Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor

Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 μm for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.     

Host T cells are the main producers of IL-17 within the central nervous system during initiation of experimental autoimmune encephalomyelitis induced by adoptive transfer of Th1 cell lines

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has long been thought to be mediated by Th1 CD4(+) T cells. Using adoptive transfer techniques, transfer of CNS specific Th1 T cells was sufficient to induce EAE in naive mice. However, recent studies found a vital role for IL-17 in induction of EAE. These studies suggested that a fraction of IL-17-producing T cells that contaminate Th1 polarized cell lines are largely responsible for initiation of EAE. In this study, we tracked the appearance and cytokine production capacity of adoptively transferred cells within the CNS of mice throughout EAE disease. IL-17-producing, adoptively transferred cells were not enriched over the low percentages present in vitro. Thus, there was no selective recruitment and/or preferential proliferation of adoptively transferred IL-17-producing cells during the induction of EAE. Instead a large number of CNS infiltrating host T cells in mice with EAE were capable of producing IL-17 following ex vivo stimulation. The IL-17-producing T cells contained both alphabeta and gammadelta TCR(+) T cells with a CD4(+)CD8(-) or CD4(-)CD8(-) phenotype. These cells concentrated within the CNS within 3 days of adoptive transfer, and appeared to play a role in EAE induction as adoptive transfer of Th1 lines derived from wild-type mice into IL-17-deficient mice induced reduced EAE clinical outcomes. This study demonstrates that an encephalitogenic Th1 cell line induces recruitment of host IL-17-producing T cells to the CNS during the initiation of EAE and that these cells contribute to the incidence and severity of disease.     

Recent environmental change in North African wetland lakes: diatom and other stratigraphic evidence from nine sites in the CASSARINA Project

All nine wetland lakes in the CASSARINA North African suite of sample sites have been disturbed strongly by human activity during the 20th century. Dated lake sediment core were used to provide evidence of the extent of recent environmental change at each site. Sedimentary diatoms at seven sites were useful for inferring salinity change trends during the last century. At two sites preservation problems severely degraded the sedimentary diatom record. Sediment core integrity was otherwise established.Lithostratigraphic measurements indicated some site specific changes in soil erosion and sediment composition but, for the Egyptian Delta lakes, no physical signal synchronous with Aswan High Dam construction was found. Sedimentary diatom assemblages were generally site characteristic and halophilous taxa were common. At two sites planktonic diatoms indicated some recent eutrophication but generally the assemblages were more indicative of salinity changes. Diatom-inferred salinity trends for the seven sites typically indicated that reductions in water salinity occurred sometime during the early or mid 20th century.Rather than climate, hydrological modification of water resources is implicated as the primary driver of salinity changes during most of the 20th century. In the western North African region these modifications were mainly local land drainage and water diversion programmes to alleviate winter flooding and/or promote summer water availability. In the Delta region, the Nile has been intensively exploited since antiquity and intensively so from the late 19th century to release more fresh water for agriculture. Here, diatom records indicate that freshening began well before the Aswan High Dam but salinity fluctuations have tended to diminish during the latter part of the 20th century. A small reversal in the water freshening trend in the 1980/90s was possibly a response to land subsidence/sea-level change or to reduced freshwater supply.Freshwater supply to the sites is generally diminishing as former freshwater surpluses switch to deficit. One site (Merja Bokka, Morocco) became completely dry in 1998 as agriculture encroached and Megene Chitane, the only acid lake in Tunisia, is currently affected by excessive inflow abstraction. At the beginning of the 21st century, eight of the nine CASSARINA sites persist as viable but modified aquatic ecosystems. They nevertheless continue to support valuable aquatic biodiversity, especially in the Delta sites and in Chitane. The modern diatom communities are clearly tolerant of considerable environmental change but the remaining sites are increasingly threatened by major hydrological disturbance. Base-line floristic data for the late 20th century are given but continuous biomonitoring combined with effective management is needed urgently to help conserve North Africa's diminishing natural wetland lake resources.     

Ionic blockade of the rat connexin40 gap junction channel by large tetraalkylammonium ions.

