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1.  Parathyroid hormone ablation alters erythrocyte parameters that are rescued by calcium-sensing receptor gene deletion 
The mechanisms by which parathyroid hormone (PTH) produces anemia, are unclear. Parathyroid hormone secretion is regulated by the extracellular Ca2+-sensing receptor. We investigated the effects of ablating PTH on hematological indices and erythrocytes volume regulation in wild-type, PTH-null and Ca2+-sensing receptor-null/PTH-null mice. The erythrocyte parameters were measured in whole mouse blood and volume regulatory systems were determined by plasma membrane K+ fluxes and osmotic fragility was measured by hemoglobin determination at varying osmolarities. We observed that the absence of PTH significantly increases mean erythrocyte volume and reticulocyte counts, while decreasing erythrocyte counts, hemoglobin, hematocrit, and mean corpuscular hemoglobin concentration. These changes were accompanied by increases in erythrocyte cation content, a denser cell population and increased K+ permeability, which were in part mediated by activation of the K+/Cl− cotransporter and Gardos channel. In addition we observed that erythrocyte osmotic fragility in PTH-null compared with wild-type mice was enhanced. When Ca2+-sensing receptor gene was deleted on the background of PTH-null mice, we observed that several of the alterations in erythrocyte parameters of PTH-null mice were largely rescued, particularly those related to erythrocyte volume, K+ fluxes and osmotic fragility, and became similar to those observed in wild-type mice. Our results demonstrate that Ca2+-sensing receptor and parathyroid hormone are functionally coupled to maintain erythrocyte homeostasis.
doi:10.1111/ejh.12110
PMCID: PMC3689863  PMID: 23528155
Parathyroid Hormone; Calcium-sensor receptor; erythrocytes; K+ channels
2.  Intracellular membrane traffic at high resolution 
Methods in cell biology  2010;96:619-648.
I. Abstract
Membrane traffic between organelles is essential for a multitude of processes that maintain cell homeostasis. Many steps in these tightly regulated trafficking pathways take place in microdomains on the membranes of organelles, which require analysis at nanometer resolution. Electron Microscopy (EM) can visualize these processes in detail and is mainly responsible for our current view of morphology on the subcellular level. This review discusses how EM can be applied to solve many questions of intracellular membrane traffic, with a focus on the endosomal system. We describe the expansion of the technique from purely morphological analysis to cryo-immuno-EM, Correlative Light Electron Microscopy (CLEM) and 3D electron tomography. In this review we go into some technical details of these various techniques. Furthermore, we provide a full protocol for immunolabeling on Lowicryl sections of high-pressure frozen cells as well as a detailed description of a simple CLEM method that can be applied to answer many membrane trafficking questions. We believe that these EM-based techniques are important tools to expand our understanding of the molecular details of endosomal sorting and intracellular membrane traffic in general.
doi:10.1016/S0091-679X(10)96026-3
PMCID: PMC4067575  PMID: 20869541
cargo sorting; endosome; electron microscopy; high-pressure freezing; freeze substitution; Tokuyasu; tomography
3.  Engineering Superficial Zone Features in Tissue Engineered Cartilage 
Biotechnology and bioengineering  2012;110(5):1476-1486.
A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here we report that hydrodynamic conditions that mimic the motion-induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features.
doi:10.1002/bit.24799
PMCID: PMC3694346  PMID: 23239161
Tissue Engineering; Bioreactors; Cartilage; Chondrocyte; Interstitial flow; Shear Stress; Two Photon Microscopy
4.  Early Impact of Social Isolation and Breast Tumor Progression in Mice 
Brain, behavior, and immunity  2012;30(Suppl):S135-S141.
Evidence from cancer patients and animal models of cancer indicates that exposure to psychosocial stress can promote tumor growth and metastasis, but the pathways underlying stress-induced cancer pathogenesis are not fully understood. Social isolation has been shown to promote tumor progression. We examined the impact of social isolation on breast cancer pathogenesis in adult female severe combined immunodeficiency (SCID) mice using the human breast cancer cell line, MDA-MB-231, a high ß-adrenergic receptor (AR) expressing line. When group-adapted mice were transferred into single housing (social isolation) one week prior to MB-231 tumor cell injection into a mammary fat pad (orthotopic), no alterations in tumor growth or metastasis were detected compared to group-housed mice. When social isolation was delayed until tumors were palpable, tumor growth was transiently increased in singly-housed mice. To determine if sympathetic nervous system activation was associated with increased tumor growth, spleen and tumor norepinephrine (NE) was measured after social isolation, in conjunction with tumor-promoting macrophage populations. Three days after transfer to single housing, spleen weight was transiently increased in tumor-bearing and non-tumor-bearing mice in conjunction with reduced splenic NE concentration and elevated CD11b+Gr-1+ macrophages. At day 10 after social isolation, no changes in spleen CD11b+ populations or NE were detected in singly-housed mice. In the tumors, social isolation increased CD11b+Gr-1+, CD11b+Gr-1-, and F4/80+ macrophage populations, with no change in tumor NE. The results indicate that a psychological stressor, social isolation, elicits dynamic but transient effects on macrophage populations that may facilitate tumor growth. The transiency of the changes in peripheral NE suggest that homeostatic mechanisms may mitigate the impact of social isolation over time. Studies are underway to define the neuroendocrine mechanisms underlying the tumor-promoting effects of social isolation, and to determine the contributions of increased tumor macrophages to tumor pathogenesis.
doi:10.1016/j.bbi.2012.05.003
PMCID: PMC3431437  PMID: 22610067
Social isolation; psychosocial stressor; breast cancer; norepinephrine; macrophages; SCID mice
5.  In Vivo Imaging of Tumors 
Cold Spring Harbor protocols  2010;2010(7):pdb.prot5452.
