Simvastatin, Its Antimicrobial Activity and Its Prevention of Alzheimer's Disease.

Simvastatin, a blockbuster drug for treating hypercholesterolemia, has multifactorial benefits as an antimicrobial agent and plays a preventative role in reducing the incidence of Alzheimer's Disease (AD). Although most of the beneficial effects of simvastatin have been attributed to its ability to reduce cholesterol levels, recent scientific studies have suggested that its benefits are largely due to its pleiotropic effects in targeting other pathways, e.g., by inhibiting protein lipidation. There are certain pleiotropic effects that can be predicted from the inhibition of the mevalonate pathway; however, some of the effects of simvastatin in proteostasis lead to reduced levels of amyloid beta, the key contributor to AD. This review discusses the use of simvastatin as an antimicrobial agent and anti-AD drug.

Molecular Mechanisms of Alzheimer's Disease III.

This Special Issue of IJMS is the third in the series: Molecular Mechanisms of Alzheimer's Disease [...].

Siccibacter turicensis from Kangaroo Scats: Possible Implication in Cellulose Digestion.

Microbiota in the kangaroo gut degrade cellulose, contributing to the kangaroo's energy and survival. In this preliminary study, to discover more about the gut microbes that contribute to the survival of kangaroos, cellulose-degrading bacteria were isolated from kangaroo scats by selection on solidified media containing carboxymethyl cellulose as the main carbon source. One frequently occurring aerobic bacterium was Siccibacter turicensis, a microbe previously isolated in fruit powder and from a patient with angular cheilitis. The whole genome sequence of the kangaroo isolate was obtained using the Illumina MiSeq platform. Its sequence shared 97.98% identity of the S. turicensis Type strain, and the ability of the Type strain to degrade cellulose was confirmed. Analysis of the genomic data focused on the cellulose operon. In addition to genes from the operon, we suggest that a gene following the operon may have an important role in regulating cellulose metabolism by signal transduction. This is the first report of S. turicensis found within microbiota of the animal gut. Because of its frequent presence in the kangaroo gut, we suggest that S. turicensis plays a role in cellulose digestion for kangaroos.

Polyphasic Characterisation of Cedecea colo sp. nov., a New Enteric Bacterium Isolated from the Koala Hindgut.

The Cedecea genus is comprised of six rarely isolated species within the Enterobacteriaceae family. Representatives are Gram-negative motile bacilli, and are typically oxidase-negative, lipase-positive and resistant to colistin and cephalothin. In this study, a putative novel Cedecea species (designated strain ZA_0188T), isolated from the koala hindgut, was characterised using a polyphasic taxonomic approach. Maximum average nucleotide identity (ANI) and 16S ribosomal RNA (rRNA) similarity scores well below thresholds of species demarcation were reported, at 81.1% and 97.9%, respectively. Multilocus phylogenetic analysis indicated strain ZA_0188T was most similar to but divergent from recognised Cedecea species. The isolate's genomic G+C content was determined as 53.0 mol%, >1% lower than previously reported in Cedecea. Phenotypically, strain ZA_0188T was distinct from recognised Cedecea species such as colistin- and cephalothin-sensitive, lipase-, sorbitol-, sucrose-, and Voges-Proskauer-negative, and melibiose-, arabinose-, arginine-, and rhamnose-positive. In preliminary experiments, strain ZA_0188T exhibited cellulase activity and high-level tolerance to eucalyptus oil compared to other enteric species surveyed. Collectively, these findings suggest that strain ZA_0188T represents a novel enteric species, for which the name Cedecea colo is proposed.

Yeast Model of Amyloid-ß and Tau Aggregation in Alzheimer's Disease.

The amyloid-ß peptide (Aß) and the phosphorylated protein tau have been widely implicated in Alzheimer's disease and are the focus of most research. Both agents have been extensively studied in mammalian cell culture and in animal studies, but new research is focusing on yeast models. Yeast are eukaryotes, just like us, and are amenable to effects and expression of Aß and tau and appear able to 'report' with considerable relevance on the effects of these biomolecules. The use of yeast enables powerful new approaches to understanding how to overcome the effects of Aß and tau, and such advances could lead to new therapies to prevent the progression of Alzheimer's disease.

Quorum protection, growth and survival.

For the growth of a cell culture, one inoculates not with one cell but with a quorum of cells. This most often a requirement, not just a convenience, and most of us take this for granted without question. Here this observation is re-examined to understand why a quorum may be required to grow cells. The importance of quorums may be widespread in the aspects of microbiology they affect. It is very likely that quorums are connected with and have a large impact on the determination of Minimal Inhibitory Concentrations. It is also possible that low cell density may adversely affect cell survival, however, this is an area where even less is known. The need for a quorum might affect other aspects of microbial cell culture, cell isolation and cell preservation. Effects also extend to mammalian cell culture. Here I seek to review studies that have been documented and speculate on how the information might be utilized in the future.

Exogenous folates stimulate growth and budding of Candida glabrata.

