A beta-helical antifreeze protein isoform with increased activity. Structural and functional insights

The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular beta-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Delta-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its beta-helical structure and hence the size of its ice-binding face.     

Overexpression of Bcl-W in the testis disrupts spermatogenesis: revelation of a role of BCL-W in male germ cell cycle control

To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken beta-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5'-bromo-2'deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.     

Trophic interactions and abiotic factors drive functional and phylogenetic structure of vertebrate herbivore communities across the Arctic tundra biome

Communities are assembled from species that evolve or colonise a given geographic region, and persist in the face of abiotic conditions and interactions with other species. The evolutionary and colonisation histories of communities are characterised by phylogenetic diversity, while functional diversity is indicative of abiotic and biotic conditions. The relationship between functional and phylogenetic diversity infers whether species functional traits are divergent (differing between related species) or convergent (similar among distantly related species). Biotic interactions and abiotic conditions are known to influence macroecological patterns in species richness, but how functional and phylogenetic diversity of guilds vary with biotic factors, and the relative importance of biotic drivers in relation to geographic and abiotic drivers is unknown. In this study, we test whether geographic, abiotic or biotic factors drive biome‐scale spatial patterns of functional and phylogenetic diversity and functional convergence in vertebrate herbivores across the Arctic tundra biome. We found that functional and phylogenetic diversity both peaked in the western North American Arctic, and that spatial patterns in both were best predicted by trophic interactions, namely vegetation productivity and predator diversity, as well as climatic severity. Our results show that both bottom–up and top–down trophic interactions, as well as winter temperatures, drive the functional and phylogenetic structure of Arctic vertebrate herbivore assemblages. This has implications for changing Arctic ecosystems; under future warming and northward movement of predators potential increases in phylogenetic and functional diversity in vertebrate herbivores may occur. Our study thus demonstrates that trophic interactions can determine large‐scale functional and phylogenetic diversity just as strongly as abiotic conditions.     

Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background

Leber hereditary optic neuropathy (LHON) is due primarily to one of three common point mutations of mitochondrial DNA (mtDNA), but the incomplete penetrance implicates additional genetic or environmental factors in the pathophysiology of the disorder. Both the 11778G-->A and 14484T-->C LHON mutations are preferentially found on a specific mtDNA genetic background, but 3460G-->A is not. However, there is no clear evidence that any background influences clinical penetrance in any of these mutations. By studying 3,613 subjects from 159 LHON-affected pedigrees, we show that the risk of visual failure is greater when the 11778G-->A or 14484T-->C mutations are present in specific subgroups of haplogroup J (J2 for 11778G-->A and J1 for 14484T-->C) and when the 3460G-->A mutation is present in haplogroup K. By contrast, the risk of visual failure is significantly less when 11778G-->A occurs in haplogroup H. Substitutions on MTCYB provide an explanation for these findings, which demonstrate that common genetic variants have a marked effect on the expression of an ostensibly monogenic mtDNA disorder.     

Geographical distribution of Allium oleraceum cytotypes in Finland and Sweden

Chromosome numbers from a total number of 226 populations of Allium oleraceum were determined in Finland, Sweden and seven other countries. Two different chromosome numbers or cytotypes were found, tetraploids (2n = 32) and pentaploids (2n = 40). In Finland, samples were collected for chromosome counts from a total of 190 populations, which fairly well covers the distribution area of the species in Finland. The majority of the populations of A. oleraceum in Finland contained only one of the two cytotypes. A clear pattern in the geographical distribution of the cytotypes could be distinguished. The pentaploid cytotype predominates in the Åland Islands and in the archipelago of Regio aboënsis. The tetraploid cytotype predominates on the mainland of the Regio aboënsis and along the coast of the Nylandia. In south Häme, all studied native populations were of the tetraploid cytotype. Of all the studied populations in Finland 43.7% were tetraploid and 51.6% pentaploid. A few mixed populations with both tetraploid and pentaploid plants occurring in the same population were also found (4.7%). The chromosome numbers of 19 populations of A. oleraceum from the southern part of Sweden were pentaploids, with the exception of one tetraploid population. It seems that the pentaploid cytotype is predominant in Sweden, but no conclusions about a geographical pattern between the two cytotypes could be drawn. Based on the occurrence of the cytotypes, it is suggested that the pentaploid cytotype might have spread to Finland from the Swedish east coast via the Åland Islands to Kaland on the Finnish west coast and along the coast of Nyland to the archipelagos of Kotka and Hamina in Karelia australis. The tetraploid populations in south Häme are clearly connected to Iron Age activity and to old inland trade routes, and may be of eastern origin.     

