Kinetic analysis of the zinc-dependent deacetylase in the lipid A biosynthetic pathway

The first committed step of lipid A biosynthesis in Gram-negative bacteria is catalyzed by the zinc-dependent hydrolase LpxC that removes an acetate from the nitrogen at the 2' '-position of UDP-3-O-acyl-N-acetylglucosamine. Recent structural characterization by both NMR and X-ray crystallography provides many important details about the active site environment of LpxC from Aquifex aeolicus, a heat-stable orthologue that displays 32% sequence identity to LpxC from Escherichia coli. The detailed reaction mechanism and specific roles of active site residues for LpxC from A. aeolicus are further analyzed here. The pH dependencies of k(cat)/K(M) and k(cat) for the deacetylation of the substrate UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc are both bell-shaped. The ascending acidic limb (pK(1)) was fitted to 6.1 +/- 0.2 for k(cat) and 5.7 +/- 0.2 for k(cat)/K(M). The descending basic limb (pK(2)) was fitted to 8.0 +/- 0.2 for k(cat) and 8.4 +/- 0.2 for k(cat)/K(M). The pH dependence of the E73A mutant exhibits loss of the acidic limb, and the mutant retains only 0.15% activity versus the wild type. The pH dependencies of the other active site mutants H253A, K227A, H253A/K227A, and D234N remain bell-shaped, although their significantly lower activities (0.25%, 0.05%, 0.007%, and 0.57%, respectively) suggest that they contribute significantly to catalysis. Our cumulative data support a mechanism for LpxC wherein Glu73 serves as the general base for deprotonation and activation of the zinc-bound water.     

A Probasin‐MerCreMer BAC allows inducible recombination in the mouse prostate

Tissue‐specific transgene expression in the prostate epithelium has previously been achieved using short prostate‐specific promoters, rendering transgenic mouse lines susceptible to integration site‐dependent effects. Here we demonstrate the applicability of bacterial artificial chromosome (BAC) technology to transgene expression in the prostate epithelium. We present mouse lines expressing an inducible Cre protein (MerCreMer) under the control of regulatory elements of the probasin gene on a BAC. These mouse lines show high organ specificity, high transgene expression in anterior, dorsal and lateral prostate lobes, no background Cre recombination using a reporter strain and adjustable amounts of Cre‐induced recombination upon tamoxifen induction. Together with two recently reported transgenic lines expressing the Cre‐ERT2 protein from small prostate‐specific promoters, these mouse lines will be useful in research focused on prostate‐specific disorders such as benign hyperplasia or cancer. genesis 47:757–764, 2009. © 2009 Wiley‐Liss, Inc.     

Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8(+) T cells sensitizing them to apoptotic cell death

The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23～24～27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.     

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv...     

Physical and catalytic properties of a peroxidase derived from cytochrome c

Except for its redox properties, cytochrome c is an inert protein. However, dissociation of the bond between methionine-80 and the heme iron converts the cytochrome into a peroxidase. Dissociation is accomplished by subjecting the cytochrome to various conditions, including proteolysis and hydrogen peroxide (H₂O₂)-mediated oxidation. In affected cells of various neurological diseases, including Parkinson's disease, cytochrome c is released from the mitochondrial membrane and enters the cytosol. In the cytosol cytochrome c is exposed to cellular proteases and to H₂O₂ produced by dysfunctional mitochondria and activated microglial cells. These could promote the formation of the peroxidase form of cytochrome c. In this study we investigated the catalytic and cytolytic properties of the peroxidase form of cytochrome c. These properties are qualitatively similar to those of other heme-containing peroxidases. Dopamine as well as sulfhydryl group-containing metabolites, including reduced glutathione and coenzyme A, are readily oxidized in the presence of H₂O₂. This peroxidase also has cytolytic properties similar to myeloperoxidase, lactoperoxidase, and horseradish peroxidase. Cytolysis is inhibited by various reducing agents, including dopamine. Our data show that the peroxidase form of cytochrome c has catalytic and cytolytic properties that could account for at least some of the damage that leads to neuronal death in the parkinsonian brain.     

Temperature dependence of binding and catalysis for the Cdc25B phosphatase

Using a combination of steady-state and single-turnover kinetics, we probe the temperature dependence of substrate association and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The transition state for substrate association is dominated by an enthalpic barrier (DeltaH(++) of 13 kcal/mol) and has a favorable entropic contribution of 4 kcal/mol at 298 K. Phosphate transfer from Cdk2-pTpY/CycA to enzyme (DeltaH(++) of 12 kcal/mol) is enthalpically more favorable than for the small molecule substrate p-nitrophenyl phosphate (DeltaH(++) of 18 kcal/mol), yet entropically less favorable (TDeltaS(++) of 2 vs. -6 kcal/mol at 298 K, respectively). By measuring the temperature dependence of binding and catalysis for several hotspot mutants involved in binding of protein substrate, we determine the enthalpy-entropy compensations for changes in rates of association and phosphate transfer compared to the wild type system. We conclude that the transition state for enzyme-substrate association involves tight and specific contacts at the remote docking site and that phospho-transfer from Cdk2-pTpY/CycA to the pre-organized active site of the enzyme is accompanied by unfavorable entropic rearrangements that promote rapid product dissociation.     

Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1–4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix–binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases.     

Effects of combined expression of antifungal barley seed proteins in transgenic wheat on powdery mildew infection

Transgenicwheat plants (variety Frisal) constitutively expressing a number of potentialantifungal proteins alone or in combinations were generated and tested forincreased resistance to Blumeria graminis f.sp. tritici(powdery mildew) in a detached leaf infection assay. The most significativerateof protection was obtained with an apoplastic ribosome-inactivation proteinfrombarley seed. Apoplastic Barnase was less efficient and individual plant linesharbouring a barley seed chitinase and -1,3-glucanase showed linespecificphenotypes from increased resistance to increased susceptibility. Combinationbycrossing of three barley seed proteins did not lead to significant improvementof protection.     

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K⁺ and Ca²⁺ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca²⁺ channels. Respective K⁺ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl^– efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 M), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 M), which inhibits K⁺ fluxes and part of the initial Ca²⁺ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.Abbreviations [Ca²⁺]_c cytosolic free Ca²⁺ - I-V current-voltage - E equilibrium potential - Pr red-light-absorbing phytochrome form - Pr far-red-light-absorbing phytochrome form - SPQ 6-methoxy-l-(3-sulphonatopropyl)quinolinium - TEA tetraethylammonium