During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks

The Smc5/6 complex belongs to the SMC (structural maintenance of chromosomes) family, which also includes cohesin and condensin. In Saccharomyces cerevisiae, the Smc5/6 complex contains six essential non-Smc elements, Nse1-6. Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 small ubiquitin-like modifier (SUMO) ligase important for Smc5 sumoylation. Characterization of two temperature-sensitive mutants, nse5-ts1 and nse5-ts2, demonstrated the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, formation of fork-associated homologous recombination intermediates, and hydroxyurea sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, and for its functionality during replication stress.     

A Secretory Golgi Bypass Route to the Apical Surface Domain of Epithelial MDCK Cells

Proteins leave the endoplasmic reticulum (ER) for the plasma membrane via the classical secretory pathway, but routes bypassing the Golgi apparatus have also been observed. Apical and basolateral protein secretion in epithelial Madin-Darby canine kidney (MDCK) cells display differential sensitivity to Brefeldin A (BFA), where low concentrations retard apical transport, while basolateral transport still proceeds through intact Golgi cisternae . We now describe that BFA-mediated retardation of glycoprotein and proteoglycan transport through the Golgi apparatus induces surface transport of molecules lacking Golgi modifications, possessing those acquired in the ER. Low concentrations of BFA induces apical Golgi bypass, while higher concentrations were required to induce basolateral Golgi bypass. Addition of the KDEL ER-retrieval sequence to model protein cores allowed observation of apical Golgi bypass in untreated MDCK cells. Basolateral Golgi bypass was only observed after the addition of BFA or upon cholesterol depletion. Thus, in MDCK cells, an apical Golgi bypass route can transport cargo from pre-Golgi organelles in untreated cells, while the basolateral bypass route is inducible.     

Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution

The crystal structure of (3R)-hydroxyacyl-CoA dehydrogenase of rat peroxisomal multifunctional enzyme type 2 (MFE-2) was solved at 2.38 A resolution. The catalytic entity reveals an alpha/beta short chain alcohol dehydrogenase/reductase (SDR) fold and the conformation of the bound nicotinamide adenine dinucleotide (NAD(+)) found in other SDR enzymes. Of great interest is the separate COOH-terminal domain, which is not seen in other SDR structures. This domain completes the active site cavity of the neighboring monomer and extends dimeric interactions. Peroxisomal diseases that arise because of point mutations in the dehydrogenase-coding region of the MFE-2 gene can be mapped to changes in amino acids involved in NAD(+) binding and protein dimerization.     

Ectopic RNase E sites promote bypass of 5'-end-dependent mRNA decay in Escherichia coli

In Escherichia coli, 5'-terminal stem-loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5'-terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two-thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. Insertion of a 12-15 residue 'ectopic' RNase E cleavage site from either the rne leader or 9S pre-rRNA into the 5'-non-coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem-loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5'-terminus, and exploit an alternative 'internal entry' pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5'-termini, the use of internal entry for bypass of barriers to decay, 'ectopic sites' and the role of translating ribosomes.     

Global gene expression in Escherichia coli biofilms

It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared with planktonic growth. Genes encoding proteins involved in adhesion (type 1 fimbriae) and, in particular, autoaggregation (Antigen 43) were highly expressed in the adhered population in a manner that is consistent with current models of sessile community development. Several novel gene clusters were induced upon the transition to biofilm growth, and these included genes expressed under oxygen-limiting conditions, genes encoding (putative) transport proteins, putative oxidoreductases and genes associated with enhanced heavy metal resistance. Of particular interest was the observation that many of the genes altered in expression have no current defined function. These genes, as well as those induced by stresses relevant to biofilm growth such as oxygen and nutrient limitation, may be important factors that trigger enhanced resistance mechanisms of sessile communities to antibiotics and hydrodynamic shear forces.     

Genetic variation and structure in the expanding moss Pogonatum dentatum (Polytrichaceae) in its area of origin and in a recently colonized area

Genetic variation in the expanding moss species Pogonatum dentatum was studied using intersimple sequence repeat (ISSR) markers. The genetic consequences of range expansion were studied by comparing source populations in a mountain area with populations from a recently colonized lowland area in Sweden. Indices of genetic variation show slightly lower number of alleles per locus in the lowlands and a similar gene diversity in both areas. Three of four lowland populations had evidence of a recently passed bottleneck. Considerably higher haplotype diversity was found in the recently colonized lowlands compared to source populations in the mountains. Patterns of allelic diversity suggest that P. dentatum experiences loss of genetic variation through founder effects and genetic drift when expanding its distribution range. Higher haplotypic diversity, less linkage disequilibrium, and fewer compatible loci indicate that sexual recombination is relatively more important in the lowlands compared to the mountains. A likely explanation is higher success of establishment from spores in the lowlands, while clonal propagation predominates in the mountains. Less genetic differentiation among lowland populations indicates more gene flow in the lowland area, involving more spores and/or fragments moving among populations.     

External forces on the limbs of jumping lemurs at takeoff and landing

Ground reaction forces were recorded for jumps of three individuals each of Lemur catta and Eulemur fulvus. Animals jumped back and forth between a ground-mounted force plate and a 0.5-m elevated platform, covering horizontal distances of 0.5-2 m. In total, 190 takeoffs and 263 landings were collected. Animals typically jumped from a run up and into a run out, during which they gained or into which they carried horizontal impulse. Correspondingly, vertical impulses dominated takeoffs and landings. Peak forces were moderate in magnitude and not much higher than forces reported for quadrupedal gaits. This is in contrast to the forces for standing jumps of specialized leapers that considerably exceed forces associated with quadrupedal gaits. Force magnitudes for the lemur jumps are more comparable to peak forces reported for other quadrupeds performing running jumps. Takeoffs are characterized by higher hindlimb than forelimb peak forces and impulses. L. catta typically landed with the hindlimbs making first contact, and the hindlimb forces and impulses were higher than the forelimb forces and impulses at landing. E. fulvus typically landed with the forelimbs striking first and also bearing the higher forces. This pattern does not fully conform to the paradigm of primate limb force distribution, with higher hindlimb than forelimb forces. However, the absolute highest forces in E. fulvus also occur at the hindlimbs, during acceleration for takeoff.     

Flumequine in Atlantic salmon Salmo salar: disposition in fish held in sea water versus fresh water

14C-labeled flumequine was administered as a single oral (5 mg kg(-1), 86 microCi kg(-1)) or intravenous (5 mg kg(-1), 82 microCi kg(-1)) dose to Atlantic salmon Salmo salar held in sea water or in fresh water. The absorption, tissue distribution and elimination were determined by means of liquid scintillation counting and whole-body autoradiography. The drug was rapidly absorbed and extensively distributed in all groups of fish. Radiolabeled compound was present in blood and muscle for more than 8 wk in the freshwater groups. In the seawater groups, however, no radioactivity was detected in the blood and muscle after 4 d and 2 wk, respectively. It was concluded that flumequine was eliminated at a substantially higher rate from Atlantic salmon in sea water than in fresh water.