Proteolytic degradation of ribonuclease A in the pretransition region of thermally and urea-induced unfolding.

The method of limited proteolysis has proven to be appropriate for the determination of unfolding rate constants (k(U)) of ribonuclease A in the transition region of thermal denaturation [Arnold, U. & Ulbrich-Hofmann, R. (1997) Biochemistry 36, 2166-2172]. The aim of the present paper was to extend this procedure to the pretransition region of thermally and urea-induced denaturation where spectroscopic methods do not allow direct measurement of k(U). The results show that the approach can be applied successfully to denaturing (free energy of unfolding Delta G < 10 kJ.mol(-1)) and to marginally native conditions (Delta G = 10-25 kJ.mol(-1)). Under moderately (Delta G = 25-30 kJ.mol(-1)) and strongly native conditions (Delta G > 30 kJ.mol(-1)), however, the determination of kU was not possible in this way as the proteolytic degradation of ribonuclease A by thermolysin or trypsin was no longer determined by global unfolding. Here, proteolysis proceeds via the native RNase A. In the presence of low concentrations of urea, the rate constants of proteolysis were, surprisingly, smaller than in the absence of urea. As the protease activity has been taken into account, this result points to a local stabilization of the RNase A molecule.     

Neutrophil Gelatinase-Associated Lipocalin (NGAL) Predicts Response to Neoadjuvant Chemotherapy and Clinical Outcome in Primary Human Breast Cancer

In our previous work we showed that NGAL, a protein involved in the regulation of proliferation and differentiation, is overexpressed in human breast cancer (BC) and predicts poor prognosis. In neoadjuvant chemotherapy (NACT) pathological complete response (pCR) is a predictor for outcome. The aim of this study was to evaluate NGAL as a predictor of response to NACT and to validate NGAL as a prognostic factor for clinical outcome in patients with primary BC. Immunohistochemistry was performed on tissue microarrays from 652 core biopsies from BC patients, who underwent NACT in the GeparTrio trial. NGAL expression and intensity was evaluated separately. NGAL was detected in 42.2% of the breast carcinomas in the cytoplasm. NGAL expression correlated with negative hormone receptor (HR) status, but not with other baseline parameters. NGAL expression did not correlate with pCR in the full population, however, NGAL expression and staining intensity were significantly associated with higher pCR rates in patients with positive HR status. In addition, strong NGAL expression correlated with higher pCR rates in node negative patients, patients with histological grade 1 or 2 tumors and a tumor size <40 mm. In univariate survival analysis, positive NGAL expression and strong staining intensity correlated with decreased disease-free survival (DFS) in the entire cohort and different subgroups, including HR positive patients. Similar correlations were found for intense staining and decreased overall survival (OS). In multivariate analysis, NGAL expression remained an independent prognostic factor for DFS. The results show that in low-risk subgroups, NGAL was found to be a predictive marker for pCR after NACT. Furthermore, NGAL could be validated as an independent prognostic factor for decreased DFS in primary human BC.     

A novel and high-throughput approach to assess photosynthetic thermal tolerance of kelp using chlorophyll α fluorometry

Foundation seaweed species are experiencing widespread declines and localized extinctions due to increased instability of sea surface temperature. Characterizing temperature thresholds are useful for predicting patterns of change and identifying species most vulnerable to extremes. Existing methods for characterizing seaweed thermal tolerance produce diverse metrics and are often time-consuming, making comparisons between species and techniques difficult, hindering insight into global patterns of change. Using three kelp species, we adapted a high-throughput method – previously used in terrestrial plant thermal biology – for use on kelps. This method employs temperature-dependent fluorescence (T–F₀) curves under heating or cooling regimes to determine the critical temperature (T_crit) of photosystem II (PSII), i.e., the breakpoint between slow and fast rise fluorescence response to changing temperature, enabling rapid assays of photosynthetic thermal tolerance using a standardized metric. This method enables characterization of T_crit for up to 48 samples per two-hour assay, demonstrating the capacity of T–F₀ curves for high-throughput assays of thermal tolerance. Temperature-dependent fluorescence curves and their derived metric, T_crit, may offer a timely and powerful new method for the field of phycology, enabling characterization and comparison of photosynthetic thermal tolerance of seaweeds across many populations, species, and biomes.     

Molecular determinants for subcellular localization of PSD-95 with an interacting K+ channel.

