Sperm production in an extremophile fish,the cave molly (<Emphasis Type="Italic">Poecilia mexicana</Emphasis>, Poeciliidae,Teleostei)

A prominent trade-off in life history theory and evolution balances the costs of reproduction with those of basic somatic needs. Hence, reproductive efforts may be reduced in environments where additional energy is required for somatic maintenance. Here, we investigated male sperm stores in Atlantic mollies (Poecilia mexicana) from a sulfidic cave and several sulfidic and non-sulfidic surface habitats. We found significant differences among populations in the number of sperm stripped per male, which was also correlated with differences in gonad weights. The largest sperm stores were detected in males from non-sulfidic surface creeks, while males from a partially sulfidic surface system had lower sperm counts, and males from completely sulfidic systems, surface as well as subterranean, had even fewer available sperm. We conclude that the extreme environmental conditions in sulfidic habitats appear to constrain male sperm production, since hydrogen sulfide as a naturally occurring toxin requires energy-demanding adaptations. Furthermore, we examined sperm counts of lab-reared cave and surface mollies in response to energy limitation. Males from stock populations were placed under high and low food treatments for a 2-week period and then stripped of sperm. Sperm counts of surface mollies tended to be reduced by low food availability, whereas sperm counts of cave mollies did not significantly vary between food treatments, which likely points towards a higher starvation resistance in cave mollies.     

Reactor Design for the Novozym 435 ® -catalysed Enantioselective Esterification of 4-methyloctanoic Acid

The bench scale Novozym 435 ® catalysed esterification of 4-methyloctanoic acid with ethanol was studied at 35°C. Esterification in a batch reactor (molar ratio of 1:8 (acid:EtOH)) resulted in the isolation of the enantiomerically enriched product (ee p =81%) and substrate (ee s =93%). In order to integrate reaction and separation, liquid-vapour equilibria calculations were performed showing that an excess of ethanol results in a very low ester fraction in the vapour phase. Since this is undesirable for an integrated process of reaction and product removal, a repeated batch reaction was performed using a molar ratio of 10:1 (acid:EtOH). After six cycles (45% conversion) the ee of 4-methyloctanoic acid ethyl ester turned out to be 80%. For different E values the ee p was calculated for batch and repeated batch reactions. It was shown that in all cases the ee p was higher for the repeated batch reaction. However, the product is not enantiopure since the E value of the reaction is rather low at the low ethanol concentration used. An alternative approach would be the continuous separation of the product during the reaction. A mathematical model was developed to describe esterification in a packed bed reactor integrated with product separation. This model shows that integration of reaction and product removal in advance is not suitable either to obtain an enantiomerically pure product. Since the optimal reaction conditions (high ethanol concentration) and the optimal separation system (low ethanol concentration) do not match in this reaction, the preference is given to the batch reaction at high ethanol concentrations because in that case the highest enantioselectivity of the enzyme is obtained.     

Giardiasis in a white stork in The Netherlands

Giardia sp. was found in the white stork (Ciconia ciconia) in The Netherlands for the first time. The Giardia sp. trophozoites that were found in the feces of a 6-wk-old white stork, were examined by light microscopy. The parasites closely resembled Giardia ardeae that had been isolated by others from several species of wading birds belonging to the order Ciconiiformes, sharing a deeply notched adhesive disk, a single caudal flagellum, and a single round median body. Serologically, the parasites did not react with anti-Giardia intestinalis monoclonal antibodies. Although no signs of intestinal disease were observed in the stork chick, the presence of parasites in all stages of development and the huge number of parasites show that the stork chick was experiencing an active infection with G. ardeae type parasites.     

RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.     

A New Spectrophotometric Method for the Detection of Lipase Activity Using 2,4-Dinitrophenyl Butyrate as a Substrate

A rapid and sensitive assay for the detection of lipase activity is described. The method is based upon the increase in absorbance at 360 nm due to the formation of the 2,4-dinitrophenolate anion during the enzymatic hydrolysis of 2,4-dinitrophenyl butyrate. The substrate is used in an emulsified form. Using a diode array spectrophotometer with internal referencing a correction can be made for absorbance changes due to clearance of the emulsion during hydrolysis. The small reaction volume and the high extinction coefficient of the product makes the method applicable for detection of both low substrate and low enzyme concentration.

Four lipases were tested: lipase from porcine pancreas, Candida cylindracea, Pseudomonas sp. and Aspergillus niger. All enzymes are readily able to catalyse the hydrolysis of 2,4-dinitrophenyl butyrate.     

Bleomycin resistance: a new dominant selectable marker for plant cell transformation

Summary Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. The expression of this determinant in plant cells confers resistance to bleomycin and allows selection of transformed plant cells.     

A Role for Neuropilins in the Interaction between Schwann Cells and Meningeal Cells

In their natural habitat, the peripheral nerve, Schwann cells (SCs) form nicely aligned pathways (also known as the bands of Büngner) that guide regenerating axons to their targets. Schwann cells that are implanted in the lesioned spinal cord fail to align in pathways that could support axon growth but form cellular clusters that exhibit only limited intermingling with the astrocytes and meningeal cells (MCs) that are present in the neural scar. The formation of cell clusters can be studied in co-cultures of SCs and MCs. In these co-cultures SCs form cluster-like non-overlapping cell aggregates with well-defined boundaries. There are several indications that neuropilins (NRPs) play an important role in MC-induced SC aggregation. Both SCs and MCs express NRP1 and NRP2 and SCs express the NRP ligands Sema3B, C and E while MCs express Sema3A, C, E and F. We now demonstrate that in SC-MC co-cultures, siRNA mediated knockdown of NRP2 in SCs decreased the formation of SC clusters while these SCs maintained their capacity to align in bands of Büngner-like columnar arrays. Unexpectedly, knockdown of NRP1 expression resulted in a significant increase in SC aggregation. These results suggest that a reduction in NRP2 expression may enhance the capacity of implanted SCs to interact with MCs that invade a neural scar formed after a lesion of the spinal cord.     

Identification and characterisation of two runx2 homologues in zebrafish with different expression patterns

Genome and gene duplications are considered to be the impetus to generate new genes, as the presence of multiple copies of a gene allows for paralogues to adopt novel function. After at least two rounds of genome/gene duplication, the Runt gene family consists of three members in vertebrates, instead of one in invertebrates. One of the family members, Runx2, plays a key role in the development of bone, a tissue that first occurs in vertebrates. The family has thus gained new gene function in the course of evolution. Two Runx2 genes were cloned in the vertebrate model system the zebrafish (Danio rerio). The expression patterns of the two genes differ and their kinetics differ up to four fold. In addition, splice forms exist that are novel when compared with mammals. Together, these findings comprise opportunities for selection and retention of the paralogues towards divergent and possibly new function.