Characterization of the Escherichia coli SecA signal peptide-binding site

SecA signal peptide interaction is critical for initiating protein translocation in the bacterial Sec-dependent pathway. Here, we have utilized the recent nuclear magnetic resonance (NMR) and Förster resonance energy transfer studies that mapped the location of the SecA signal peptide-binding site to design and isolate signal peptide-binding-defective secA mutants. Biochemical characterization of the mutant SecA proteins showed that Ser226, Val310, Ile789, Glu806, and Phe808 are important for signal peptide binding. A genetic system utilizing alkaline phosphatase secretion driven by different signal peptides was employed to demonstrate that both the PhoA and LamB signal peptides appear to recognize a common set of residues at the SecA signal peptide-binding site. A similar system containing either SecA-dependent or signal recognition particle (SRP)-dependent signal peptides along with the prlA suppressor mutation that is defective in signal peptide proofreading activity were employed to distinguish between SecA residues that are utilized more exclusively for signal peptide recognition or those that also participate in the proofreading and translocation functions of SecA. Collectively, our data allowed us to propose a model for the location of the SecA signal peptide-binding site that is more consistent with recent structural insights into this protein translocation system.     

Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.     

Hyaluronidase increases the biodistribution of acid alpha-1,4 glucosidase in the muscle of Pompe disease mice: an approach to enhance the efficacy of enzyme replacement therapy

Pompe disease (glycogen storage disease type II) is a glycogen storage disease caused by a deficiency of the lysosomal enzyme, acid maltase/acid alpha-1,4 glucosidase (GAA). Deficiency of the enzyme leads primarily to intra-lysosomal glycogen accumulation, primarily in cardiac and skeletal muscles, due to the inability of converting glycogen into glucose. Enzyme replacement therapy (ERT) has been applied to replace the deficient enzyme and to restore the lost function. However, enhancing the enzyme activity to the muscle following ERT is relatively insufficient. In order to enhance GAA activity into the muscle in Pompe disease, efficacy of hyaluronidase (hyase) was examined in the heart, quadriceps, diaphragm, kidney, and brain of mouse model of Pompe disease. Administration of hyase 3000 U/mouse (intravenous) i.v. or i.p. (intraperitoneal) and 10 min later recombinant human GAA (rhGAA) 20 mg/kg i.v. showed more GAA activity in hyase i.p. injected mice compared to those mice injected with hyase via i.v. Injection of low dose of hyase (3000 U/mouse) or high dose of hyase (10,000 U/mouse) i.p. and 20 min or 60 min later 20 mg/kg rhGAA i.v. increased GAA activity into the heart, diaphragm, kidney, and quadriceps compared to hyase untreated mice. These studies suggest that hyase enhances penetration of enzyme into the tissues including muscle during ERT and therefore hyase pretreatment may be important in treating Pompe disease.     

Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor

The microbial diversity of a deteriorated biological phosphorus removal reactor was investigated by methods not requiring direct cultivation. The reactor was fed with media containing acetate and high levels of phosphate (P/C weight ratio, 8:100) but failed to completely remove phosphate in the effluent and showed very limited biological phosphorus removal activity. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA was used to investigate the bacterial diversity. Up to 11 DGGE bands representing at least 11 different sequence types were observed; DNA from the 6 most dominant of these bands was further isolated and sequenced. Comparative phylogenetic analysis of the partial 16S rRNA sequences suggested that one sequence type was affiliated with the alpha subclass of the Proteobacteria, one was associated with the Legionella group of the gamma subclass of the Proteobacteria, and the remaining four formed a novel group of the gamma subclass of the Proteobacteria with no close relationship to any previously described species. The novel group represented approximately 75% of the PCR-amplified DNA, based on the DGGE band intensities. Two oligonucleotide rRNA probes for this novel group were designed and used in a whole-cell hybridization analysis to investigate the abundance of this novel group in situ. The bacteria were coccoid and 3 to 4 microm in diameter and represented approximately 35% of the total population, suggesting a relatively close agreement with the results obtained by the PCR-based DGGE method. Further, based on electron microscopy and standard staining microscopic analysis, this novel group was able to accumulate granule inclusions, possibly consisting of polyhydroxyalkanoate, inside the cells.     

Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a role in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.     

