Changes in microbial diversity in industrial wastewater evaporation ponds following artificial salination

The salinity of industrial wastewater evaporation ponds was artificially increased from 3-7% to 12-16% (w/v), in an attempt to reduce the activity of sulfate-reducing bacteria (SRB) and subsequent emission of H2S. To investigate the changes in bacterial diversity in general, and SRB in particular, following this salination, two sets of universal primers targeting the 16S rRNA gene and the functional apsA [adenosine-5'-phosphosulfate (APS) reductase alpha-subunit] gene of SRB were used. Phylogenetic analysis indicated that Proteobacteria was the most dominant phylum both before and after salination (with 52% and 68%, respectively), whereas Firmicutes was the second most dominant phylum before (39%) and after (19%) salination. Sequences belonging to Bacteroidetes, Spirochaetes and Actinobacteria were also found. Several groups of SRB from Proteobacteria and Firmicutes were also found to inhabit this saline environment. Comparison of bacterial diversity before and after salination of the ponds revealed both a shift in community composition and an increase in microbial diversity following salination. The share of SRB in the 16S rRNA gene was reduced following salination, consistent with the reduction of H2S emissions. However, the community composition, as shown by apsA gene analysis, was not markedly affected.     

STAT-1- and IRF-3-dependent pathways are not essential for repression of ICP0-null mutant herpes simplex virus type 1 in human fibroblasts

Efficient herpes simplex virus type 1 (HSV-1) infection of human fibroblasts (HFs) is highly dependent on the viral immediate-early regulatory protein ICP0 unless the infection is conducted at a high multiplicity. ICP0-null mutant HSV-1 exhibits a plaque-forming defect of up to 3 orders of magnitude in HFs, whereas in many other cell types, this defect varies between 10- and 30-fold. The reasons for the high ICP0 requirement for HSV-1 infection in HFs have not been established definitively. Previous studies using other cell types suggested that ICP0-null mutant HSV-1 is hypersensitive to interferon and that this sensitivity is dependent on the cellular promyelocytic leukemia (PML) protein. To investigate the roles of two important aspects of interferon signaling in the phenotype of ICP0-null mutant HSV-1, we isolated HFs depleted of STAT-1 or interferon regulatory factor 3 (IRF-3). Surprisingly, plaque formation by the mutant virus was not improved in either cell type. We found that the sensitivity to interferon pretreatment of both ICP0-null mutant and wild-type (wt) HSV-1 was highly dependent on the multiplicity of infection. At a low multiplicity in virus yield experiments, both viruses were extremely susceptible to interferon pretreatment of HFs, but the sensitivity of the wild type but not the mutant could be overcome at higher multiplicities. We found that both wt and ICP0-null mutant HSV-1 remained sensitive to interferon in PML-depleted HFs albeit to an apparently lesser extent than in control cells. The data imply that the substantial reduction in ICP0-null HSV-1 infectivity at a low multiplicity in HFs does not occur through the activities of STAT-1- and IRF-3-dependent pathways and cannot be explained solely by enhanced sensitivity to interferon. We suggest that antiviral activities induced by interferon may be separable from and additive to those resulting from PML-related intrinsic resistance mechanisms.     

Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.     

Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice

Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.     

Operational considerations for augmentation of Trichogramma exiguum (Hymenoptera: Trichogrammatidae) for suppression of Rhyacionia frustrana (Lepidoptera: Tortricidae) in Pinus taeda plantations

Studies were performed to assess the operational feasibility of Trichogramma exiguum Pinto & Platner (Hymenoptera: Trichogrammatidae) augmentation for suppression of the Nantucket pine tip moth, Rhyacionia frustrana (Comstock) (Lepidoptera: Tortricidae), in commercial loblolly pine, Pinus taeda L., plantations. Single inundative releases containing two cohorts of encapsulated T. exiguum at a potential rate of 224,200 +/- 27,600 females per ha per cohort were made into two 4-ha plots during the second R. frustrana generation in 2000. Augmentation failed to increase parasitism rates above those occurring naturally; yet, 10% fewer shoots were attacked by R. frustrana, but not below acceptable levels. Quality control data suggest that low emergence levels and intense predation by ants upon developing T. exiguum lowered actual release rates to 13,000 +/- 900 females per ha per cohort. The effect of capsule distribution and microclimate on the discovery of capsules by predators (indicated by some E. kuehniella egg removal), parasitoid predation (percentage of eggs removed or destroyed), and subsequent parasitoid emergence was investigated. Uniformly distributed capsules experienced significantly higher predation levels than clustered capsules, and capsules exposed to field conditions for 5 d experienced higher predation than those exposed for 3 d, independently of distribution. Discovery of capsules by predators was unaffected by distribution or exposure period. Microhabitat significantly impacted average maximum daily temperature, the number of consecutive hours per day at or above 35 degrees C, and parasitoid emergence percentages. Parasitoid emergence declined significantly in response to increasing number of consecutive hours per day above 35 degrees C. Microclimate did not impact capsule discovery by predators or predation levels. Augmentation of T. exiguum for suppression of R. frustrana damage may not be practical within P. taeda plantations.     

