A novel series of potent and selective IKK2 inhibitors

A novel series of aminopyrimidine IKK2 inhibitors have been developed which show excellent in vitro inhibition of this enzyme and good selectivity over the IKK1 isoform. The relative potency and selectivity of these compounds has been rationalized using QSAR and structure-based modelling.     

Working memory: imaging the magic number four

Many brain regions have been implicated in memory performance, but the relationship between memory capacity and neural activity has not been clear. Recent studies show that activity in the posterior parietal cortex increases with working memory load, implicating this region in the storage of representations in visual memory.     

A new interpretation of the Keller-Segel model based on multiphase modelling

In this paper an alternative derivation and interpretation are presented of the classical Keller-Segel model of cell migration due to random motion and chemotaxis. A multiphase modelling approach is used to describe how a population of cells moves through a fluid containing a diffusible chemical to which the cells are attracted. The cells and fluid are viewed as distinct components of a two-phase mixture. The principles of mass and momentum balance are applied to each phase, and appropriate constitutive laws imposed to close the resulting equations. A key assumption here is that the stress in the cell phase is influenced by the concentration of the diffusible chemical. By restricting attention to one-dimensional cartesian geometry we show how the model reduces to a pair of nonlinear coupled partial differential equations for the cell density and the chemical concentration. These equations may be written in the form of the Patlak-Keller-Segel model, naturally including density-dependent nonlinearities in the cell motility coefficients. There is a direct relationship between the random motility and chemotaxis coefficients, both depending in an inter-related manner on the chemical concentration. We suggest that this may explain why many chemicals appear to stimulate both chemotactic and chemokinetic responses in cell populations. After specialising our model to describe slime mold we then show how the functional form of the chemical potential that drives cell locomotion influences the ability of the system to generate spatial patterns. The paper concludes with a summary of the key results and a discussion of avenues for future research.     

Membrane-bound matrix metalloproteinase-8 on activated polymorphonuclear cells is a potent, tissue inhibitor of metalloproteinase-resistant collagenase and serpinase

Little is known about the cell biology or the biologic roles of polymorphonuclear cell (PMN)-derived matrix metalloproteinase-8 (MMP-8). When activated with proinflammatory mediators, human PMN release only approximately 15-20% of their content of MMP-8 ( approximately 60 ng/10(6) cells) exclusively as latent pro-MMP-8. However, activated PMN incubated on type I collagen are associated with pericellular collagenase activity even when bathed in serum. PMN pericellular collagenase activity is attributable to membrane-bound MMP-8 because: 1) MMP-8 is expressed in an inducible manner in both pro- and active forms on the surface of human PMN; 2) studies of activated PMN from mice genetically deficient in MMP-8 (MMP-8(-/-)) vs wild-type (WT) mice show that membrane-bound MMP-8 accounts for 92% of the MMP-mediated, PMN surface type I collagenase activity; and 3) human membrane-bound MMP-8 on PMN cleaves types I and II collagens, and alpha(1)-proteinase inhibitor, but is substantially resistant to inhibition by tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Binding of MMP-8 to the PMN surface promotes its stability because soluble MMP-8 has t(1/2) = 7.5 h at 37 degrees C, but membrane-bound MMP-8 retains >80% of its activity after incubation at 37 degrees C for 18 h. Studies of MMP-8(-/-) vs WT mice given intratracheal LPS demonstrate that 24 h after intratracheal LPS, MMP-8(-/-) mice have 2-fold greater accumulation of PMN in the alveolar space than WT mice. Thus, MMP-8 has an unexpected, anti-inflammatory role during acute lung injury in mice. TIMP-resistant, active MMP-8 expressed on the surface of activated PMN is likely to be an important form of MMP-8, regulating lung inflammation and collagen turnover in vivo.     

Mathematical modelling of the use of macrophages as vehicles for drug delivery to hypoxic tumour sites

Poor drug delivery and low rates of cell proliferation are two factors associated with hypoxia that diminish the efficacy of many chemotherapeutic drugs. Since macrophages are known to migrate specifically towards, and localize within, hypoxic tumour regions, a promising resolution to these problems involves genetically engineering macrophages to perform such anti-tumour functions as inducing cell lysis and inhibiting angiogenesis. In this paper we outline a modelling approach to characterize macrophage infiltration into early avascular solid tumours, and extensions to study the interaction of these cells with macrophages already present within the tumour. We investigate the role of chemotaxis and chemokine production, and the efficacy of macrophages as vehicles for drug delivery to hypoxic tumour sites. The model is based upon a growing avascular tumour spheroid, in which volume is filled by tumour cells, macrophages and extracellular material, and tumour cell proliferation and death is regulated by nutrient diffusion. Crucially, macrophages occupy volume, and hence contribute to the volume balance and hence the size of the tumour. We also include oxygen-dependent production of macrophage chemokines, which can lead to accumulations in the hypoxic region of the tumour. We find that the macrophage chemotactic sensitivity is a key determinant of macrophage infiltration and tumour size. Although increased infiltration should be beneficial from the point of view of macrophage-based therapies, such infiltration in fact leads to increased tumour sizes. Finally, we include terms representing the induced death of tumour cells by hypoxic engineered macrophages. We demonstrate that reductions in tumour size can be achieved, but predict that a combination of therapies would be required for complete eradication. We also highlight some counter-intuitive predictions-for example, absolute and relative measures of tumour burden lead to different conclusions about prognosis. In summary, this paper illustrates how mathematical models may be used to investigate promising macrophage-based therapies.     

Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola 1448A reveals divergence among pathovars in genes involved in virulence and transposition

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis. Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.     

Definition of the M-conotoxin superfamily: characterization of novel peptides from molluscivorous Conus venoms

Most of the >50,000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.     

Description and behavioural biology of two Ufens species (Hymenoptera: Trichogrammatidae), egg parasitoids of Homalodisca species (Hemiptera: Cicadellidae) in southern California

The morphology, distribution, and hosts of two egg parasitoids, Ufens principalis Owen sp. n. and U. ceratus Owen sp. n. (Hymenoptera: Trichogrammatidae), are described for the first time. These species are compared to U. niger (Ashmead), the only species of Ufens s. str. currently described from the Nearctic, and diagnostic differences are presented. The behavioural biology of U. principalis, and U. ceratus to a smaller extent, is also described for the first time. Ufens principalis exhibited a rapid and long-distance response in the form of directional flight toward freshly laid eggs of Homalodisca species, its primary hosts in southern California. Parasitism involved aggregations of female U. principalis on fresh Homalodisca egg masses, which remained attractive to U. principalis for a relatively short time. The level of oviposition by U. principalis females was low during most of the day and peaked before sunset in tandem with a peak in Homalodisca oviposition. Oviposition behaviour of U. principalis is described and the distribution of ovipositor probe durations showed that most probes were generally of very short duration. Mating of both Ufens species occurred on the egg mass, with males showing aggressive behaviour towards each other as they competed for emerging females. Ufens ceratus males displayed greater aggression towards other males than U. principalis males. By contrast, fights among U. principalis males involved more individuals and lasted longer than corresponding fights between U. ceratus males.     

Functional dissection of an AP-2 beta2 appendage-binding sequence within the autosomal recessive hypercholesterolemia protein

The autosomal recessive hypercholesterolemia (ARH) protein plays a critical role in regulating plasma low density lipoprotein (LDL) levels. Inherited defects in ARH lead to a hypercholesterolemia that closely phenocopies that caused by a defective LDL receptor. The elevated serum LDL-cholesterol levels typical of ARH patients and the pronounced accumulation of the LDL receptor at the cell surface of hepatocytes in ARH-null mice argue that ARH operates by promoting the internalization of the LDL receptor within clathrin-coated vesicles. ARH contains an amino-terminal phosphotyrosine-binding domain that associates physically with the LDL receptor internalization sequence and with phosphoinositides. The carboxyl-terminal half of ARH contains a clathrin-binding sequence and a separate AP-2 adaptor binding region providing a plausible mechanism for how ARH can act as an endocytic adaptor or CLASP (clathrin-associated sorting protein) to couple LDL receptors with the clathrin machinery. Because the interaction with AP-2 is highly selective for the independently folded appendage domain of the beta2 subunit, we have characterized the ARH beta2 appendage-binding sequence in detail. Unlike the known alpha appendage-binding motifs, ARH requires an extensive sequence tract to bind the beta appendage with comparably high affinity. A minimal 16-residue sequence functions autonomously and depends upon ARH residues Asp253, Phe259, Leu262, and Arg266. We suggested that biased beta subunit engagement by ARH and the only other beta2 appendage selective adaptor, beta-arrestin, promotes efficient incorporation of this mechanistically distinct subset of CLASPs into clathrin-coated buds.     

Neither the A- nor B-repeat regions of the fibrinogen-binding protein of Streptococcus equi subsp. equi are essential for fibrinogen binding

The major cell wall-associated protein (FgBP) of Streptococcus equi subsp. equi possesses two internal blocks of repeated sequence (A and B) and binds horse fibrinogen (Fg) avidly through residues located in the N-terminal half of the molecule. In the present study, we investigated the roles of the two repeats blocks in Fg binding through construction of recombinant FgBP proteins containing defined internal deletions of sequence. Ligand binding experiments clearly showed that neither repeat is essential for Fg binding. However, residues within the B repeats seem to play a major role in the aberrant mobility observed for FgBP following sodium dodecyl sulfate polyacrylamide gel electrophoresis.