Selection of the midcell division site in Bacillus subtilis through MinD-dependent polar localization and activation of MinC

Bacterial cell division commences with the assembly of the tubulin-like protein, FtsZ, at midcell to form a ring. Division site selection in rod-shaped bacteria is mediated by MinC and MinD, which form a division inhibitor. Bacillus subtilis DivIVA protein ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at midcell. We have examined the localization of MinC protein and show that it is targeted to midcell and retained at the mature cell poles. This localization is reminiscent of the pattern previously described for MinD. Localization of MinC requires both early (FtsZ) and late (PbpB) division proteins, and it is completely dependent on MinD. The effects of a divIVA mutation on localization of MinC now suggest that the main role of DivIVA is to retain MinCD at the cell poles after division, rather than recruitment to nascent division sites. By overexpressing minC or minD, we show that both proteins are required to block division, but that only MinD needs to be in excess of wild-type levels. The results suggest a mechanism whereby MinD is required both to pilot MinC to the cell poles and to constitute a functional division inhibitor.     

Fas-induced changes in cdc2 and cdk2 kinase activity are not sufficient for triggering apoptosis in HUT-78 cells

Recent evidence suggested a role for the cell cycle dependent kinases cdc2 and cdk2 in apoptosis. An important mechanism by which many cell types could undergo apoptosis is through the activation of the Fas molecule on the cell membrane. To investigate whether Fas-induced cell death activated cdc2 and cdk2 kinases inappropriately, the human T lymphoma cells HUT-78, which express a high copy number of Fas, and two other previously characterized subclones of the same cell line which express mutant, cell death-deficient dominant-negative forms of Fas, were Fas-challenged and the changes in cdc2 and cdk2 kinase activity monitored. In both wild-type and Fas-mutated HUT-78 cells, apoptosis was associated simultaneously with decreased cdc2 and increased cdk2 activity. This association suggested that changes in cdc2 and cdk2 kinase activity are secondary events in cell death mediated by Fas. J. Cell. Biochem. 64:579–585. © 1997 Wiley-Liss, Inc.     

Palladium(II) derivatives of alkylsulfanyl substituted thiophenes as precursors of inorganic polymers: Spectroscopic, electrochemical investigations and X-ray crystal structure of trans-PdCl2[3-(butylsulfanyl)thiophene]2

Palladium(II) complexes with two thiophene derivatives bearing alkylsulfanyl chains, i.e., 3-(n-butylsulfanyl)thiophene and 4,4′-bis(n-butylsulfanyl)-2,2′-bithiophene, are synthesised and spectroscopically and electrochemically characterised. The molecular structure of PdCl₂[3-(n-butylsulfanyl)thiophene]₂ was determined by X-ray analysis. The properties of the complexes have been compared with those of PdCl₂[3,3′-bis(n-butylsulfanyl)-2,2′-bithiophene] and of a conductive polymer partially coordinated with Pd(II), previously synthesised by us. We found that Pd(II) ions can coordinate sulfanyl sulfur atoms both in cis and trans configuration, leading to a reticulate material, where some kind of interchain bridging may be reasonably supposed to enhance the bulk conductivity.     

A shotgun proteomic method for the identification of membrane-embedded proteins and peptides

Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome.     

Dendritic cells: driving the differentiation programme of T cells in viral infections

Protective immunity against viral pathogens depends on the generation and maintenance of a small population of memory CD8(+) T cells. Successful memory cell generation begins with early interactions between na?ve T cell and dendritic cells (DCs) within the inflammatory milieu of the secondary lymphoid tissues. Recent insights into the role of different populations of DCs, and kinetics of antigen presentation, during viral infections have helped to understand how DCs can shape the immune response. Here, we review the recent progress that has been made towards defining how specific DC subsets drive effector CD8(+) T-cell expansion and differentiation into memory cells. Further, we endeavour to examine how the molecular signals imparted by DCs coordinate to generate protective CD8(+) T-cell immunity.     

