bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of glial fibrillary acidic protein expression on neurite outgrowth from retinal explants in a permissive environment
Authors: Toops Kimberly A , Hagemann Tracy L , Messing Albee , Nickells Robert W
Published: December 2012

Background

Increased expression of glial fibrillary acidic protein (GFAP) within macroglia is commonly seen as a hallmark of glial activation after damage within the central nervous system, including the retina. The increased expression of GFAP in glia is also considered part of the pathologically inhibitory environment for regeneration of axons from damaged neurons. Recent studies have raised the possibility that reactive gliosis and increased GFAP cannot automatically be assumed to be negative events for the surrounding neurons and that the context of the reactive gliosis is critical to whether neurons benefit or suffer. We utilized transgenic mice expressing a range of Gfap to titrate the amount of GFAP in retinal explants to investigate the relationship between GFAP concentration and the regenerative potential of retinal ganglion cells.

Findings

Explants from Gfap

-/-
and Gfap

+/-
mice did not have increased neurite outgrowth compared with Gfap

+/+
or Gfap over-expressing mice as would be expected if GFAP was detrimental to axon regeneration. In fact, Gfap over-expressing explants had the most neurite outgrowth when treated with a neurite stimulatory media. Transmission electron microscopy revealed that neurites formed bundles, which were surrounded by larger cellular processes that were GFAP positive indicating a close association between growing axons and glial cells in this regeneration paradigm.

Conclusions

We postulate that glial cells with increased Gfap expression support the elongation of new neurites from retinal ganglion cells possibly by providing a scaffold for outgrowth.
Evaluation of efficacy of iodine prophylaxis in Poland based on the examination of schoolchildren living in Opoczno Town (Lodz Voivodship)
Authors: Zygmunt Arkadiusz , Adamczewski Zbigniew , Wojciechowska-Durczyńska Katarzyna , Cyniak-Magierska Anna , Krawczyk-Rusiecka Kinga , Zygmunt Agnieszka , Karbownik-Lewińska Małgorzata , Lewiński Andrzej
Published: December 2012

Background

In 1997 a currently obligatory model of iodine prophylaxis, based on mandatory iodization of household salt with 30 mg KI/kg, was introduced. The aim of our study was to assess the iodine intake among school-age children living in Opoczno in 3 subsequent time points – in 1994, before establishment of currently operating model of iodine prophylaxis, in 1999 – 2 years after implementation of iodine prophylaxis and in 2010, – 14 years after its implementation.

Methods

We assessed goitre incidence and urine iodine concentration (UIC) in 104 children in 1994, 207 children in 1999 and 174 children in 2012. Age of examined children ranged from 6 to 15 years. The thyroid volumes evaluated by ultrasound were compared to reference values for thyroid volume proposed by Zimmermann at al. Moreover, we have introduced a new index – V/BSA ratio (comparison of thyroid volume to the body surface area), which to our belief allows for more accurate assessment of thyroid volume.

Results

The median of UICs was 45.5 μg/L (1994), 101.1 μg/L (1999) and 100.6 μg/L (2010). The distribution of obtained results has changed as well – iodine concentrations below 50 μg/L were present in 59.1% children in 1994, in 12.6% children – in 1999 and in 7.1% children – in 2010.

Although a significant decrease in goitre incidence with regard to age – 92.6% (1994) vs 18.5% (1999) and 15.8% (2010), as well as with regard to BSA – 95.4% (1994) vs 15.2% (1999) and 11.6% (2010) was observed, it still points to the iodine deficiency, which is in contradiction with UICs as they are within normal limits. V/BSA ratio avoids such discrepancy. The values of ratio V/BSA were higher in 1994 (7.079 ± 2.775) than in 1999 (2.935 ± 1.112) (p<0.05) and in 2010 (2.846 ± 1.029) (p<0.05).