PMCID: PMC3891051  PMID: 20647356
6.  Meta-Analysis of Genome-Wide Association Studies Identifies Six New Loci for Serum Calcium Concentrations 
O'Seaghdha, Conall M. | Wu, Hongsheng | Yang, Qiong | Kapur, Karen | Guessous, Idris | Zuber, Annie Mercier | Köttgen, Anna | Stoudmann, Candice | Teumer, Alexander | Kutalik, Zoltán | Mangino, Massimo | Dehghan, Abbas | Zhang, Weihua | Eiriksdottir, Gudny | Li, Guo | Tanaka, Toshiko | Portas, Laura | Lopez, Lorna M. | Hayward, Caroline | Lohman, Kurt | Matsuda, Koichi | Padmanabhan, Sandosh | Firsov, Dmitri | Sorice, Rossella | Ulivi, Sheila | Brockhaus, A. Catharina | Kleber, Marcus E. | Mahajan, Anubha | Ernst, Florian D. | Gudnason, Vilmundur | Launer, Lenore J. | Mace, Aurelien | Boerwinckle, Eric | Arking, Dan E. | Tanikawa, Chizu | Nakamura, Yusuke | Brown, Morris J. | Gaspoz, Jean-Michel | Theler, Jean-Marc | Siscovick, David S. | Psaty, Bruce M. | Bergmann, Sven | Vollenweider, Peter | Vitart, Veronique | Wright, Alan F. | Zemunik, Tatijana | Boban, Mladen | Kolcic, Ivana | Navarro, Pau | Brown, Edward M. | Estrada, Karol | Ding, Jingzhong | Harris, Tamara B. | Bandinelli, Stefania | Hernandez, Dena | Singleton, Andrew B. | Girotto, Giorgia | Ruggiero, Daniela | d'Adamo, Adamo Pio | Robino, Antonietta | Meitinger, Thomas | Meisinger, Christa | Davies, Gail | Starr, John M. | Chambers, John C. | Boehm, Bernhard O. | Winkelmann, Bernhard R. | Huang, Jie | Murgia, Federico | Wild, Sarah H. | Campbell, Harry | Morris, Andrew P. | Franco, Oscar H. | Hofman, Albert | Uitterlinden, Andre G. | Rivadeneira, Fernando | Völker, Uwe | Hannemann, Anke | Biffar, Reiner | Hoffmann, Wolfgang | Shin, So–Youn | Lescuyer, Pierre | Henry, Hughes | Schurmann, Claudia | Munroe, Patricia B. | Gasparini, Paolo | Pirastu, Nicola | Ciullo, Marina | Gieger, Christian | März, Winfried | Lind, Lars | Spector, Tim D. | Smith, Albert V. | Rudan, Igor | Wilson, James F. | Polasek, Ozren | Deary, Ian J. | Pirastu, Mario | Ferrucci, Luigi | Liu, Yongmei | Kestenbaum, Bryan | Kooner, Jaspal S. | Witteman, Jacqueline C. M. | Nauck, Matthias | Kao, W. H. Linda | Wallaschofski, Henri | Bonny, Olivier | Fox, Caroline S. | Bochud, Murielle
PLoS Genetics  2013;9(9):e1003796.
Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. We characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression in bone in response to dietary calcium in mice. Our results shed new light on the genetics of calcium homeostasis.
Author Summary
Calcium is vital to many biological processes and its serum concentration is tightly regulated. Family studies have shown that serum calcium is under strong genetic control. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤21,679 additional individuals. We identified seven loci (six new regions) as being robustly associated with serum calcium. Three loci implicate regions involved in rare monogenic diseases including disturbances of serum calcium levels. Several of the newly identified loci harbor genes linked to the hormonal control of serum calcium. In mice experiments, we characterized the expression of these genes in gut, kidney, and bone, and explored the influence of dietary calcium intake on the expression of these genes in these organs. Our results shed new light on the genetics of calcium homeostasis and suggest a role for dietary calcium intake in bone-specific gene expression.
doi:10.1371/journal.pgen.1003796
PMCID: PMC3778004  PMID: 24068962
7.  Stromal matrix metalloprotease-13 knockout alters Collagen I structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic E0771 mammary tumor cells 
BMC Cancer  2013;13:411.
Background
Matrix metalloproteases and collagen are key participants in breast cancer, but their precise roles in cancer etiology and progression remain unclear. MMP13 helps regulate collagen structure and has been ascribed largely harmful roles in cancer, but some studies demonstrate that MMP13 may also protect against tumor pathology. Other studies indicate that collagen’s organizational patterns at the breast tumor-host interface influence metastatic potential. Therefore we investigated how MMP13 modulates collagen I, a principal collagen subtype in breast tissue, and affects tumor pathology and metastasis in a mouse model of breast cancer.