Folate, vitamin B9, is well recognized as being essential for cell growth. The utilization of folate is common to all cells, but the source of it may be quite different. For example, mammalian cells depend on exogenous uptake of folates, while plants and microbes can synthesize them. There has been little consideration of uptake of folate in microbial cells, and studies on the effects of folates in mammalian cells, where conditions are restricted. This study shows that exogenous folates (folic acid or folinic acid), causes Candida glabrata cells suspended in water alone to undergo two cycles of cell division and to form multiple buds. The effect was limited to cells in the stationary phase and more profound in quiescent cells. These data indicate a novel response of yeast to folates that may increase the utility of yeast as a model to study folate transport and signaling.

Pretreatment of chemically-synthesized Aß42 affects its biological activity in yeast.

The tendency of amyloid ß (Aß42) peptide to misfold and aggregate into insoluble amyloid fibrils in Alzheimer's disease (AD) has been well documented. Accumulation of Aß42 fibrils has been correlated with abnormal apoptosis and unscheduled cell division which can also trigger the death of neuronal cells, while oligomers can also exhibit similar activities. While investigations using chemically-synthesized Aß42 peptide have become common practice, there appear to be differences in outcomes from different preparations. In order to resolve this inconsistency, we report 2 separate methods of preparing chemically-synthesized Aß42 and we examined their effects in yeast. Hexafluoroisopropanol pretreatment caused toxicity while, ammonium hydroxide treated Aß42 induced cell proliferation in both C. glabrata and S. cerevisiae. The hexafluoroisopropanol prepared Aß42 had greater tendency to form amyloid on yeast cells as determined by thioflavin T staining followed by flow cytometry and microscopy. Both quiescent and non-quiescent cells were analyzed by these methods of peptide preparation. Non-quiescent cells were susceptible to the toxicity of Aß42 compared with quiescent cells (p < 0.005). These data explain the discrepancy in the previous publications about the effects of chemically-synthesized Aß42 on yeast cells. The effect of Aß42 on yeast cells was independent of the size of the peptide aggregates. However, the Aß42 pretreatment determined whether the molecular conformation of peptide resulted in proliferation or toxicity in yeast based assays.

Application of yeast to study the tau and amyloid-ß abnormalities of Alzheimer's disease.

The major molecules associated with Alzheimer's disease, the phosphorylated protein tau and the 42 amino acid peptide, amyloid-ß (Aß), have recently been analyzed in yeast. These yeast studies have provided major new insights into the effects of tau and Aß and, at the same time, offered new approaches to rapidly search for chemicals that may be involved in prevention of Alzheimer's disease. The following review summarizes the role of yeast and its contribution in Alzheimer's disease research, and highlights important studies that have been conducted in this model organism.

Lipid constituents of the edible mushroom, Pleurotus giganteus demonstrate anti-Candida activity.

Different solvent extracts of Pleurotus giganteus fruiting bodies were tested for antifungal activities against Candida species responsible for human infections. The lipids extracted from the ethyl acetate fraction significantly inhibited the growth of all the Candida species tested. Analysis by GC/MS revealed lipid components such as fatty acids, fatty acid methyl esters, ergosterol, and ergosterol derivatives. The sample with high amounts of fatty acid methyl esters was the most effective antifungal agent. The samples were not cytotoxic to a mammalian cell line, mouse embryonic fibroblasts BALB/c 3T3 clone A31. To our knowledge, this is the first report of antifungal activity of the lipid components of Pleurotus giganteus against Candida species.

Latrepirdine (dimebon) enhances autophagy and reduces intracellular GFP-Aß42 levels in yeast.

Latrepirdine (Dimebon), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer's disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model, Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of amyloid-ß (Aß)42 aggregates. Latrepirdine was shown to upregulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro green fluorescent protein (GFP) tagged Aß yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aß42. GFP-Aß42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aß42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aß42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aß42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aß42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aß42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aß42.

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor.

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K(d), of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC(50) of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and (15)N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,ß-methylene adenosine 5'-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered--an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including (15)N-(1)H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.

Inhibition of respiratory growth and survival in yeast by dopamine and counteraction with ascorbate or glutathione.

Dopamine is a key monamine neurotransmitter, yet it can also exhibit toxicity to neuronal cells. There are suggestions that dopamine may be neurotoxic due to its propensity to induce the formation of reactive oxygen species, which may in turn adversely affect mitochondrial function and cell viability. In this study, the effects of dopamine or a dopamine reaction product on yeast growth and survival have been explored. Yeast is ideal for such a study because, unlike mammalian cells, yeast cells can be grown even when respiratory function is totally absent. Indeed, dopamine was found to be inhibitory to yeast growth in media where respiratory function was required and cytotoxic to yeast cells suspended in water. The inhibitory effects of dopamine were reduced greatly by the antioxidants ascorbate and glutathione, suggesting the involvement of reactive oxygen species in dopamine-mediated toxicity. It would appear that yeast may offer a convenient model to perform screens for further compounds that may provide protection against dopamine-mediated growth inhibition and toxicity.

