The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.     

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c ₆ are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b ₆ f and Photosystem I. Despite plastocyanin and cytochrome c ₆ differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c ₆ reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c ₆ does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of osmotic stress on the growth of epicotyls of Cicer arietinum in relation to changes in the autolytic process and glycanhydrolytic cell wall enzymes

Simulation of drought by polyethylene glycol (PEG) inhibited elongation of epicotyls of Cicer arietinum L. cv. Castellana but had no effect on growth capacity since growth was restored once the inhibitory condition had been removed. The amount of proteins in the cell wall was correlated with the elongation of the epicotyls and decreased when elongation was inhibited. PEG-induced inhibition of elongation had different effects on the various glycanhydrolytic cell wall enzymes. Only α-galactosidase (EC 3. 2. 1. 22) seemed related to the lack of elongation, increasing its activity when elongation was inhibited. The β-galactosidase (EC 3. 2. 1. 23) and β-glucosidase (EC 3. 2. 1. 21) studied did not show changes in their specific activities during the inhibition of elongation. β-Galactosidase is responsible for the autolytic process in Cicer arietinum . This enzyme hydrolyzes specified linkages in the cell wall, releasing sugar constituents. Our present results show that β-galactosidase is not directly related with elongation because no changes could be observed during inhibition of elongation. The autolytic process is related with chemical processes taking place in the cell wall and preceding elongation of the epicotyls, i. e. the loosening process. Cell wall loosening is necessary for elongation to take place but elongation does not necessarily follow loosening if the osmotic conditions are unfavorable     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.     

Comparative morphology of porpoise (Cetacea: Phocoenidae) pterygoid sinuses: Phylogenetic and functional implications

High‐resolution X‐ray computed tomographic scans were used to examine pterygoid sinus morphology within extant porpoise species and one delphinid (Tursiops truncatus), in order to consider: 1) intraspecific and interspecific variation among the studied species; 2) the most parsimonious sequence of character acquisition; and 3) the potential functional roles of the preorbital lobes of the sinuses in sound reflection. Scans revealed that the pterygoid/palatine regions are mediolaterally broader in the earliest diverging phocoenid (Neophocaena phocaenoides) and Tursiops truncatus than the dorsoventrally elongated sinuses observed in other species. Rostrocaudal lengths of the sphenoidal regions of the sinuses in all individuals studied are proportionally similar, indicating conservatism in this region across species. The neonate Phocoena phocoena has shorter preorbital lobes than adults, but they are still proportionally longer than Neophocaena phocaenoides and Phocoena spinipinnis. The preorbital lobes broaden mediolaterally to varying degrees across species; in particular, Phocoenoides dalli has the largest dorsal and lateral expansion of this region. Assuming the highest pulse frequency produced by porpoises is 150 kHz, all regions of the preorbital lobes are thick enough to reflect the wavelengths produced. In addition, the neonate preorbital lobes are not as elongated as they are in adults, and the dorsal third of this region may not reflect sound to the same extent. This study reinforces the importance of using nondestructive methods to quantify variation in endocranial anatomy and the value of CT data for recovering phylogenetically useful information, as well as functional roles sinuses play in concert with the soft tissue head anatomy for biosonar. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

RAC1 induces nuclear alterations through the LINC complex to enhance melanoma invasiveness

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.     

Brain-derived neurotrophic factor alleviates the oxidative stress induced by oxygen and glucose deprivation in an ex vivo brain slice model

Brain-derived neurotrophic factor (BDNF) is considered as a putative therapeutic agent against stroke. Since BDNF role on oxidative stress is uncertain, we have studied this role in a rat brain slice ischemia model, which allows BDNF reaching the neural parenchyma. Hippocampal and cerebral cortex slices were subjected to oxygen and glucose deprivation (OGD) and then returned to normoxic conditions (reperfusion-like, RL). OGD/RL increased a number of parameters mirroring oxidative stress in the hippocampus that were reduced by the BDNF presence. BDNF also reduced the OGD/RL-increased activity in a number of antioxidant enzymes in the hippocampus but no effects were observed in the cerebral cortex. In general, we conclude that alleviation of oxidative stress by BDNF in OGD/RL-exposed slices relies on decreasing cPLA2 activity, rather than modifying antioxidant enzyme activities. Moreover, a role for the oxidative stress in the differential ischemic vulnerability of cerebral cortex and hippocampus is also supported.     

Enterococcal Infective Endocarditis following Periodontal Disease in Dogs

In humans, one of the major factors associated with infective endocarditis (IE) is the concurrent presence of periodontal disease (PD). However, in veterinary medicine, the relevance of PD in the evolution of dogs’ endocarditis remains poorly understood. In order to try to establish a correlation between mouth-associated Enterococcus spp. and infective endocarditis in dogs, the present study evaluated the presence and diversity of enterococci in the gum and heart of dogs with PD. Samples were collected during necropsy of 32 dogs with PD and visually diagnosed with IE, which died of natural causes or euthanasia. Enterococci were isolated, identified and further characterized by Pulsed-Field Gel Electrophoresis (PFGE); susceptibility to antimicrobial agents and pathogenicity potential was also evaluated. In seven sampled animals, PFGE-patterns, resistance and virulence profiles were found to be identical between mouth and heart enterococci obtained from the same dog, allowing the establishment of an association between enterococcal periodontal disease and endocarditis in dogs. These findings represent a crucial step towards understanding the pathogenesis of PD-driven IE, and constitute a major progress in veterinary medicine.     

Different baseline sister chromatid exchange levels in density fractionated human lymphocytes

Summary Different activation states of B and T lymphocytes, as manifested by differences in cell density, were obtained by Percoll density centrifugation of unstimulated human lymphocytes. Four different density fractions were defined: B cells with low (1.043 g/ml) and high (1.056) density, and T cells with low (1.067) and high (1.077) density, respectively. Sister chromatid exchange (SCE) conditions and proliferation rates were determined. Total B cells, stimulated by the bacterial mitogen Branhamella, had 4.6 SCE per cell, the lowest mean baseline SCE level recorded among lymphocytes. The growth rate was intermediate between that of low and high density T cells. The two T cell fractions stimulated by phytohemagglutinin (PHA) had different baseline SCE frequencies and different growth characteristics: the low density cells had 5.7 SCEs per cell and a short cell cycle, whereas high density cells had 12.5 SCEs per cell and a longer cell cycle. The differences in baseline SCE frequency and growth characteristics between the two T cell fractions seem to be correlated with the differences in the activation state as reflected by the cell density. Both high and low density T cell are G₀ populations which supposedly differ with respect to previous history in vivo such as age and contact with antigens. The reason why these cells react differently to bromodeoxyuridine (BrdU) is unknown, but differences in intracellular DNA precursor pools and enzyme activities might play a role.