Methods
Tumors were implanted into murine mammary tissues, and their growth analyzed in Wildtype and MMP13 KO mice. Following extraction, tumors were analyzed for collagen I levels and collagen I macro- and micro-structural properties at the tumor-host boundary using immunocytochemistry and two-photon and second harmonic generation microscopy. Lungs were analyzed for metastases counts, to correlate collagen I changes with a clinically significant functional parameter. Statistical analyses were performed by t-test, analysis of variance, or Wilcoxon-Mann–Whitney tests as appropriate.
Results
We found that genetic ablation of host stromal MMP13 led to: 1. Increased mammary tumor collagen I content, 2. Marked changes in collagen I spatial organization, and 3. Altered collagen I microstructure at the tumor-host boundary, as well as 4. Increased metastasis from the primary mammary tumor to lungs.
Conclusions
These results implicate host MMP13 as a key regulator of collagen I structure and metastasis in mammary tumors, thus making it an attractive potential therapeutic target by which we might alter metastatic potential, one of the chief determinants of clinical outcome in breast cancer. In addition to identifying stromal MMP13 is an important regulator of the tumor microenvironment and metastasis, these results also suggest that stromal MMP13 may protect against breast cancer pathology under some conditions, a finding with important implications for development of chemotherapies directed against matrix metalloproteases.
doi:10.1186/1471-2407-13-411
PMCID: PMC3766650  PMID: 24010522
Two-photon; Microscopy; Cancer; Second harmonic generation; Collagen; SHG; Tumor; MMP; Matrix metalloprotease; MMP-13; Intravital; Imaging; In vivo; Multiphoton; Intrinsic; Fluorophore
8.  Bone Loss following Spinal Cord Injury in a Rat Model 
Journal of Neurotrauma  2012;29(8):1676-1682.
Abstract
The current study was undertaken to follow the time course of bone loss in the proximal tibia of rats over several weeks following thoracic contusion spinal cord injury (SCI) of varying severity. It was hypothesized that bone loss would be more pronounced in the more severely injured animals, and that hindlimb weight bearing would help prevent bone loss. Twenty-six female Sprague-Dawley rats (200–225 g, 6–7 weeks old) received standard thoracic (T9) injuries at energies of 6.25, 12.5, 25, or 50 g-cm. The rats were scored weekly for hindlimb function during locomotion. At 0, 2 or 3, and 8 weeks, high-resolution micro-CT images of each right tibia were obtained. Mechanical indentation testing was done to measure the compressive strength of the cancellous bone structure. The 6.25 g-cm group showed near normal locomotion, the 12.5 and 25 g-cm groups showed the ability to frequently or occasionally generate weight-supported plantar steps, respectively, and the 50 g-cm group showed only movement without weight-supported plantar stepping. The 6.25, 12.5 and 25 g-cm groups remained at the same level of bone volume fraction (cancBV/TV=0.24±0.07), while the 50 g-cm group experienced severe bone loss (67%), resulting in significantly lower (p<0.05) bone volume fraction (cancBV/TV=0.11±0.05) at 8 weeks. Proximal tibia cancellous bone strength was reduced by approximately 50% in these severely injured rats. Instead of a linear proportionality between injury severity and bone loss, there appears to be a distinct functional threshold, marked by occasional weight-supported stepping, above which bone loss does not occur.
doi:10.1089/neu.2011.2037
PMCID: PMC3353757  PMID: 22181016
bone loss; cancellous; density; locomotion; spinal cord injury
9.  Two-Photon and Second Harmonic Microscopy in Clinical and Translational Cancer Research 
Annals of Biomedical Engineering  2012;40(2):277-291.
Application of two-photon microscopy (TPM) to translational and clinical cancer research has burgeoned over the last several years, as several avenues of pre-clinical research have come to fruition. In this review, we focus on two forms of TPM—two-photon excitation fluorescence microscopy, and second harmonic generation microscopy—as they have been used for investigating cancer pathology in ex vivo and in vivo human tissue. We begin with discussion of two-photon theory and instrumentation particularly as applicable to cancer research, followed by an overview of some of the relevant cancer research literature in areas that include two-photon imaging of human tissue biopsies, human skin in vivo, and the rapidly developing technology of two-photon microendoscopy. We believe these and other evolving two-photon methodologies will continue to help translate cancer research from the bench to the bedside, and ultimately bring minimally invasive methods for cancer diagnosis and treatment to therapeutic reality.
doi:10.1007/s10439-012-0512-9
PMCID: PMC3342697  PMID: 22258888
Two-photon microscopy; Cancer; Second harmonic generation; Collagen; SHG; Endoscopy
10.  Measuring intranodal pressure and lymph viscosity to elucidate mechanisms of arthritic flare and therapeutic outcomes 
Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints, whose etiology is largely unknown. Recent studies in mice demonstrated alterations in lymphatics from affected joints precede flares. Thus, we aimed to develop novel methods for measuring lymph node pressure and lymph viscosity in limbs of mice. Pressure measurements were performed by inserting a glass micropipette connected to a pressure transducer into popliteal lymph nodes (PLN) or axillary lymph nodes (ALN) of mice and determined that the lymphatic pressures were 9 and 12 cm of water, respectively. We are also developing methods for measuring lymph viscosity in lymphatic vessels afferent to PLN, which can be measured by multi-photon fluorescence recovery after photobleaching (MP-FRAP) of FITC-BSA injected into the hind footpad. These results demonstrate the potential of lymph node pressure and lymph viscosity measurements, and warrant future studies to test these outcomes as biomarkers of arthritic flare.