Sex-dependent effect of BAG1 in ameliorating motor deficits of Huntington disease transgenic mice

The pathogenesis of Huntington disease (HD) is attributed to the misfolding of huntingtin (htt) caused by an expanded polyglutamine (polyQ) domain. Considerable effort has been devoted to identifying molecules that can prevent or reduce htt misfolding and the associated neuropathology. Although overexpression of chaperones is known to reduce htt cytotoxicity in cellular models, only modest protection is seen with Hsp70 overexpression in HD mouse models. Because the activity of Hsp70 is modulated by co-chaperones, an interesting issue is whether the in vivo effects of chaperones on polyQ protein toxicity are dependent on other modulators. In the present study, we focused on BAG1, a co-chaperone that interacts with Hsp70 and regulates its activity. Of htt mice expressing the N171-82Q mutant, we found that male N171-82Q mice show a greater deficit in rotarod performance than female N171-82Q mice. This sex-dependent motor deficit was improved by crossing N171-82Q mice with transgenic mice overexpressing BAG1 in neurons. Transgenic BAG1 also reduces the levels of mutant htt in synaptosomal fraction of male HD mice. Overexpression of BAG1 augmented the effects of Hsp70 by reducing aggregation of mutant htt in cultured cells and improving neurite outgrowth in htt-transfected PC12 cells. These findings suggest that the effects of chaperones on HD pathology are influenced by both their modulators and sex-dependent factors.     

The membrane-anchoring domain of epidermal growth factor receptor ligands dictates their ability to operate in juxtacrine mode

All ligands of the epidermal growth factor (EGF) receptor (EGFR) are synthesized as membrane-anchored precursors. Previous work has suggested that some ligands, such as EGF, must be proteolytically released to be active, whereas others, such as heparin-binding EGF-like growth factor (HB-EGF) can function while still anchored to the membrane (i.e., juxtacrine signaling). To explore the structural basis for these differences in ligand activity, we engineered a series of membrane-anchored ligands in which the core, receptor-binding domain of EGF was combined with different domains of both EGF and HB-EGF. We found that ligands having the N-terminal extension of EGF could not bind to the EGFR, even when released from the membrane. Ligands lacking an N-terminal extension, but possessing the membrane-anchoring domain of EGF, still required proteolytic release for activity, whereas ligands with the membrane-anchoring domain of HB-EGF could elicit full biological activity while still membrane anchored. Ligands containing the HB-EGF membrane anchor, but lacking an N-terminal extension, activated EGFR during their transit through the Golgi apparatus. However, cell-mixing experiments and fluorescence resonance energy transfer studies showed that juxtacrine signaling typically occurred in trans at the cell surface, at points of cell-cell contact. Our data suggest that the membrane-anchoring domain of ligands selectively controls their ability to participate in juxtacrine signaling and thus, only a subclass of EGFR ligands can act in a juxtacrine mode.     

Novel ryanodine-binding properties in mammalian retina

The ryanodine receptor (RyR)/Ca2+ release channel mobilizes Ca2+ from internal calcium stores to support a variety of neuronal functions. To investigate the presence of such a protein in mammalian retina, we applied ryanodine binding, PCR and antibodies against known RyRs. Surprisingly, ryanodine-binding properties of retinal endoplasmic reticulum-enriched membrane fraction were vastly different from those of skeletal and cardiac muscles ryanodine-binding proteins. In common with the skeletal and cardiac muscle, ryanodine bound with high-affinity to two or more types of binding site (Kd1 = 20.6 and Kd2 = 114 nM); binding was strongly stimulated by high concentrations of NaCl; it was inhibited by tetracaine and the protein appeared to possess an ATP-binding site. Unlike cardiac and skeletal muscle, RyRs in retina binding was Ca2+-independent; inhibited by caffeine and dantrolene; less sensitive to ruthenium red; and unaffected by La3+. Also, in retina, ryanodine rapidly associated to and dissociated from its binding sites. Furthermore, although the protein bound the ATP analog BzATP, retinal ryanodine binding was not stimulated by nucleotides. Immunostaining of bovine retinal sections with anti-RyR2 showed a strong staining of amacrine, horizontal and ganglion cells. Finally, using RT-PCR, the three known RyR isoforms were identified in retina. However, consistent with the novel binding properties, the peptide maps yielded by trypsin treatment and Western blotting demonstrate different patterns. Together, the results suggest that retina expresses a novel ryanodine-binding protein, likely to be a ryanodine receptor. Its presence in retina suggests that this protein might play a role in controlling intracellular Ca2+ concentration.     

Evaluating eukaryotic secreted protein prediction

Background  

Improvements in protein sequence annotation and an increase in the number of annotated protein databases has fueled development of an increasing number of software tools to predict secreted proteins. Six software programs capable of high throughput and employing a wide range of prediction methods, SignalP 3.0, SignalP 2.0, TargetP 1.01, PrediSi, Phobius, and ProtComp 6.0, are evaluated.     

The distribution of fitness effects among beneficial mutations

We know little about the distribution of fitness effects among new beneficial mutations, a problem that partly reflects the rarity of these changes. Surprisingly, though, population genetic theory allows us to predict what this distribution should look like under fairly general assumptions. Using extreme value theory, I derive this distribution and show that it has two unexpected properties. First, the distribution of beneficial fitness effects at a gene is exponential. Second, the distribution of beneficial effects at a gene has the same mean regardless of the fitness of the present wild-type allele. Adaptation from new mutations is thus characterized by a kind of invariance: natural selection chooses from the same spectrum of beneficial effects at a locus independent of the fitness rank of the present wild type. I show that these findings are reasonably robust to deviations from several assumptions. I further show that one can back calculate the mean size of new beneficial mutations from the observed mean size of fixed beneficial mutations.