Conclusions

Hitherto model of iodine prophylaxis has proved to be effective in eliminating the iodine deficiency. The iodine intake is now more even, homogenous, which translates into smaller scatter of UICs and less percentage of children, in whom UIC is less than 50 μg/L. However, the iodine intake only slightly exceeds the recommended values, so median of UICs oscillates around the lower limit of references values.
ABT-737 reverses the acquired radioresistance of breast cancer cells by targeting Bcl-2 and Bcl-xL
Authors: Li Ji-Yu , Li Yu-Yang , Jin Wei , Yang Qing , Shao Zhi-Ming , Tian Xing-Song
Published: December 2012

Background

Acquired radioresistance of cancer cells remains a fundamental barrier to attaining the maximal efficacy of radiotherapy for the treatment of breast cancer. Anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, play an important role in the radioresistance of cancer cells. In the present study, we aimed to determine if ABT-737, a BH3-only mimic, could reverse the acquired radioresistance of the breast cancer cell line MDA-MB-231R by targeting Bcl-2 and Bcl-xL.

Methods

The radiosensitivity of MDA-MB-231 and MDA-MB-231R cells was compared using colony formation assays. Reverse-transcription PCR and western blot were performed to detect the expression of Bcl-2 and Bcl-xL in the cancer cell lines. Annexin V flow cytometric analysis and caspase-3 colorimetric assay were used to evaluate apoptosis of the cancer cells. Cell viability was measured using the Cell Counting Kit-8. The animals used in this study were 4 to 6-week-old athymic female BALB/c nu/nu mice.

Results

The MDA-MB-231R cells were more radioresistant than the MDA-MB-231 cells, and Bcl-2 and Bcl-xL were overexpressed in the MDA-MB-231R cells. While ABT-737 was able to restore the radiosensitivity of the MDA-MB-231R cells in vitro and in vivo experiment, it was not able to enhance the radiosensitivity of the MDA-MB-231 cells. In addition, ABT-737 increased radiation-induced apoptosis in the MDA-MB-231R cells. Bcl-2 and Bcl-xL were down regulated in the MDA-MB-231R cells following treatment with ABT-737.

Conclusions

Targeting of the anti-apoptotic proteins Bcl-2 and Bcl-xL with ABT-737 may reverse the acquired radioresistance of MDA-MB-231R cells in vitro and in vivo. These findings suggest an attractive strategy for overcoming the acquired radioresistance of breast cancer cells.
Influence of birth order, birth weight, colostrum and serum immunoglobulin G on neonatal piglet survival
Authors: Cabrera Rafael A , Lin Xi , Campbell Joy M , Moeser Adam J , Odle Jack
Published: December 2012

Background

Intake of colostrum after birth is essential to stimulate intestinal growth and function, and to provide systemic immunological protection via absorption of Immunoglobulin G (IgG). The birth order and weight of 745 piglets (from 75 litters) were recorded during a one-week period of farrowing. Only pigs weighing greater than 0.68 kg birth weight were chosen for the trial. Sow colostrum was collected during parturition, and piglets were bled between 48 and 72 hours post-birth. Piglet serum IgG and colostral IgG concentrations were determined by radial immunodiffusion.

Results

Sow parity had a significant (P < 0.001) effect on sow colostral IgG concentration, being 5% higher in multiparous females. Sow colostral IgG concentration explained 6% and piglet birth order accounted for another 4% of the variation observed in piglet serum IgG concentration (P < 0.05); however, birth weight had no detectable effect. Piglet serum IgG concentration had both a linear (P < 0.05) and quadratic effect (P < 0.05) on % survival. Piglets with 1,000 mg/dl serum IgG or less (n=24) had a 67% survival; whereas, piglets with IgG concentrations between 2250 to 2500 mg/dl (n=247) had a 91% survival. Birth order had no detectable effect on survival, but birth weight had a positive linear effect (P < 0.05). Piglets weighing 0.9 kg (n = 107) at birth had a 68% survival rate, and those weighing 1.6 kg (n = 158) had an 89% survival.

