Glycogenin protein and mRNA expression in response to changing glycogen concentration in exercise and recovery

Glycogenin (GN-1) is essential for the formation of a glycogen granule; however, rarely has it been studied when glycogen concentration changes in exercise and recovery. It is unclear whether GN-1 is degraded or is liberated and exists as apoprotein (apo)-GN-1 (unglycosylated). To examine this, we measured GN-1 protein and mRNA level at rest, at exhaustion (EXH), and during 5 h of recovery in which the rate of glycogen restoration was influenced by carbohydrate (CHO) provision. Ten males cycled (65% VO2 max) to volitional EXH (117.8 +/- 4.2 min) on two separate occasions. Subjects were administered carbohydrate (CHO; 1 g.kg(-1).h(-1) Gatorlode) or water [placebo (PL)] during 5 h of recovery. Muscle biopsies were taken at rest, at EXH, and following 30, 60, 120, and 300 min of recovery. At EXH, total glycogen concentration was reduced (P < 0.05). However, GN-1 protein and mRNA content did not change. By 5 h of recovery, glycogen was resynthesized to approximately 60% of rest in the CHO trial and remained unchanged in the PL trial. GN-1 protein and mRNA level did not increase during recovery in either trial. We observed modest amounts of apo-GN-1 at EXH, suggesting complete degradation of some granules. These data suggest that GN-1 is conserved, possibly as very small, or nascent, granules when glycogen concentration is low. This would provide the ability to rapidly restore glycogen during early recovery.     

Primary human astrocytes produce 24(S),25-epoxycholesterol with implications for brain cholesterol homeostasis

Cholesterol is an essential component of the CNS and its metabolism in the brain has been implicated in various neurodegenerative diseases. The oxysterol produced from cholesterol, 24( S )-hydroxycholesterol, is known to be an important regulator of brain cholesterol homeostasis. In this study, we focussed on another oxysterol, 24( S ),25-epoxycholesterol (24,25EC), which has not been studied before in a neurological context. 24,25EC is unique in that it is synthesized in a shunt in the mevalonate pathway, parallel to cholesterol and utilizing the same enzymes. Considering that all the cholesterol present in the brain is derived from de novo synthesis, we investigated whether or not primary human neurons and astrocytes can produce 24,25EC. We found that astrocytes produced more 24,25EC than neurons under basal conditions, but both cell types had the capacity to synthesize this oxysterol when the enzyme 2,3-oxidosqualene cyclase was partially inhibited. Furthermore, both added 24,25EC and stimulated cellular production of 24,25EC (by partial inhibition of 2,3-oxidosqualene cyclase) modulated expression of key cholesterol-homeostatic genes regulated by the liver X receptor and the sterol regulatory element-binding protein-2. Moreover, we found that 24,25EC synthesized in astrocytes can be taken up by neurons and exert downstream effects on gene regulation. In summary, we have identified 24,25EC as a novel neurosterol which plays a likely role in brain cholesterol homeostasis.     

Both linear and discontinuous ribosome scanning are used for translation initiation from bicistronic human immunodeficiency virus type 1 env mRNAs

Human immunodeficiency virus type 1 (HIV-1) generates 16 alternatively spliced isoforms of env mRNA that contain the same overlapping open reading frames for Vpu and Env proteins but differ in their 5' untranslated regions (UTR). A subset of env mRNAs carry the extra upstream Rev initiation codon in the 5' UTR. We explored the effect of the alternative UTR on the translation of Vpu and Env proteins from authentic env mRNAs expressed from cDNA constructs. Vpu expression from the subset of env mRNA isoforms with exons containing an upstream Rev AUG codon was minimal. However, every env mRNA isoform expressed similar levels of Env protein. Mutations that removed, altered the strength of, or introduced upstream AUG codons dramatically altered Vpu expression but had little impact on the consistent expression of Env. These data show that the different isoforms of env mRNA are not redundant but instead regulate Vpu production in HIV-1-infected cells. Furthermore, while the initiation of Vpu translation conforms to the leaky ribosome-scanning model, the consistent Env synthesis infers a novel, discontinuous ribosome-scanning mechanism to translate Env.     

