EARLY STARVATION1 specifically affects the phosphorylation action of starch‐related dikinases

Starch phosphorylation by starch‐related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50‐kDa starch‐binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α‐glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.     

Nutrient Leaching and End Product Accumulation in Plastic Composite Supports for L-(+)-Lactic Acid Biofilm Fermentation

Investigations on the leachate bioavailability, leaching rate, and lactic acid accumulation properties of plastic composite supports (PCS) were essential for large-scale or long-term lactic acid fermentation. Leachates from PCS and polypropylene discs (controls) were analyzed by the micro-Kjeldahl method; by absorbances at 260, 275, and 280 nm; and by bioassays with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The amount of leached nitrogen in a 20-ml initial soaking solution had a high correlation with the soaking solution's cell density (r = 0.87) and absorbance at 260 nm (r = 0.95). Leaching rates of various PCS were evaluated by 20 20-ml simulated repeated-batch fermentations (RBF). PCS with only yeast extract as the minor agricultural ingredient had a high leaching rate and leached out 51 to 60% of the total nitrogen during the first RBF. PCS blended with dried bovine albumin, dried bovine erythrocytes, and/or soybean flour had slowed nutrient leaching (20 to 30% of the initial leached nitrogen). Hence, they could still maintain 1 g of lactic acid per liter and measurable cell density (absorbance at 620 nm, 0.4 to 0.6) at the 20th 20-ml RBF. Lactic acid accumulation properties of PCS were evaluated by soaking the supports in a 30% lactic acid solution for 72 h at 45(deg)C. The lactic acid-soaked supports were rinsed three times and then heat treated (121(deg)C, 15 min) in 15 ml of deionized water. The results showed that lactic acid accumulation in PCS was mainly due to absorption and had no correlation with lactic acid production or biofilm formation.     

Characterization of Plant Alcohol Dehydrogenase

The final activity of the alcohol dehydrogenase (E.C.1.1.1.1, abbreviated ADH) from germinating pea, isolated by fractionating with ammonium sulphate, chromatography on DEAE cellulose and gel filtration, was 80,000, from bean 25,000 and from lentil 13,500 units per mg protein. Molecular weights of the ADHs are close to each other: pea and bean ADH 60,000, lentil ADH 70,000. The K_m values are mutually similar with three enzymes, i.e. of the order of 10⁻⁴M for NAD and 10⁻²M for ethanol. The pH optima lie in the alkaline region. These enzymes catalyse oxidation of a number of monovalent alcohols. At temperatures above 60°C the enzymes are thermally unstable. Stability is enhanced slowly by ethanol but not by NAD. Pyrazol, imidazol and pyridine inhibit plant ADH similarly to the enzyme from horse liver. There is a similarity between plant alcohol dehydrogenases and animal and yeast enzymes.     

Improvement of llama (Lama glama) seminal characteristics using collagenase

Llama semen is characterized by great structural viscosity and minimal sperm progressive motility. These characteristics, inherent to South American Camelid ejaculates, have slowed down the development of assisted reproductive techniques in these species. The aim of the present research was to evaluate the effect of different combinations of dilutions and incubation time with H-TALP-BSA medium, with and without adding 0.1% collagenase, on the qualitative and quantitative semen characteristics, for its use in assisted fertility techniques. Ejaculates (n = 8; r = 3) were obtained using electroejaculation. Each ejaculate was evaluated and then split into four aliquots. Two of these were diluted 4:1 and 8:1 in 0.1% collagenase in H-TALP-BSA (treatments 1 and 3) and the other two 4:1 and 8:1 in H-TALP-BSA without collagenase (treatments 2 and 4). Treatments 1 and 2 were incubated 4 min at 37 °C while treatments 3 and 4 were incubated 8 min. All aliquots were centrifuged at 800 × g for 4 min immediately after incubation. Supernatants were pipetted to observe thread formation and pellets were re-diluted in H-TALP-BSA. Supernatants from samples treated with collagenase did not form a thread when pipetted, while the ones from samples that were not treated with the enzyme did. Only semen samples treated with collagenase showed progressive sperm motility, with averages over 40%. There were no significant differences (P > 0.05) for the percentage of live spermatozoa and for the percentage of detached heads between raw and treated semen samples. Percentages of spermatozoa with functional membranes were significantly higher (P ≤ 0.05) in samples treated with collagenase than in raw semen and in samples incubated without collagenase. These results show that treating semen with 0.1% collagenase in H-TALP-BSA improves semen rheological properties while facilitates the separation of spermatozoa from seminal plasma in llama; it also promotes sperm progressive motility, while maintaining sperm membrane functionality and integrity. Consequently, this protocol could be used for in vitro llama embryo production with ejaculated spermatozoa.     

A new cardiid bivalve from the Pliocene Baklan Basin (Turkey) and the origin of modern Ponto‐Caspian taxa

Abstract: We present the first record of the cardiid genus Monodacna from the Pliocene of Anatolia, Turkey. Monodacna imrei sp. nov. was found in the Pliocene Killik Formation from the western margin of the Baklan Basin, in very marginal brackish to freshwater lacustrine deposits. The new record extends the stratigraphic range of the modern Ponto‐Caspian genus back into the Pliocene and adds to earlier evidence that modern Ponto‐Caspian taxa originated in the Pliocene of south‐western Turkey.     

Climatic niche and neutral genetic diversity of the six Iberian pine species: a retrospective and prospective view

Quaternary climatic fluctuations have left contrasting historical footprints on the neutral genetic diversity patterns of existing populations of different tree species. We should expect the demography, and consequently the neutral genetic structure, of taxa less tolerant to particular climatic extremes to be more sensitive to long‐term climate fluctuations. We explore this hypothesis here by sampling all six pine species found in the Iberian Peninsula (2464 individuals, 105 populations), using a common set of chloroplast microsatellite markers, and by looking at the association between neutral genetic diversity and species‐specific climatic requirements. We found large variation in neutral genetic diversity and structure among Iberian pines, with cold‐enduring mountain species (Pinus uncinata, P. sylvestris and P. nigra) showing substantially greater diversity than thermophilous taxa (P. pinea and P. halepensis). Within species, we observed a significant positive correlation between population genetic diversity and summer precipitation for some of the mountain pines. The observed pattern is consistent with the hypotheses that: (i) more thermophilous species have been subjected to stronger demographic fluctuations in the past, as a consequence of their maladaptation to recurrent glacial cold stages; and (ii) altitudinal migrations have allowed the maintenance of large effective population sizes and genetic variation in cold‐tolerant species, especially in more humid regions. In the light of these results and hypotheses, we discuss some potential genetic consequences of impending climate change.