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.     

Cadmium-2-acetylaminofluorene interaction in isolated rat hepatocytes

Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. Evidence exists that Cd can affect parameters which are of great importance in the response towards xenobiotics. However, there is a lack of information about the mechanisms that take place at the cellular and molecular levels upon dual exposure to Cd and other toxins. The purpose of the present work was therefore to examine the biochemical interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF) in isolated rat hepatocytes. The cells were incubated for 10 hr with a sub-cytotoxic concentration (0.22 M) of ¹⁰⁹Cd. This was followed by a 10 hr exposure to 1 M [³H]AAF. Cellular distribution of Cd and ³H was determined. Sephadex G-75 elution profiles of the cytosol showed that Cd was almost entirely associated with the intermediate molecular weight (IMW) fractions containing metallothionein (MT) (>80%), and with high molecular weight proteins. In parallel, the highest proportion of ³H was found in the low molecular weight components. Further analysis of IMW fractions by DEAE A-25 anion-exchange chromatography revealed that, in addition to Cd, there was some ³H which coeluted along with MT-I and MT-II isoforms, but preferentially with MT-I. Moreover, Cd pretreatment caused a 1.6-fold increase in MT level, as measured by the silver-saturation assay. Under these conditions, there was a 17% lower binding of ³H to the DNA. This reduced binding was neither accompanied by diminished AAF uptake nor by inhibition of cytochrome P-450 activity. Taken together, these results suggest that Cd exposure has a protective effect against the genotoxicity of AAF. MT, whose synthesis is induced, could play a role in the Cd-AAF interaction through scavenging of reactive metabolites.Abbreviations AAF 2-acetylaminofluorene - Cd cadmium - DMSO dimethyl sulfoxide - HBSS Hank's balanced salt solution - LDH lactate dehydrogenase - MT metallothionein - UDS unscheduled DNA synthesis     

Metabolism of Benzyladenine is Impaired in a Mutant of Arabidopsis thaliana Lacking Adenine Phosphoribosyltransferase Activity

Formation of the riboside-5′-monophosphate is a general feature of the metabolism of cytokinins in plants. As part of a study of the biological significance of the nucleotide form of cytokinins, we analyzed a mutant of Arabidopsis thaliana deficient in adenine phosphoribosyltransferase (APRT) activity for its ability to metabolize N⁶-benzyladenine (BA). Formation of N⁶-benzyladenosine-5′-monophosphate (BAMP) was assayed in vivo, by feeding tritiated BA to wild-type and mutant plantlets, and in crude plantlet extracts. Metabolites were separated by high performance liquid chromatography and quantitated by on-line liquid scintillation spectrometry. BA was rapidly absorbed by A. thaliana plantlets and primarily converted to BAMP and to BA 7- and 9-glucosides. BA was also rapidly absorbed by APRT-deficient plantlets, but its conversion to BAMP was strongly reduced. Formation of BAMP from N⁶-benzyladenosine was not affected in the mutant plantlets. In vitro conversion of BA to its nucleoside-5′-monophosphate was detected in crude extracts of wild-type plantlets, but not in extracts of APRT-deficient plantlets. Therefore, results of both assays indicate that APRT-deficient tissue does not convert BA to BAMP to a significant extent. Further, nondenaturing isoelectric focusing analysis of APRT activity in leaf extracts indicated that the enzyme activities which metabolize adenine and BA into their corresponding riboside-5′-monophosphate in extracts of wild-type plantlets have the same apparent isoelectric point. These activities were not detected in extracts prepared from APRT-deficient plantlets. Thus, these results demonstrate that APRT is the main enzyme which converts BA to its nucleotide form in young A. thaliana plants and that the ribophosphorylation of BA is not a prerequisite of its absorption by the plantlets.     

Adaptations to reduction of endometrial surface available for placental development in sheep

On Day 5 of pregnancy, before the blastocyst migrates to the uterus, one uterine horn was ligated to restrict the trophoblast to the lumen ipsilateral to the corpus luteum. The numbers of placentomes (caruncles and cotyledons) were reduced by half, but neither at 120 nor at 140 days of pregnancy (term 147 days) did the weights of placentae and fetuses of treated ewes differ significantly from those of control ewes. Amongst uterus-ligated animals prepared for chronic study, the rate of uterine blood flow (electromagnetic flow transducer, ml/min) to the pregnant horn was higher than in control ewes, as was the concentration of progestagens in maternal peripheral blood. There may be a compensatory response that causes hypertrophy of placentomes and that increases blood flow to the uterine horn containing placental tissue.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

Synthesis of 4′-methoxyadenosine and related compounds

Addition of iodine and methanol to N⁶,N⁶-dibenzoyl-9(2,3-O-carbonyl-5-deoxy-β-d-erythro-pent-4-enofuranosyl)adenine (4) selectively gives N⁶,N⁶-dibenzoyl-2′,3′-O-carbonyl-5′-deoxy-5′-iodo-4′-methoxyadenosine (5). Compound 5 can be converted into 4′-methoxyadenosine via hydrolysis of the carbonate followed by benzoylation, displacement of the 5′-iodo function by benzoate ion, and hydrolysis with ammonia. Configurational assignments are based upon comparisons of ¹H- and ¹³C-n.m.r. spectra with those of previously characterised analogues in the uracil series and by borate electrophoresis. Intermediates in the above scheme have also been converted into 5′-amino-5′-deoxy-4′-methoxyadenosine, 4′-methoxy-5′-O-sulfamoyladenosine, and ethyl 4′-methoxyadenosine-5′-carboxylate, each of which is a 4′-methoxy analogue of biologically active derivatives of adenosine.