Autoactivation of human ADAM8: a novel pre-processing step is required for catalytic activity

Members of the ADAM (a disintegrin and metalloproteinase) family of proteins possess a multidomain architecture which permits functionalities as adhesion molecules, signalling intermediates and proteolytic enzymes. ADAM8 is found on immune cells and is induced by multiple pro-inflammatory stimuli suggesting a role in inflammation. Here we describe an activation mechanism for recombinant human ADAM8 that is independent from classical PC (pro-protein convertase)-mediated activation. N-terminal sequencing revealed that, unlike other ADAMs, ADAM8 undergoes pre-processing at Glu(158), which fractures the Pro (pro-segment)-domain before terminal activation takes place to remove the putative cysteine switch (Cys(167)). ADAM8 lacking the DIS (disintegrin) and/or CR (cysteine-rich) and EGF (epidermal growth factor) domains displayed impaired ability to complete this event. Thus pre-processing of the Pro-domain is co-ordinated by DIS and CR/EGF domains. Furthermore, by placing an EK (enterokinase) recognition motif between the Pro- and catalytic domains of multiple constructs, we were able to artificially remove the pro-segment prior to pre-processing. In the absence of pre-processing of the Pro-domain a marked decrease in specific activity was observed with the autoactivated enzyme, suggesting that the Pro-domain continued to associate and inhibit active enzyme. Thus, pre-processing of the Pro-domain of human ADAM8 is important for enzyme maturation by preventing re-association of the pro-segment with the catalytic domain. Given the observed necessity of DIS and CR/EGF for pre-processing, we conclude that these domains are crucial for the proper activation and maturation of human ADAM8.     

Genetic control of wheat quality: interactions between chromosomal regions determining protein content and composition, dough rheology, and sponge and dough baking properties

While the genetic control of wheat processing characteristics such as dough rheology is well understood, limited information is available concerning the genetic control of baking parameters, particularly sponge and dough (S&D) baking. In this study, a quantitative trait loci (QTL) analysis was performed using a population of doubled haploid lines derived from a cross between Australian cultivars Kukri × Janz grown at sites across different Australian wheat production zones (Queensland in 2001 and 2002 and Southern and Northern New South Wales in 2003) in order to examine the genetic control of protein content, protein expression, dough rheology and sponge and dough baking performance. The study highlighted the inconsistent genetic control of protein content across the test sites, with only two loci (3A and 7A) showing QTL at three of the five sites. Dough rheology QTL were highly consistent across the 5 sites, with major effects associated with the Glu-B1 and Glu-D1 loci. The Glu-D1 5 + 10 allele had consistent effects on S&D properties across sites; however, there was no evidence for a positive effect of the high dough strength Glu-B1-al allele at Glu-B1. A second locus on 5D had positive effects on S&D baking at three of five sites. This study demonstrated that dough rheology measurements were poor predictors of S&D quality. In the absence of robust predictive tests, high heritability values for S&D demonstrate that direct selection is the current best option for achieving genetic gain in this product category. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Efficacy of chemical and fluorescent protein markers in studying plant colonization by endophytic non-pathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolates

In studying plant colonization by inoculated Fusarium oxysporum endophytes, it is important to be able to distinguish inoculated isolates from saprophytic strains. In the current study, F. oxysporum isolates were transformed with the green (GFP) and red fluorescent protein (DsRed) genes, and benomyl- and chlorate-resistant mutant isolates were also developed. The benomyl- and chlorate-resistant mutants, and the fluorescently labelled transformants, were able to grow on potato dextrose agar amended with 20 mg Benlate^® l^?1, 30 g chlorate l^?1 and 150 μg hygromycin ml^?1, respectively. Single spores of all mutants remained stable after several transfers on non-selective media. Most mutants and transformants produced colony diameters that did not differ significantly from that of their wild-type progenitors after 7 days of growth on non-selective media. Few mutants, however, had growth rates that were either slower or faster than for their wild-types. Plant colonization studies showed that root and rhizome tissue colonization by most benomyl- and chlorate-resistant mutants was similar to that of their wild-type isolates. Unlike GFP transformants, DsRed transformants were difficult to visualize in planta. Both the mutants and transformants can be used for future studies to investigate colonization, distribution and survival of biocontrol F. oxysporum endophytes in banana plants.