doi:10.1111/j.1749-6632.2011.06237.x
PMCID: PMC3334848  PMID: 22172039
Rheumatoid Arthritis; Lymph Node; Flare; Lymphatic Pressure; Lymph Viscosity
11.  β-Adrenergic receptors (β-AR) regulate VEGF and IL-6 production by divergent pathways in high β-AR-expressing breast cancer cell lines 
Activation of β-adrenergic receptors (β-AR) drives proangiogenic factor production in several types of cancers. To examine β-AR regulation of breast cancer pathogenesis, β-AR density, signaling capacity, and functional responses to β-AR stimulation were studied in four human breast adenocarcinoma cell lines. β-AR density ranged from very low in MCF7 and MB-361 to very high in MB-231 and in a brain-seeking variant of MB-231, MB-231BR. Consistent with β-AR density, β-AR activation elevated cAMP in MCF7 and MB-361 much less than in MB-231 and MB-231BR. Functionally, β-AR stimulation did not markedly alter vascular endothelial growth factor (VEGF) production by MCF7 or MB-361. In the two high β-AR-expressing cell lines MB-231 and MB-231BR, β-AR-induced cAMP and VEGF production differed considerably, despite similar β-AR density. The β2-AR-selective agonist terbutaline and the endogenous neurotransmitter norepinephrine decreased VEGF production by MB-231, but increased VEGF production by MB-231BR. Moreover, β2-AR activation increased IL-6 production by both MB-231 and MB-231BR. These functional alterations were driven by elevated cAMP, as direct activation of adenylate cyclase by forskolin elicited similar alterations in VEGF and IL-6 production. The protein kinase A antagonist KT5720 prevented β-AR-induced alterations in MB-231 and MB-231BR VEGF production, but not IL-6 production.
Conclusions
β-AR expression and signaling is heterogeneous in human breast cancer cell lines. In cells with high β-AR density, β-AR stimulation regulates VEGF production through the classical β-AR-cAMP-PKA pathway, but this pathway can elicit directionally opposite outcomes. Furthermore, in the same cells, β-AR activate a cAMP-dependent, PKA-independent pathway to increase IL-6 production. The complexity of breast cancer cell β-AR expression and functional responses must be taken into account when considering β-AR as a therapeutic target for breast cancer treatment.
doi:10.1007/s10549-011-1348-y
PMCID: PMC3126869  PMID: 21234673
Breast cancer; β-Adrenergic receptors; VEGF; IL-6; Norepinephrine; cAMP
12.  Measurement of the ratio of forward-propagating to back-propagating second harmonic signal using a single objective 
Optics express  2010;18(10):10538-10550.
In this paper, we present a method to determine, for the first time, the ratio of forward-propagating second harmonic (SHG) signal to back-propagating SHG signal (F/B) in vivo on the surface of intact tissue samples without any biopsy or tissue sectioning, using only epidetection (i.e., via a single objective lens). This method has the additional benefit of using the confocal detection apparatus already contained within common commercially available two-photon laser-scanning microscopes, and hence can allow the measurement of the SHG F/B ratio in vivo with minimal purchase of new equipment.
PMCID: PMC3427947  PMID: 20588906
13.  Multiphoton fluorescence recovery after photobleaching in bounded systems 
Multiphoton fluorescence recovery after photobleaching (MP-FRAP) is a laser microscopy technique used to measure diffusion coefficients of macromolecules in biological systems. The three-dimensional resolution and superior depth penetration within scattering samples offered by MP-FRAP make it an important tool for investigating both in vitro and in vivo systems. However, biological systems frequently confine diffusion within solid barriers, and to date the effect of such barriers on the measurement of absolute diffusion coefficients via MP-FRAP has not been studied. We have used Monte Carlo simulations of diffusion and MP-FRAP to understand the effect of barriers of varying geometries and positions relative to the two-photon focal volume. Furthermore, we supply ranges of barrier positions within which MP-FRAP can confidently be employed to measure accurate diffusion coefficients. Finally, we produce two new MP-FRAP models that can produce accurate diffusion coefficients in the presence of a single plane boundary or parallel infinite plane boundaries positioned parallel to the optical axis, up to the resolution limit of the multiphoton laser scanning microscope.
PMCID: PMC3413246  PMID: 21728580
14.  The Calcium-Sensing Receptor Mediates Bone Turnover Induced by Dietary Calcium and Parathyroid Hormone in Neonates 
Journal of Bone and Mineral Research  2010;26(5):1057-1071.
