Misfolding and aggregation of protein molecules are major threats to all living organisms. Therefore, cells have evolved quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins. DnaK/DnaJ and GroES/ GroEL are the best-characterized molecular chaperone systems in bacteria. In Caulobacter crescentus these chaperone machines are the products of essential genes, which are both induced by heat shock and cell cycle regulated. In this work, we characterized the viabilities of conditional dnaKJ and groESL mutants under different types of environmental stress, as well as under normal physiological conditions. We observed that C. crescentus cells with GroES/EL depleted are quite resistant to heat shock, ethanol, and freezing but are sensitive to oxidative, saline, and osmotic stresses. In contrast, cells with DnaK/J depleted are not affected by the presence of high concentrations of hydrogen peroxide, NaCl, and sucrose but have a lower survival rate after heat shock, exposure to ethanol, and freezing and are unable to acquire thermotolerance. Cells lacking these chaperones also have morphological defects under normal growth conditions. The absence of GroE proteins results in long, pinched filamentous cells with several Z-rings, whereas cells lacking DnaK/J are only somewhat more elongated than normal predivisional cells, and most of them do not have Z-rings. These findings indicate that there is cell division arrest, which occurs at different stages depending on the chaperone machine affected. Thus, the two chaperone systems have distinct roles in stress responses and during cell cycle progression in C. crescentus.Caulobacter crescentus, an aquatic bacterium and a member of the ␣ subdivision of the Proteobacteria, produces two cell types: motile, DNA replication-quiescent "swarmer cells" and sessile, DNA replication-competent "stalked cells." The former are important for dispersion, and the latter are important for reproduction (65).Each motile swarmer cell has a single polar flagellum and several pili at one pole. CtrA, a DNA-binding response regulator that directly controls transcription of at least 95 genes in 55 operons (48), is present in swarmer cells, where it binds to the C. crescentus origin of replication and blocks replication initiation (61). Simultaneously, CtrA directly represses transcription of gcrA (35), ftsZ (44), and podJ (12), blocking the early steps in cell division and polar development. Swarmer cells undergo differentiation to stalked cells, during which the polar pili, flagellum, and chemotaxis apparatus are lost and are replaced by a stalk that grows at the pole previously occupied by the flagellum. Concurrent with the swarmer cell-stalked cell transition, CtrA is degraded (60), while DnaA levels increase (31). The presence of DnaA, not just the absence of CtrA, is required to trigger an increase in GcrA levels and start the next wave of cell cycle transcription, which includes exp...

We investigated the antioxidant activity of phenylpropionic acids--caffeic (CAF), ferulic (FER), para-coumaric (COU) and cinnamic (CIN)--and phenolic acids and related compounds--gallic (GAL), methyl gallate (meGAL), vanillic (VAN) and gentisic (GEN)--using visible spectroscopy, inhibition of nitroblue tetrazolium (NBT) reduction, and electrochemical methods including cyclic voltammetry and potentiometry. In the spectroscopic assays, only CAF, GAL and meGAL were able to inhibit NBT reduction. The same compounds showed the lowest oxidation potentials (Epa) and the highest redox potentials deltaE) in the cyclic voltammetric and potentiometric studies, respectively. In addition, it was observed that the greater the number of hydroxyls linked to the aromatic ring, the greater was the antioxidant activity of the analysed compounds. The correlations of Spermann--used to compare the methods between themselves and the methods with the relationship structure-antioxidant activity--were r = -0.9762 for the cyclic voltammetric-potentiometric methods. r = 0.8333 for the inhibition of NBT reduction-potentiometric methods and r = -0.8095 for the inhibition of NBT reduction-cyclic voltammetric methods. The correlations for cyclic voltammetric, potentiometric and inhibition of NBT reduction methods-number of hydroxyls linked to the aromatic ring were r = -0.9636, 0.9636 and 0.9142, respectively. These findings indicate that the electrochemical methods together with spectroscopic studies are a good tool to evaluate the antioxidant activity of substances.

Summary Expression of heat shock genes in Gram

We have evaluated the development of antibodies in response to donor allograft valve implant in patients who received cellularized and decellularized allografts and determined possible immunogenic epitopes considered responsible for antibodies reactivity. Serum samples from all recipients who received cellularized allografts or decellularized allografts were collected before valve replacement and at 5, 10, 30 and 90 days post-operatively and frozen until required. Tests were performed using the Luminex-based single human leukocyte antigen (HLA)-A, -B, -C and HLA-DR, -DQ antigen microsphere assay. To determine possible immunogenic epitopes, we used the HLAMatchmaker (HLAMM) software if applicable. Decellularized grafts elicited lower levels of anti-HLA class I and II antibody formation after implantation than cellularized allografts. All patients from cellularized group presented donor-specific antibodies class I and II within 3 months of observation period. In HLAMM analysis, the cellularized group had significantly higher numbers of immunogenic epitopes than decellularized group for both class I and II (p: 0.002 - cl I / p: 0.009 - cl II / p: 0.004 - cl I and II). Our findings demonstrate that the anti-HLA antibodies detected in the cellularized group were against donor HLA possible immunogenic epitopes and that in the decellularized group the anti-HLA antibodies were not against donor HLA possible immunogenic epitopes. These findings lead us to suggest that choosing sodium dodecyl sulfate decellularization process is the best alternative to decrease the immunogenicity of allograft valve transplant.

SummaryThe heat shock response in Caulobacter crescentus was previously shown to be positively regulated by the alternative sigma factor of RNA polymerase (RNAP) s s s s 32 , and negatively modulated by DnaK during the induction phase of the heat shock response but not during the recovery phase. In the present work we have investigated the involvement of the chaperone ClpB in the control of the heat shock response in C. crescentus . Data obtained indicated a role of ClpB in downregulation of heat shock protein (HSP) synthesis, as cells lacking this chaperone showed a prolonged shutoff phase of the heat shock response. In Escherichia coli , it has been proposed that the DnaK chaperone system switches transcription back to constitutively expressed genes through simultaneous reactivation of heat-aggregated s s s s 70 , as well as sequestration of s s s s 32 away from RNAP. In C. crescentus , results obtained with a clpB null mutant indicate that ClpB could be involved in the reactivation of the major sigma factor s s s s 73 . In support of this hypothesis, we showed that transcription directed from s s s s 73 -dependent promoters is not switched back in the clpB null mutant during the recovery phase. Furthermore, we observed that resolubilization of heat-aggregated s s s s 73 is dependent on the presence of ClpB. Our findings also indicated that the absence of ClpB made cells more sensitive to heat shock and ethanol but not to other stresses, and unable to acquire thermotolerance.

A large number of bacteria regulate chaperone gene expression during heat shock by the HrcA-CIRCE system, in which the DNA element called CIRCE serves as binding site for the repressor protein HrcA under nonstress conditions. In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction is controlled by a 32 -dependent promoter and the HrcA-CIRCE system plays a role in regulation of groESL expression under physiological temperatures. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein, and specific binding to the CIRCE element was obtained by gel shift assays. The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that the GroE chaperonin machine modulates HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells, producing different amounts of GroES/GroEL. In addition, we identified the putative DNA-binding domain of HrcA through extensive protein sequence comparison and constructed various HrcA mutant proteins containing single amino acid substitutions in or near this region. In vitro and in vivo experiments with these mutated proteins indicated several amino acids important for repressor activity.Caulobacter crescentus, a member of the ␣-subdivision of proteobacteria, responds to heat shock by elevating the levels of synthesis of over 20 polypeptides (16). Several heat shockinducible genes in C. crescentus have been characterized, including dnaKJ (15), groESL (1), lon (37), hrcA/grpE (30), and ftsH (11), and they all are positively regulated by the alternative sigma factor of RNA polymerase 32 . The rpoH gene encoding 32 in C. crescentus has also been characterized, and one of its promoters was shown to be 32 dependent, indicating an autogenous control of rpoH transcription (28, 38). Furthermore, the levels of 32 were shown to increase transiently during heat shock, and the increased transcription of rpoH seems to account for the induction of 32 levels (28, 38). Recent studies have shown that, as described for Escherichia coli (for a review, see reference 39), the heat shock protein (HSP) DnaK is a negative modulator of the heat shock response in C. crescentus, which acts by inhibiting 32 activity and stimulating its degradation (7). However, despite the strong effect of DnaK levels on the induction phase of the response, downregulation of HSP synthesis is not affected by changes in the amount of this chaperone. Competition between 32 and 73 , the major sigma factor in C. crescentus which was shown to be heat shock inducible, has been proposed as the most important factor controlling the shutoff of HSP synthesis during the recovery phase (7). Another important negative modulator of the heat shock response, the repressor protein HrcA in C. crescentus, has also been described (30). This protein, which is absent in E. coli, has been found to occur in a growing number of diverse bacterial species and pr...

BackgroundThe identification of low-level antibodies by single-antigen bead methodology has brought advancements to risk evaluation of kidney transplant recipients. However, the use of mean fluorescence intensity (MFI) to quantify antibodies and to guide therapy is not enough. Notably, immunoglobulin G (IgG) subclass switching is hypothesized to follow a programmed sequence after an emergency signal from the germinal center. In transplantation this process is not clear yet. In the present study, we sequentially evaluate anti-HLA donor specific antibody (DSA) subclasses, their profile changes, and C1q-binding ability and the influence of those characteristics on antibody mediated rejection (AMR) occurrence and allograft function.MethodsA total of 30 DSA-positive patients were tested for IgG subclass content and C1q-binding in sequential serum samples.ResultsTwenty-one patients were DSA-positive before transplant; patients sensitized only by transfusion or pregnancies had IgG1 and/or IgG3, and patients sensitized by both transfusion and pregnancies or previous transplant showed a broader range of IgG subclasses. C1q binding was detected in high MFI made up of IgG1 or multiple IgG subclasses. Only 4 patients were positive for C1q posttransplantation and 3 of these showed an increase in MFI, changes in subclasses patterns, AMR, and allograft dysfunction.ConclusionsPosttransplant evaluation of DSA subclasses and the ability to bind C1q may be informative for both AMR occurrence and allograft dysfunction. Monitoring these events may help to better define risk and interventional time points.

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