Ion channels and PSD-95 are colocalized in specific neuronal subcellular locations by an unknown mechanism. To investigate mechanisms of localization, we used biolistic techniques to express GFP-tagged PSD-95 (PSD-95:GFP) and the K(+)-selective channel Kv1.4 in slices of rat cortex. In pyramidal cells, PSD-95:GFP required a single PDZ domain and a region including the SH3 domain for localization to postsynaptic sites. When transfected alone, PSD-95:GFP was present in dendrites but absent from axons. When cotransfected with Kv1.4, PSD-95:GFP appeared in both axons and dendrites, while Kv1.4 was restricted to axons. When domains that mediate the interaction of Kv1.4 and PSD-95 were disrupted, Kv1.4 localized nonspecifically. Our results provide evidence that Kv1.4 itself may determine its subcellular location, while an associated MAGUK protein is a necessary but not sufficient cofactor.     

Cell surface properties correlated with cohesion in Myxococcus xanthus.

The gliding behavior of Myxococcus xanthus cells is controlled by two multigene systems, A and S, which encode information for adventurous and social behaviors, respectively. The S system can be genetically disrupted through mutation, such as a dsp mutation, or phenotypically disrupted by treating cells with the diazo dye Congo red (Arnold and Shimkets, J. Bacteriol. 170:5765-5770, 1988). One of the functions controlled by the S system is cell agglutination. Immediately after the induction of agglutination, wild-type cells begin to form aggregates, and within 30 min the cells are packed side-to-side in clumps containing thousands of cells. Changes in the cohesive properties of S+ cells are correlated with changes in the topology of the cell surface observed by electron microscopy. Two types of cell-associated appendages were observed on wild-type cells: thin filaments (ca. 5 nm in diameter), which have been called fimbriae or pili, at one cell pole, and thick, flaccid filaments (ca. 50 nm in diameter), referred to as fibrils, at both the sides and tips of cells. Cohesion was correlated with the secretion of the thick fibrils, which coat the cell surface and form an extracellular matrix in which the cells are interconnected. Several lines of evidence suggest that these thick fibrils are involved in cohesion. First, Dsp cells were unable to agglutinate or secrete this extracellular material. Second, wild-type cells which were treated with Congo red neither agglutinated nor secreted the extracellular fibrils. Finally, removal of the Congo red from wild-type cells restored cohesion and also restored production of the thick fibrils. Attempts to estimate the efficiency with which two cells cohered following collision suggested that under optimal conditions, one in three collisions resulted in stable contact. The collision efficiency decreased linearly as the cell density increased, suggesting a cell density-dependent regulation of cohesion. Some aspects of gliding behavior can be explained in terms of an inducer and an inhibitor of S motility.     

Polycomb group protein Bmi1 negatively regulates IL-10 expression in activated macrophages

Macrophages exert a wide variety of functions, which necessitate a high level of plasticity on the chromatin level. In the work presented here, we analyzed the role of the polycomb group protein Bmi1 during the acute response of bone marrow derived macrophages (BMDM) to lipopolysaccharide (LPS). Unexpectedly, we observed that Bmi1 was rapidly induced at the protein level and transiently phosphorylated upon LPS treatment. The induction of Bmi1 was dependent on MAP-kinase signaling. LPS treatment of BMDM in the absence of Bmi1 resulted in a pronounced increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Our results identify Bmi1 as a repressor of IL-10 expression during macrophage activation.     

A tetrameric allyl complex of sodium, and computational modeling of the Na-allyl chemical shift

Transmetallation of Li[A′] (A′ = [1,3-(SiMe₃)₂C₃H₃]⁻) with sodium tert-butoxide produces the corresponding sodium salt, which crystallizes from THF/toluene in the form of a cyclic tetramer, {Na[A′](thf)}₄. The Na atoms are in a square planar arrangement, bridged with π-bound allyl ligands; the Na-C distances range from 2.591(3)-2.896(3) Å, with an average of 2.70 Å. The geometries of several model organosodium complexes containing cyclopentadienyl and allyl ligands were optimized with density functional theory methods. The optimized structures were used with the gauge-including atomic orbital (GIAO) method to calculate their ²³Na NMR magnetic shielding values. Unlike the case with NaCp, the chemical shift of unsubstituted Na(C₃H₅) is very sensitive to the presence of coordinated THF (causing a 20 ppm upfield shift); silyl substitution has an even larger effect (30 ppm upfield shift). The observed ²³Na shift of δ −3.3 ppm for Na[A′] in THF-d₈, however, cannot be reliably distinguished from that calculated for the [Na(thf)₄]⁺ cation alone.