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Author(s): Jaroslaw Marszalek [1]; Krzysztof Liberek [1]; Igor Konieczny [1] Sir Eastern European countries have a tradition of stressing the importance of solid scientific education and research. Even under communism, [...]

Presents information on a study which tested the accessibility of the short-lived lambda O protein to the ClpP/ClpX protease during the various stages of lambda DNA replication. Bacteriophage lambda proteins involved in DNA replication; Materials and methods; Results and discussion.

The Escherichia coli σ32 transcriptional regulator has been shown to be degraded both in vivo and in vitro by the FtsH (HflB) protease, a member of the AAA protein family. In our attempts to study this process in detail, we found that two σ32 mutants lacking 15–20 C-terminal amino acids had substantially increased half-lives in vivo or in vitro, compared with wild-type σ32. A truncated version of σ32, σ32CΔ, was purified to homogeneity and shown to be resistant to FtsH-dependent degradation in vitro, suggesting that FtsH initiates σ32 degradation from its extreme C-terminal region. Purified σ32CΔ interacted with the DnaK and DnaJ chaperone proteins in a fashion similar to that of wild-type σ32. However, in contrast to wild-type σ32, σ32CΔ was largely deficient in its in vivo and in vitro interaction with core RNA polymerase. As a consequence, the truncated σ32 protein was completely non-functional in vivo, even when overproduced. Furthermore, it is shown that wild-type σ32 is protected from degradation by FtsH when complexed to the RNA polymerase core, but sensitive to proteolysis when in complex with the DnaK chaperone machine. Our results are in agreement with the proposal that the capacity of the DnaK chaperone machine to autoregulate its own synthesis negatively is simply the result of its ability to sequester σ32 from RNA polymerase, thus making it accessible to degradation by the FtsH protease. [ABSTRACT FROM AUTHOR]

Background: One of the main limitations of peritoneal dialysis (PD) is deterioration of functional and morphological characteristics of the peritoneum. This complication appears to be related to the low biocompatibility profile of PD fluids. Recently, induction of the heat shock protein (HSP) stress response was demonstrated in cultured human mesothelial cells exposed to PD fluid in vitro. We investigated whether expression of heat shock protein 72 (HSP-72) in peritoneal macrophages is induced upon exposure to PD fluid during continuous ambulatory PD., Methods: Peritoneal leukocytes were isolated from 4-hour dwell dialysate; peripheral blood mononuclear cells (PBMC) and peripheral blood monocytes isolated from the same patients were used as a control. In separate experiments, PBMC from healthy individuals were exposed in vitro to different PD fluids or to culture media. Expression of HSP-72 was assessed by Western immunoblotting, flow cytometry, and reverse-transcription polymerase chain reaction analysis., Results: Macrophages and leukocytes isolated from dialysis effluent expressed significantly increased HSP-72 and mRNA levels compared to blood monocytes and PBMC of the same patients. In vitro exposure of PBMC to fresh PD fluids resulted in significantly higher expression of HSP-72 compared to those incubated in culture medium. PBMC exposed in vitro to standard lactate-buffered dialysis fluids also expressed significantly more HSP-72 compared to cells exposed to bicarbonate/lactate-buffered fluids., Conclusion: Our results indicate that exposure to PD fluids during dialysis triggers a shock response in peritoneal cells, which is manifested by significantly increased HSP-72 expression at both protein and mRNA levels. Analysis of this protein expression in peritoneal macrophages could be a new, convenient, and relevant way to assess the biocompatibility of PD fluids ex vivo.