Development of the CELLOP optimisation model for plant cell cultivation

A mathematical computer-aided model CELLOP was constructed in which the desirability functions in a three-dimensional experimental design can be used to find the optimal growing conditions for plant cells. CELLOP is intended for the optimisation of 3 to 6 physical, chemical, or biological variables in the cultivation conditions of plant cell cultures. The model was used to optimise the culturing conditions (calcium, inorganic nitrogen, and sucrose concentrations) for coumarin-producing, spontaneously embryogenic cell lines of angelica Angelica archangelica L. subsp. archangelica and hogs fennel Peucedanum palustre (L.) Moench. For A. archangelica the overall optimum concentrations were 0.47 mM Ca²⁺, 5.06 mM NO₃ ⁻, 0.40 mM NH₄ ⁺, and 96.25 mM sucrose. The dry mass was 24.7 % higher and the coumarin content 40.5 % higher than those achieved in the standard 75 % Gamborg B5 medium. For A. archangelica the highest embryogenic activity was reached in the media containing 1.25 mM Ca²⁺. In the case of P. palustre the overall optimum concentrations were 1.60 mM Ca²⁺, 2.84 mM NO³ ₋, 0.23 mM NH₄ ⁺, and 85.10 mM sucrose. For P. palustre the dry mass production increased by 61.8 % and the coumarin content by 58.1 % compared to the values achieved in the Gamborg B5 medium. For P. palustre the highest embryogenic activity was reached in the presence of 50.0 mM NO₃ ⁻ and 4.01 mM NH₄ ⁺.     

A possible active segment on the inactive human X chromosome

An idic(Xp-) in which the two X chromosomes are attached short arm to short arm, and which thus has two b regions (the Q-dark segment next to the centromere on Xp) between the inactivation centers, assumed to be situated on the Q-dark region next to the centromere on Xq, showed 63.8% bipartite Barr bodies as compared with 22.2% formed by idic(Xq-). In addition, the mean distance of the two parts of the Barr bodies in the fibroblasts of a patient with idic(Xp-) is significantly greater than in the cases with one or no b region. Contrary to the other patients with abnormal X chromosomes, the buccal cells of a woman idic(Xp-) showed a number of bipartite Barr bodies. — To explain these observations we have put forward the hypothesis that the b region on the Xp always remains active and thus, when the rest of the chromosome forms a Barr body, this segment is extended, allowing the two parts of the X chromatin to get farther apart and at the same time increasing the percentage of bipartite bodies.     

Jasmonates induce over-accumulation of methylputrescine and conjugated polyamines in Hyoscyamus muticus L. root cultures

 Jasmonic acid (JA) and its methyl ester (MeJA) at concentrations ranging from 0.001 to 10 μM provoked large increases in methylputrescine levels in normal and hairy roots of Hyoscyamus muticus L.; generally, levels of free putrescine and perchloric acid-soluble conjugated putrescine, spermidine and spermine also increased dramatically. More ¹⁴C-putrescine was formed when hairy roots were incubated with labelled ornithine than with arginine; conjugated ¹⁴C-putrescine was also rapidly formed. In accord with these results, ornithine decarboxylase (EC 4.1.1.17) activity was higher than that of arginine decarboxylase (EC 4.1.1.19), and MeJA enhanced these activities about two- and fourfold, respectively. Although treatment of root cultures with jasmonates enhanced precursor (putrescine, methylputrescine) levels and accumulation of secondary metabolites such as acid-soluble conjugated di-/polyamines, it provoked only modest increases in tropane alkaloid tissue levels. Received: 24 March 1999 / Revision received: 5 October 1999 / Accepted: 26 October 1999