Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.     

Long-term bird colonization and turnover in restored woodlands

The long-term effectiveness of restored areas for biodiversity is poorly known for the majority of restored ecosystems worldwide. We quantified temporal changes in bird occurrence in restoration plantings of different ages and geometries, and compared observed patterns with a reference dataset from woodland remnants on the same farms as our plantings. Over time, bird species richness remained unchanged in spring but exhibited modest increases in winter. We found that wider plantings supported significantly greater bird species richness in spring and winter than narrow plantings. There was no evidence of a significant interaction between planting width and time. We recorded major temporal changes in the occurrence of a range of individual species that indicated a clear turnover of species as plantings matured. Our results further revealed marked differences in individual species occurrence between plantings and woodland remnants. Life-history attributes associated with temporal changes in the bird assemblage were most apparent in winter survey data, and included diet, foraging and nesting patterns, movement behaviour (e.g. migratory vs. dispersive), and body size. Differences in bird assemblages between plantings of different ages suggest that it is important that farms support a range of age classes of planted woodland, if the aim is to maximize the number of native bird species in restored areas. Our data also suggest that changes in the bird species occupying plantings of different ages can be anticipated in a broadly predictable way based on planting geometry (especially width) and key life-history attributes, particularly movement patterns and habitat and diet specialisation.     

Semidry electrophoretic transfer of RNA to membranes.

We describe a method using a semi-dry gel electro-blotter to transfer RNA from standard agarose-formaldehyde denaturing gels in less than 30 min. The method requires equilibrating the gel in a low ionic strength buffer. The transfer is done under high-current and low-voltage conditions. This method maintains the overall sharpness of the bands on the final autoradiogram while shortening the time required for Northern transfer by approximately 12 hours.     

The analysis of recombinant interleukin-2 by thermospray liquid chromatography-mass spectrometry

The complete sequence of recombinant human interleukin-2 expressed in Escherichia coli has been confirmed by thermospray liquid chromatography-mass spectrometry (TS-LC-MS) of a tryptic digest derived from 100 micrograms (7 nmol) of reduced carboxymethylated interleukin-2. The preparation was shown by this method to contain predominantly unprocessed N-terminal initiator Met, with some authentic N-terminal Ala; the rest of the protein was as predicted from the DNA sequence, though some deamidated material was noted. TS-LC-MS proved to be a rapid and efficient method for surveying the protein tryptic peptide products allowing all the data to be collected in one chromatographic run; all tryptic fragments were identified by their molecular ions including those for the larger peptides (Mr 1500-3500) which, due to the presence of doubly and triply charged molecular ions, were brought within the mass range of the instrument (1800 Da). It is proposed that TS-LC-MS is a good general method for analyzing recombinant protein digests with respect to sequence confirmation, processing, and post-translational modification, and since each chromatographic peak is identified allows for subsequent monitoring of the protein by LC using uv detection. The method suffers from the disadvantages that all the sample is consumed during the experiment and that no fragment (sequence) ions are generally observed.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Subcellular localization and internalization of the four human leptin receptor isoforms.

There are four known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains (designated by the number of unique C-terminal amino acids). In cells expressing HLR-5, -15, or -274, 15-25% of the leptin binding sites were located at the plasma membrane. In contrast, in cells expressing HLR-67, only 5% of the total binding sites were at the plasma membrane. Immunofluorescent microscopy showed that all four isoforms partially co-localized with calnexin and beta-COP, markers of the endoplasmic reticulum and the Golgi, respectively. All isoforms were also detected in an unidentified punctate compartment. All isoforms were internalized via clathrin-mediated endocytosis, but at different rates. After 20 min at 37 degrees C, 45% of a bound cohort of labeled ligand had been internalized by HLR-15, 30% by HLR-67, 25% by HLR-274, and 15% by HLR-5. Degradation of internalized leptin occurred in lysosomes. Overnight exposure to leptin down-regulated all isoforms, but to a variable extent. HLR-274 displayed the greatest down-regulation and also appeared to reach lysosomes more quickly than the other isoforms. The faster degradation of HLR-274 may help to terminate leptin signaling.     

Protein degradation in aged nematodes (Turbatrix aceti).

The rates of degradation of total soluble proteins in the free-living nematode, $\text{Turbatrix aceti}$, have been estimated by following the loss of acid-insoluble insoluble radioactivity from protein during a nonradioactive chase period after initial labeling with [³⁵S]methionine. These proteins appear to lose label kinetically as a homogeneous class in age-synchronized nematode populations. However, proteins are degraded more slowly in senescent cultures than in young cultures. Protein degradation rates decline progressively during nematode aging. These findings suggest that the protein degradative system in T. aceti may become partially defective with advancing age which may result in the accumulation of aberrant protein molecules in senescent organisms.