Alzheimer's amyloid-beta rescues yeast from hydroxide toxicity.

Amyloid-beta(Abeta42), which is known to be toxic to neuronal cells, protects yeast cells from severe sodium hydroxide toxicity. More than 85% cell death was caused by treatment with 1 mM NaOH and approximately 95% was observed at a 2 mM concentration. However, greater than 55% cells survived the treatment in the presence of Abeta42. A strong protective effect of the peptide was also evident from the differential staining of the treated culture with propidium iodide.

Abeta aggregation and possible implications in Alzheimer's disease pathogenesis.

Amyloid beta protein (Abeta) has been associated with Alzheimer's disease (AD) because it is a major component of the extracellular plaque found in AD brains. Increased Abeta levels correlate with the cognitive decline observed in AD. Sporadic AD cases are thought to be chiefly associated with lack of Abeta clearance from the brain, unlike familial AD which shows increased Abeta production. Abeta aggregation leading to deposition is an essential event in AD. However, the factors involved in Abeta aggregation and accumulation in sporadic AD have not been completely characterized. This review summarizes studies that have examined the factors that affect Abeta aggregation and toxicity. By necessity these are studies that are performed with recombinant-derived or chemically synthesized Abeta. The studies therefore are not done in animals but in cell culture, which includes neuronal cells, other mammalian cells and, in some cases, non-mammalian cells that also appear susceptible to Abeta toxicity. An understanding of Abeta oligomerization may lead to better strategies to prevent AD.

A new method to measure cellular toxicity of non-fibrillar and fibrillar Alzheimer's Abeta using yeast.

The 42 amino acid amyloid-beta (Abeta) can exist in multiple physical states including oligomers and fibrils. This study shows that fibril formation is hastened by the biological buffers required to support the growth of mammalian cells, but is prevented if Abeta is maintained in water. Here we describe a method to produce Abeta in oligomeric form and the comparison of stable fibrillar and non-fibrillar forms in cell toxicity studies in water, achieved through the use of yeast. We show that extracellular, non-fibrillar Abeta causes a dose dependent loss of cell viability while fibrillar Abeta has low toxicity.

Copper transport and Alzheimer's disease.

This brief review discusses copper transport in humans, with an emphasis on knowledge learned from one of the simplest model organisms, yeast. There is a further focus on copper transport in Alzheimer's Disease (AD). Copper homeostasis is essential for the well-being of all organisms, from bacteria to yeast to humans: survival depends on maintaining the required supply of copper for the many enzymes, dependent on copper for activity, while ensuring that there is no excess free copper, which would cause toxicity. A virtual orchestra of proteins are required to achieve copper homeostasis. For copper uptake, Cu(II) is first reduced to Cu(I) via a membrane-bound reductase. The reduced copper can then be internalised by a copper transporter where it is transferred to copper chaperones for transport and specific delivery to various organelles. Of significance are internal copper transporters, ATP7A and ATP7B, notable for their role in disorders of copper deficiency and toxicity, Menkes and Wilson's disease, respectively. Metallothioneins and Cu/Zn superoxide dismutase can protect against excess copper in cells. It is clear too, increasing age, environmental and lifestyle factors impact on brain copper. Studies on AD suggest an important role for copper in the brain, with some AD therapies focusing on mobilising copper in AD brains. The transport of copper into the brain is complex and involves numerous players, including amyloid precursor protein, A beta peptide and cholesterol.

Simvastatin reduces ergosterol levels, inhibits growth and causes loss of mtDNA in Candida glabrata.

Statins are widely used for lowering cholesterol levels through their action on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Yeasts use HMG-CoA reductase for the same enzymatic step as humans, but in yeasts the main end-product of the pathway is ergosterol rather than cholesterol. We considered that insights into the effects of statins in humans could be gained by examination of the effects of simvastatin on the petite-positive yeast Candida glabrata. Simvastatin was found to inhibit growth, and this was associated with lower ergosterol levels. As simvastatin-treated cultures of yeast were passaged, the frequencies of petite cells (respiratory-deficient yeast mutants with deletions in the mitochondrial genome) increased with time and with simvastatin concentration. DNA staining of the petite mutants showed that they were devoid of mtDNA, suggesting a defect in the maintenance of mtDNA. These observations in C. glabrata may provide further insights into the molecular effects of statins in humans undergoing treatment for hypercholesterolemia. In addition, if C. glabrata is a valid model for studying statin treatments, it would be very useful for the preliminary screening of agents to reduce statin side-effects.

Design of endoperoxides with anti-Candida activity.

Broad antifungal structure-activity relationships governing epoxy-endoperoxides 2 and 3 and their parent endoperoxides 1 are reported. Their inhibitory activity against Candida albicans in conjunction with hemolytic activity and/or growth inhibition of cultured mammalian cells are reported. This information provided guidance for the further development of endoperoxide and epoxy-endoperoxides as topical antifungal agents.