Estrogen depletion alters mineralization regulation mechanisms in an ovariectomized monkey animal model

Ovariectomized animal models have been extensively used in osteoporosis research due to the resulting loss of bone mass. The purpose of the present study was to test the hypothesis that estrogen depletion alters mineralization regulation mechanisms in an ovariectomized monkey animal model. To achieve this we used Raman microspectroscopy to analyze humeri from monkeys that were either SHAM-operated or ovariectomized (N = 10 for each group). Measurements were made as a function of tissue age and cortical surface (periosteal, osteonal, endosteal) based on the presence of calcein fluorescent double labels. In the present work we focused on osteoid seams (defined as a surface with evident calcein labels, 1 μm distance away from the mineralizing front, and for which the Raman spectra showed the presence of organic matrix but not mineral), as well as the youngest mineralized tissue between the second fluorescent label and the mineralizing front, 1 μm inwards from the front with the phosphate mineral peak evident in the Raman spectra (TA1). The spectroscopically determined parameters of interest were the relative glycosaminoglycan (GAG) and pyridinoline (Pyd) contents in the osteoid, and the mineral content in TA1. At all three cortical surfaces, significant correlations were evident in the SHAM-operated animals between osteoid GAG (negative) and Pyd content, and mineral content, unlike the OVX animals. These results suggest that in addition to the well-established effects on turnover rates and bone mass, estrogen depletion alters the regulation of mineralization by GAGs and Pyd

Essential role of histidine for rapid copper(II)-mediated disassembly of neurokinin B amyloid

Neurokinin B is a tachykinin peptide involved in a diverse range of neuronal functions. It rapidly forms an amyloid, which is considered physiologically important for efficient packing into dense core secretory vesicles within hypothalamic neurons. Disassembly of the amyloid is thought to require the presence of copper ions, which interact with histidine at the third position in the peptide sequence. However, it is unclear how the histidine is involved in the amyloid structure and why copper coordination can trigger disassembly. In this work, we demonstrate that histidine contributes to the amyloid structure via π-stacking interactions with nearby phenylalanine residues. The ability of neurokinin B to form an amyloid is dependent on any aromatic residue at the third position in the sequence; however, only the presence of histidine leads to both amyloid formation and rapid copper-induced disassembly

Elucidating the mechanisms underlying GATA-1 activity

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Breaking the Gingival Barrier in Periodontitis

The break of the epithelial barrier of gingiva has been a subject of minor interest, albeit playing a key role in periodontal pathology, transitory bacteraemia, and subsequent systemic lowgrade inflammation (LGI). The significance of mechanically induced bacterial translocation in gingiva (e.g., via mastication and teeth brushing) has been disregarded despite the accumulated knowledge of mechanical force effects on tight junctions (TJs) and subsequent pathology in other epithelial tissues. Transitory bacteraemia is observed as a rule in gingival inflammation, but is rarely observed in clinically healthy gingiva. This implies that TJs of inflamed gingiva deteriorate, e.g., via a surplus of lipopolysaccharide (LPS), bacterial proteases, toxins, Oncostatin M (OSM), and neutrophil proteases. The inflammation-deteriorated gingival TJs rupture when exposed to physiological mechanical forces. This rupture is characterised by bacteraemia during and briefly after mastication and teeth brushing, i.e., it appears to be a dynamic process of short duration, endowed with quick repair mechanisms. In this review, we consider the bacterial, immune, and mechanical factors responsible for the increased permeability and break of the epithelial barrier of inflamed gingiva and the subsequent translocation of both viable bacteria and bacterial LPS during physiological mechanical forces, such as mastication and teeth brushing

Pannexin-1-dependent caspase-1 activation and secretion of IL-1β is regulated by zinc

Inflammatory processes induced by IL-1β are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase-1 activation and IL-1β secretion. One of the endogenous mediators of IL-1β secretion is adenosine triphosphate, acting via the P2X7-receptor and caspase-1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre-treatment with the zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylene diamine (TPEN). These effects on IL-1β secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase-1 activity, and upstream of caspase-1 activation. TPEN did however inhibit the activity of pannexin-1, a hemi-channel critical for adenosine triphosphate and nigericin-induced IL-1β release. These data provide new insights into the mechanisms of caspase-1 activation and how zinc metabolism contributes to inflammatory mechanisms

hSSB1 phosphorylation is dynamically regulated by DNA-PK and PPP-family protein phosphatases

This work was supported by a National Health and Medical Research Council project grant [1066550], an Australian Research Council project grant [DP 120103099] and by a Queensland Health Senior Clinical Research Fellowship awarded to K.J.O. This work was also supported by the Wellcome Trust [094476/Z/10/Z], which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews. NWA was supported by a scholarship awarded by Cancer Council Queensland. E.B. is supported by an Advance Queensland Research Fellowship.The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 is also directly phosphorylated by DNA-PK at serine residue 134. While this modification is largely suppressed in undamaged cells by PPP-family protein phosphatases, S134 phosphorylation is enhanced following the disruption of replication forks and promotes cellular survival. Together, these data thereby represent a novel mechanism for hSSB1 regulation following the inhibition of replication.Publisher PDFPeer reviewe

