In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and β, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol β inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and β are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and β.     

Induced resistance in mountain birch: defence against leaf-chewing insect guild and herbivore competition

Summary The effect of leaf damage simulating the feeding of early season insect herbivore species, e.g. Epirrita autumnata, to mountain birch, Betula pubescens ssp. tortuosa, on the performance of insect larvae was studied with eleven leaf-chewing sawfly species. I found variation in the results that was due to short- and long-term inducible responses and to the phenology of herbivore species. In general, early and mid-season species were more strongly affected by induced reactions than late-season species. This finding is in accordance with earlier results but I could show that the persistance of induced reactions rather than the influence of timing of damage is responsible for the result. The growth of the larvae of mid-season sawfly species was affected by both short- and long-term induced reactions. This result shows that early season species may escape short-term induced reactions of mountain birch in current year but may not avoid long-term effects. It is supposed that seasonal deterioration of leaf quality either masks the effects of induced defences or late-season species are better adapted to low-quality leaves. Some species show variation in their response to induced defence in different years. This may be due to yearly differences in induced reactions as well as to species-specific responses. Induced defence reactions may play a role in competitive interactions between herbivore species in leaf-chewing guild of mountain birch.     

Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer

Wnt signaling regulates embryonic pattern formation and morphogenesis of most organs. Aberrations of regulation of Wnt signaling may lead to cancer. Here, we have used positional cloning to identify the causative mutation in a Finnish family in which severe permanent tooth agenesis (oligodontia) and colorectal neoplasia segregate with dominant inheritance. Eleven members of the family lacked at least eight permanent teeth, two of whom developed only three permanent teeth. Colorectal cancer or precancerous lesions of variable types were found in eight of the patients with oligodontia. We show that oligodontia and predisposition to cancer are caused by a nonsense mutation, Arg656Stop, in the Wnt-signaling regulator AXIN2. In addition, we identified a de novo frameshift mutation 1994-1995insG in AXIN2 in an unrelated young patient with severe tooth agenesis. Both mutations are expected to activate Wnt signaling. The results provide the first evidence of the importance of Wnt signaling for the development of dentition in humans and suggest that an intricate control of Wnt-signal activity is necessary for normal tooth development, since both inhibition and stimulation of Wnt signaling may lead to tooth agenesis. Our findings introduce a new gene for hereditary colorectal cancer and suggest that tooth agenesis may be an indicator of cancer susceptibility.     

Performance of the cyclic autumnal moth,<Emphasis Type="Italic"> Epirrita autumnata</Emphasis>, in relation to birch mast seeding

The mast depression hypothesis has been put forward to explain the 9- to 10-year population cycle of the autumnal moth (Epirrita autumnata; Lepidoptera: Geometridae) in northern Fennoscandia. We analysed long-term data from Finnish Lapland in order to evaluate the critical assumption of the mast depression hypothesis: that better individual performance of herbivores, followed by high annual growth rate of populations, occurs in the year following mast seeding of the host, the mountain birch ( Betula pubescens ssp. czerepanovii). Since mast seeding has been suggested to occur at the expense of chemical defence against herbivores, we bioassayed the quality of birch leaves from the same trees by means of yearly growth trials with autumnal moth larvae. We also measured the size of wild adults as a determinant of potential fecundity of the species in different years. The relative growth rate of larvae was poorer in post-mast years compared to other years, rather than better as assumed by the hypothesis. Conversely, a slight indication of the increase in potential fecundity was observed due to the somewhat larger adult size in post-mast years. Population growth rate estimates, however, showed that the increase in fecundity would have to be much higher to facilitate population increase towards a cyclic peak with outbreak density. Accordingly, our two data sets do not support the assumption of a higher annual growth rate in autumnal moth populations subsequent to mast seeding of the host, thereby contradicting the predictions of the mast depression hypothesis. Temperatures, when indexed by the North Atlantic Oscillation and accumulated thermal sums, were observed to correlate with the abundance or rate of population change of the autumnal moth. The factors underlying the regular population cycles of the autumnal moth, however, remain unidentified. Overall, we suggest that the causal agents in cyclic insect population dynamics should be clarified by field experimentation, since trophic interactions are complex and are further modified by abiotic factors such as climate.     

The role of microtubules in the regulation of proteoglycan synthesis in chondrocytes under hydrostatic pressure

Chondrocytes of the articular cartilage sense mechanical factors associated with joint loading, such as hydrostatic pressure, and maintain the homeostasis of the extracellular matrix by regulating the metabolism of proteoglycans (PGs) and collagens. Intermittent hydrostatic pressure stimulates, while continuous high hydrostatic pressure inhibits, the biosynthesis of PGs. High continuous hydrostatic pressure also changes the structure of cytoskeleton and Golgi complex in cultured chondrocytes. Using microtubule (MT)-affecting drugs nocodazole and taxol as tools we examined whether MTs are involved in the regulation of PG synthesis in pressurized primary chondrocyte monolayer cultures. Disruption of the microtubular array by nocodazole inhibited [(35)S]sulfate incorporation by 39-48%, while MT stabilization by taxol caused maximally a 17% inhibition. Continuous hydrostatic pressure further decreased the synthesis by 34-42% in nocodazole-treated cultures. This suggests that high pressure exerts its inhibitory effect through mechanisms independent of MTs. On the other hand, nocodazole and taxol both prevented the stimulation of PG synthesis by cyclic 0. 5 Hz, 5 MPa hydrostatic pressure. The drugs did not affect the structural and functional properties of the PGs, and none of the treatments significantly affected cell viability, as indicated by the high level of PG synthesis 24-48 h after the release of drugs and/or high hydrostatic pressure. Our data on two-dimensional chondrocyte cultures indicate that inhibition of PG synthesis by continuous high hydrostatic pressure does not interfere with the MT-dependent vesicle traffic, while the stimulation of synthesis by cyclic pressure does not occur if the dynamic nature of MTs is disturbed by nocodazole. Similar phenomena may operate in cartilage matrix embedded chondrocytes.     