We have investigated, in neonates, whether the calcium-sensing receptor (CaR) mediates the effects of dietary calcium on bone turnover and/or modulates parathyroid hormone (PTH)–induced bone turnover. Wild-type (WT) pups and pups with targeted deletion of the Pth (Pth–/–) gene or of both Pth and CaR (Pth–/–CaR–/–) genes were nursed by dams on a normal or high-calcium diet. Pups nursed by dams on a normal diet received daily injections of vehicle or of PTH(1–34) (80 µg/kg) for 2 weeks starting from 1 week of age. In pups receiving vehicle and fed by dams on a normal diet, trabecular bone volume, osteoblast number, type 1 collagen–positive area, and mineral apposition rate, as well as the expression of bone-formation-related genes, all were reduced significantly in Pth–/– pups compared with WT pups and were decreased even more dramatically in Pth–/–CaR–/– pups. These parameters were increased in WT and Pth–/– pups but not in Pth–/–CaR–/– pups fed by dams on a high-calcium diet compared with pups fed by dams on a normal diet. These parameters also were increased in WT, Pth–/–, and Pth–/–CaR–/– pups following exogenous PTH treatment; however, the percentage increase was less in Pth–/–CaR–/– pups than in WT and Pth–/– pups. In vehicle-treated pups fed by dams on either the normal or high-calcium diet and in PTH-treated pups fed by dams on a normal diet, the number and surfaces of osteoclasts and the ratio of RANKL/OPG were reduced significantly in Pth–/– pups and less significantly in Pth–/–CaR–/– pups compared with WT pups. These parameters were further reduced significantly in WT and Pth–/– pups from dams fed a high-calcium diet but did not decrease significantly in similarly treated Pth–/–CaR–/– pups, and they increased significantly in PTH-treated pups compared with vehicle-treated, genotype-matched pups fed by dams on the normal diet. These results indicate that in neonates, the CaR mediates alterations in bone turnover in response to changes in dietary calcium and modulates PTH-stimulated bone turnover. © 2011 American Society for Bone and Mineral Research.
doi:10.1002/jbmr.300
PMCID: PMC3179300  PMID: 21542007
CALCIUM-SENSING RECEPTOR; PARATHYROID HORMONE; DIETARY CALCIUM; BONE TURNOVER
15.  The cation channel mucolipin-1 is a bifunctional protein that facilitates membrane remodeling via its serine lipase domain 
Experimental cell research  2011;317(6):691-705.
Phospholipase modulators have been shown to affect the topology of lipid bilayers and the formation of tubulo-vesicular structures, but the specific endogenous phospholipases involved have yet to be identified. Here we show that TRPML1 (MLN1), a Ca2+-permeable channel contributes to membrane remodeling through a serine-lipase consensus domain, and thus represents a novel type of bifunctional protein. Remarkably, this serine lipase active site determines the ability of MLN1 to generate tubulo-vesicular extensions in mucolipin-1-expressing oocytes, human fibroblasts and model membrane vesicles. Our demonstration that MLN1 is involved in membrane remodeling and the formation of extensions suggests that it may play a role in the formation of cellular processes linked to the late endosome/lysosome (LE/L) pathway. MLN1 is absent or mutated in patients with mucolipidosis IV (MLIV), a lysosomal disorder with devastating neurological and other consequences. This study provides potential insight into the pathophysiology of MLIV.
doi:10.1016/j.yexcr.2011.01.008
PMCID: PMC3103141  PMID: 21256127
membrane remodeling; phospholipase; TRPML1; mucolipin; mucolipidosis IV; tubulo-vesicular
16.  Common variants in the calcium-sensing receptor gene are associated with total serum calcium levels 
Human Molecular Genetics  2010;19(21):4296-4303.
Serum calcium levels are tightly regulated. We performed genome-wide association studies (GWAS) in population-based studies participating in the CHARGE Consortium to uncover common genetic variations associated with total serum calcium levels. GWAS of serum calcium concentrations was performed in 20 611 individuals of European ancestry for ∼2.5 million genotyped and imputed single-nucleotide polymorphisms (SNPs). The SNP with the lowest P-value was rs17251221 (P = 2.4 * 10−22, minor allele frequency 14%) in the calcium-sensing receptor gene (CASR). This lead SNP was associated with higher serum calcium levels [0.06 mg/dl (0.015 mmol/l) per copy of the minor G allele] and accounted for 0.54% of the variance in serum calcium concentrations. The identification of variation in CASR that influences serum calcium concentration confirms the results of earlier candidate gene studies. The G allele of rs17251221 was also associated with higher serum magnesium levels (P = 1.2 * 10−3), lower serum phosphate levels (P = 2.8 * 10−7) and lower bone mineral density at the lumbar spine (P = 0.038), but not the femoral neck. No additional genomic loci contained SNPs associated at genome-wide significance (P < 5 * 10−8). These associations resemble clinical characteristics of patients with familial hypocalciuric hypercalcemia, an autosomal-dominant disease arising from rare inactivating mutations in the CASR gene. We conclude that common genetic variation in the CASR gene is associated with similar but milder features in the general population.
doi:10.1093/hmg/ddq342
PMCID: PMC2951868  PMID: 20705733
17.  The Abnormal Phenotypes of Cartilage and Bone in Calcium-Sensing Receptor Deficient Mice Are Dependent on the Actions of Calcium, Phosphorus, and PTH 
PLoS Genetics  2011;7(9):e1002294.
