The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins

The Escherichia coli MukB, MukE, and MukF proteins form a bacterial condensin (MukBEF) that contributes to chromosome management by compacting DNA. MukB is an ATPase and DNA-binding protein of the SMC superfamily; however, the structure and function of non-SMC components, such as MukF, have been less forthcoming. Here, we report the crystal structure of the N-terminal 287 amino acids of MukF at 2.9 A resolution. This region folds into a winged-helix domain and an extended coiled-coil domain that self-associate to form a stable, doubly domain-swapped dimer. Protein dissection and affinity purification data demonstrate that the region of MukF C-terminal to this fragment binds to MukE and MukB. Our findings, together with sequence analyses, indicate that MukF is a kleisin subunit for E. coli condensin and suggest a means by which it may organize the MukBEF assembly.     

Genome screen for twinning rate QTL in four North American Holstein families

The objective of this study was to identify twinning rate quantitative trait loci (QTL) by typing pooled samples in a preliminary screening followed by interval mapping to test QTL effects. Four elite North American Holstein half-sib sire families with high twinning rate predicted transmitting abilities (PTA) were used in this study. Chromosomes 5, 7, 19 and 23 were not genotyped as these chromosomes were scanned for QTL in these families in a previous study. DNA was extracted from phenotypically extreme sons in each sire family. Two pools were prepared from sons of sires in each phenotypic tail, two each for high and low PTA levels for twinning rates. Each pool contained DNA from 4 to 15% of all sons of the sire depending on family. A total of 268 fluorescently labelled microsatellite markers were tested for heterozygosity in sires. About 135--170 informative markers per family were genotyped using pooled DNA samples. Based on the preliminary evidence for potential twinning rate QTL from pooled typing, interval mapping was performed subsequently on 12 chromosomal regions by family combinations. Evidence of QTL for twinning rate was found in one family on BTA 21 and 29 at a chromosome-wide P<0.05 and on BTA 8, 10 and 14 with a chromosome-wide P<0.01.     

Differential effects of tumor necrosis factor-alpha and CD40L on NF-kappa B inhibitory proteins I kappa B alpha, beta and epsilon and on the induction of the Jun amino-terminal kinase pathway in Ramos Burkitt lymphoma cells

Interaction between the CD40 ligand and its cognate receptor is known to affect various aspects of B-cell biology. Less is known about the biological consequences of B-cell signaling through tumor necrosis factor alpha (TNF-alpha) and its two receptors. We have used Ramos germinal center (GC)-derived Burkitt's lymphoma (BL) cells as a model system to compare some of the early signaling events of TNF-alpha and CD40L on the NF-kappaB and c-Jun amino-terminal kinase (JNK) pathways. We have previously found that both TNF-alpha and CD40L induced enhanced cell aggregation, adherence and modified cell surface morphology of Ramos cells. In the present report, it was found that treatment with either TNF-alpha or CD40L resulted in a rapid degradation (within 15 min) of IkappaBalpha, followed by a recovery period lasting up to a few hours. The level of IkappaBbeta, another inhibitory molecule of the NF-kappaB pathway, also decreased following treatment with CD40L or TNF-alpha. However, whereas CD40L induced a rapid drop without significant recovery within 2 h, TNF-alpha caused a slow and gradual decline of IkappaBbeta. In addition, treatment with CD40L resulted in a gradual and modest decline of up to 60% of the level of IkappaBepsilon within 2 h, whereas a much smaller decline was seen with TNF-alpha (approx. 20%) Our results thus show that in Ramos cells, TNF-alpha and CD40L have common, as well as differential, signaling effects on the IkappaBalpha, IkappaBbeta and IkappaBepsilon, which form inhibitory complex(es) with the NF-kappaB cytosolic proteins. We also found that CD40L, but not TNF-alpha activates the JNK pathway through transient phosphorylation of its threonine183/tyrosine185 residues. As expected, c-Jun, which is known to be a substrate of JNK, was also phosphorylated at serine residue 73 by treatment with CD40L, but not by TNF-alpha.     

