Increased Cytotoxicity and Decreased In Vivo Toxicity of FdUMP[10] Relative to 5-FU

Abstract

The efficacy of treatment with 5-Fluorouracil (5-FU) is limited, in part, by its inefficient conversion to 5-Fluoro-2′-deoxyuridine-5-O-monophosphate (FdUMP). We present data indicating that FdUMP[10], designed as a pro-drug for intracellular release of FdUMP, is cytotoxic as a consequence of uptake of the multimeric form. FdUMP[10] is stable in cell culture medium, with more than one-half of the material persisting as multimers of at least six nucleotides after a 48 h incubation at 37°C. FdUMP[10] is more than 400 times more cytotoxic than 5-FU towards human colorectal tumor cells (H630). FdUMP[10] also has decreased toxicity in vivo, with doses as high as 200 mg/kg/day (qdx3) administered to Balb/c mice without morbidity, compared to a maximum tolerated dose of 45 mg/kg/day for 5-FU using the same protocol. FdUMP[10] shows reduced sensitivity to OPRTase-and TK-mediated drug resistance, relative to 5-FU and FdU, respectively, and is much more cytotoxic than 5-FU towards cells that overexpress thymidylate synthase. Thus, FdUMP[10] is less susceptible to resistance mechanisms that limit the clinical utility of 5-FU. The increased cytotoxicity, decreased toxicity in vivo, and reduced sensitivity to drug resistance of FdUMP[10], relative to 5-FU, indicates multimeric FdUMP is potentially valuable as an antineoplastic agent, either as a single agent, or in combination with 5-FU.     

Synthesis of (2′,5′)-Oligoadenylate Antisense Chimeras Targeting Steroid 5α-Reductase

Abstract

2–5A antisense chimeras have been synthesized which target human steroid 5α-reductase mRNA. To enhance the stability of the chimera towards degradative enzymes the terminal phosphodiester bond was isomerized from 3′,5′ to 3′,3′ and the 5′-phosphate group was thiolated.     

A Solid-Phase Synthesis of 2′,5′-Linked Oligoadenylates (2-5A)

Abstract

2′,5′-Oligoadenylate 5′-triphosphates (2-5A) as products of 2-5A synthetase and activators of ribonuclease L (RNase L), are mediators in one of the mechanisms of interferon′s antiviral action. Upon activation, RNase L inhibits protein synthesis due to the degradation of RNAs. This activity of 2-5A could possibly find an application in virus or cancer chemotherapy, but two major barriers prevent the use of 2′,5′-linked oligoadenylates as therapeutic agents. The 2-5A is readily degraded by a 2′,5′ phosphodiesterase and as a highly negatively charged molecule, is not readily taken up by cells. One possible solution to this latter limitation might be found in chemical modifications of the 2-5A structure. Many analogues of 2-5A have been already obtained with modified base, ribose or phosphate moieties. While these have provided some important information about the enzyme- activator interactions, the cell permeability problem still remains unsolved. One of the major obstacles in this study is lack of a convenient method of synthesis of 2′,5′ ribonucleotides of widely varying structure.     

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small “scars” on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of “provisional clot”-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.     

Solution structures of Mycobacterium tuberculosis thioredoxin C and models of intact thioredoxin system suggest new approaches to inhibitor and drug design

Here, we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C‐terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared with the solution structure. On the basis of these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti‐mycobacterial agents, or as chemical genetic probes of function. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Experimental infections of Orchitophrya stellarum (Scuticociliata) in American blue crabs (Callinectes sapidus) and fiddler crabs (Uca minax)