Patients with neonatal severe hyperparathyroidism (NSHPT) are homozygous for the calcium-sensing receptor (CaR) mutation and have very high circulating PTH, abundant parathyroid hyperplasia, and severe life-threatening hypercalcemia. Mice with homozygous deletion of CaR mimic the syndrome of NSHPT. To determine effects of CaR deficiency on skeletal development and interactions between CaR and 1,25(OH)2D3 or PTH on calcium and skeletal homeostasis, we compared the skeletal phenotypes of homozygous CaR–deficient (CaR−/−) mice to those of double homozygous CaR– and 1α(OH)ase–deficient [CaR−/−1α(OH)ase−/−] mice or those of double homozygous CaR– and PTH–deficient [CaR−/−PTH−/−] mice at 2 weeks of age. Compared to wild-type littermates, CaR−/− mice had hypercalcemia, hypophosphatemia, hyperparathyroidism, and severe skeletal growth retardation. Chondrocyte proliferation and PTHrP expression in growth plates were reduced significantly, whereas trabecular volume, osteoblast number, osteocalcin-positive areas, expression of the ALP, type I collagen, osteocalcin genes, and serum ALP levels were increased significantly. Deletion of 1α(OH)ase in CaR−/− mice resulted in a longer lifespan, normocalcemia, lower serum phosphorus, greater elevation in PTH, slight improvement in skeletal growth with increased chondrocyte proliferation and PTHrP expression, and further increases in indices of osteoblastic bone formation. Deletion of PTH in CaR−/− mice resulted in rescue of early lethality, normocalcemia, increased serum phosphorus, undetectable serum PTH, normalization in skeletal growth with normal chondrocyte proliferation and enhanced PTHrP expression, and dramatic decreases in indices of osteoblastic bone formation. Our results indicate that reductions in hypercalcemia play a critical role in preventing the early lethality of CaR−/− mice and that defects in endochondral bone formation in CaR−/− mice result from effects of the marked elevation in serum calcium concentration and the decreases in serum phosphorus concentration and skeletal PTHrP levels, whereas the increased osteoblastic bone formation results from direct effects of PTH.
Author Summary
Mice with homozygous deletion of the calcium-sensing receptor (CaR) mimic the syndrome of neonatal severe hyperparathyroidism (NSHPT) in humans with very high circulating parathyroid hormone (PTH) and severe life-threatening hypercalcemia. To determine effects of CaR deficiency on skeletal development and interactions between CaR and 1,25(OH)2D3 or PTH on calcium and skeletal homeostasis, we compared the skeletal phenotypes of homozygous CaR–deficient mice to those of double homozygous CaR– and 1,25(OH)2D3–deficient mice or those of double homozygous CaR– and PTH–deficient mice. CaR–deficient mice had hypercalcemia, hypophosphatemia, hyperparathyroidism, severe skeletal growth retardation, and abnormalities; and most died within 2 weeks of age. Deletion of 1,25(OH)2D3 in CaR–deficient mice resulted in a longer lifespan, normocalcemia, lower serum phosphorus, greater elevation in PTH, and slight improvement in skeletal growth. Deletion of PTH in CaR–deficient mice resulted in rescue of early lethality, normocalcemia, increased serum phosphorus, and normalization in skeletal growth. Our results indicate that reductions in hypercalcemia reduce the early lethality of CaR–deficient mice and that deletion of PTH in patients with NSHPT may normalize skeletal growth and development.
doi:10.1371/journal.pgen.1002294
PMCID: PMC3178615  PMID: 21966280
18.  Mitochondrial membrane potential probes and the proton gradient: a practical usage guide 
BioTechniques  2011;50(2):98-115.
Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function, particularly in the context of cell fate determination in biological and biomedical research. However, valid interpretation of results obtained with such probes requires careful consideration of numerous controls, as well as possible effects of non-protonic charges on dye behavior. In this context, we provide an overview of some of the important technical considerations, controls, and parallel complementary assays that can be employed to help ensure appropriate interpretation of results, thus providing a practical usage guide for monitoring mitochondrial membrane potentials with cationic probes. In total, this review will help illustrate both the strengths and potential pitfalls of common mitochondrial membrane potential dyes, and highlight best-usage approaches for their efficacious application in life sciences research.
doi:10.2144/000113610
PMCID: PMC3115691  PMID: 21486251
Mitochondria; hyperpolarization; depolarization; membrane potential; calcium; proton; cationic; fluorescent; probe; dye; pH
19.  Mapping of Human Autoantibody Binding Sites on the Calcium-Sensing Receptor 
Previously, we have demonstrated the presence of anti-calcium-sensing receptor (CaSR) antibodies in patients with autoimmune polyglandular syndrome type 1 (APS1), a disease that is characterized in part by hypoparathyroidism involving hypocalcemia, hyperphosphatemia, and low serum levels of parathyroid hormone. The aim of this study was to define the binding domains on the CaSR of anti-CaSR antibodies found in APS1 patients and in one patient suspected of having autoimmune hypocalciuric hypercalcemia (AHH). A phage-display library of CaSR peptides was constructed and used in biopanning experiments with patient sera. Selectively enriched IgG-binding peptides were identified by DNA sequencing, and subsequently, immunoreactivity to these peptides was confirmed in ELISA. Anti-CaSR antibody binding sites were mapped to amino acid residues 41–69, 114–126, and 171–195 at the N-terminal of the extracellular domain of the receptor. The major autoepitope was localized in the 41–69 amino acid sequence of the CaSR with antibody reactivity demonstrated in 12 of 12 (100%) APS1 patients with anti-CaSR antibodies and in 1 AHH patient with anti-CaSR antibodies. Minor epitopes were located in the 114–126 and 171–195 amino acid domains, with antibody reactivity shown in 5 of 12 (42%) and 4 of 12 (33%) APS1 patients, respectively. The results indicate that epitopes for anti-CaSR antibodies in the AHH patient and in the APS1 patients who were studied are localized in the N-terminal of the extracellular domain of the receptor. The present work has demonstrated the successful use of phage-display technology in the discovery of CaSR-specific epitopes targeted by human anti-CaSR antibodies. © 2010 American Society for Bone and Mineral Research.
