Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.     

Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.     

Cloning and Sequencing of an Alkaline Protease Gene from Bacillus lentus and Amplification of the Gene on the B. lentus Chromosome by an Improved Technique

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.     

Influence of added microbial enzymes on energy and protein availability of selected feed ingredients

Addition of enzymes to feed ingredients results in an improved energy availability that reduces the difference between gross and metabolisable energy of the raw material. The level of improvement seen is related to enzyme type and to dosage, and correlates well with the substrate specificity of the various enzymes present. Thus the values found in this study are specific for the enzymes and dosages used. Nitrogen retention is also improved by enzyme addition but this did not show a clear dosage response with all materials. An improved nitrogen retention cannot be directly correlated with protein digestibility but an improved retention of protein may offer the possibility to reduce protein levels in the diet.     

Non-enzymatic depolymerization of cotton cellulose by fungal mimicking metabolites

Small, low molecular weight, non-enzymatic compounds have been linked to the early stages of brown rot decay as the enzymes involved with holocellulose degradation are too large to penetrate the S3 layer of intact wood cells. We investigated the most notable of these compounds, i.e. hydrogen peroxide, iron, and oxalic acid. The former two are involved in the Fenton reaction in which they react to form hydroxyl radicals, which cause an accelerated depolymerization in cotton cellulose. We found the same reaction to be caused by both iron Fe³⁺ and Fe²⁺. A 10 mM oxalic acid solution showed significant depolymerization effect on cotton cellulose. An oxalic acid/sodium oxalate buffered pH gradient had an inhibitory effect on the reduction of cellulose polymers at increased pH values. The organic iron chelator, EDTA, was found to promote depolymerization of cellulose in combination with Fenton’s reagents, but inhibited the effect of oxalic acid in the absence of iron and hydrogen peroxide. Manganese was tested to see if metals other than iron could generate a significant impact on the degree of polymerization (DP) in cotton cellulose. Depolymerizing properties comparable to iron were seen. The results confirm that low molecular weight metabolites are capable of depolymerizing cellulose and suggest an importance of these mechanisms during incipient decay by brown rot fungi.     

Collaborative Moments. Expanding the Anthropological Field through Cross-Disciplinary Practice

ABSTRACT

This article explores some implications of cross-disciplinarity, as experienced in practice. Anthropologists are used to fuse different styles of reasoning by integrating the points of view and unspoken certainties of their partners in the field into their analysis. Fieldwork can be seen as an experiment in real time, where insights gained intersubjectively gradually shape up as knowledge through analysis. This line of thought is brought to bear on a discussion of collaboration between anthropologists, archaeologists, and biologists in North West Greenland. Through actual experiences from the field, this article shows how knowledge generated on the edge of one’s familiar disciplinary territory may both expand and intensify the anthropological field. Collaborative moments are seen to make new anthropological insights emerge through the co-presence of several analytical perspectives in the field.     

Zn(2+) site engineering at the oligomeric interface of the dopamine transporter

The hetN gene plays an important role in heterocyst differentiation and pattern formation. An immunoblotting study showed that the hetN gene in Anabaena sp. PCC 7120 was expressed in vegetative cells grown with combined nitrogen. After a switch to a medium without combined nitrogen, hetN expression first declined and was then followed by a rapid increase in its product, HetN, which was only present in mature heterocysts. HetN is located on both thylakoid membranes and plasma membranes as determined by immunoblotting using purified membranes. Overexpression of hetN completely prevented hetR up-regulation under nitrogen-deprivation conditions, suggesting that its role in pattern control may depend on its inhibition of hetR expression.     

Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.     

Functional importance of GLP-1 receptor species and expression levels in cell lines

Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands.