Novel renal replacement strategies for the elimination of serum free light chains in patients with kappa light chain nephropathy

Multiple myeloma (MM) is a malignancy with excessive production of monoclonal proteins. At disease presentation 30% of MM patients have significant renal impairment which may progress to renal failure requiring dialysis. Besides chemotherapy extracorporeal elimination procedures such as plasma exchange have been applied as adjuvant strategies to eliminate free light chains from circulating blood, however the efficacy was poor with older techniques. We report about a highly efficient method to eliminate serum free light chain (sFLC) using a newly designed protein leaking membrane in patients suffering from sFLC induced acute renal failure. The protein leaking membrane (HCO 1100) is characterized by increased pore size facilitating elimination of middle molecules such as sFLC kappa (22.5 kD). The HCO 1100 membrane was applied in a hemodialysis and hemodiafiltration mode and compared to standard procedures (high flux hemodialysis, hemodiafiltration and plasma exchange). Hemodiafiltration with the protein leaking membrane HCO 1100 was superior to all other extracorporeal replacement strategies in eliminating sFLC-kappa from circulating blood. A median blood reduction rate of 40.8% (range 13.9% - 66.4%) was achieved during hemodiafiltration. The corresponding peak clearance rate was 25 ml/min. Importantly, the poorest elimination rate was achieved by plasma exchange followed by standard high flux hemodialysis. Extracorporeal elimination strategies with the protein leaking membrane HCO 1100 may be a promising adjuvant treatment strategy for patients with sFLC nephropathy requiring dialysis. Hemodiafiltration and to lesser extend also hemodialysis with the HCO 1100 hemofilter are able to eliminate substantial amounts of sFLC kappa in MM patients

Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His(6) tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L(−1) could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process

Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1+ gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1¿ strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression