Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor’s abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced – indicating viral protein-induced alterations in the cofactors’ binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.     

Stabilization of DNA triple helices by a series of mono- and disubstituted amidoanthraquinones.

We have used quantitative DNase I footprinting to measure the relative affinities of four disubstituted and two monosubstituted amidoanthraquinone compounds for intermolecular DNA triplexes, and have examined how the position of the attached base-functionalized substituents affects their ability to stabilize DNA triplexes. All four isomeric disubstituted derivatives examined stabilize DNA triplexes at micromolar or lower concentrations. Of the compounds studied the 2,7-disubstituted amidoanthraquinone displayed the greatest triplex affinity. The order of triplex affinity for the other disubstituted ligands decreases in the order 2,7 > 1,8 = 1,5 > 2,6, with the equivalent monosubstituted compounds being at least an order of magnitude less efficient. The 1,5-disubstituted derivative also shows some interaction with duplex DNA. These results have been confirmed by molecular modelling studies, which provide a rational basis for the structure-activity relationships. These suggest that, although all of the compounds bind through an intercalative mode, the 2,6, 2,7 and 1,5 disubstituted isomers bind with their two side groups occupying adjacent triplex grooves, in contrast with the 1,8 isomer which is positioned with both side groups in the same triplex groove.     

HISTORICAL BIOGEOGRAPHY IN NORTH AMERICAN ARID REGIONS: AN APPROACH USING MITOCHONDRIAL-DNA PHYLOGENY IN GRASSHOPPER MICE (GENUS ONYCHOMYS)

Restriction-endonuclease-site variation of mitochondrial DNA (mtDNA) was used to investigate patterns of geographic and phylogenetic divergence within the rodent genus Onychomys. Onychomys has occupied arid habitats in the western North American deserts, shrub-steppes, and grasslands since the late Tertiary. A phylogenetic analysis of the total mtDNA restriction-site variation throughout the range of Onychomys suggests that the distribution of this genus has been affected by the same Quaternary pluvial-interpluvial climatic fluctuations that have resulted in the periodic fragmentation of arid habitats in western North America. Onychomys mtDNA haplotypes define at least five discrete geographical subsets, suggesting that there are five areas of endemism for biota restricted to arid and semiarid habitats in North America. The mtDNA-haplotype phylogeny can be used to infer an hypothesis of historical relationships among the five areas of endemism as follows: ([{(Wyoming Basin + Interior Plains + Colorado Plateaus) + (Columbia Basin + Great Basin)} + Gulf Coastal Plain] + Chihuahuan) + Western Deserts. The results of this study point to the potential use of mtDNA-haplotype phylogenies to reconstruct historical biogeographic events in Quaternary time. The utility of mtDNA variation depends in part on the ecology and distribution of the species being examined. Therefore, our hypothesized area cladogram can be tested by investigating regional relationships in other western North American taxa with distributions similar to Onychomys.     

Red–blue plots for detecting clusters in count data

A new index and four new graphical displays, termed "red–blue" plots, are presented to study and measure clustering in spatially referenced count data. The index can detect clusters in the form of patches, comprising several nearby large counts, and in the form of gaps, comprising several nearby small counts. The new methods quantify the degree to which the count for each sample unit contributes towards the overall degree of clustering, either as part of a patch or as a gap; provide tests of nonrandomness to detect clustering; and facilitate a comprehensive definition of the size and dimension of a cluster. The methods are illustrated using aphid field data.     

Restriction fragment length polymorphism at the HLA-C locus

We have used the HLA-C-specific DNA probe pC250 to investigate restriction fragment length polymorphism (RFLP) at the HLA-C locus. Genomic Southern blot hybridization included DNA prepared from a panel of homozygous typing cells representing serological specificities Cw1 to Cw8 and also from samples representing Cw blanks. Although many restriction nucleases failed to reveal any polymorphism, RFLPs were evident with Taq I, Pvu II, Bst XI, Nde 1, and Nci I in addition to the previously reported Eco RI. In the case of Bst XI, a unique RFLP defined a subset of serologically defined Cw blanks. Comparison of RFLP sizes with restriction fragment lengths obtained from the known HLA-Cw3 gene sequence permitted the localization of intragenic C locus RFLLs and the identification of a variable Taq I site in the second intron, a variable Nci I site near the end of the fourth exon, and a variable Pvu lI site in the fifth intron.     

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in Human Plasma and Cerebrospinal Fluid

The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120-500-fold) between total BChE activity in the CSF and the BChE-rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF--both lumbar and ventricular--was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.     

A revision of the family Zoogonidae Odhner, 1902 (Platyhelminthes: Digenea): Subfamily Lepidophyllinae and comments on some aspects of biology

Summary A detailed revision of the zoogonid subfamily Lepidophyllinae is presented, using morphological characters discussed in an earlier paper. Twelve genera and 50 species are treated in detail with keys and cladograms to genera and species. The genera and species covered are: Lepidophyllum steenstrupi, L. appyi, L. armatum, L. brachycladium, L. cameroni, L. pleuronectini, L. pyriforme, L. schantaricum, Urinatrema hispidum, U. hirudinacea, Panopula cavernossa, P. bridgeri, P. spinosa, Limnoderetrema minutum (Manter, 1954) [formerly Deretrema] n.g. (in freshwater fishes; genital pore at oral sucker or pharynx level), n. comb., Brachyenteron peristedioni, B. acropomatis, B. campbelli, B. doederleiniae, B. magnibursatum, B. parexocoeti, B. pycnorganum, Steganodermatoides kergeleni, S. agassizi, S. allocytti, S. maceri, Neosteganoderma glandulosum, N. infundibulum, Proctophantastes abyssorum, P. gillissi, Deretrema (Deretrema) fusillus, D. (D.) cholaeum, D. (D.) pacificum, D. (Spinoderetrema) plotosi, D. (S.) acutum, D. (S.) fellis, D. (S.) ovale, D.(S.) sebastodis, D. (Luxitrema) philippinensis, D. plagiorchis, Pseudochetosoma salmonicola, Overstreetia sodwanaensis, Steganoderma (Steganoderma) formosum, S. (S.) atherinae, S. (Lecithostaphylus) retroflexum, S. (L.) depauperati (Yamaguti, 1970) n. comb., S. (L.) hemirhamphi, S. (L.) nitens, S. (L.) parexocoeti, S. macrophallos, S. oviformis. Most zoogonids were found to exhibit some level of predilection for a particular piscine host group, but little general information on the zoogeography of the group was discovered. Ultrastructural evidence is presented suggesting that the membranous egg-capsule of the zoogonines shows vestiges of the three layers of a normal tanned egg-shell.     

Transport of Ca2+ by Yersinia pestis.

Low-calcium-response, or Lcr, plasmids of yersiniae are known to promote an in vitro nutritional requirement for 2.5 mM Ca2+ at 37 degrees C which, if not fulfilled, results in cessation of growth with induction of virulence functions (Lcr+). The mechanism whereby Ca2+ regulates this metabolic shift is unknown. Radioactive Ca2+ was not actively accumulated by yersiniae but was excluded by an exit reaction analogous to those described for other bacteria. Nevertheless, cultivation at 37 degrees C with 0.1 mM Ca2+, a level insufficient to prevent restriction of cell division, promoted significantly more binding of the cation by Lcr+ organisms than by plasmid-deficient Lcr- mutants. According, Lcr+ yersiniae may possess unique ligands capable of recognizing Ca2+.