Evaluation of antimicrobial effects of novel implant materials by testing the prevention of biofilm formation using a simple small scale medium-throughput growth inhibition assay.

Staphylococcal colonization of implants is a serious complication of orthopaedic surgery. Anti-infectious modification of implant surfaces may serve to prevent bacterial colonization. The authors set out to develop an in vitro test system for the analysis of prevention of biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus on implant materials. Biofilm growth was monitored over 10 days on titanium disks in order to develop appropriate test parameters. Bacterial cell counts following ultrasonic treatment of the colonized samples were compared with scanning electron microscope images of the specimens. Copper ion containing surfaces (ie copper [Cu] and inter-metallic Ti-Cu films) were used for growth inhibition assays: copper ion releasing specimens led to reduced bacterial numbers in biofilms and decreased bacterial persistence in the model used. The assay used represents an inexpensive and quick in vitro screen for the antibacterial effects of novel implant surface materials.

Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress.

Loss of parkin function is responsible for the majority of autosomal recessive parkinsonism. Here, we show that parkin is not only a stress-protective, but also a stress-inducible protein. Both mitochondrial and endoplasmic reticulum (ER) stress induce an increase in parkin-specific mRNA and protein levels. The stress-induced upregulation of parkin is mediated by ATF4, a transcription factor of the unfolded protein response (UPR) that binds to a specific CREB/ATF site within the parkin promoter. Interestingly, c-Jun can bind to the same site, but acts as a transcriptional repressor of parkin gene expression. We also present evidence that mitochondrial damage can induce ER stress, leading to the activation of the UPR, and thereby to an upregulation of parkin expression. Vice versa, ER stress results in mitochondrial damage, which can be prevented by parkin. Notably, the activity of parkin to protect cells from stress-induced cell death is independent of the proteasome, indicating that proteasomal degradation of parkin substrates cannot explain the cytoprotective activity of parkin. Our study supports the notion that parkin has a role in the interorganellar crosstalk between the ER and mitochondria to promote cell survival under stress, suggesting that both ER and mitochondrial stress can contribute to the pathogenesis of Parkinson's disease.

Parkin interacts with the proteasome subunit alpha4.

Mutations in the parkin gene encoding an E3 ligase are responsible for autosomal recessive Parkinson's disease. Putative parkin substrates and interacting partners have been identified, but the molecular mechanism underlying parkin-related neurodegeneration is still unclear. We have identified the 20S proteasomal subunit alpha4 (synonyms: PSMA7, XAPC7, subunit alpha type 7) as a new interacting partner of parkin. The C-terminal IBR-RING domain of parkin and the C-terminal part of alpha4 were essential for the interaction. Biochemical studies revealed that alpha4 was not a substrate for parkin-dependent ubiquitylation. Putative functions of the interaction might therefore be substrate presentation to the proteasome or regulation of proteasomal activity. Full-length parkin and parkin lacking the N-terminal ubiquitin-like domain slightly increased the proteasomal activity in HEK 293T cells, in line with the latter hypothesis.

The fla gene cluster is involved in the biogenesis of flagella in Halobacterium salinarum.

In this study, a flagella-related protein gene cluster is described for Halobacterium salinarum. The fla gene cluster is located upstream of the flagellin genes flgB1-3 and oriented in the opposite direction. It consists of nine open reading frames (ORFs): htpIX, a member of the halobacterial transducer protein gene family, and the genes flaD-K. The genes flaD, E, G, H, I and J share high homologies with genes from other Archaea. Interestingly, flaK shows similarities to bacterial genes involved in the regulation of flagellar synthesis. The ORFs of flaH, flaI and flaK contain sequences coding for nucleotide binding sites. Furthermore, flaI contains a motif called the bacterial type II secretion protein E signature, indicating a functional relation to members of the bacterial pili type IV-type II secretion protein superfamily. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the genes flaE to flaK are transcribed into one polycistronic message. In frame deletion mutants of flaI were generated by gene replacement. The deletion strain lacks motility and belongs to the fla(-) mutant class, indicating that it is deficient in flagellar biogenesis. The overall amount of flagellin protein in Delta flaI cells is reduced, although transcription of the flagellin genes is unaffected. Therefore, the flaI gene product is involved in the biosynthesis, transport or assembly of flagella in H. salinarum.

