Loss of exocrine pancreatic stimulation during parenteral feeding suppresses digestive enzyme expression and induces Hsp70 expression

Luminal nutrients are essential for the growth and maintenance of digestive tissue including the pancreas and small intestinal mucosa. Long-term loss of luminal nutrients such as during animal hibernation has been shown to result in mucosal atrophy and a corresponding stress response characterized by the induction of heat shock protein (Hsp)70 expression. This study was conducted to determine if the loss of luminal nutrients during total parenteral nutrition (TPN) would result in atrophy of the exocrine pancreas and small intestinal mucosa as well as an induction of Hsp70 expression in rats. In experiment 1, the treatment groups included an orally fed control, a saline-infused surgical control, or TPN treatment for 7 days. In experiment 2, the treatment groups included an orally fed control and TPN alone or coinfused with varying doses of glucagon-like peptide (GLP)-2, a mucosal proliferation agent, for 7 days. In experiment 1, TPN resulted in a 40% reduction in pancreatic mass that was associated with a dramatic reduction in digestive enzyme expression, enhanced apoptosis, and a 200% increase in Hsp70 expression. Conversely, heat shock cognate 70, Hsp27, and Hsp60 expression was not changed in the pancreas. In experiment 2, TPN resulted in a 30% reduction in jejunal mucosa mass and a similar induction of Hsp70 expression. The inclusion of GLP-2 during TPN attenuated jejunal mucosal atrophy and inhibited Hsp70 expression, suggesting that Hsp70 induction is sensitive to cell growth. These data indicate that pancreatic and intestinal mucosal atrophy caused by a loss of luminal nutrient stimulation is accompanied by a compensatory response involving Hsp70.     

Peptide stabilized amphotericin B nanodisks

Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic alpha-helical peptide, termed 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH(2)), solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids). Characterization studies revealed that interaction with DMPC induced a near doubling of 4F tryptophan fluorescence emission quantum yield (excitation 280 nm) and a approximately 7 nm blue shift in emission wavelength maximum. Inclusion of AMB in the vesicle substrate resulted in formation of 4F AMB-ND. Spectra of AMB containing particles revealed the antibiotic is a highly effective quencher of 4F tryptophan fluorescence emission, giving rise to a Ksv=7.7 x 10(4). Negative stain electron microscopy revealed that AMB-ND prepared with 4F possessed a disk shaped morphology similar to ND prepared without AMB or prepared with apoA-I. In yeast and pathogenic fungi growth inhibition assays, 4F AMB-ND was as effective as apoA-I AMB-ND. The data indicate that AMB-ND generated using an amphipathic peptide in lieu of apoA-I form a discrete population of particles that possess potent biological activity. Given their intrinsic versatility, peptides may be preferred for scale up and clinical application of AMB-ND.     

Characterization of the OxyR regulon of Neisseria gonorrhoeae

OxyR regulates the expression of the majority of H(2)O(2) responses in Gram-negative organisms. In a previous study we reported the OxyR-dependent derepression of catalase expression in the human pathogen Neisseria gonorrhoeae. In the present study we used microarray expression profiling of N. gonorrhoeae wild-type strain 1291 and an oxyR mutant strain to define the OxyR regulon. In addition to katA (encoding catalase), only one other locus displayed a greater than two-fold difference in expression in the wild type : oxyR comparison. This locus encodes an operon of two genes, a putative peroxiredoxin/glutaredoxin (Prx) and a putative glutathione oxidoreductase (Gor). Mutant strains were constructed in which each of these genes was inactivated. A previous biochemical study in Neisseria meningitidis had confirmed function of the glutaredoxin/peroxiredoxin. Assay of the wild-type 1291 cell free extract confirmed Gor activity, which was lost in the gor mutant strain. Phenotypic analysis of the prx mutant strain in H(2)O(2) killing assays revealed increased resistance, presumably due to upregulation of alternative defence mechanisms. The oxyR, prx and gor mutant strains were deficient in biofilm formation, and the oxyR and prx strains had decreased survival in cervical epithelial cells, indicating a key role for the OxyR regulon in these processes.     

Quinazoline derivatives as MC-I inhibitors: evaluation of myocardial uptake using Positron Emission Tomography in rat and non-human primate

Several quinazoline derivatives were made as mitochondrial complex 1 inhibitors. Compound 4 showed an IC(50) of 11.3 nM and was the most potent compound of this series. The (18)F analog of 4, [(18)F] 4, was injected in the rat and showed high and rapid heart uptake, fast liver clearance, and low blood uptake. Images obtained using a microPET showed clear delineation of the myocardium in normal rats and perfusion deficit in ischemic rats. In the non-human primate, [(18)F] 4 showed rapid uptake and clearance from the myocardium and high liver uptake.     

Unusual Cu(I)/Ag(I) coordination of Escherichia coli CusF as revealed by atomic resolution crystallography and X-ray absorption spectroscopy

Elevated levels of copper or silver ions in the environment are an immediate threat to many organisms. Escherichia coli is able to resist the toxic effects of these ions through strictly limiting intracellular levels of Cu(I) and Ag(I). The CusCFBA system is one system in E. coli responsible for copper/silver tolerance. A key component of this system is the periplasmic copper/silver-binding protein, CusF. Here the X-ray structure and XAS data on the CusF-Ag(I) and CusF-Cu(I) complexes, respectively, are reported. In the CusF-Ag(I) structure, Ag(I) is coordinated by two methionines and a histidine, with a nearby tryptophan capping the metal site. EXAFS measurements on the CusF-Cu(I) complex show a similar environment for Cu(I). The arrangement of ligands effectively sequesters the metal from its periplasmic environment and thus may play a role in protecting the cell from the toxic ion.     