The Proportion of Three Foundation Plant Species and Their Genotypes Influence an Arthropod Community: Restoration Implications for the Endangered Southwestern Willow Flycatcher

As part of a restoration project, multiple genotypes of two tree species, Fremont cottonwood (Populus fremontii) and Goodding's willow (Salix gooddingii), and one shrub species, Coyote willow (S. exigua), were experimentally planted in different proportions at the Palo Verde Ecological Reserve near Blythe, California, U.S.A. These common woody plant species are important to the endangered southwestern willow flycatcher, providing perch, nesting, and foraging habitat. We conducted this study to evaluate plant species proportion and plant genotype effects on the arthropod community, the prey base for the endangered southwestern willow flycatcher. Three patterns emerged. First, plant species proportions were important; the arthropod community had the greatest richness and diversity (H′) when Goodding's willow proportion was high and Fremont cottonwood proportion was lower; that is, fewer Fremont cottonwoods are required to positively affect overall arthropod diversity. Second, we found significant genotypic effects, for all three plant species, on arthropod species accumulation. Third, while both planting proportion and genotype effects were significant, we found that the effect of planting proportion on arthropod richness was about twice as large as the effect of plant genotype. This shows that both plant species proportions and genotype should be utilized in restoration projects to maximize habitat heterogeneity and arthropod richness. Similar studies can determine which planting proportion and specific genotypes may result in a more favorable arthropod prey base for the southwestern willow flycatcher and other species of concern. Greater attention to planting design and genotype can result in significant gains in diversity at little or no additional project cost.     

MICROBODIES IN EXPERIMENTALLY ALTERED CELLS

A rapid and sustained increase in the number of microbodies in liver and kidney cells can be induced in male rats by ethyl chlorophenoxyisobutyrate (CPIB), a hypolipidemic drug. This phenomenon permits investigation of several aspects of microbody behavior in experimental conditions. Reversal experiments demonstrate that liver cells revert to normal between 2 and 3 weeks after withdrawal of CPIB and that one of the mechanisms for removal of excess microbodies is their incorporation into structures indistinguishable from lysosomes. In a state of rapid cell division, such as that present during liver regeneration, microbody proliferation apparently occupies a high biological priority. In necrotic or degenerating cells microbody structure remains relatively normal. The increase in microbodies induced by CPIB is inhibited by chloramphenicol. No increase in microbodies occurred in female rats or in chickens, guinea pigs, or rabbits at the dosage used (0.25% in diet). No changes in microbodies were seen in monkey liver. Catalase activity was generally parallel to the numerical response in microbodies. Additional observations suggest that the microbody response to CPIB is not related to hepatomegaly induced by this agent but may be related to the hypolipidemic effect of CPIB, though hypolipidemia per se is not a specific or sufficient cause of microbody proliferation.     

Rosiglitazone improves survival and hastens recovery from pancreatic inflammation in obese mice

Obesity increases severity of acute pancreatitis (AP) by unclear mechanisms. We investigated the effect of the PPAR-gamma agonist rosiglitazone (RGZ, 0.01% in the diet) on severity of AP induced by administration of IL-12+ IL-18 in male C57BL6 mice fed a low fat (LFD) or high fat diet (HFD), under the hypothesis that RGZ would reduce disease severity in HFD-fed obese animals. In both LFD and HFD mice without AP, RGZ significantly increased body weight and % fat mass, with significant upregulation of adiponectin and suppression of erythropoiesis. In HFD mice with AP, RGZ significantly increased survival and hastened recovery from pancreatic inflammation, as evaluated by significantly improved pancreatic histology, reduced saponification of visceral adipose tissue and less severe suppression of erythropoiesis at Day 7 post-AP. This was associated with significantly lower circulating and pancreas-associated levels of IL-6, Galectin-3, osteopontin and TIMP-1 in HFD + RGZ mice, particularly at Day 7 post-AP. In LFD mice with AP, RGZ significantly worsened the degree of intrapancreatic acinar and fat necrosis as well as visceral fat saponification, without affecting other parameters of disease severity or inflammation. Induction of AP lead to major suppression of adiponectin levels at Day 7 in both HFD and HFD + RGZ mice. In conclusion, RGZ prevents development of severe AP in obese mice even though it significantly increases adiposity, indicating that obesity can be dissociated from AP severity by improving the metabolic and inflammatory milieu. However, RGZ worsens selective parameters of AP severity in LFD mice.