The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum.

Investigations of transcriptional regulation and the characterization of promoters in homologous expression systems are most easily performed using suitable reporter genes. Presumably because of the high internal salt concentration in halophilic Archaea, the successful application of the commonly used reporter genes has not been reported so far. Recently, the gene for an extremely halophilic beta-galactosidase (bgaH) from Haloferax alicantei has become available. After transformation of Halobacterium salinarum with a vector-carrying bgaH, the enzyme activity in cell lysates could be readily determined by a simple colorimetric assay and colonies could be screened for activity on plates containing Xgal substrate. Expression of bgaH under the control of various halobacterial promoters of known strength led to different specific beta-galactosidase activities in the lysates. Using Northern blot hybridization and semiquantitative RT-PCR, it was shown that the bgaH transcript level corresponded to the specific enzyme activity. Therefore, the bgaH gene of Haloferax alicantei appears to be a useful tool for in vivo studies of gene expression in Halobacterium salinarum and possibly other halophilic Archaea.

Myasthenia gravis: selective enrichment of antiacetylcholine receptor antibody production in untransformed human B cell cultures.

B cells producing antibodies against the acetylcholine receptor (AchR) play a central role in the pathogenesis of myasthenia gravis (MG). Although anti-AchR autoantibodies have been studied extensively, not much is known about autoimmune B cells and their antigen-driven activation. This has mainly been due to difficulties in establishing and maintaining untransformed antigen-specific B cells in vitro. In this study, we show that highly enriched B cells from peripheral blood and thymus of MG patients can be maintained in culture over a period of 4 weeks when grown on the AchR-expressing rhabdomyosarcoma cell line TE671 together with an anti-CD40 stimulus and lymphokines. Anti-AchR antibody secretion could be detected in the majority of B cell cultures on TE671 cells up to 4 weeks. In contrast, B cells cultured on CDw32-transfected L cells binding anti-CD40 antibodies (the CD40 system) produced only small amounts of anti-AchR antibodies at single time points, whereas the overall IgG production was higher than on TE671 cells. The expression of the relevant autoantigen on the adherent cell line in addition to other growth stimuli could account for this difference and may provide a useful tool for investigating antigen-dependent B cell activation in MG and other B cell-mediated autoimmune conditions.

Extensive proteolysis inhibits high-level production of eukaryal G protein-coupled receptors in the archaeon Haloferax volcanii.

In this study the usage of the halophilic archaeon Haloferax volcanii as a production system for eukaryal G protein-coupled receptors (GPCRs) was characterized. The genes of four GPCRs were fused to the dihydrofolate reductase gene of H. volcanii. In Northern blots both 5' fragments and full-length fusion transcripts were found. In contrast, only C-terminal fusion protein fragments could be detected in Western blot analyses. Ligand binding experiments revealed that a minor amount of correctly folded human beta 2 adrenergic receptor was inserted into the membrane. The introduction of different modifications at the 5' and the 3' end of the receptor genes did not significantly increase the production level. Determination of the subcellular localization showed that fusion protein fragments containing one or more receptor helices were located in the membrane. The results indicate that neither transcription, translation nor membrane translocation but the activity of one or more proteases limits the level of GPCR production in H. volcanii.

hyp gene products in Alcaligenes eutrophus are part of a hydrogenase-maturation system.

In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD-reducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB [Dernedde, J., Eitinger, M. & Friedrich, B. (1993) Arch. Microbiol. 159, 545-553]. Re-examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. Immunoblot analysis showed the presence of the H2-activating SH subunit (HoxH) at levels comparable to those observed in the wild-type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. 63Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.