Cortical granule matrix disassembly during exocytosis in sea urchin eggs.

Cortical granule exocytosis in sea urchins was studied using hyperosmotic and polymer-containing seawater to halt granule matrix dispersal. Addition of Na2SO4-containing seawater (2.5 osmole/kg) to Strongylocentrotus purpuratus eggs 10 to 40 sec after insemination resulted in arrest of the exocytic wave during propagation. EM examination of these eggs revealed that matrix disassembly occurred in distinct stages. In the earliest stage, granule-plasma membrane fusion had occurred, but the matrix remained completely intact. This early stage was observed in hyperosmotic media, either ionic or nonionic, suggesting that matrix hydration is required for disassembly and exocytic pore widening, but not for membrane fusion. Subsequent stages, in which partially disassembled matrices remained within omega-configured pockets, were captured by activating eggs in 30% dextran in seawater. Stability of these intermediates stages required the presence of Ca2+ and Mg2+; in the absence of divalent cations the matrices completely disassembled and the exocytic pockets flattened. Divalent cations appeared to prevent fragmentation of the matrix lamellae. Late stages of matrix disassembly, in which the lamellae fragmented and formed small particles, were inhibited by media of high ionic strength. Hyperosmolality alone, provided by sucrose, was unable to halt these late stages suggesting that water availability does not play an important role once a critical point in matrix dispersal has been reached.     

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata)

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240- bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.      

Characterisation of a novel gene family of putative cyclic nucleotide- and calmodulin-regulated ion channels in Arabidopsis thaliana

In plants, cyclic GMP is involved in signal transduction in response to light and gibberellic acid. For cyclic AMP, a potential role during the plant cell cycle was recently reported. However, cellular targets for cyclic nucleotides in plants are largely unknown. Here we report on the identification and characterisation of a new gene family in Arabidopsis, which share features with cyclic nucleotide-gated channels from animals and inward-rectifying K⁺ channels from plants. The identified gene family comprises six members (Arabidopsis thaliana cyclic nucleotide-gated channels, AtCNGC1–6) with significant homology among the deduced proteins. Hydrophobicity analysis predicted six membrane-spanning domains flanked by hydrophilic amino and carboxy termini. A putative cyclic nucleotide binding domain (CNBD) which contains several residues that are invariant in other CNBDs was located in the carboxy terminus. This domain overlaps with a predicted calmodulin (CaM) binding site, suggesting interaction between cyclic nucleotide and CaM regulation. We demonstrated interaction of the carboxy termini of AtCNGC1 and AtCNGC2 with CaM in yeast, indicating that the CaM binding sites are functional. Furthermore, it was shown that both AtCNGC1 and AtCNGC2 can partly complement the K⁺-uptake-deficient yeast mutant CY162. Therefore, we propose that the identified genes constitute a family of plant cyclic nucleotide- and CaM-regulated ion channels.     

Optimization of pH,Temperature and Inoculum Ratio for the Production of β‐D‐Galactosidase by Kluyveromyces marxianus Using a Lactose‐free Medium

The pH, temperature and inoculum ratio for the production of β‐galactosidase by Kluyveromyces marxianus CDB 002 were optimized using sugar‐cane molasses (100 g/l) in a lactose‐free medium. The temperature optimum was evaluated in the range from 28–37 °C. Lactase production was initiated after substrate consumption indicating a reversible enzyme inhibition or catabolic repression. The specific enzyme activity after 45 h was between 456.3 U/g cell mass (37 °C) and 733.3 U/g (34 °C), whereas the highest volumetric activity was obtained at 30 °C: 21.8 U/ml. This is generally consistent with results from other authors that used whey as a carbon source. Ethanol as a by‐product reached its maximum concentration after 10–14 h (31.1–40.5 g/l), but was completely consumed afterwards. A pH of 5.5 without further control gave the best production rate for lactase (484.4 U/l × h). In this process, the pH was stable during cell growth at 5.5 and then went up to pH 7.2 after 45 h. At a fixed pH of 5.5 or 6.5, the production rates achieved 313.3 U/l × h and 233.3 U/ l × h, respectively. These results differed from those of other authors, who suggested a fixed pH at 7.0 using whey as a carbon source. There were no significant differences between inoculum ratios of 1% [v/v] and 10% [v/v] so that 1% is the preferable ratio as it is cheaper. Yeast extract (10 g/l) and peptone (20 g/l) were used as the vitamin and nitrogen source, respectively, for the studies of temperature and pH. These were substituted by corn steep liquor (100 g/l) for inoculum ratio experiments. Production of lactase using sugar cane molasses in a lactose‐free medium gave better enzyme productivity rates than obtained by other authors using whey. The optimum conditions for β‐galactosidase synthesis were a temperature of 30–34 °C and an inoculum ratio of 1% [v/v], an initial pH of 5.5 without any further control or a control of 5.5 during cell growth. Then the pH was raised up to 7.     

Energy benefits and emergent space use patterns of an empirically parameterized model of memory‐based patch selection

Many species frequently return to previously visited foraging sites. This bias towards familiar areas suggests that remembering information from past experience is beneficial. Such a memory‐based foraging strategy has also been hypothesized to give rise to restricted space use (i.e. a home range). Nonetheless, the benefits of empirically derived memory‐based foraging tactics and the extent to which they give rise to restricted space use patterns are still relatively unknown. Using a combination of stochastic agent‐based simulations and deterministic integro‐difference equations, we developed an adaptive link (based on energy gains as a foraging currency) between memory‐based patch selection and its resulting spatial distribution. We used a memory‐based foraging model developed and parameterized with patch selection data of free‐ranging bison Bison bison in Prince Albert National Park, Canada. Relative to random use of food patches, simulated foragers using both spatial and attribute memory are more efficient, particularly in landscapes with clumped resources. However, a certain amount of random patch use is necessary to avoid frequent returns to relatively poor‐quality patches, or avoid being caught in a relatively poor quality area of the landscape. Notably, in landscapes with clumped resources, simulated foragers that kept a reference point of the quality of recently visited patches, and returned to previously visited patches when local patch quality was poorer than the reference point, experienced higher energy gains compared to random patch use. Furthermore, the model of memory‐based foraging resulted in restricted space use in simulated landscapes and replicated the restricted space use observed in free‐ranging bison reasonably well. Our work demonstrates the adaptive value of spatial and attribute memory in heterogeneous landscapes, and how home ranges can be a byproduct of non‐omniscient foragers using past experience to minimize temporal variation in energy gains.