent dithiothreitol was found to abolish the cAMP dependence of nAChR phosphorylation, whereas the less potent reagent 2-mercaptoethanol did not affect the assay. Concentrations in the millimolar range of the chelators EDTA and EGTA were found to inhibit nAChR phosphorylation effectively. This inhibition was not due to a withdrawal of Ca2+ by the chelators, but rather to a reversible inhibition by the Mg2+ complexes. These observations may explain some of the discrepancies found in the literature concerning endogenous and exogenous nAChR phosphorylation.     

On selective partial sterility as an explanation of the behavior of the double-throwing stock and the Petunia

Ohne Zusammenfassung

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On selective partial sterility as an explanation of the behavior of the double-throwing stock and the Petunia

     

Insulin regulates menin expression, cytoplasmic localization, and interaction with FOXO1

Menin is the ubiquitously expressed nuclear protein product of the MEN1 gene, which interacts with PKB/Akt in the cytoplasm to inhibit its activity. This study describes a novel insulin-dependent mechanism of menin regulation and interaction with other metabolic proteins. We show that insulin downregulated menin in a time-dependent manner via the human insulin receptor. Inhibition analysis indicated a critical role for the protein kinase Akt in regulation of menin expression and localization. Insulin-mediated decrease in menin expression was abrogated by the PI3K/Akt inhibitor LY-294002 at early time points, from 2 to 7 h. Furthermore, exposure to insulin resulted in the cytoplasmic localization of menin and increased interaction with FOXO1. Fasting followed by refeeding modulates serum insulin levels, which corresponded to an increase in menin interaction with FOXO1 in the liver. Liver-specific hemizygous deletion of menin resulted in increased expression of FOXO1 target genes, namely IGFBP-1, PGC-1α, insulin receptor, Akt, and G-6-Pase. This study provides evidence that menin expression and localization are regulated by insulin signaling and that this regulation triggers an increase in its interaction with FOXO1 via Akt with metabolic consequences.     

High-yield production of hydrophobins RodA and RodB from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins in Pichia pastoris and present fed-batch fermentation yields of 200–300 mg/l fermentation broth. Protein bands of expected sizes were detected by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy and fed-batch production using P. pastoris may be transferred to hydrophobins from other species.