Outbreaks of an unidentified ciliate have occurred on several occasions in blue crabs from Chesapeake Bay held during winter months in flow-through systems. The parasite was initially thought to be Mesanophrys chesapeakensis, but molecular analysis identified it as Orchitophyra stellarum, a facultative parasite of sea stars (Asteroidea). We investigated the host-parasite association of O. stellarum in the blue crab host. Crabs were inoculated with the ciliate, or they were held in bath exposures after experimentally induced autotomy of limbs in order to determine potential mechanisms for infection. Crabs inoculated with the ciliate, or exposed to it after experimental autotomy, rapidly developed fatal infections. Crabs that were not experimentally injured, but were exposed to the ciliate, rarely developed infections; thus, indicating that the parasite requires a wound or break in the cuticle as a portal of entry. For comparative purposes, fiddler crabs, Uca minax, were inoculated with the ciliate in a dose-titration experiment. Low doses of the ciliate (10 per crab) were sometimes able to establish infections, but high intensity infections developed quickly at doses over 500 ciliates per crab. Chemotaxis studies were initiated to determine if the ciliate preferentially selected blue crab serum (BCS) over other nutrient sources. Cultures grown on medium with BCS or fetal bovine serum showed some conditioning in their selection for different media, but the outcome in choice experiments indicated that the ciliate was attracted to BCS and not seawater. Our findings indicate that O. stellarum is a facultative parasite of blue crabs. It can cause infections in exposed crabs at 10–15 °C, but it requires a portal of entry for successful host invasion, and it may find injured hosts using chemotaxis.     

Effects of Soil Compaction and Meloidogyne incognita on Cotton Root Architecture and Plant Growth

The effects of a soil hardpan and Meloidogyne incognita on cotton root architecture and plant growth were evaluated in microplots in 2010 and 2011. Soil was infested with M. incognita at four different levels with or without a hardpan. The presence of a hardpan resulted in increased plant height, number of main stem nodes, and root fresh weight for cotton seedlings both years. Meloidogyne incognita decreased height and number of nodes for seedlings in 2010. Nematode infestation increased seedling root length and enhanced root magnitude, altitude, and exterior path length in 2010. This was also the case for root length and magnitude in 2011 at lower infestation levels suggesting compensatory growth. A hardpan had no consistent effect on these root parameters but increased root volume in both years. A hardpan hastened crop maturity and increased the number of fruiting branches that were produced, while M. incognita infection delayed crop development and reduced plant height and number of bolls. Both M. incognita infection and a hardpan reduced taproot length and root dry weight below the hardpan in both years. Root topological indices under all the treatments ranged from 1.71 to 1.83 both years indicating that root branching followed a herringbone pattern. The techniques for characterizing root architecture that were used in this study provide a greater understanding of changes that result from disease and soil abiotic parameters affecting root function and crop productivity.     

A 3-D geometric morphometric study of intraspecific variation in the ontogeny of the temporal bone in modern Homo sapiens

This study addresses how the human temporal bone develops the population-specific pattern of morphology observed among adults and at what point in ontogeny those patterns arise. Three-dimensional temporal bone shape was captured using 15 landmarks on ontogenetic series of specimens from seven modern human populations. Discriminant function analysis revealed that population-specific temporal bone morphology is evident early in ontogeny, with significant shape differences among many human populations apparent prior to the eruption of the first molar. As early as five years of age, temporal bone shape reflects population history and can be used to reliably sort populations, although those in closer geographic proximity and molecular affinity are more likely to be misclassified. The deviation of cold-adapted populations from this general pattern of congruence between temporal bone morphology and genetic distances, identified in previous work, was confirmed here in adult and subadult specimens, and was revealed to occur earlier in ontogeny than previously recognized. Significant differences exist between the ontogenetic trajectories of some pairs of populations, but not among others, and the angles of these trajectories do not reflect genetic relationships or final adult temporal bone size. Significant intrapopulation differences are evident early in ontogeny, with differences becoming amplified by divergent trajectories in some groups. These findings elucidate how the congruence between adult human temporal bone morphology and population history develops, and reveal that this pattern corresponds closely to that described previously for facial ontogeny.     

