Cohesin Is Limiting for the Suppression of DNA Damage–Induced Recombination between Homologous Chromosomes

Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage–induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G₂/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G₂/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage–induced recombinants in G₂/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.     

Comments on controversial tick (Acari: Ixodida) species names and species described or resurrected from 2003 to 2008

There are numerous discrepancies in recent published lists of the ticks of the world. Here we review the controversial names, presenting evidence for or against their validity and excluding some altogether. We also address spelling errors and present a list of 17 species described or resurrected during the years 2003–2008. We consider the following 35 tick species names to be invalid: Argas fischeri Audouin, 1826, Ornithodoros boliviensis Kohls and Clifford, 1964, Ornithodoros steini (Schulze, 1935), Amblyomma acutangulatum Neumann, 1899, Amblyomma arianae Keirans and Garris, 1986, Amblyomma bibroni (Gervais, 1842), Amblyomma colasbelcouri (Santos Dias, 1958), Amblyomma concolor Neumann, 1899, Amblyomma cooperi Nuttall and Warburton, 1908, Amblyomma curruca Schulze, 1936, Amblyomma cyprium Neumann, 1899, Amblyomma decorosum (Koch, 1867), Amblyomma nocens Robinson, 1912, Amblyomma perpunctatum (Packard, 1869), Amblyomma striatum Koch, 1844, Amblyomma superbum Santos Dias, 1953, Amblyomma testudinis (Conil, 1877), Amblyomma trinitatis Turk, 1948, Dermacentor confractus (Schulze 1933), Dermacentor daghestanicus Olenev, 1928, Haemaphysalis himalaya Hoogstraal, 1966, Haemaphysalis vietnamensis Hoogstraal and Wilson, 1966, Hyalomma detritum Schulze, 1919, Ixodes apteridis Maskell, 1897, Ixodes donarthuri Santos Dias, 1980, Ixodes kempi Nuttall, 1913, Ixodes neotomae Cooley, 1944, Ixodes rangtangensis Teng, 1973, Ixodes robertsi Camicas, Hervy, Adam and Morel, 1998, Ixodes serrafreirei Amorim, Gazetta, Bossi and Linhares, 2003, Ixodes tertiarius Scudder, 1885, Ixodes uruguayensis Kohls and Clifford, 1967, Ixodes zealandicus Dumbleton, 1961, Ixodes zumpti Arthur, 1960 and Rhipicephalus camelopardalis Walker and Wiley, 1959. We consider the following 40 names valid: Argas delicatus Neumann, 1910, Argas vulgaris Filippova, 1961, Ornithodoros aragaoi Fonseca, 1960, Ornithodoros dugesi Mazzoti, 1943, Ornithodoros knoxjonesi Jones and Clifford, 1972, Ornithodoros marocanus Velu, 1919, Ornithodoros nattereri Warburton, 1927, Amblyomma beaurepairei Vogelsang and Santos Dias, 1953, Amblyomma crassipes (Neumann, 1901), Amblyomma echidnae Roberts, 1953, Amblyomma fuscum Neumann, 1907, Amblyomma orlovi (Kolonin, 1995), Amblyomma parkeri Fonseca and Arag?o, 1952, Amblyomma pseudoconcolor Arag?o, 1908, Bothriocroton oudemansi (Neumann, 1910), Bothriocroton tachyglossi (Roberts, 1953), Dermacentor abaensis Teng, 1963, Dermacentor confragus (Schulze 1933), Dermacentor ushakovae Filippova and Panova, 1987, Haemaphysalis anomaloceraea Teng, 1984, Haemaphysalis filippovae Bolotin, 1979, Haemaphysalis pavlovskyi Pospelova-Shtrom, 1935, Hyalomma excavatum Koch, 1844, Hyalomma isaaci Sharif, 1928, Hyalomma rufipes Koch, 1844, Hyalomma turanicum Pomerantzev, 1946, Ixodes arabukiensis Arthur, 1959, Ixodes boliviensis Neumann, 1904, Ixodes columnae Takada and Fujita, 1992, Ixodes maslovi Emel′yanova and Kozlovskaya, 1967, Ixodes sachalinensis Filippova, 1971, Ixodes siamensis Kitaoka and Suzuki, 1983, Ixodes sigelos Keirans, Clifford and Corwin, 1976, Ixodes succineus Weidner, 1964, Rhipicephalus aurantiacus Neumann, 1907, Rhipicephalus cliffordi Morel, 1965, Rhipicephalus pilans Schulze, 1935, Rhipicephalus pseudolongus Santos Dias, 1953, Rhipicephalus serranoi Santos Dias, 1950 and Rhipicephalus tetracornus Kitaoka and Suzuki, 1983.     

Swimming response of goldfish,Carassius auratus,and the tetra,Hemigrammus caudovittatus,larvae to individual food items and patches

Synopsis Swimming speed and swimming path of goldfish and tetra larvae were studied in aquaria containing food patches composed of decapsulated cysts and immobilized nauplii of Artemia salina or sparsely distributed prey. The mean swimming speed of starved larvae in the medium without food was about four times higher than the speed of larvae feeding in a patch. Satiated larvae swam about 1.5 times slower than hungry fish. Consumption of single prey items by starved larvae caused the following sequence of swimming responses: handling pause (cessation of swimming), slow swimming in a restricted area, and fast swimming (approximately twice as fast as hungry larvae before encountering food) accompanied by a widening of the area searched (area increased searching). Mean swimming speed was constant over a broad range (10¹–10³ ind·1^–1 of food density, although at extreme (high or low) values of food density it depended on swimming responses of the predator. Frequency of visits to the different parts of the aquarium strongly depended on encounters of hungry fish with food particles or patches.     

Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta

X-ray analysis of enzyme–DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9–10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (ΔG° ~ −8.7 to −9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1–DNA interactions provide only one order of magnitude (ΔG° ~ −1.1 to −1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.     

A gradient of silent substitution rate in the human pseudoautosomal region

It has been demonstrated that recombination in the human p-arm pseudoautosomal region (p-PAR) is at least twenty times more frequent than the genomic average of approximately 1 cM/Mb, which may affect substitution patterns and rates in this region. Here I report the analysis of substitution patterns and rates in 10 human, chimpanzee, gorilla, and orangutan genes across the p-PAR. Between species silent divergence in the p-PAR forms a gradient, increasing toward the telomere. The correlation of silent divergence with distance from the p-PAR boundary is highly significant (rho = 0.911, P < 0.001). After exclusion of the CpG dinucleotides this correlation is still significant (rho = 0.89, P < 0.01), thus the substitution rate gradient cannot be explained solely by the differences in the extent of methylation across the p-PAR. Frequent recombination in the PAR may result in a relatively strong effect of biased gene conversion (BGC), which, because of the increased probability of fixation of the G or C nucleotides at (A or T)/(G or C) segregating sites, may affect substitution rates. BGC, however, does not seem to be the factor creating the substitution rate gradient in the p-PAR, because the only gradient is still detactable if only A<-->T and G<-->C substitutions are taken into account (rho = 0.82, P < 0.01). I hypothesize that the substitution rate gradient in the p-PAR is due to the mutagenic effect of recombination, which is very frequent in the distal human p-PAR and might be lower near the p-PAR boundary.     

NAADP triggers the fertilization potential in starfish oocytes

In invertebrates oocytes or eggs, the fertilization or activation potential establishes the fast electrical block to polyspermy and, in some species, provides the Ca2+ influx which contributes to the following intracellular Ca2+ wave. In echinoderms, the molecule triggering the activation potential is still unknown. The aim of this study was to assess whether nicotinic acid-adenine dinucleotide phosphate (NAADP) elicited the fertilization potential in starfish oocytes. The changes in membrane potential induced by the sperm were measured in oocytes held at a low resting potential, so that the Ca2+-action potential was inactivated and only the initial slower depolarization caused by the sperm could be studied. Decreasing extracellular Na+ concentration did not prevent the onset of the fertilization potential, while removal of external Ca2+ abolished it. The pre-incubation with SK&F 96365 and verapamil and the pre-injection of BAPTA inhibited the fertilization potential, while the injection of heparin only reduced its duration. The biophysical and pharmacological properties of the sperm-elicited depolarization were similar to those displayed by the NAADP-activated Ca2+-mediated current recently described in starfish oocytes. Indeed, the desensitization of NAADP-receptors prevented the onset of the fertilization potential. Taken together, these data suggest that NAADP could trigger the fertilization potential in starfish oocytes.     

New fatty acid oxidation inhibitors with increased potency lacking adverse metabolic and electrophysiological properties

New inhibitors of palmitoylCoA oxidation were synthesized based on a structurally novel lead, CVT-3501 (1). Investigation of structure-activity relationships was conducted with respect to potency of inhibition of cardiac mitochondrial palmitoylCoA oxidation and metabolic stability. Potent and metabolically stable analogues 33, 42, and 43 were evaluated in vitro for cytochrome P450 inhibition and potentially adverse electrophysiological effects. Compound 33 was also found to have favorable pharmacokinetic properties in rat.     

CVT-4325: a potent fatty acid oxidation inhibitor with favorable oral bioavailability

New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the ‘Western Portion’ of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC₅₀ = 380 nM rat mitochondria) with favorable PK properties (F = 93%, t_1/2 = 13.6 h, dog).     

High resolution mass spectrometric alveolar proteomics: identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micropreparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products. The high resolution mass spectrometric proteome analysis should facilitate the unequivocal identification of subunits, aggregations, modifications and degradation products of surfactant proteins and hence contribute to the understanding of the mechanistic basis of lung disease pathogenesis.     

Transgenic expression of Cre recombinase from the tyrosine hydroxylase locus

Catecholaminergic neurons are affected in several neurological and psychiatric diseases. Tyrosine hydroxylase (TH) is the first, rate-limiting enzyme in catecholamine synthesis. We report a knockin mouse expressing Cre-recombinase from the 3'-untranslated region of the endogenous Th gene by means of an internal ribosomal entry sequence (IRES). The resulting Cre expression matches the normal pattern of TH expression, while the pattern and level of TH are not altered in the knockin mouse. Crossings with two different LacZ reporter mice demonstrated Cre-mediated genomic recombination in TH expressing tissues. In addition, LacZ was found in some unexpected cell populations (including oocytes), indicating recombination due to transient developmental TH expression. Our novel knockin mouse can be used for generation of tissue-specific or general knockouts (depending on scheme of crossing) in mice carrying genes flanked by loxP sites. This knockin mouse can also be used for tracing cell lineages expressing TH during development.