MEPD: a resource for medaka gene expression patterns

The Medaka Expression Pattern Database (MEPD) is a database for gene expression patterns determined by in situ hybridization in the small freshwater fish medaka (Oryzias latipes). Data have been collected from various research groups and MEPD is developing into a central expression pattern depository within the medaka community. Gene expression patterns are described by images and terms of a detailed medaka anatomy ontology of over 4000 terms, which we have developed for this purpose and submitted to Open Biological Ontologies. Sequences have been annotated via BLAST match results and using Gene Ontology terms. These new features will facilitate data analyses using bioinformatics approaches and allow cross-species comparisons of gene expression patterns. Presently, MEPD has 19,757 entries, for 1024 of them the expression pattern has been determined.     

Proteolytic susceptibility of the serine protease inhibitor trappin-2 (pre-elafin): evidence for tryptase-mediated generation of elafin

A number of serine, cysteine, metallo- and acid proteases were evaluated for their ability to proteolytically cleave the serine protease inhibitor trappin-2, also known as pre-elafin, and to release elafin from its precursor. None of the metalloproteases or acid proteases examined cleaved trappin-2, while serine and cysteine proteases preferentially cleaved trappin-2 within its non-inhibitory N-terminal moiety. Cathepsin L, cathepsin K, plasmin, trypsin and tryptase were able to release elafin by cleaving the Lys 38 -Ala 39 peptide bond in trappin-2. However, purified tryptase appeared to be efficient at releasing elafin. Incubation of trappin-2 with purified mast cells first challenged with anti-immunoglobulin E or calcium ionophore A23187 resulted in the rapid generation of elafin. This proteolytic release of elafin from trappin-2 was inhibited in the presence of a tryptase inhibitor, suggesting that this mast cell enzyme was involved in the process. Finally, ex vivo incubation of trappin-2 with sputum from cystic fibrosis patients indicated the production of a proteolytic immunoreactive fragment with the same mass as that of native elafin. This cleavage did not occur when preincubating the sputum with polyclonal antibodies directed against tryptase. Taken together, these findings indicate that tryptase could likely be involved in the maturation of trappin-2 into elafin under physiological conditions.     

An extracellular carboxylesterase from the basidiomycete Pleurotus sapidus hydrolyses xanthophyll esters

An extracellular enzyme capable of efficient hydrolysis of xanthophyll esters was purified from culture supernatants of the basidiomycete Pleurotus sapidus. Under native conditions, the enzyme exhibited a molecular mass of 430 kDa, and SDS-PAGE data suggested a composition of eight identical subunits. Biochemical characterisation of the purified protein showed an isoelectric point of 4.5, and ideal hydrolysis conditions were observed at pH 5.8 and 40 degrees C. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. An 1861-bp cDNA containing an open reading frame of 1641 bp was cloned from a cDNA library that showed ca. 40% homology to Candida rugosa lipases. The P. sapidus carboxylesterase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus that has been characterised at the molecular level.     

Structural dissection of ATP turnover in the prototypical GHL ATPase TopoVI

GHL proteins are functionally diverse enzymes defined by the presence of a conserved ATPase domain that self-associates to trap substrate upon nucleotide binding. The structural states adopted by these enzymes during nucleotide hydrolysis and product release, and their consequences for enzyme catalysis, have remained unclear. Here, we have determined a complete structural map of the ATP turnover cycle for topoVI-B, the ATPase subunit of the archaeal GHL enzyme topoisomerase VI. With this ensemble of structures, we show that significant conformational changes in the subunit occur first upon ATP binding, and subsequently upon release of hydrolyzed P(i). Together, these data provide a structural framework for understanding the role of ATP hydrolysis in the type II topoisomerase reaction. Our results also suggest that the GHL ATPase module is a molecular switch in which ATP hydrolysis serves as a prerequisite but not a driving force for substrate-dependent structural transitions in the enzyme.