An active site-tyrosine-containing heptapeptide from D-amino acid oxidase

The flavoenzyme D-amino acid oxidase (Eo) is rapidly chlorinated by N-chloro-D-leucine (Rudie, N.G., Porter, D.J.T., and Bright, H.J. (1980) J. Biol. Chem. 255, 498-508). We have carried out chymotryptic digestion of E0-36Cl2 and find that all of the radiolabel is located in a heptapeptide having [3.5-36Cl2]chlorotyrosine as the COOH-terminal residue. This heptapeptide, having the sequence -Asp-Leu-Glu-Arg-Gly-Ile-Tyr-, is located within a larger fragment obtained previously from cyanogen bromide cleavage of E0. These results demonstrate that the target for chlorination in E0 must be a single tyrosine residue and provide, when taken together with previous findings, the first clear evidence for the identity and location of an active site residue in the polypeptide chain of D-amino oxidase.     

Peptide N-alkylamides by solid phase synthesis

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH2CH3, NH2CH2CH3, NH2CH2CF3) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [DGlu6, Pro9-NHCH2CH3]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [DGlu6]-GnRH. Other analogs including [DTrp6, Pro9-NHCH2CH3]-GnRH, [DAla6, Pro9-NHCH2CF3]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.     

Local grassland restoration affects insect communities

1. It is hypothesised that ecological restoration in grasslands can induce an alternative stable state shift in vegetation. The change in vegetation influences insect community assemblages and allows for greater functional redundancy in pollination and refuge for native insect species. 2. Insect community assemblages at eight coastal California grassland sites were evaluated. Half of these sites had undergone restoration through active revegetation of native grassland flora and half were non‐restored. Insects were collected from Lupinus bicolor (Fabaceae) within 2 × 2‐m² plots in spring 2017. Lupinus bicolor is a common native species that is used in California restoration projects, and home and state landscaping projects. 3. Ordination demonstrated that insect community assemblages were different between restored and non‐restored sites. These differences were seen in insect functional groups as well as taxa‐specific differences and were found to be driven by environmental characteristics such as non‐native forb cover. 4. Functional redundancy of herbivores decreased at restored sites, while pollinators became more redundant compared with non‐restored sites. The assemblages of the common species found at restoration sites contained more native insects than those found at non‐restored sites, including species such as Bombus vosnesenskii. 5. Local grassland restoration has the potential to induce an alternative stable state change and affect insect community assemblages. Additionally, it was found that grassland restoration can be a potential conservation tool to provide refugia for bumblebees (Bombus), but additional studies are required to fully understand its broader applicability.     

Characterization of the microtubule movement produced by sea urchin egg kinesin

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.     

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.     

Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

How Multidisciplinary Are the Multidisciplinary Journals Science and Nature?

Interest in cross-disciplinary research knowledge interchange runs high. Review processes at funding agencies, such as the U.S. National Science Foundation, consider plans to disseminate research across disciplinary bounds. Publication in the leading multidisciplinary journals, Nature and Science, may signify the epitome of successful interdisciplinary integration of research knowledge and cross-disciplinary dissemination of findings. But how interdisciplinary are they? The journals are multidisciplinary, but do the individual articles themselves draw upon multiple fields of knowledge and does their influence span disciplines? This research compares articles in three fields (Cell Biology, Physical Chemistry, and Cognitive Science) published in a leading disciplinary journal in each field to those published in Nature and Science. We find comparable degrees of interdisciplinary integration and only modest differences in cross-disciplinary diffusion. That said, though the rate of out-of-field diffusion might be comparable, the sheer reach of Nature and Science, indicated by their potent Journal Impact Factors, means that the diffusion of knowledge therein can far exceed that of leading disciplinary journals in some fields (such as Physical Chemistry and Cognitive Science in our samples).     

Effects of variable water motion on regeneration of Hemipholis elongata (Echinodermata, Ophiuroidea)

Abstract. To determine whether increased water motion affects patterns of regeneration in the subtidal burrowing brittlestar Hemipholis elongata (phylum Echinodermata), individuals were subjected to laboratory-controlled turbulence conditions. Half of each replicate aquarium experienced oscillatory (wave-like) turbulence while the other half had no turbulence. Individual brittlestars from which arm-tips had been removed were allowed to burrow and to regenerate. Regenerated arm-tip length and weight were tested for differences between organisms in calm and turbulent conditions. Regenerated arm-tip length differed significantly between control and treatment, but arm-tip dry weight and skeleton/tissue ratio of regenerated arm-tips did not. To quantify plasticity in the skeleton, 15 morphological measurements made on the proximal face of vertebral ossicles (using scanning electron microscopy) were integrated as an index of overall ossicle size. We found a significant difference in the overall size index of the vertebral ossicles between treatments, but could not determine which of the measurements contributed most to the difference. The results indicate that regeneration in H. elongata is a complex process that can be modified by environmental conditions.