The devil to pay: a cost of mutualism with Myrmelachista schumanni ants in 'devil's gardens' is increased herbivory on Duroia hirsuta trees

'Devil's gardens' are nearly pure stands of the myrmecophyte, Duroia hirsuta, that occur in Amazonian rainforests. Devil's gardens are created by Myrmelachista schumanni ants, which nest in D. hirsuta trees and kill other plants using formic acid as an herbicide. Here, we show that this ant-plant mutualism has an associated cost; by making devil's gardens, M. schumanni increases herbivory on D. hirsuta. We measured standing leaf herbivory on D. hirsuta trees and found that they sustain higher herbivory inside than outside devil's gardens. We also measured the rate of herbivory on nursery-grown D. hirsuta saplings planted inside and outside devil's gardens in ant-exclusion and control treatments. We found that when we excluded ants, herbivory on D. hirsuta was higher inside than outside devil's gardens. These results suggest that devil's gardens are a concentrated resource for herbivores. Myrmelachista schumanni workers defend D. hirsuta against herbivores, but do not fully counterbalance the high herbivore pressure in devil's gardens. We suggest that high herbivory may limit the spread of devil's gardens, possibly explaining why devil's gardens do not overrun Amazonian rainforests.     

Characterization and <Emphasis Type="Italic">in vitro</Emphasis> Cytotoxicity Testing of Ethanolamine-derived Cadmium Chelating Agents

We have synthesized and characterized the new cadmium chelating agent potassium bis(2-hydroxyethyl)aminoethyldithiocarbonate hemihydrate, K[bhexan] · 0.5H₂O (2), that is structurally related to the known effective in vivo cadmium chelating agent potassium bis(2-hydroxyethyl)dithiocarbamate, K[bhedtc] (1). The corresponding cadmium complex of 2 differs from di(bis(2-hydroxyethyl)dithiocarbamato)cadmium(II), Cd(bhedtc)₂ (3), in that the insoluble compound exhibits an elemental composition consistent with a cadmium:ligand ratio of 2:1. The cytotoxicity of the 1–3 was investigated using the human osteoblast-like cell line, Saos-2. Compounds 1 or 2 did not affect cell adherence or cell viability in the 100–500 μM concentration range studied, whereas 3 resulted in a concentration-dependent increase in loss of cell adherence and decrease in cell viability. Overall, the results of the loss of cell adherence, trypan blue exclusion and MTT assays showed that administration of 3 (cadmium complex of 1) resulted in cytotoxicity lower than that of cadmium chloride, but higher than that of the chelator 1 alone. The effect of simultaneous addition of cadmium chloride and 1 or 2 on cell viability was also assessed using the MTT assay. For the 100 μM cadmium chloride experiments, cell viability comparable to control cells was achieved for both 1 and 2 in the 100–500 μM concentration range studied. Cell viability comparable to control cells was achieved for 1 but not 2 in the 100–500 μM concentration range studied for the 200 μM cadmium chloride experiments. Thus 1 appears more effective than 2 in the ability to mediate the cytotoxic effects of cadmium in vitro upon concomitant administration.     

Humming in bears: a peculiar sustained mammalian vocalization

A peculiar sustained vocalization has long been known under various names in several bear species but not been studied in sufficient detail. Based on a critical survey of the relevant literature, analyses of tape recordings and pertinent own observations we tried to clarify the presence of this vocalization in the species of the Ursidae as well as its structural characteristics and specific mode of sound production. To avoid confusion with other vocalization types we introduce the term humming for it. Furthermore we discuss its communicatory and functional significance and formulate hypotheses as to its evolutionary origin against the background of acoustic communication signal repertoires known in the terrestrial Carnivora. Humming is present in all extant species of the Ursidae with the exception of the giant panda. It has similar structural characteristics and the same mode of sound production in all species. It is also known in adult bears but its occurrence is largely restricted to cubs. Humming is a synapomorphic vocalization type of the Ursidae which is not phylogenetically related to another vocalization type known in the terrestrial Carnivora. It is a rapid sequence of very short single sounds; long bouts of sustained exhalatory sound production are interrupted by very short inhalatory phases without sound. Both the sound and the body vibration accompanying its production are highly likely to be communication signals. Yet, controlled physiological experiments on bear mothers and cubs are still necessary to formulate and test specific hypotheses as to the communicatory function and adaptive significance of humming.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

Desert Survivors: the design and implementation of a television program to enhance local scientific literacy

Outreach efforts by faculty members are oftentimes limited in scope due to hectic schedules. We developed a program to enhance science literacy in elementary school children that allows experts to reach a tremendous audience while minimizing their time commitment. The foundation of the program is a television series entitled "Desert Survivors." The episodes air on local cable access television and are available to teachers on DVD. Each episode features a guest expert who spotlights a particular organism and how that organism overcomes the myriad of hardships inherent to desert survival. Local classrooms are visited to solicit questions from students regarding the organism of interest. These videotaped questions are integrated into Desert Survivors television production and provide the guest expert with the basis to discuss the ecology, physiology, and evolutionary biology of the organism. The program is bolstered through the use of an interactive website. Assessment strategies are in place to ensure program efficacy. Herein, we describe the development of the program as a model for innovative outreach opportunities.