Zinc as an anti-tumor agent in prostate cancer and in other cancers

Human prostate glandular epithelial cells have the unique capability of accumulating high levels of zinc. This is essential to inhibit m-aconitase activity so that citrate can accumulate for secretion into prostatic fluid, which is a major function of the prostate gland. As a result, the Krebs cycle is truncated with the consequence of the lost ATP production that would result from citrate oxidation. The cellular accumulation of zinc also inhibits mitochondrial terminal oxidation and respiration. In addition to these metabolic effects, zinc accumulation exhibits anti-proliferative effects via its induction of mitochondrial apoptogenesis. Zinc accumulation also inhibits the invasive/migration activities in malignant prostate cells. The anti-proliferative effects and the effects on invasion and migration occur through zinc activation of specific intracellular signaling pathways. Consequently, these effects impose anti-tumor actions by zinc. The ability of prostate cells to accumulate zinc is due to the expression and activity of the zinc uptake transporter, ZIP1. To avoid the anti-tumor effects of zinc, in prostate cancer the malignant prostate cells exhibit a silencing of ZIP1 gene expression accompanied by a depletion of cellular zinc. Therefore we regard ZIP1 as a tumor suppressor gene in prostate cancer. In addition to prostate cells, similar tumor suppressor effects of zinc have been identified in several other types of tumors.     

Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.     

Functional mimicry of the LDL receptor-associated protein through multimerization of a minimized third domain

The LDL receptor-associated protein (RAP) is a ligand for the LDL receptor-related protein (LRP1). The first and third domains of RAP can each bind to one of many sequence-related pairs of complement-type repeats (CR) found within the LRP1 ectodomain. Multiple sites of interaction between the multivalent RAP ligand and the multivalent LRP1 receptor yield strong binding avidity for the complex. The third domain of RAP can be significantly truncated, with material retention of monovalent CR pair-binding affinity, provided that the minimized sequence is stabilized with an intramolecular disulfide bond. We demonstrate that the avidity of full-length RAP for LRP1 in vitro can be partially reconstituted by assembly of truncated, disulfide-linked RAP peptides on tetravalent streptavidin or bivalent immunoglobulin scaffolds. The peptide complex with streptavidin shows pronounced hepatotropism in vivo, replicating the biodistribution of full-length RAP.     

Oxygenase domain of Drosophila melanogaster nitric oxide synthase: unique kinetic parameters enable a more efficient NO release

Although nitric oxide (NO) is important for cell signaling and nonspecific immunity in the fruit fly Drosophila melanogaster, little is known about its single NO synthase (dNOS). We expressed the oxygenase domain of dNOS (dNOSoxy), characterized its spectroscopic, kinetic, and catalytic properties, and interpreted them in light of a global kinetic model for NO synthesis. Single turnover reactions with ferrous dNOSoxy showed it could convert Arg to N'omega-hydroxy-l-arginine (NOHA), or NOHA to citrulline and NO, when it was given 6R-tetrahydrobiopterin and O2. The dNOSoxy catalyzed Arg hydroxylation and NOHA oxidation at rates that matched or exceeded the rates catalyzed by the three mammalian NOSoxy enzymes. Consecutive heme-dioxy, ferric heme-NO, and ferric heme species were observed in the NOHA reaction of dNOSoxy, indicating that its catalytic mechanism is the same as in the mammalian NOS. However, NO dissociation from dNOSoxy was 4 to 9 times faster than that from the mammalian NOS enzymes. In contrast, the dNOSoxy ferrous heme-NO complex was relatively unreactive toward O2 and in this way was equivalent to the mammalian neuronal NOS. Our data show that dNOSoxy has unique settings for the kinetic parameters that determine its NO synthesis. Computer simulations reveal that these unique settings should enable dNOS to be a more efficient and active NO synthase than the mammalian NOS enzymes, which may allow it to function more broadly in cell signaling and immune functions in the fruit fly.     

Reductase domain of Drosophila melanogaster nitric-oxide synthase: redox transformations, regulation, and similarity to mammalian homologues

The nitric oxide synthase of Drosophila melanogaster (dNOS) participates in essential developmental and behavioral aspects of the fruit fly, but little is known about dNOS catalysis and regulation. To address this, we expressed a construct comprising the dNOS reductase domain and its adjacent calmodulin (CaM) binding site (dNOSr) and characterized the protein regarding its catalytic, kinetic, and regulatory properties. The Ca2+ concentration required for CaM binding to dNOSr was between that of the mammalian endothelial and neuronal NOS enzymes. CaM binding caused the cytochrome c reductase activity of dNOSr to increase 4 times and achieve an activity comparable to that of mammalian neuronal NOS. This change was associated with decreased shielding of the FMN cofactor from solvent and an increase in the rate of NADPH-dependent flavin reduction. Flavin reduction in dNOSr was relatively slow following the initial 2-electron reduction, suggesting a slow inter-flavin electron transfer, and no charge-transfer complex was observed between bound NADP+ and reduced FAD during the process. We conclude that dNOSr catalysis and regulation is most similar to the mammalian neuronal NOS reductase domain, although differences exist in their flavin reduction behaviors. The apparent conservation between the fruit fly and mammalian enzymes is consistent with dNOS operating in various signal cascades that involve NO.     

