Correct identification of the chloroplastic protoporphyrinogen IX oxidase N-terminus places the biochemical data in frame

CTLA-4 gene constructs were designed to express CTLA-4 exclusively in the endoplasmic reticulum (ER). Four different CTLA-4 gene constructs were transfected into HEK 293 (human embryonic kidney) and A20 (Balb/c mouse B lymphoma) cells. All constructs contained an ER retention signal and coded for CTLA-4 expression in the ER. One of the constructs, which contained the membrane part of CTLA-4, coded for an expression both on the cell surface and in the ER. Three of the expressed CTLA-4 types (including the ER-membrane-expressed form) caused a reduced surface expression of B7 in the A20 cells. Only constructs which allow dimerization of CTLA-4 showed this effect. It is assumed that intracellular CTLA-4 bound B7 and inhibited therefore the transport of B7 to the surface. The binding obviously caused also an enhanced degradation of the complexes because both proteins showed a low concentration in the transfected cell lines. CTLA-4-transfected and B7-reduced A20 cells showed a diminished costimulating activity upon T cells. This was demonstrated by a reduced proliferation of T cells from ovalbumin-immunized Balb/c mice, incubated with ovalbumin peptide-primed CTLA-4-transfected A20 cells.     

Interleukin-1 Receptor Antagonist Has a Novel Function in the Regulation of Matrix Metalloproteinase-13 Expression

Interleukin-1 receptor antagonist (IL-1Ra) is an IL-1 family member, which binds to IL-1 receptors but does not induce any intracellular signaling. We addressed whether IL-1Ra has a novel function in regulation of the extracellular matrix or adhesion molecules. Polymerase chain reaction array analysis demonstrated a ~5-fold increase in matrix metalloproteinase 13 (MMP-13) mRNA expression of IL-1Ra siRNA-transfected Ca9-22 human oral squamous epithelial carcinoma cells compared with the control. In fact, MMP-13 mRNA and protein expression as well as its activity in IL-1Ra siRNA-transfected Ca9-22 cell lines were significantly higher than those in the control. IL-1Ra siRNA treatment resulted in strong elevation of MMP-13 expression, whereas addition of rhIL-1Ra (40 ng/ml) suppressed MMP-13 expression, suggesting that IL-1Ra had a specific effect on MMP-13 induction. IL-1Ra siRNA could potently suppress IL-1α. No significant difference was found between the MMP-13 mRNA expression of IL-1Ra siRNA-transfected cells and those treated with anti-IL-1α or anti-IL-1β antibodies. These results suggested that continuous supply of IL-1 had no effect on the induction of MMP-13 by IL-1Ra siRNA. Histopathological investigation of MMP-13 in periodontal tissue showed specific localization in the junctional epithelial cells of IL-1Ra knockout (KO) mice. Furthermore, infection with Aggregatibacter actinomycetemcomitans to establish an experimental periodontitis model resulted in predominant localization of MMP-13 along apical junctional epithelial cells. Laminin-5, which is degraded by MMP-13, was found in the internal basal lamina of wild-type mice, whereas the internal basal lamina of IL-1Ra KO mice did not show obvious laminin-5 localization. In particular, laminin-5 localization almost disappeared in the internal basal lamina of IL-1Ra KO mice infected with A. actinomycetemcomitans, suggesting that the suppression of IL-1Ra resulted in strong induction of MMP-13 that degraded laminin-5. In conclusion, IL-1Ra is associated with MMP-13 expression and has a novel function in such regulation without interference of the IL-1 signaling cascade.     

High Cardiorespiratory Fitness Is Negatively Associated with Daily Cortisol Output in Healthy Aging Men

Physical fitness has salutary psychological and physical effects in older adults by promoting neuroplasticity and adaptation to stress. In aging, however, the effects of fitness on the hypothalamic-pituitary-adrenal (HPA) axis are mixed. We investigated the association between cardiorespiratory fitness and HPA activity in healthy elderly men (n = 22, mean age 68 y; smokers, obese subjects, those taking drugs or reporting recent stressful events were excluded), by measuring in saliva: i) daily pattern of cortisol secretion (6 samples: 30’ post-awakening, and at 12.00, 15.00, 18.00, 21.00, 24.00 h); and ii) the cortisol response to a mental challenge. Cardiorespiratory fitness (VO_2max) was estimated using the Rockport Walking Test and the participants were assigned to high-fit (HF, ≥60°, n = 10) and low-fit (LF, ≤35°, n = 12) groups according to age-specific percentiles of VO_2max distribution in the general population. At all daytimes, basal cortisol levels were lower in the HF than the LF group, most notably in the evening and midnight samples, with a significant main effect of physical fitness for cortisol levels overall; the area-under-the-curve for total daily cortisol output was significantly smaller in the HF group. Among the subjects who responded to mental stress (baseline-to-peak increment >1.5 nmol/L; n = 13, 5 LF, 8 HF), the amplitude of cortisol response and the steepness of recovery decline displayed an increasing trend in the HF subjects, although between-group differences failed to reach the threshold for significance. In conclusion, cardiorespiratory fitness in healthy aging men is negatively correlated with daily cortisol output and contributes to buffering the HPA dysregulation that occurs with advancing age, thus possibly playing a beneficial role in contrasting age-related cognitive and physical decline.     

