Glycolaldehyde is a precursor of pyridoxal phosphate in Escherichia coli B

Carbon-labeled glycolaldehyde prepared from [(14)C]serine was used to supply the nutritional requirement of a pyridoxineless auxotroph of Escherichia coli. Pyridoxal phosphate isolated from bacteria so grown was found to have incorporated the radioactive glycolaldehyde with little dilution. The radioactivity which was unincorporated into pyridoxal phosphate was recovered almost entirely in the culture fluid. The results establish for the first time that glycolaldehyde is indeed a natural precursor of pyridoxal phosphate or it is readily converted to such a precursor.     

Direct kinetic evidence for folding via a highly compact, misfolded state.

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein.     

Convergence of IL-1β and VDR Activation Pathways in Human TLR2/1-Induced Antimicrobial Responses

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.     

The hERG K(+) channel S4 domain L532P mutation: characterization at 37°C

hERG (human Ether-à-go-go Related Gene) is responsible for ion channels mediating rapid delayed rectifier potassium current, I(Kr), which is key to cardiac action potential repolarization. Gain-of-function hERG mutations give rise to the SQT1 variant of the Short QT Syndrome (SQTS). Reggae mutant zebrafish, with a S4 zERG mutation (Leucine499Proline; L499P), display arrhythmic features analogous to those seen in the SQTS. The affected S4 domain ERG residue is highly conserved. This study was executed to determine how the homologous hERG mutation (L532P) influences channel function at 37°C. Whole-cell measurements of current (I(hERG)) were made from HEK 293 cells expressing WT or L532P hERG. The half maximal activation voltage (V(0.5)) of L532P I(hERG) was positively shifted by ~+36mV compared to WT I(hERG); however at negative voltages a pronounced L532P I(hERG) was observed. Both activation and deactivation time-courses were accelerated for L532P I(hERG). The inactivation V(0.5) for L532P I(hERG) was shifted by ~+32mV. Under action potential (AP) voltage-clamp, L532P I(hERG) exhibited a dome-shaped current peaking at ~+16mV, compared to ~-31mV for WT-I(hERG). The L532P mutation produced an ~5-fold increase in the IC(50) for dronedarone inhibition of I(hERG). Homology modeling indicated that the L532 residue within the S4 helix lies closely apposed to the S5 region of an adjacent hERG subunit. Alterations to the S4 domain structure and, potentially, to interactions between adjacent hERG subunits are likely to account for the functional effects of this mutation.     

Increasing leaf glutathione through stem feeding does not acclimate Eucalyptus camaldulensis seedlings towards high-light stress

Elevated foliar concentrations of glutathione (GSH) are a common stress response and potentially crucial in conferring increased stress tolerance. The present study addressed the following questions: can increased foliar GSH levels be achieved in the short term by applying a stem feeding technique to tree seedlings? If yes, will elevated GSH concentrations provide improved tolerance to the adverse effects of high-light stress? To this end Eucalyptus camaldulensis seedlings were stem fed a 5 mM GSH solution for 6–7 h before subjecting them to high-light exposure designed to induce photoinhibition. GSH in leaves was measured using a standard photometric method, and the effect of the high-light treatment was evaluated by the decrease in the optimum quantum efficiency of photosystem II (PSII) measured by chlorophyll fluorescence (F _v/F _m). Stem feeding GSH significantly increased GSH concentrations in the leaves up to 40% above control plants. Exposure to artificial high-light intensity for 3 h induced significant photoinhibition in leaves, measured by a 15% decrease in F _v/F _m. At the same time, photosynthesis and stomatal conductance measurements indicated that leaf physiology was not disrupted as a result of the stem feeding technique. However, we have no indication that elevated GSH increased tolerance; neither did it increase sensitivity of plants to high light-induced photoinhibition. This result was accompanied by maintained rates of photochemistry before and after light stress. Unlike previous GSH-related experiments increased tolerance by increasing the rate of photochemistry was not achieved in the present experiment.     

IGFBP-3 Inhibits the Proliferation of Neural Progenitor Cells

Insulin like growth factor-1 (IGF-1) plays an important role in the proliferation and differentiation of neural progenitor cells. The effects of IGF-1 can be regulated by insulin like growth factor binding protein-3 (IGFBP-3) which can either inhibit or stimulate the proliferation of cells depending on the expression of proteases that can release IGF-1 from IGF1-IGFBP3 complex. Although IGF-1 is essential for the development of brain, both IGFBP-3 and IGF-1 are elevated in the brains of children younger than 6 months of age. Likewise, IGFBP-3 is also upregulated following cerebral ischemia and hypoxia. However, the role of IGFBP-3 in neurogenesis is not clear. Using an in vitro culture system of rat neural progenitor cells, we demonstrate that IGFBP-3 specifically regulates the IGF-1 mediated neural progenitor cell proliferation via down regulation of phopho-Akt, and cyclin D1. In addition, IGFBP-3 also decreased the content of nestin in the neural progenitor cells indicating its potential role in neurogenesis.     

A WXW Motif Is Required for the Anticancer Activity of the TAT-RasGAP317–326 Peptide

TAT-RasGAP_317–326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP_317–326 sequence for the anticancer activities of TAT-RasGAP_317–326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP_317–326.