Genetic analysis of varicella-zoster virus ORF0 to ORF4 by use of a novel luciferase bacterial artificial chromosome system

To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZV(BAC)) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZV(Luc)). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZV(Luc) genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.     

Virus-specific CD8+ T cells in the liver: armed and ready to kill

Influenza A virus infection of C57BL/6 mice is a well-characterized model for studying CD8+ T cell-mediated immunity. Analysis of primary and secondary responses showed that the liver is highly enriched for CD8+ T cells specific for the immunodominant H2D(b)NP(366-374) (D(b)NP(366)) epitope. Functional analysis established that these liver-derived virus-specific CD8+ T cells are fully competent cytotoxic effectors and IFN-gamma secretors. In addition, flow cytometric analysis of early apoptotic cells showed that these influenza-specific CD8+ T cells from liver are as viable as those in the spleen, bronchoalveolar lavage, mediastinal lymph nodes, or lung. Moreover, cytokine profiles of the influenza-specific CD8+ T cells recovered from different sites were consistent with the bronchoalveolar lavage, rather than liver population, being the most susceptible to activation-induced cell death. Importantly, adoptively transferred influenza virus-specific CD8+ T cells from the liver survived and were readily recalled after virus challenge. Together, these results show clearly that the liver is not a "graveyard" for influenza virus-specific CD8+ T cells.     

RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity

Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.     

Yeast sphingolipids do not need to contain very long chain fatty acids

Synthesis of VLCFAs (very long chain fatty acids) and biosynthesis of DHS (dihydrosphingosine) both are of vital importance for Saccharomyces cerevisiae. The bulk of VLCFAs and DHS are used for ceramide synthesis by the Lag1p (longevity-assurance gene 1)/Lac1p (longevity-assurance gene cognate 1)/Lip1p (Lag1p/Lac1p interacting protein) ceramide synthase. LAG1 and LAC1 are redundant but LIP1 is essential. Here we show that 4Delta (lag1Deltalac1Deltaypc1Deltaydc1Delta) cells devoid of all known endogenous ceramide synthesis pathways are unviable but can be rescued by the expression of Lass5, a mouse LAG1 homologue. Ceramide synthase activity of 4Delta.Lass5 cells only utilizes C16 and C18 fatty acids and does not require the help of Lip1p, an essential cofactor of Lag1p/Lac1p. HPLC-electrospray ionization-MS/MS analysis demonstrated that in IPCs (inositolphosphorylceramides) of 4Delta.Lass5, the very long chain fatty acids (C26 and C24) account for <1% instead of the normal >97%. Notwithstanding, IPCs incorporated into glycosylphosphatidylinositol anchors of 4Delta.Lass5 show normal mobility on TLC and the ceramide- and raft-dependent traffic of Gas1p (glycophospholipid-anchored surface protein) from endoplasmic reticulum to Golgi remains almost normal. Moreover, the biosynthesis of C24:0 fatty acids remains essential. Thus, C(24:0) and dihydrosphingosine are both necessary for survival of yeast cells even if they utilize C16 and C18 fatty acids for sphingolipid biosynthesis.     

CTCF mediates insulator function at the CFTR locus

Regulatory elements that lie outside the basal promoter of a gene may be revealed by local changes in chromatin structure and histone modifications. The promoter of the CFTR (cystic fibrosis transmembrane conductance regulator) gene is not responsible for its complex pattern of expression. To identify important regulatory elements for CFTR we have previously mapped DHS (DNase I-hypersensitive sites) across 400 kb spanning the locus. Of particular interest were two DHS that flank the CFTR gene, upstream at -20.9 kb with respect to the translational start site, and downstream at +15.6 kb. In the present study we show that these two DHS possess enhancer-blocking activity and bind proteins that are characteristic of known insulator elements. The DHS core at -20.9 kb binds CTCF (CCCTC-binding factor) both in vitro and in vivo; however, the +15.6 kb core appears to bind other factors. Histone-modification analysis across the CFTR locus highlights structural differences between the -20.9 kb and +15.6 kb DHS, further suggesting that these two insulator elements may operate by distinct mechanisms. We propose that these two DHS mark the boundaries of the CFTR gene functional unit and establish a chromatin domain within which the complex profile of CFTR expression is maintained.