doi:10.1359/jbmr.090703
PMCID: PMC3153323  PMID: 19580466
autoantibody; autoimmune polyendocrine syndrome type 1; parathyroid; calcium-sensing receptor; hypoparathyroidism
20.  Swim Training Initiated Acutely after Spinal Cord Injury Is Ineffective and Induces Extravasation In and Around the Epicenter 
Journal of Neurotrauma  2009;26(7):1017-1027.
Abstract
Activity-based rehabilitation is a promising strategy for improving functional recovery following spinal cord injury (SCI). While results from both clinical and animal studies have shown that a variety of approaches can be effective, debate still exists regarding the optimal post-injury period to apply rehabilitation. We recently demonstrated that rats with moderately severe thoracic contusive SCI can be re-trained to swim when training is initiated 2 weeks after injury and that swim training had no effect on the recovery of overground locomotion. We concluded that swim training is a task-specific model of post-SCI activity-based rehabilitation. In the present study, we ask if re-training initiated acutely is more or less effective than when initiated at 2 weeks post-injury. Using the Louisville Swim Scale, an 18-point swimming assessment, supplemented by kinematic assessment of hindlimb movement during swimming, we report that acute re-training is less effective than training initiated at 2 weeks. Using the bioluminescent protein luciferase as a blood-borne macromolecular marker, we also show a significant increase in extravasation in and around the site of SCI following only 8 min of swimming at 3 days post-injury. Taken together, these results suggest that acute re-training in a rat model of SCI may compromise rehabilitation efforts via mechanisms that may involve one or more secondary injury cascades, including acute spinal microvascular dysfunction.
doi:10.1089/neu.2008.0829
PMCID: PMC2848951  PMID: 19331515
activity-based rehabilitation; microvascular; rat; spinal cord injury; swimming
21.  Swim Training Initiated Acutely after Spinal Cord Injury Is Ineffective and Induces Extravasation In and Around the Epicenter 
Journal of neurotrauma  2009;26(7):10.1089/neu.2008-0829.
Activity-based rehabilitation is a promising strategy for improving functional recovery following spinal cord injury (SCI). While results from both clinical and animal studies have shown that a variety of approaches can be effective, debate still exists regarding the optimal post-injury period to apply rehabilitation. We recently demonstrated that rats with moderately severe thoracic contusive SCI can be re-trained to swim when training is initiated 2 weeks after injury and that swim training had no effect on the recovery of overground locomotion. We concluded that swim training is a task-specific model of post-SCI activity-based rehabilitation. In the present study, we ask if re-training initiated acutely is more or less effective than when initiated at 2 weeks post-injury. Using the Louisville Swim Scale, an 18-point swimming assessment, supplemented by kinematic assessment of hindlimb movement during swimming, we report that acute re-training is less effective than training initiated at 2 weeks. Using the bioluminescent protein luciferase as a blood-borne macromolecular marker, we also show a significant increase in extravasation in and around the site of SCI following only 8 min of swimming at 3 days post-injury. Taken together, these results suggest that acute re-training in a rat model of SCI may compromise rehabilitation efforts via mechanisms that may involve one or more secondary injury cascades, including acute spinal microvascular dysfunction.
doi:10.1089/neu.2008-0829
PMCID: PMC2848951  PMID: 19331515
activity-based rehabilitation; microvascular; rat; spinal cord injury; swimming
22.  Anti-Parathyroid and Anti-Calcium Sensing Receptor Antibodies in Autoimmune Hypoparathyroidism 
The parathyroid glands are an infrequent target for autoimmunity; the exception being in the autoimmune polyglandular syndrome type 1, where autoimmune hypoparathyroidism is the rule. Antibodies that are directed against the parathyroid cell-surface calcium-sensing receptor (CaSR) have recently been recognised to be present in the serum of patients with autoimmune hypoparathyroidism. In some individuals, these anti-CaSR antibodies have also been shown to produce functional activation of the receptor, suggesting a direct pathogenic role in hypocalcemia. Additionally, a few hypercalcemic patients with autoimmune hyperparathyroidism owing to anti-CaSR antibodies that inhibit receptor activation have now been identified. Other novel parathyroid autoantigens are starting to be elucidated. These findings suggest that new approaches to treatment, such as CaSR antagonists or agonists (calcilytics/calcimimetics) may be worthwhile.
doi:10.1016/j.ecl.2009.01.001
PMCID: PMC2700354  PMID: 19328421
Parathyroid; calcium-sensing receptor; CaSR; autoantibody; anti-parathyroid antibody; ant-CaSR antibody; autoimmune; hypoparathyroidism; autoimmune hypocalciuric hypercalcemia
23.  Identification and Dissection of Ca2+-binding Sites in the Extracellular Domain of Ca2+-sensing Receptor* 
The Journal of biological chemistry  2007;282(26):19000-19010.
Ca2+-sensing receptors (CaSRs) represent a class of receptors that respond to changes in the extracellular Ca2+ concentration ([Ca2+]o) and activate multiple signaling pathways. A major barrier to advancing our understanding of the role of Ca2+ in regulating CaSRs is the lack of adequate information about their Ca2+-binding locations, which is largely hindered by the lack of a solved three-dimensional structure and rapid off rates due to low Ca2+-binding affinities. In this paper, we have reported the identification of three potential Ca2+-binding sites in a modeled CaSR structure using computational algorithms based on the geometric description and surface electrostatic potentials. Mutation of the predicted ligand residues in the full-length CaSR caused abnormal responses to [Ca2+]o, similar to those observed with naturally occurring activating or inactivating mutations of the CaR, supporting the essential role of these predicted Ca2+-binding sites in the sensing capability of the CaSR. In addition, to probe the intrinsic Ca2+-binding properties of the predicted sequences, we engineered two predicted continuous Ca2+-binding sequences individually into a scaffold protein provided by a non-Ca2+-binding protein, CD2. We report herein the estimation of the metal-binding affinities of these predicted sites in the CaSR by monitoring aromatic-sensitized Tb3+ fluorescence energy transfer. Removing the predicted Ca2+-binding ligands resulted in the loss of or significantly weakened cation binding. The potential Ca2+-binding residues were shown to be involved in Ca2+/Ln3+ binding by high resolution NMR and site-directed mutagenesis, further validating our prediction of Ca2+-binding sites within the extracellular domain of the CaSR.
doi:10.1074/jbc.M701096200
PMCID: PMC2867057  PMID: 17478419
24.  Pilot case-control investigation of risk factors for hip fractures in the urban Indian population 
Background
Despite the reported high prevalence of osteoporosis in India, there have been no previous studies examining the risk factors for hip fracture in the Indian population.
Methods
We carried out a case control investigation comprising 100 case subjects (57 women and 43 men) admitted with a first hip fracture into one of three hospitals across New Delhi. The 100 controls were age and sex matched subjects who were either healthy visitors not related to the case patients or hospital staff. Information from all subjects was obtained through a questionnaire based interview.
Results
There was a significant increase in the number of cases of hip fracture with increasing age. There were significantly more women (57%) than men (43%). Univariate analysis identified protective effects for increased activity, exercise, calcium and vitamin supplements, almonds, fish, paneer (cottage cheese), curd (plain yogurt), and milk. However, tea and other caffeinated beverages were significant risk factors. In women, hormone/estrogen therapy appeared to have a marginal protective effect. For all cases, decreased agility, visual impairment, long term medications, chronic illnesses increased the risk of hip fracture. The multivariate analysis confirmed a protective effect of increased activity and also showed a decrease in hip fracture risk with increasing body mass index (odds ratio (OR) 0.024, 95% confidence interval (CI) 0.006-0.10 & OR 0.81, 95% CI 0.68-0.97 respectively). Individuals who take calcium supplements have a decreased risk of hip fracture (OR 0.076; CI 0.017-0.340), as do individuals who eat fish (OR 0.094; CI 0.020-0.431), and those who eat paneer (OR 0.152; 0.031-0.741). Tea drinkers have a higher risk of hip fracture (OR 22.8; 95% CI 3.73-139.43). Difficulty in getting up from a chair also appears to be an important risk factor for hip fractures (OR 14.53; 95% CI 3.86-54.23).
Conclusions
In the urban Indian population, dietary calcium, vitamin D, increased body mass index, and higher activity levels have a significant protective effect on hip fracture. On the other hand, caffeine intake and decreased agility increase the risk of hip fracture. Future studies should be done in order to direct primary preventive programs for hip fracture in India.
doi:10.1186/1471-2474-11-49
PMCID: PMC2845554  PMID: 20226091
25.  Swimming as a Model of Task-Specific Locomotor Retraining After Spinal Cord Injury in the Rat 
Background
The authors have shown that rats can be retrained to swim after a moderately severe thoracic spinal cord contusion. They also found that improvements in body position and hindlimb activity occurred rapidly over the first 2 weeks of training, reaching a plateau by week 4. Overground walking was not influenced by swim training, suggesting that swimming may be a task-specific model of locomotor retraining.
Objective
To provide a quantitative description of hindlimb movements of uninjured adult rats during swimming, and then after injury and retraining.
Methods
The authors used a novel and streamlined kinematic assessment of swimming in which each limb is described in 2 dimensions, as 3 segments and 2 angles.
Results
The kinematics of uninjured rats do not change over 4 weeks of daily swimming, suggesting that acclimatization does not involve refinements in hindlimb movement. After spinal cord injury, retraining involved increases in hindlimb excursion and improved limb position, but the velocity of the movements remained slow.
Conclusion
These data suggest that the activity pattern of swimming is hardwired in the rat spinal cord. After spinal cord injury, repetition is sufficient to bring about significant improvements in the pattern of hindlimb movement but does not improve the forces generated, leaving the animals with persistent deficits. These data support the concept that force (load) and pattern generation (recruitment) are independent and may have to be managed together with respect to postinjury rehabilitation.
doi:10.1177/1545968308331147
PMCID: PMC2836886  PMID: 19270266
Spinal cord injury; Swimming; Task-specific learning; Rat; Locomotor retraining; Rehabilitation

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