Occurrence of N-acetylglucosamine 6-phosphate in complex carbohydrates. Characterization of a phosphorylated sialyl oligosaccharide from bovine colostrum

The occurrence of phosphate-containing sialyl oligosaccharides in bovine colostrum was investigated. Two major sialyl oligosaccharide phosphates were identified, one of which was structurally similar to the previously characterized sialyl oligosaccharide 1-phosphates of human urine. The second sialyl oligosaccharide phosphate of bovine colostrum was found to be of a novel type. Structural studies including monosaccharide and phosphate analysis, glycosidase and phosphatase treatments, methylation analysis, and periodate treatment indicated the structure of this compound to be NeuAc alpha 2-6Gal beta 1-4GlcNAc-6-P. This provides the first evidence for the occurrence of N-acetylglucosamine 6-phosphate as an integral component in complex carbohydrates.     

Candidate Gene Analysis of Tooth Agenesis Identifies Novel Mutations in Six Genes and Suggests Significant Role for WNT and EDA Signaling and Allele Combinations

Failure to develop complete dentition, tooth agenesis, is a common developmental anomaly manifested most often as isolated but also as associated with many developmental syndromes. It typically affects third molars or one or few other permanent teeth but severe agenesis is also relatively prevalent. Here we report mutational analyses of seven candidate genes in a cohort of 127 probands with non-syndromic tooth agenesis. 82 lacked more than five permanent teeth excluding third molars, called as oligodontia. We identified 28 mutations, 17 of which were novel. Together with our previous reports, we have identified two mutations in MSX1, AXIN2 and EDARADD, five in PAX9, four in EDA and EDAR, and nine in WNT10A. They were observed in 58 probands (44%), with a mean number of missing teeth of 11.7 (range 4 to 34). Almost all of these probands had severe agenesis. Only few of the probands but several relatives with heterozygous genotypes of WNT10A or EDAR conformed to the common type of non-syndromic tooth agenesis, incisor-premolar hypodontia. Mutations in MSX1 and PAX9 affected predominantly posterior teeth, whereas both deciduous and permanent incisors were especially sensitive to mutations in EDA and EDAR. Many mutations in EDAR, EDARADD and WNT10A were present in several families. Biallelic or heterozygous genotypes of WNT10A were observed in 32 and hemizygous or heterozygous genotypes of EDA, EDAR or EDARADD in 22 probands. An EDARADD variant were in seven probands present together with variants in EDAR or WNT10A, suggesting combined phenotypic effects of alleles in distinct genes.     

Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding

Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single-module, cellulose-degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello-oligomers: cellobiose (Glc₂), cellotriose (Glc₃), and cellotetraose (Glc₄), at high resolution (1.6–2.1 Å). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β-sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from −5 to +2. In four cellotriose and one cellotetraose complex structures, the cello-oligosaccharide also spanned over the cleavage site (−1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello-oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (⁴C₁) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.     

A comparison of unheated loose housing with stables on the respiratory health of weaned-foals in cold winter conditions: an observational field-study

Background

Newly weaned horses in Finland are often moved to unheated loose housing systems in which the weanlings have free access to a paddock and a shelter. This practice is considered to be good for the development of young horses. The daily temperatures can stay below ? 20 °C in Finland for several consecutive weeks during the winter season. However, the effect of unheated housing in a cold climatic environment on the respiratory health of weanlings under field conditions has not been studied before. This investigation was an observational field-study comprising 60 weanlings among 11 different voluntary participant rearing farms in Finland. Weanlings were either kept in unheated loose housing systems (n = 36) or in stables (n = 24) and were clinically examined on two separate occasions 58 days apart in cold winter conditions.

Results

The odds of clinical respiratory disease were lower in the older foals (log_e days); OR = 0.009, P = 0.044). The plasma fibrinogen concentration was higher when the available space (m²/weanling) in the sleeping hall was smaller (P = 0.014) and it was lower when the sleeping hall was not insulated (P = 0.010). The plasma fibrinogen concentrations at the second examination were lower with a body condition score above 3 (P = 0.070). Standardbreds kept in loose housing systems had a lower body condition score than Finnhorses or Standardbreds kept in stables at both examinations (P = 0.026 and P = 0.007, respectively). Haemoglobin level was lower in weanlings in loose housing systems compared to their counterparts at the first examination (P = 0.037). Finnhorses had higher white blood cell count than Standardbreds at first (P = 0.002) and at the second examination (P = 0.001).

Conclusions

Keeping weanling horses in cold loose housing systems does not seem to increase the occurrence of respiratory disease, but special attention should be focused on ventilation, air quality and feeding-practices. Our field study data suggest it might be advantageous to keep Standardbred foals born late in the season in a stable over the Finnish winter.