Accuracy in mineral identification: image spectral and spatial resolutions and mineral spectral properties

Problems related to airborne hyperspectral image data are reviewed and the requirements for data analysis applied to mineralogical (rocks and soils) interpretation are discussed. The variability of mineral spectral features, including absorption position, shape and depth is considered and interpreted as due to chemical composition, grain size effects and mineral association. It is also shown how this variability can be related to well defined geologic processes. The influence of sensor noise and diffuse atmospheric radiance in classification accuracy is also analyzed

hSSB1 (NABP2/OBFC2B) is regulated by oxidative stress

The maintenance of genome stability is an essential cellular process to prevent the development of diseases including cancer. hSSB1 (NABP2/ OBFC2A) is a critical component of the DNA damage response where it participates in the repair of double-strand DNA breaks and in base excision repair of oxidized guanine residues (8-oxoguanine) by aiding the localization of the human 8-oxoguanine glycosylase (hOGG1) to damaged DNA. Here we demonstrate that following oxidative stress, hSSB1 is stabilized as an oligomer which is required for hSSB1 to function in the removal of 8-oxoguanine. Monomeric hSSB1 shows a decreased affinity for oxidized DNA resulting in a cellular 8-oxoguanine-repair defect and in the absence of ATM signaling initiation. While hSSB1 oligomerization is important for the removal of 8-oxoguanine from the genome, it is not required for the repair of double-strand DNA-breaks by homologous recombination. These findings demonstrate a novel hSSB1 regulatory mechanism for the repair of damaged DNA.Publisher PDFPeer reviewe

The molecular details of a novel phosphorylation-dependent interaction between MRN and the SOSS complex

The repair of double-strand DNA breaks (DSBs) by homologous recombination is crucial in the maintenance of genome integrity. While the key role of the Mre11-Rad50-Nbs1 (MRN) complex in repair is well known, hSSB1 (SOSSB and OBFC2B), one of the main components of the sensor of single-stranded DNA (SOSS) protein complex, has also been shown to rapidly localize to DSB breaks and promote repair. We have previously demonstrated that hSSB1 binds directly to Nbs1, a component of the MRN complex, in a DNA damage-independent manner. However, recruitment of the MRN complex has also been demonstrated by an interaction between Integrator Complex Subunit 3 (INTS3; also known as SOSSA), another member of the SOSS complex, and Nbs1. In this study, we utilize a combined approach of in silico, biochemical, and functional experiments to uncover the molecular details of INTS3 binding to Nbs1. We demonstrate that the forkhead-associated domain of Nbs1 interacts with INTS3 via phosphorylation-dependent binding to INTS3 at Threonine 592, with contributions from Serine 590. Based on these data, we propose a model of MRN recruitment to a DSB via INTS3

Phenotypic spectrum in osteogenesis imperfecta due to mutations in TMEM38B: unravelling a complex cellular defect.

Context: Recessive mutations in TMEM38B cause type XIV osteogenesis imperfecta (OI) by dysregulating intracellular calcium flux. Objectives: Clinical and bone material phenotype description and osteoblast differentiation studies. Design and Setting: Natural history study in paediatric research centres. Patients: Eight patients with type XIV OI. Main Outcome Measures: Clinical examinations included: bone mineral density, radiographs, echocardiography and muscle biopsy. Bone biopsy samples (n=3) were analysed using histomorphometry, quantitative backscattered electron microscopy and Raman microspectroscopy. Cellular differentiation studies were performed on proband and control osteoblasts and normal murine osteoclasts. Results: The clinical phenotype of type XIV OI ranges from asymptomatic to severe. Previously unreported features include vertebral fractures, periosteal cloaking, coxa vara and extraskeletal features (muscular hypotonia, cardiac abnormalities). Proband L1-L4 bone density Z-score was reduced (median -3.3 [range -4.77 to +0.1; n=7]), and increased by +1.7 (1.17 to 3.0; n=3) following bisphosphonate therapy. TMEM38B mutant bone has reduced trabecular bone volume, osteoblast and particularly osteoclast numbers, with >80% reduction in bone resorption. Bone matrix mineralization is normal and nanoporosity low. We demonstrate a complex osteoblast differentiation defect with decreased expression of early markers and increased late and mineralization-related markers. Predominance of TRIC-B over TRIC-A expression in murine osteoclasts supports an intrinsic osteoclast defect underlying low bone turnover. Conclusions: OI type XIV has a bone histology, matrix mineralization and osteoblast differentiation pattern that is distinct from OI with collagen defects. Probands are responsive to bisphosphonates and some show muscular and cardiovascular features possibly related to intracellular calcium flux abnormalities