The rat connexin40 gap junction channel is permeable to monovalent cations including tetramethylammonium and tetraethylammonium ions. Larger tetraalkyammonium (TAA(+)) ions beginning with tetrabutylammonium (TBA(+)) reduced KCl junctional currents disproportionately. Ionic blockade by tetrapentylammonium (TPeA(+)) and tetrahexylammonium (THxA(+)) ions were concentration- and voltage-dependent and occurred only when TAA(+) ions were on the same side as net K(+) efflux across the junction, indicative of block of the ionic permeation pathway. The voltage-dependent dissociation constants (K(m)(V(j))) were lower for THxA(+) than TPeA(+), consistent with steric effects within the pore. The K(m)-V(j) relationships for TPeA(+) and THxA(+) were fit with different reaction rate models for a symmetrical (homotypic) connexin gap junction channel and were described by either a one- or two-site model that assumed each ion traversed the entire V(j) field. Bilateral addition of TPeA(+) ions confirmed a common site of interaction within the pore that possessed identical K(m)(V(j)) values for cis-trans concentrations of TPeA(+) ions as indicated by the modeled I-V relations and rapid channel block that precluded unitary current measurements. The TAA(+) block of K(+) currents and bilateral TPeA(+) interactions did not alter V(j)-gating of Cx40 gap junctions. N-octyl-tributylammonium and -triethylammonium also blocked rCx40 channels with higher affinity and faster kinetics than TBA(+) or TPeA(+), indicative of a hydrophobic site within the pore near the site of block.     

Identification of an Overlapping Binding Domain on Cdc20 for Mad2 and Anaphase-Promoting Complex: Model for Spindle Checkpoint Regulation

Activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis in mammalian cells. Cdc20, which specifically recognizes APC substrates involved in the metaphase-to-anaphase transition, plays a pivotal role in APC activation through direct interaction with the APC. The activation of the APC by Cdc20 is prevented by the interaction of Cdc20 with Mad2 when the spindle checkpoint is activated. Using deletion mutagenesis and peptide mapping, we have identified the sequences in Cdc20 that target it to Mad2 and the APC, respectively. These sequences are distinct but overlapping, providing a possible structural explanation for the internal modulation of the APC-Cdc20 complex by Mad2. In the course of these studies, a truncation mutant of Cdc20 (1-153) that constitutively binds Mad2 but fails to bind the APC was identified. Overexpression of this mutant induces the formation of multinucleated cells and increases their susceptibility to undergoing apoptosis when treated with microtubule-inhibiting drugs. Our experiments demonstrate that disruption of the Mad2-Cdc20 interaction perturbs the mitotic checkpoint, leading to premature activation of the APC, sensitizing the cells to the cytotoxic effects of microtubule-inhibiting drugs.     

Chlorination of Indicator Bacteria and Viruses in Primary Sewage Effluent

Wastewater disinfection is used in many countries for reducing fecal coliform levels in effluents. Disinfection is therefore frequently used to improve recreational bathing waters which do not comply with microbiological standards. It is unknown whether human enteric viruses (which are responsible for waterborne disease) are simultaneously inactivated alongside fecal coliforms. This laboratory study focused on the chlorination of primary treated effluent with three doses (8, 16, and 30 mg/liter) of free chlorine as sodium hypochlorite. Seeding experiments showed that inactivation (>5 log₁₀ units) of Escherichia coli and Enterococcus faecalis was rapid and complete but that there was poor inactivation (0.2 to 1.0 log₁₀ unit) of F⁺-specific RNA (FRNA) bacteriophage (MS2) (a potential virus indicator) at all three doses. However, seeded poliovirus was significantly more susceptible (2.8 log₁₀ units) to inactivation by chlorine than was the FRNA bacteriophage. To ensure that these results were not artifacts of the seeding process, comparisons were made between inactivation rates of laboratory-seeded organisms in sterilized sewage and inactivation rates of organisms occurring naturally in sewage. Multifactorial analysis of variance showed that there was no significant difference (P > 0.05) between the inactivation rates for seeded and naturally occurring FRNA bacteriophage. However, laboratory-grown poliovirus was inactivated much more rapidly than were naturally occurring, indigenous enteroviruses (P < 0.001). This may reflect differences in the way indigenous virus is presented to the disinfectant. Inactivation rates for indigenous enteroviruses were quite similar to those seen for FRNA bacteriophage at lower doses of chlorine. These results have significance for the effectiveness of chlorination as a sewage treatment process, particularly where virus contamination is of concern, and suggest that FRNA bacteriophage would be an appropriate indicator of such viral inactivation under field conditions.     

Interobserver error in human skin colorimetry

Twenty light-skinned adults were measured at the upper inner arm site using two commonly used reflectance spectrophotometers. Each subject was measured by each of three investigators to assess the influence of interobserver error on the reflectance readings. A repeated measures design analysis of variance showed no significant variance component due to observers.