Dramatic phenotypic plasticity within species of Siphomutabilus n. g. (Digenea: Cryptogonimidae) from Indo-Pacific caesionines (Perciformes: Lutjanidae)

A survey of Indo-Pacific lutjanids of the subfamily Caesioninae revealed the presence of Siphodera gurukun Machida, 1910 and two new cryptogonimid taxa from off Heron and Lizard Islands on the Great Barrier Reef, Australia, Ningaloo Reef, Western Australia and Rasdhoo Atoll, Maldives. A combined morphological and genetic characterisation of these species shows that they form a clade distinct from the type-species of Siphodera Linton, 1910, S. vinaledwardsii (Linton, 1901), and warrants the proposal of a new genus. Here we propose Siphomutabilus n. g. and transfer Siphodera gurukun Machida, 1986 as the type-species, Siphomutabilus gurukun (Machida, 1986) n. comb. Siphodera aegyptensis Hassanine & Gibson, 2005 is transferred to Siphomutabilus as S. aegyptensis (Hassanine & Gibson, 2005) n. comb. based on morphological and ecological similarities. Siphomutabilus raritas n. sp. is described from Caesio cuning (Bloch) off Lizard Island and S. bitesticulatus n. sp. is described from Pterocaesio marri Schultz off Heron Island. The two new species are unique in that they have two testes, making their morphology broadly consistent with that of Metadena Linton, 1910, yet the molecular analyses conducted here indicates that they are unequivocally united with Siphomutabilus gurukun (which has multiple testes) to the exclusion of Metadena lutiani (Yamaguti, 1942), which was sequenced here. The dramatic phenotypic plasticity observed among such closely related species of Siphomutabilus suggests a secondary modification of what is generally considered a robust generic diagnostic character within this and other digenean families, highlighting the need for a combined morphological and molecular diagnostic approach when characterising these taxa. Siphodera Linton, 1910 is amended to include just two species, the type-species S. vinaledwardsii (Linton, 1901) Linton, 1910 and S. cirrhiti Yamaguti, 1970, which are distinguished by their lack of oral spines and multiple testes that are primarily extracaecal. Siphodera ghanensis Fischthal & Thomas, 1968 is considered a species incertae sedis here based on significant morphological and ecological differences compared with species of Siphodera and Siphomutabilus n. g.     

Synthesis and characterization of 123I-CMICE-013: A potential SPECT myocardial perfusion imaging agent

Coronary artery disease (CAD) is a major cause of death in Canada and the United States. Single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) is a useful diagnostic test in the management of patients with CAD. The widely used SPECT MPI agents, ^99mTc sestamibi and ^99mTc tetrofosmin, exhibit less than ideal pharmacokinetic properties with decreasing uptake with higher flows. ¹²³I has a similar energy as ^99mTc, an ideal half life, and is readily available from cyclotrons. The objective of this study was to develop an ¹²³I labeled MPI agent based on rotenone, a mitochondrial complex I inhibitor, as an alternative to currently available SPECT MPI agents. Methods: ¹²³I-CMICE-013 was synthesized by radiolabeling rotenone with ¹²³I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60 °C for 45 min, followed by RP-HPLC purification. The product was formulated in 5% EtOH in 10 mM NaOAc pH 6.5. The inactive analog ¹²⁷I-CMICE-013 was isolated and characterized by NMR and mass spectrometry, and the structure determined. Micro-SPECT imaging studies were carried out in normal and infarcted rats. Biodistribution studies were performed in normal rats at 2 h (n = 6) and 24 h (n = 8) post injection (p.i.). Results: ¹²³I-CMICE-013 was isolated with >95% radiochemical purity and high specific activity (14.8–111 GBq/μmol; 400–3000 mCi/μmol). Structural analysis showed that rotenone was iodinated at 7′-position, with removal of the 6′,7′-double bond, and addition of a hydroxy group at 6′-position. MicroSPECT images in normal rats demonstrated homogeneous and sustained myocardial uptake with minimal interference from lung and liver. Absent myocardial perfusion was clearly identified in rats with permanent left coronary artery ligation and ischemia-reperfusion injury. In vivo biodistribution studies in normal rats at 2 h p.i. showed significant myocardial uptake (2.01 ± 0.48%ID/g) and high heart to liver (2.98 ± 0.93), heart to lung (4.11 ± 1.04) and heart to blood (8.37 ± 3.97) ratios. At 24 h p.i., the majority of ¹²³I-CMICE-013 was cleared from tissues, and a significant amount of tracer was found in the thyroid, indicating in vivo deiodination of the tracer. Conclusion: ¹²³I-CMICE-013 is a promising new radiotracer for SPECT MPI with high myocardial uptake, very good target to background ratios and favorable biodistribution characteristics.