Physical and chemical perturbations induce the formation of protein aggregates with different structural features

The in vitro aggregation of the model GST–GFP fusion protein was induced by several effectors, including those mimicking variations occurring under cell stress conditions. In particular, we examined the effects of thermal treatments, redox state and pH variations, salt addition, and freezing and thawing cycles. The resulting aggregates displayed different morphologies as seen by electron microscopy, and different secondary and tertiary structures, as indicated by Fourier transform infrared spectroscopy and fluorescence. Therefore, proteins can be forced to undergo multiple aggregation pathways that lead to assemblies with different molecular structures and, possibly, specific physiological and pathological roles.In conclusion, great caution should be taken in inferring conclusions on protein aggregation and disaggregation in vivo from results obtained using aggregates produced under non-physiological perturbations.     

Response regulator YycF essential for bacterial growth: X-ray crystal structure of the DNA-binding domain and its PhoB-like DNA recognition motif

A response regulator YycF and its cognate sensor kinase YycG constitute the two-component signal transduction system essential for growth of Gram-positive bacteria with a low GC content. We have determined the X-ray crystal structure of the effector domain of Bacillus subtilis YycF involved in DNA binding. The structure, containing a winged helix-turn-helix motif, was found to be very similar to that of the response regulator PhoB from Escherichia coli. Specific binding of YycF to the PhoB-regulated alkaline phosphatase promoter was also demonstrated.     

A comprehensive characterization of genome-wide copy number aberrations in colorectal cancer reveals novel oncogenes and patterns of alterations

To develop a comprehensive overview of copy number aberrations (CNAs) in stage-II/III colorectal cancer (CRC), we characterized 302 tumors from the PETACC-3 clinical trial. Microsatellite-stable (MSS) samples (n = 269) had 66 minimal common CNA regions, with frequent gains on 20 q (72.5%), 7 (41.8%), 8 q (33.1%) and 13 q (51.0%) and losses on 18 (58.6%), 4 q (26%) and 21 q (21.6%). MSS tumors have significantly more CNAs than microsatellite-instable (MSI) tumors: within the MSI tumors a novel deletion of the tumor suppressor WWOX at 16 q23.1 was identified (p<0.01). Focal aberrations identified by the GISTIC method confirmed amplifications of oncogenes including EGFR, ERBB2, CCND1, MET, and MYC, and deletions of tumor suppressors including TP53, APC, and SMAD4, and gene expression was highly concordant with copy number aberration for these genes. Novel amplicons included putative oncogenes such as WNK1 and HNF4A, which also showed high concordance between copy number and expression. Survival analysis associated a specific patient segment featured by chromosome 20 q gains to an improved overall survival, which might be due to higher expression of genes such as EEF1B2 and PTK6. The CNA clustering also grouped tumors characterized by a poor prognosis BRAF-mutant-like signature derived from mRNA data from this cohort. We further revealed non-random correlation between CNAs among unlinked loci, including positive correlation between 20 q gain and 8 q gain, and 20 q gain and chromosome 18 loss, consistent with co-selection of these CNAs. These results reinforce the non-random nature of somatic CNAs in stage-II/III CRC and highlight loci and genes that may play an important role in driving the development and outcome of this disease.     

Dimerization properties of a Xenopus laevis kinesin-II carboxy-terminal stalk fragment

We have analysed the structural and physical properties of the carboxy-terminal stalk region of a kinesin-II, Xenopus kinesin-like protein 3A/B (Xklp3A/B), which we showed to be essential for heterodimerization in a previous work (De Marco et al., 2001). We expressed the corresponding A-stalk and B-stalk fragments and investigated their modes of interaction by analytical ultracentrifugation (AUC), circular dichroism spectroscopy, denaturation assays and electron microscopy. Co-expression of the A-stalk and B-stalk produced the properly folded, hetero-dimeric coiled coil at high yields. The dimeric nature of the complex was confirmed by AUC. We also found that the isolated A-stalk fragment forms a stable helix by itself and shows a significant tendency towards homodimer and higher-order complex formation. In the absence of the corresponding A-stalk fragment, the isolated B-stalk fragment remains partially unfolded, which suggests that the A-stalk provides a template structure for the B-stalk in order to recompose the complete heterodimeric coiled coil.