Segregation of a single outboard left-end origin is essential for the viability of parvovirus minute virus of mice

During DNA replication, the hairpin telomeres of Minute Virus of Mice (MVM) are extended and copied to create imperfectly palindromic duplex junction sequences that bridge adjacent genomes in concatameric replicative-form DNA. These are resolved by the viral initiator protein, NS1, but mechanisms employed at the two telomeres differ. Left-end:left-end junctions are resolved asymmetrically at a single site, OriLTC, by NS1 acting in concert with a host factor, parvovirus initiation factor (PIF). Replication segregates doublet and triplet sequences, initially present as unpaired nucleotides in the bubble region of the left-end hairpin stem, to either side of the junction. These act as spacers between the NS1 and PIF binding sites, and their asymmetric distribution sets up active (OriLTC) and inactive (OriLGAA) forms of OriL. We used a reverse genetic approach to disrupt this asymmetry and found that neither opposing doublets nor triplets in the hairpin bubble were tolerated. Viable mutants were isolated at low frequency and found to contain second-site mutations that either restored the asymmetry or crippled one PIF binding site. These mutations either inactivated the inboard or activated the outboard form of OriL, a polarity that strongly suggests that, in the genus Parvovirus, an active inboard OriL is lethal.     

Identification of presenilin 1-selective γ-secretase inhibitors with reconstituted γ-secretase complexes

Accumulation of the β-amyloid (Aβ) peptides is one of the major pathologic hallmarks in the brains of Alzheimer's disease (AD) patients. Aβ is generated by sequential proteolytic cleavage of the amyloid precursor protein (APP) catalyzed by β- and γ-secretases. Inhibition of Aβ production by γ-secretase inhibitors (GSIs) is thus being pursued as a target for treatment of AD. In addition to processing APP, γ-secretase also catalyzes proteolytic cleavage of other transmembrane substrates, with the best characterized one being the cell surface receptor Notch. GSIs reduce Aβ production in animals and humans but also cause significant side effects because of the inhibition of Notch processing. The development of GSIs that reduce Aβ production and have less Notch-mediated side effect liability is therefore an important goal. γ-Secretase is a large membrane protein complex with four components, two of which have multiple isoforms: presenilin (PS1 or PS2), aph-1 (aph-1a or aph-1b), nicastrin, and pen-2. Here we describe the reconstitution of four γ-secretase complexes in Sf9 cells containing PS1--aph-1a, PS1--aph-1b, PS2--aph-1a, and PS2--aph-1b complexes. While PS1--aph-1a, PS1--aph-1b, and PS2--aph-1a complexes displayed robust γ-secretase activity, the reconstituted PS2--aph-1b complex was devoid of detectable γ-secretase activity. γ-Secretase complexes containing PS1 produced a higher proportion of the toxic species Aβ42 than γ-secretase complexes containing PS2. Using the reconstitution system, we identified MRK-560 and SCH 1500022 as highly selective inhibitors of PS1 γ-secretase activity. These findings may provide important insights into developing a new generation of γ-secretase inhibitors with improved side effect profiles.     

Association of peak aerobic power with capillary density but not oxidative potential in human vastus lateralis muscle

In this study, we have postulated that in healthy males, peak aerobic power ([Formula: see text]) would associate with muscle capillary density rather than oxidative potential, regardless of fibre type or subtype. To test this, active but untrained volunteers (n = 11) were separated into high (HI) and low (LO) groups based on [Formula: see text] obtained during a progressive cycle task to fatigue. The 26% higher (P?< 0.05) [Formula: see text] observed in HI (40.8?± 1.5?mL·kg(-1)·min(-1), mean?± SE) compared with LO ( 51.4?± 0.90?mL·kg(-1)·min(-1), mean?± SE) was not accompanied by differences in age (21.3?± 1.2 compared with 21.1?± 0.63?years, respectively) or body mass (72.4?± 4.6 compared with 71.6?± 1.9?kg, respectively). Tissue samples obtained from the vastus lateralis indicated greater (P?< 0.05) capillary counts per fibre (CC;?+24%) in HI compared with LO, regardless of fibre type (I, IIA, IIX, IIAX). Capillary density (CD) as measured in a field of defined area was also elevated (+22%; P?< 0.05), as was the number of capillaries per fibre (+22%; P?< 0.05). No differences were observed between the 2 groups in the distribution, area, and the CC/fibre area ratio in the different fibre types and subtypes. Similarly, there was no difference between the HI and LO groups in oxidative potential, as measured by succinic dehydrogenase activity in the different fibre types. It is concluded that the higher capillary density may contribute to improved vascular conductance and the elevated [Formula: see text] observed in the untrained participants.     

Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family

Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm–egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.     

Urinary C-type natriuretic peptide excretion: a potential novel biomarker for renal fibrosis during aging

Renal aging is characterized by structural changes in the kidney including fibrosis, which contributes to the increased risk of kidney and cardiac failure in the elderly. Studies involving healthy kidney donors demonstrated subclinical age-related nephropathy on renal biopsy that was not detected by standard diagnostic tests. Thus there is a high-priority need for novel noninvasive biomarkers to detect the presence of preclinical age-associated renal structural and functional changes. C-type natriuretic peptide (CNP) possesses renoprotective properties and is present in the kidney; however, its modulation during aging remains undefined. We assessed circulating and urinary CNP in a Fischer rat model of experimental aging and also determined renal structural and functional adaptations to the aging process. Histological and electron microscopic analysis demonstrated significant renal fibrosis, glomerular basement membrane thickening, and mesangial matrix expansion with aging. While plasma CNP levels progressively declined with aging, urinary CNP excretion increased, along with the ratio of urinary to plasma CNP, which preceded significant elevations in proteinuria and blood pressure. Also, CNP immunoreactivity was increased in the distal and proximal tubules in both the aging rat and aging human kidneys. Our findings provide evidence that urinary CNP and its ratio to plasma CNP may represent a novel biomarker for early age-mediated renal structural alterations, particularly fibrosis. Thus urinary CNP could potentially aid in identifying subjects with preclinical structural changes before the onset of symptoms and disease, allowing for the initiation of strategies designed to prevent the progression of chronic kidney disease particularly in the aging population.     

Indoleamine 2,3-diooxygenase in periaortic fat: mechanisms of inhibition of contraction

Indoleamine 2,3-dioxygenase (IDO) metabolizes L-tryptophan to L-kynurenine, promotes immunosuppression, and has been described as a consumer of superoxide. We discovered IDO expression in periaortic fat and tested the hypothesis that periarterial IDO functionally reduces agonist-induced contraction. Our model was the thoracic aorta, abdominal aorta, and superior mesenteric artery of the male Sprague-Dawley rat. Periaortic fat from the thoracic aorta stained intensely for IDO, the brown fat marker uncoupling protein-1, and oil red O as a general lipid marker. White fat around the mesenteric artery and abdominal aorta stained less for IDO; brown fat was less abundant. IDO activity (kynurenine-to-tryptophan ratio via HPLC) was detected in visceral and mesenteric artery fat (ratio: ～4) but was highest in perithoracic aortic fat (ratio: 10 ± 1.1). In isometric contractile experiments, periadventitial fat reduced ANG II-induced thoracic aortic (with fat: 34% of without fat) and mesenteric artery (with fat: 63% of without fat) maximal contraction. In contrast, periadventitial fat did not reduce agonist-induced contraction in the abdominal aorta. The IDO inhibitor 1-L-methyltryptophan (1-MT) reversed the fat-induced reduction of ANG II-induced contraction in the thoracic aorta but not in the mesenteric artery. The IDO metabolite kynurenine relaxed the thoracic aorta only at high (9 mM) concentrations, whereas the downstream metabolite quinolinic acid (1 mM) relaxed the contracted thoracic aorta (～80%). 1-MT did not correct the reduction in basal superoxide levels observed in the presence of perithoracic aortic fat. We conclude that IDO is an enzyme active primarily in brown fat surrounding the thoracic aorta and depresses aortic contractility.     

Identification of a sporozoite-specific antigen from Toxoplasma gondii

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.     

Automation of Molecular-Based Analyses: A Primer on Massively Parallel Sequencing

Recent advances in genetics have been enabled by new genetic sequencing techniques called massively parallel sequencing (MPS) or next-generation sequencing. Through the ability to sequence in parallel hundreds of thousands to millions of DNA fragments, the cost and time required for sequencing has dramatically decreased. There are a number of different MPS platforms currently available and being used in Australia. Although they differ in the underlying technology involved, their overall processes are very similar: DNA fragmentation, adaptor ligation, immobilisation, amplification, sequencing reaction and data analysis. MPS is being used in research, translational and increasingly now also in clinical settings. Common applications include sequencing of whole genomes, whole exomes or targeted genes for disease-causing gene discovery, genetic diagnosis and targeted cancer therapy. Even though the revolution that is occurring with MPS is exciting due to its increasing use, improving and emerging technologies and new applications, significant challenges still exist. Particularly challenging issues are the bioinformatics required for data analysis, interpretation of results and the ethical dilemma of ‘incidental findings’.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.