Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells

Thiamine-dependent enzymes (TDEs) control metabolic pathways that are frequently altered in cancer and therefore present cancer-relevant targets. We have previously shown that the recombinant enzyme thiaminase cleaves and depletes intracellular thiamine, has growth inhibitory activity against leukemia and breast cancer cell lines, and that its growth inhibitory effects were reversed in leukemia cell lines by rapamycin. Now, we first show further evidence of thiaminase therapeutic potential by demonstrating its activity against breast and leukemia xenografts, and against a primary leukemia xenograft. We therefore further explored the metabolic effects of thiaminase in combination with rapamycin in leukemia and breast cell lines. Thiaminase decreased oxygen consumption rate and increased extracellular acidification rate, consistent with the inhibitory effect of acute thiamine depletion on the activity of the TDEs pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes; these effects were reversed by rapamycin. Metabolomic studies demonstrated intracellular thiamine depletion and the presence of the thiazole cleavage product in thiaminase-treated cells, providing validation of the experimental procedures. Accumulation of ribose and ribulose in both cell lines support the thiaminase-mediated suppression of the TDE transketolase. Interestingly, thiaminase suppression of another TDE, branched chain amino ketoacid dehydrogenase (BCKDH), showed very different patterns in the two cell lines: in RS4 leukemia cells it led to an increase in BCKDH substrates, and in MCF-7 breast cancer cells it led to a decrease in BCKDH products. Immunoblot analyses showed corresponding differences in expression of BCKDH pathway enzymes, and partial protection of thiaminase growth inhibition by gabapentin indicated that BCKDH inhibition may be a mechanism of thiaminase-mediated toxicity. Surprisingly, most of thiaminase-mediated metabolomic effects were also reversed by rapamycin. Thus, these studies demonstrate that acute intracellular thiamine depletion by recombinant thiaminase results in metabolic changes in thiamine-dependent metabolism, and demonstrate a previously unrecognized role of mTOR signaling in the regulation of thiamine-dependent metabolism.     

Synthesis and Triplex Forming Properties of an Acyclic N7-Glycosylated Guanine Nucleoside

Abstract

A chiral acyclic nucleoside, one in which the ribose carbohydrate has been replaced with a glycerol-based linker, is prepared by glycosylating guanine at the N⁷-nitrogen. The stereochemically pure derivative is converted to a DMT-protected phosphoramidite for incorporation into DNA sequences. Sequence containing the acyclic N⁷-dG nucleoside are capable of forming DNA triplexes in which it is likely that the N¹-H and N²-amino groups of the N⁷-dG are involved in recognition of the guanine base in G-C base pairs.     

Isolation of the β-galactosphingolipid coniferoside using a tumor cell proteome reverse affinity protocol

New approaches are vital to the development of marine natural products (MNP) as therapeutic leads. One of the more time consuming aspects of MNP research arises in the connection between structure and function. Here, we describe an isolation protocol that adapts tumor cell proteomes as a vehicle for MNP isolation therein uniting structural and functional analysis. Application of this method to extracts of the sponge Agelas conifera led to the isolation of a unique poly-hydroxybutyrated β-galactosphingolipid, coniferoside.     

Evaluation of NTF1836 as an inhibitor of the mycothiol biosynthetic enzyme MshC in growing and non-replicating Mycobacterium tuberculosis

The mycothiol biosynthesis enzyme MshC catalyzes the ligation of cysteine with the pseudodisaccharide GlcN-Ins and has been identified as an essential enzyme in Mycobacterium tuberculosis. We now report on the development of NTF1836 as a micromolar inhibitor of MshC. Using commercial libraries, we conducted preliminary structure-activity relationship (SAR) studies on NTF1836. Based on this data, NTF1836 and five structurally related compounds showed similar activity towards clinical strains of M. tuberculosis. A gram scale synthesis was developed to provide ample material for biological studies. Using this material, we determined that inhibition of M. tuberculosis growth by NTF1836 was accompanied by a fall in mycothiol and an increase in GlcN-Ins consistent with the targeting of MshC. We also determined that NTF1836 kills non-replicating M. tuberculosis in the carbon starvation model of latency.     

Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti

The recent global Zika epidemics have revealed the significant threat that mosquito-borne viruses pose. There are currently no effective vaccines or prophylactics to prevent Zika virus (ZIKV) infection. Limiting exposure to infected mosquitoes is the best way to reduce disease incidence. Recent studies have focused on targeting mosquito reproduction and immune responses to reduce transmission. Previous work has evaluated the effect of insulin signaling on antiviral JAK/STAT and RNAi in vector mosquitoes. Specifically, insulin-fed mosquitoes resulted in reduced virus replication in an RNAi-independent, ERK-mediated JAK/STAT-dependent mechanism. In this work, we demonstrate that targeting insulin signaling through the repurposing of small molecule drugs results in the activation of both RNAi and JAK/STAT antiviral pathways. ZIKV-infected Aedes aegypti were fed blood containing demethylasterriquinone B1 (DMAQ-B1), a potent insulin mimetic, in combination with AKT inhibitor VIII. Activation of this coordinated response additively reduced ZIKV levels in Aedes aegypti. This effect included a quantitatively greater reduction in salivary gland ZIKV levels up to 11 d post-bloodmeal ingestion, relative to single pathway activation. Together, our study indicates the potential for field delivery of these small molecules to substantially reduce virus transmission from mosquito to human. As infections like Zika virus are becoming more burdensome and prevalent, understanding how to control this family of viruses in the insect vector is an important issue in public health.     

Wood properties and chemical composition of the eccentric growth branch of Viburnum odoratissimum var. awabuki

To clarify the wood properties and chemical composition of branches of Viburnum odoratissimum produced by unusual eccentric growth, we investigated growth strain (GS), basic density (D _b), microfibril angle (MFA), elastic moduli (E _L and E _L/D _b), creep deformation, cellulose crystalline features, and lignin structure in upper and lower sides of the branches, and considered the correlations among these factors. In most measuring positions, the distribution of GS showed that higher tensile GS was in the upper side and compressive GS was in the lower side of the branch, which combines GS features of reaction wood. However, the generation of GS in the lower side was different from that in compression wood, because E _L/D _b and MFA had a negative correlation. The creep compliance curves show that the upper-side wood had low rigidity and high viscosity, whereas the lower-side wood had large rigidity and low viscosity. Relative creep had a negative relation with MFA in the upper side, which is unusual. The cellulose crystalline features showed no obvious difference between both sides of the branch; however, the lignin with less β-O-4 proportion and less S units but more G units seemed to exist in the lower side because of a decreased syringyl/guaiacyl (S/G) molar ratio. This suggests that cell wall could be reinforced by lignin resulting in lower viscosity in the lower side of the branch. Additionally, the S/G ratio showed a relatively high correlation with GS in the lower side. These results suggest that lignin structure plays an important role in adapting to environmental changes during eccentric growth for V. odoratissimum.     

Acyclovir triphosphate is a suicide inactivator of the herpes simplex virus DNA polymerase

The triphosphate form of 9-[(2-hydroxyethoxy)-methyl]guanine (acyclovir), ACVTP, inactivates the herpes simplex virus type 1 DNA polymerase. ACVTP does not innately inactivate resting polymerase, but becomes an inactivator only while being processed as an alternative substrate. Pseudo first-order rates of inactivation were measured at varying concentrations of ACVTP and fixed concentrations of the natural substrate, deoxyguanosine triphosphate. These studies indicated that a reversible enzyme-ACVTP (Michaelis-type) complex is formed at the active site prior to inactivation. The formation of this complex was competitively retarded by deoxyguanosine triphosphate. An apparent dissociation constant (KD) of 3.6 +/- 0.2 (S.D.) nM was determined for ACVTP from this reversible complex. A second method for the estimation of the KD which used the extrapolated initial velocities produced a value of 5.9 +/- 0.4 (S.D.) nM. The rate of conversion of the reversible complex to the inactivated complex, at saturating ACVTP, was calculated to be 0.24 min-1. No reactivation of enzyme activity was detected following isolation of the inactivated complex by rapid desalting on Sephadex G-25. Under these conditions, an overall reactivation rate of 1.5 X 10(-5) min-1 could have been easily detected. Therefore, the overall inhibition constant must have been less than 3 pM. In contrast, when host DNA polymerase alpha was incubated with 14 microM ACVTP, only 60% inhibition of enzyme activity was observed, but inactivation was not detected. These data indicate that ACVTP functions as a suicide inactivator of the herpes simplex virus type 1 DNA polymerase, and is only a weak reversible inhibitor of DNA polymerase alpha.     

Mn porphyrin-based superoxide dismutase (SOD) mimic, MnIIITE-2-PyP5+, targets mouse heart mitochondria

The Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnIIITE-2-PyP5+ (AEOL-10113) has proven effective in treating oxidative stress-induced conditions including cancer, radiation damage, diabetes, and central nervous system trauma. The ortho cationic pyridyl nitrogens of MnTE-2-PyP5+ are essential for its high antioxidant potency. The exceptional ability of MnIIITE-2-PyP5+ to dismute O2.- parallels its ability to reduce ONOO- and CO3-. Decreasing levels of these species are considered its predominant mode of action, which may also involve redox regulation of signaling pathways. Recently, Ferrer-Sueta at al. (Free Radic. Biol. Med. 41:503-512; 2006) showed, with submitochondrial particles, that>or=3 microM MnIIITE-2-PyP5+ was able to protect components of the mitochondrial electron transport chain from peroxynitrite-mediated damage. Our study complements their data in showing, for the first time that micromolar mitochondrial concentrations of MnIIITE-2-PyP5+ are obtainable in vivo. For this study we have developed a new and sensitive method for MnIIITE-2-PyP5+ determination in tissues. The method is based on the exchange of porphyrin Mn2+ with Zn2+, followed by the HPLC/fluorescence detection of ZnIITE-2-PyP4+. At 4 and 7 h after a single 10 mg/kg intraperitoneal administration of MnIIITE-2-PyP5+, the mice (8 in total) were anesthetized and perfused with saline. Mitochondria were then isolated by the method of Mela and Seitz (Methods Enzymol.55:39-46; 1979). We found MnIIITE-2-PyP5+ localized in heart mitochondria to 2.95 ng/mg protein. Given the average value of mitochondrial volume of 0.6 microL/mg protein, the calculated MnIIITE-2-PyP5+ concentration is 5.1 microM, which is sufficient to protect mitochondria from oxidative damage. This study establishes, for the first time, that MnIIITE-2-PyP5+, a highly charged metalloporphyrin, is capable of entering mitochondria in vivo at levels sufficient to exert there its antioxidant action; such a result encourages its development as a prospective therapeutic agent.     

How to put all your eggs in one basket: empirical patterns of offspring provisioning throughout a mother's lifetime

Maternal effects arise when a mother's phenotype or the environment she experiences influences the phenotype of her progeny. Most studies of adaptive maternal effects are a "snapshot" of a mother's lifetime offspring provisioning and do not generally consider the effects of earlier siblings on those produced later. Here we show that in soil mites, offspring provisioning strategies are dynamic, changing from an emphasis on egg number in young females to egg size in older females. This pattern may be adaptive if it increases the survival of younger offspring that must compete with older, larger siblings. The dynamic shift in egg provisioning was greater in high-food environments in which females lived longer, creating increasing asymmetry in offspring competitive abilities. Females reared in isolation and in the presence of a high-density colony had identical provisioning strategies, suggesting that, unlike males in this species, females do not use pheromones to assess colony size. Our findings suggest that the adaptive significance of maternal effects may be misinterpreted when studies consider only a snapshot of a female's offspring provisioning strategy or when components of the offspring provisioning strategy are studied in isolation.     

Spatiotemporal railway use by grizzly bears in Canada's Rocky Mountains

Railway networks contribute to the direct mortality of wildlife through collisions with trains, which can threaten vulnerable wildlife populations even in protected areas, including grizzly bears (Ursus arctos) in Banff and Yoho National Parks, Canada. Mitigation to reduce bear-train collisions requires information about how grizzly bears use the railway spatially and temporally and how particular types of use might increase collision vulnerability. We used data from 27 grizzly bears fitted with global positioning system (GPS) collars between 2000 and 2016 to relate railway use by bears via resource selection functions to variables that described land cover, human use, and topography. We used the same suite of explanatory variables to distinguish pairs of 4 types of steps, in which 3 successive GPS points (with 2-hr fix rates) included ≥1 within 30 m of the rail (hereafter on) and 2 others that defined locations where bears effectively entered the railway (first fix off rail, next 2 on), crossed it (only the middle fix on the rail), continued along the railway (all 3 fixes on), or exited the railway corridor (first 2 on, last off). We compared both sites of higher use and each of these 4 step types to the relative frequency of bear-train collisions, predicting a positive correlation for continue step types. Relative to available locations, bears were more likely to use the railway close to railroad sidings (sections of twinned track where trains sometimes stop), at intermediate distances from human-use features (e.g., town sites, highways, trails), in areas with lower values of the compound topographic index (a proxy for wetness; within 500 m), and within 90 m of rugged terrain. Seasonally, bears made greater use of the railway in spring and fall. Among 1,515 sequences of 3 steps, crossing locations comprised >50% and were most distinct from continue locations (about 20%), which occurred in areas with more rugged terrain (within 300 m), closer to railway sidings, in spring and fall, and with steps that were 60% shorter. Contrary to our prediction, past reports of bear-train collisions were negatively correlated with continue locations and unrelated to overall use or any other movement type. Our results suggest that railway use by bears increased where it provided increased forage or easier travel, particularly in spring and fall, but more work will be needed to determine the mechanistic basis of bear-train collisions. Meanwhile, mitigation efforts such as habitat alteration or warning systems might target locations where past strikes are concentrated for grizzly bears or other sensitive populations. © 2019 The Wildlife Society.