Why biomanipulation can be effective in peaty lakes

The effects of fish stock reduction (biomanipulation) was studied in an 85 ha shallow peaty turbid lake. The lake cleared in a 4-week period in April–May 2004, which demonstrated that biomanipulation can be effective in peaty lakes. We demonstrated that it is possible to reduce the fish stock to <25 kg ha⁻¹ benthivorous fish and <15 kg ha⁻¹ planktivorous fish, sufficiently low to switch the lake from a turbid to a clear state. Knowledge of lake morphology, fish stock, fish behaviour, and a variety of fishing methods was necessary to achieve this goal. It is expected that continuation of fisheries to remove young of the year planktivorous species is needed for several years, until macrophytes provide sufficient cover for zooplankton and can compete with phytoplankton. Cladocerans developed strongly after fish removal. The clearing of the lake coincided with a sudden decrease of filamentous cyanobacteria and suspended detritus, and a strong increase of Bosmina. We assume that Bosmina was able to reduce filamentous prokaryotes and detritus. After the disappearance of the cyanobacteria, Bosmina disappeared too. After the clearing of the lake Daphnia dominated in zooplankton and apparently was able to keep phytoplankton levels low. In our case, wind resuspension did not prevent biomanipulation from being successful. No correlation between windspeed and turbidity was found, neither in an 85 ha nor in a 230 ha shallow peaty lake. Regression analysis showed that on average 50% of the amount of suspended detritus can be explained by resuspension by fish and 50% by phytoplankton decomposition. The main goal of this biomanipulation experiment, clear water and increased submerged plant cover in a shallow peaty lake, was reached.     

Temporal patterns of occurrence and transmission of the blood parasite Haemoproteus payevskyi in the great reed warbler Acrocephalus arundinaceus

We studied the prevalence and intensity of the haemosporidian blood parasite Haemoproteus payevskyi in great reed warblers at Lake Kvismaren (6 years) and Lake Segersj? (3 years) in Sweden. Based on microscopic inspection of slides from 282 adult birds, 20.6% showed infection of H. payevskyi in circulating red blood cells in at least 1 year. For parasite prevalence, there was no difference between years, sex, and age classes. However, parasite intensity was higher in females than in males, and this was most pronounced in 1-year-old birds. Individuals scored to carry parasites in year _n were more likely to show parasite infection year _n + 1 than birds scored to be parasite-free in year _n . None of 99 juvenile birds examined at the breeding site in late summer, 4–9 weeks after hatching, showed infection of H. payevskyi. Parasite intensity in infected adult birds decreased in the course of the breeding season and no new or relapse infections were observed during this period. Thus, our data imply that in the great reed warbler, a long-distance migrant to tropical Africa, transmission of H. payevskyi occurs on wintering sites or at stopover sites during migration.     

Iteron-binding ORF157 and FtsZ-like ORF156 proteins encoded by pBtoxis play a role in its replication in Bacillus thuringiensis subsp. israelensis

We recently identified a minireplicon of pBtoxis from Bacillus thuringiensis subsp. israelensis that contained an operon encoding two novel proteins (ORF156 and ORF157), both of which are required for replication. ORF157 contains a helix-turn-helix motif and shares no homology with known plasmid replication proteins (Rep), and ORF156 contains the signature motif present in FtsZ/tubulin proteins, the latter of which are known to function in cell division and chromosome segregation. Here we show that the minimal sequence composed of four 12-bp imperfect direct repeats (iterons) in the pBtoxis minireplicon was sufficient to replicate a reporter plasmid in B. thuringiensis subsp. israelensis when ORF156 and ORF157 functions were provided in trans. To further investigate the roles of ORF156 and ORF157 in pBtoxis replication, six-histidine-tagged recombinant rORF156 and rORF157 proteins were purified from Escherichia coli and used in electrophoretic mobility shift assays. Our results demonstrated that rORF157, but not rORF156, binds specifically to the pBtoxis iterons, suggesting that ORF157 functions as a Rep protein. Although rORF156 did not bind to the iteron sequence, we showed that it bound to rORF157-DNA complexes. In addition, we showed that rORF156 has GTPase activity characteristic of the FtsZ/tubulin superfamily of proteins. Taken together, these results suggest that the iterons compose the minimal replication origin (ori) of pBtoxis and that ORF157 and ORF156 are involved in the initiation of pBtoxis replication and possibly in the segregation and partitioning of this plasmid to daughter cells.     

Identification of critical residues in the propeptide of LasA protease of Pseudomonas aeruginosa involved in the formation of a stable mature protease

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.