Structural and Molecular Biology of Type I Galactosemia: Disease-associated Mutations

Type I galactosemia results from reduced galactose 1-phosphate uridylyltransferase (GALT) activity. Signs of disease include damage to the eyes, brain, liver, and ovaries. However, the exact nature and severity of the pathology depends on the mutation(s) in the patient's genes and his/her environment. Considerable enzymological and structural knowledge has been accumulated and this provides a basis to explain, at a biochemical level, impairment in the enzyme in the more than 230 disease-associated variants, which have been described. The most common variant, Q188R, occurs close to the active site and the dimer interface. The substitution probably disrupts both UDP-sugar binding and homodimer stability. Other alterations, for example K285N, occur close to the surface of the enzyme and most likely affect the folding and stability of the enzyme. There are a number of unanswered questions in the field, which require resolution. These include the possibility that the main enzymes of galactose metabolism form a supramolecular complex and the need for a high resolution crystal structure of human GALT. (C) 2011 IUBMB IUBMB Life, 63(11): 949-954, 2011

Development of a bovine collagen-apatitic calcium phosphate cement for potential fracture treatment through vertebroplasty

The aim of this study was to examine the potential of incorporating bovine fibres as a means of reinforcing a typically brittle apatite calcium phosphate cement for vertebroplasty. Type I collagen derived from bovine Achilles tendon was ground cryogenically to produce an average fibre length of 0.96 ± 0.55 mm and manually mixed into the powder phase of an apatite-based cement at 1, 3 or 5 wt.%. Fibre addition of up to 5 wt.% had a significant effect (P = 0.001) on the fracture toughness, which was increased by 172%. Adding =1 wt.% bovine collagen fibres did not compromise the compressive properties significantly, however, a decrease of 39-53% was demonstrated at =3 wt.% fibre loading. Adding bovine collagen to the calcium phosphate cement reduced the initial and final setting times to satisfy the clinical requirements stated for vertebroplasty. The cement viscosity increased in a linear manner (R = 0.975) with increased loading of collagen fibres, such that the injectability was found to be reduced by 83% at 5 wt.% collagen loading. This study suggests for the first time the potential application of a collagen-reinforced calcium phosphate cement as a viable option in the treatment of vertebral fractures, however, issues surrounding efficacious cement delivery need to be addressed. © 2012 Acta Materialia Inc.

Modeling the FeK line profiles in type i active galactic nuclei with a compton-thick disk wind

We have modeled a small sample of Seyfert galaxies that were previously identified as having simple X-ray spectra with little intrinsic absorption. The sources in this sample all contain moderately broad components of FeK-shell emission and are ideal candidates for testing the applicability of a Compton-thick accretion disk wind model to active galactic nucleus (AGN) emission components. Viewing angles through the wind allow the observer to see the absorption signature of the gas, whereas face-on viewing angles allow the observer to see the scattered light from the wind. We find that the FeK emission line profiles are well described with a model of a Compton-thick accretion disk wind of solar abundances, arising tens to hundreds of gravitational radii from the central black hole. Further, the fits require a neutral component of FeKa emission that is too narrow to arise from the inner part of the wind, and likely comes from a more distant reprocessing region. Our study demonstrates that a Compton-thick wind can have a profound effect on the observed X-ray spectrum of an AGN, even when the system is not viewed through the flow. © 2012. The American Astronomical Society. All rights reserved..

Misfolding of galactose 1-phosphate uridylyltransferase can result in type I galactosemia

Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant's level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.

Interaction of very-low-density lipoprotein isolated from type I (insulin-dependent) diabetic subjects with human monocyte-derived macrophages

Very-low-density lipoproteins (VLDL) (density less than 1.006 g/mL) were isolated from type I (insulin-dependent) diabetic patients in good to fair glycemic control and from age-, sex-, and race-matched, nondiabetic, control subjects. VLDL were incubated with human, monocyte-derived macrophages obtained from nondiabetic donors, and the rates of cellular cholesteryl ester synthesis and cholesterol accumulation were determined. VLDL isolated from diabetic patients stimulated significantly more cholesteryl ester synthesis than did VLDL isolated from control subjects (4.04 +/- 1.01 v 1.99 +/- 0.39 nmol 14C-cholesteryl oleate synthesized/mg cell protein/20 h; mean +/- SEM, P less than .05). The stimulation of cholesteryl ester synthesis in macrophages incubated with VLDL isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (P less than .05). The increase in cholesteryl ester synthesis and accumulation in macrophages were mediated by a significant increase in the receptor mediated, high affinity degradation (2.55 +/- 0.23 v 2.12 +/- 0.20 micrograms degraded/mg cell protein/20 h) and accumulation (283 +/- 35 v 242 +/- 33 ng/mg cell protein/20 h) of 125I-VLDL isolated from diabetic patients compared with VLDL from control subjects. To determine if changes in VLDL apoprotein composition were responsible for the observed changes in cellular rates of cholesteryl ester synthesis and accumulation, we also examined the apoprotein composition of the VLDL from both groups. There were no significant differences between the apoproteins B, E, and C content of VLDL from both groups. We also determined the chemical composition of VLDL isolated from both groups of subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3-and viral-induced expression of type i interferons

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression. © 2012 Nature America, Inc. All rights reserved.

Type-I interferons induce lung protease responses following respiratory syncytial virus infection via RIG-I-like receptors

The role of proteases in viral infection of the lung is poorly understood. Thus, we examined matrix metalloproteinases (MMPs) and cathepsin proteases in respiratory syncytial virus (RSV)-infected mouse lungs. RSV-induced gene expression for MMPs -2, -3, -7, -8, -9, -10, -12, -13, -14, -16, -17, -19, -20, -25, -27, and -28 and cathepsins B, C, E, G, H, K, L1, S, W, and Z in the airways of Friend leukemia virus B sensitive strain mice. Increased proteases were present in the bronchoalveolar lavage fluid (BALF) and lung tissue during infection. Mitochondrial antiviral-signaling protein (MAVS) and TIR-domain-containing adapter-inducing interferon-β-deficient mice were exposed to RSV. <i>Mavsi>-deficient mice had significantly lower expression of airway MMP-2, -3, -7, -8, -9, -10, -12, -13, and -28 and cathepsins C, G, K, S, W, and Z. In lung epithelial cells, retinoic acid-inducible gene-1 (RIG-I) was identified as the major RIG-I-like receptor required for RSV-induced protease expression via MAVS. Overexpression of RIG-I or treatment with interferon-β in these cells induced MMP and cathepsin gene and protein expression. The significance of RIG-1 protease induction was demonstrated by the fact that inhibiting proteases with batimastat, E64 or ribavirin prevented airway hyperresponsiveness and enhanced viral clearance in RSV-infected mice.

PESSTO monitoring of SN 2012hn: further heterogeneity among faint Type I supernovae

We present optical and infrared monitoring data of SN 2012hn collectedby the Public European Southern Observatory Spectroscopic Survey forTransient Objects. We show that SN 2012hn has a faint peak magnitude(MR ˜ -15.65) and shows no hydrogen and no clearevidence for helium in its spectral evolution. Instead, we detectprominent Ca II lines at all epochs, which relates this transient topreviously described `Ca-rich' or `gap' transients. However, thephotospheric spectra (from -3 to +32 d with respect to peak) of SN2012hn show a series of absorption lines which are unique and a redcontinuum that is likely intrinsic rather than due to extinction. Linesof Ti II and Cr II are visible. This may be a temperature effect, whichcould also explain the red photospheric colour. A nebular spectrum at+150 d shows prominent Ca II, O I, C I and possibly Mg I lines whichappear similar in strength to those displayed by core-collapsesupernovae (SNe). To add to the puzzle, SN 2012hn is located at aprojected distance of 6 kpc from an E/S0 host and is not close to anyobvious star-forming region. Overall SN 2012hn resembles a group offaint H-poor SNe that have been discovered recently and for which aconvincing and consistent physical explanation is still missing. Theyall appear to explode preferentially in remote locations offset from amassive host galaxy with deep limits on any dwarf host galaxies,favouring old progenitor systems. SN 2012hn adds heterogeneity to thissample of objects. We discuss potential explosion channels includingHe-shell detonations and double detonations of white dwarfs as well aspeculiar core-collapse SNe.

Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype

The objective of this study was to investigate the nature and biomechanical properties of collagen fibers within the human myocardium. Targeting cardiac interstitial abnormalities will likely become a major focus of future preventative strategies with regard to the management of cardiac dysfunction. Current knowledge regarding the component structures of myocardial collagen networks is limited, further delineation of which will require application of more innovative technologies. We applied a novel methodology involving combined confocal laser scanning and atomic force microscopy to investigate myocardial collagen within ex-vivo right atrial tissue from 10 patients undergoing elective coronary bypass surgery. Immuno-fluorescent co-staining revealed discrete collagen I and III fibers. During single fiber deformation, overall median values of stiffness recorded in collagen III were 37±16% lower than in collagen I [p<0.001]. On fiber retraction, collagen I exhibited greater degrees of elastic recoil [p<0.001; relative percentage increase in elastic recoil 7±3%] and less energy dissipation than collagen III [p<0.001; relative percentage increase in work recovered 7±2%]. In atrial biopsies taken from patients in permanent atrial fibrillation (n=5) versus sinus rhythm (n=5), stiffness of both collagen fiber subtypes was augmented (p<0.008). Myocardial fibrillar collagen fibers organize in a discrete manner and possess distinct biomechanical differences; specifically, collagen I fibers exhibit relatively higher stiffness, contrasting with higher susceptibility to plastic deformation and less energy efficiency on deformation with collagen III fibers. Augmented stiffness of both collagen fiber subtypes in tissue samples from patients with atrial fibrillation compared to those in sinus rhythm are consistent with recent published findings of increased collagen cross-linking in this setting.

Natural history of markers of collagen turnover in patients with early diastolic dysfunction and impact of eplerenone

OBJECTIVES: This study was designed to evaluate the impact of eplerenone on collagen turnover in preserved systolic function heart failure (HFPSF).

BACKGROUND: Despite growing interest in abnormal collagen metabolism as a feature of HFPSF with diastolic dysfunction, the natural history of markers of collagen turnover and the impact of selective aldosterone antagonism on this natural history remains unknown.

METHODS: We evaluated 44 patients with HFPSF, randomly assigned to control (n = 20) or eplerenone 25 mg daily (n = 24) for 6 months, increased to 50 mg daily from 6 to 12 months. Serum markers of collagen turnover and inflammation were analyzed at baseline and at 6 and 12 months and included pro-collagen type-I and -III aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha. Doppler-echocardiographic assessment of diastolic filling indexes and tissue Doppler analyses were also obtained.

RESULTS: The mean age of the patients was 80 +/- 7.8 years; 46% were male; 64% were receiving an angiotensin-converting enzyme inhibitor, 34% an angiotensin-II receptor blocker, and 68% were receiving beta-blocker therapy. Pro-collagen type-III and -I aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha increased with time in the control group. Eplerenone treatment had no significant impact on any biomarker at 6 months but attenuated the increase in pro-collagen type-III aminoterminal peptide at 12 months (p = 0.006). Eplerenone therapy was associated with modest effects on diastolic function without any impact on clinical variables or brain natriuretic peptide.

CONCLUSIONS: This study demonstrates progressive increases in markers of collagen turnover and inflammation in HFPSF with diastolic dysfunction. Despite high background utilization of renin-angiotensin-aldosterone modulators, eplerenone therapy prevents a progressive increase in pro-collagen type-III aminoterminal peptide and may have a role in management of this disease. (The Effect of Eplerenone and Atorvastatin on Markers of Collagen Turnover in Diastolic Heart Failure; NCT00505336).

DES14X3taz: A Type I Superluminous Supernova Showing a Luminous, Rapidly Cooling Initial Pre-peak Bump

We present DES14X3taz, a new hydrogen-poor superluminous supernova (SLSN-I) discovered by the Dark Energy Survey (DES) supernova program, with additional photometric data provided by the Survey Using DECam for Superluminous Supernovae. Spectra obtained using Optical System for Imaging and low-Intermediate-Resolution Integrated Spectroscopy on the Gran Telescopio CANARIAS show DES14X3taz is an SLSN-I at <i style="margin: 0px; padding: 0px; border: 0px; font-variant-numeric: inherit; font-stretch: inherit; font-size: 18px; line-height: 27px; font-family: minion-pro, Georgia, "Times New Roman", STIXGeneral, serif; vertical-align: baseline; color: rgb(51, 51, 51);">zi> = 0.608. Multi-color photometry reveals a double-peaked light curve: a blue and relatively bright initial peak that fades rapidly prior to the slower rise of the main light curve. Our multi-color photometry allows us, for the first time, to show that the initial peak cools from 22,000 to 8000 K over 15 rest-frame days, and is faster and brighter than any published core-collapse supernova, reaching 30% of the bolometric luminosity of the main peak. No physical 56Ni-powered model can fit this initial peak. We show that a shock-cooling model followed by a magnetar driving the second phase of the light curve can adequately explain the entire light curve of DES14X3taz. Models involving the shock-cooling of extended circumstellar material at a distance of 400  are preferred over the cooling of shock-heated surface layers of a stellar envelope. We compare DES14X3taz to the few double-peaked SLSN-I events in the literature. Although the rise times and characteristics of these initial peaks differ, there exists the tantalizing possibility that they can be explained by one physical interpretation.

A Rare Loss-of-Function Variant in Scavenger Receptor Class B Type I (SCARB1) Raises HDL Cholesterol and Increases Risk of Coronary Disease

Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL)
cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a
lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI
knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased
atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains
unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328
individuals with extremely high plasma HDL-C levels, we identified a homozygote for a lossof-function
variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene
encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and
abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells
derived from induced pluripotent stem cells from the homozygous subject, and in mice.
Large population-based studies revealed that subjects who are heterozygous carriers of
the P376L variant have significantly increased levels of plasma HDL-C. P376L carriers have
a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is
statistically significant).

STAT3, p38 MAP Kinase and NF-{kappa}B Drive Unopposed Monocyte-dependent Fibroblast MMP-1 Secretion in Tuberculosis.

Tissue destruction characterizes infection with Mycobacterium tuberculosis (Mtb). Type I collagen provides the lung's tensile strength, is extremely resistant to degradation, but is cleaved by matrix metalloproteinase (MMP)-1. Fibroblasts potentially secrete quantitatively more MMP-1 than other lung cells. We investigated mechanisms regulating Mtb-induced collagenolytic activity in fibroblasts in vitro and in patients. Lung fibroblasts were stimulated with conditioned media from Mtb-infected monocytes (CoMTb). CoMTb induced sustained increased MMP-1 (74 versus 16 ng/ml) and decreased tissue inhibitor of metalloproteinase (TIMP)-1 (8.6 versus 22.3 ng/ml) protein secretion. CoMTb induced a 2.7-fold increase in MMP-1 promoter activation and a 2.5-fold reduction in TIMP-1 promoter activation at 24 hours (P = 0.01). Consistent with this, TIMP-1 did not co-localize with fibroblasts in patient granulomas. MMP-1 up-regulation and TIMP-1 down-regulation were p38 (but not extracellular signal–regulated kinase or c-Jun N-terminal kinase) mitogen-activated protein kinase–dependent. STAT3 phosphorylation was detected in fibroblasts in vitro and in tuberculous granulomas.STAT3 inhibition reduced fibroblast MMP-1 secretion by 60% (P = 0.046). Deletion of the MMP-1 promoter NF-B–binding site abrogated promoter induction in response to CoMTb. TNF-, IL-1ß, or Oncostatin M inhibition in CoMTb decreased MMP-1 secretion by 65, 63, and 25%, respectively. This cytokine cocktail activated the same signaling pathways in fibroblasts and induced MMP-1 secretion similar to that induced by CoMTb. This study demonstrates in a cellular model and in patients with tuberculosis that in addition to p38 and NF-B, STAT3 has a key role in driving fibroblast-dependent unopposed MMP-1 production that may be key in tissue destruction in patients.

Low vitamin D status adversely affects bone health parameters in adolescents

Background: The effects of subclinical vitamin D deficiency on bone mineral density (BMD) and bone turnover in adolescents, especially in boys, are unclear.

Objective: We aimed to investigate the relations of different stages of vitamin D status and BMD and bone turnover in a representative sample of adolescent boys and girls.

Design: BMD was measured by dual-energy X-ray absorptiometry at the nondominant forearm and dominant heel in a random sample of 12- (n = 260) and 15-y-old (n = 239) boys and 12- (n = 266) and 15-y-old (n = 250) girls. Serum 25-hydroxyvitamin D, parathyroid hormone, osteocalcin, and type I collagen cross-linked C-telopeptide were assessed by using enzyme-linked immunoassays. Relations between vitamin D status and bone health indexes were assessed by using regression modeling.

Results: Using multivariate regression to adjust for potential physical, lifestyle, and dietary confounding factors, we observed that 12-and 15-y-old girls with high vitamin D status (>= 74.1 nmol/L) had significantly greater forearm (but not heel) BMD (beta = 0.018; SE = 0.008; P < 0.05 for each age group) and lower serum parathyroid hormone concentrations and bone turnover markers than did those with low vitamin D status. These associations were evident in subjects sampled throughout the year and in winter only. There was no significant relation between vitamin D status and BMD in boys.

Conclusions: Maintaining serum 25-hydroxyvitamin D concentrations above approximate to 50 nmol/L throughout the year may be a cost-effective means of improving bone health. Increased emphasis on exploring strategies for improving vitamin D status in adolescents is needed.