Conclusion

We found that the combination of sow colostrum IgG concentration and birth order can account for 10% of the variation of piglet serum IgG concentration and that piglets with less than 1,000 mg/dl IgG serum concentration and weight of 0.9 kg at birth had low survival rate when compared to their larger siblings. The effective management of colostrum uptake in neonatal piglets in the first 24 hrs post-birth may potentially improve survival from birth to weaning.
cnvHiTSeq: integrative models for high-resolution copy number variation detection and genotyping using population sequencing data
Authors: Bellos Evangelos , Johnson Michael R , M Coin Lachlan J
Published: December 2012

Recent advances in sequencing technologies provide the means for identifying copy number variation (CNV) at an unprecedented resolution. A single next-generation sequencing experiment offers several features that can be used to detect CNV, yet current methods do not incorporate all available signatures into a unified model. cnvHiTSeq is an integrative probabilistic method for CNV discovery and genotyping that jointly analyzes multiple features at the population level. By combining evidence from complementary sources, cnvHiTSeq achieves high genotyping accuracy and a substantial improvement in CNV detection sensitivity over existing methods, while maintaining a low false discovery rate. cnvHiTSeq is available at http://sourceforge.net/projects/cnvhitseq
A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts
Authors: Suh Eric J , Remillard Matthew Y , Legesse-Miller Aster , Johnson Elizabeth L , Lemons Johanna MS , Chapman Talia R , Forman Joshua J , Kojima Mina , Silberman Eric S , Coller Hilary A
Published: December 2012

Background

Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.

Results

Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.

Conclusions

microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.
Technical Advance: Measurement of iNKT cell responses at the single-cell level against rare HIV-1-infected dendritic cells in a mixed culture
Authors: Andersson Sofia K , Paquin-Proulx Dominic , Kroll Mirko , Sandberg Johan K , Moll Markus
Published: December 2012

A microscopy-based assay to measure iNKT cell responses against rare HIV-1 infected dendritic cells, infected with an eGFP expressing reporter virus.

iNKT cells recognize lipid antigens, such as α-GalCer, presented in complex with CD1d expressed by DCs. Exposure of DCs to HIV-1 can lead to productive infection, and it was demonstrated recently that HIV-1 inhibits CD1d surface expression in an apparent mode of immune evasion. However, studies of the interaction between T cells, including iNKT cells and HIV-infected DCs in vitro, are hampered by the low frequency of productive infection in DCs. Here, we demonstrate the utility of full-length HIV-1 modified to express eGFP to address this problem. This virus allowed identification of single, rare productively infected cells in a mixed DC population by fluorescence microscopy and enabled detailed studies of the interaction of such cells with individual iNKT cells. iNKT cell responses to α-GalCer presented by HIV-1-positive and -negative DCs were quantified by intracellular IFN-γ staining in iNKT cells forming conjugates with DCs. Whereas complex formation was observed between iNKT cells and uninfected and infected DCs, only iNKT cells in contact with uninfected DCs produced IFN-γ. This microscopy assay, based on full-length HIV-1 modified to express eGFP, thus allows detailed evaluation of HIV-1 immune-evasion mechanisms in rare virus-infected live DCs.
Health Care Law versus Constitutional Law
Authors: Hall Mark A.
Published: December 2012

National Federation of Independent Business v. Sebelius, the Supreme Court's ruling on the Patient Protection and Affordable Care Act, is a landmark decision — both for constitutional law and for health care law and policy. Others will study its implications for constitutional limits on a range of federal powers beyond health care. This article considers to what extent the decision is also about health care law, properly conceived. Under one view, health care law is the subdiscipline that inquires how courts and government actors take account of the special features of medicine that make legal or policy issues especially problematic — rather than regarding health care delivery and finance more generically, like most any other economic or social enterprise. Viewed this way, the opinions from the Court's conservative justices are mainly about general constitutional law principles. In contrast, Justice Ruth Bader Ginsburg's dissenting opinion for the four more liberal justices is just as much about health care law as it is about constitutional law. Her opinion gives detailed attention to the unique features of health care finance and delivery in order to inform her analysis of constitutional precedents and principles. Thus, the Court's multiple opinions give a vivid depiction of the compelling contrasts between communal versus individualistic conceptions of caring for those in need, and between health care and health insurance as ordinary commodities versus ones that merit special economic, social, and legal status.
“Our Federalism” Moves Indoors
Authors: Ruger Theodore W.
Published: December 2012

A great deal of the US Supreme Court's federalism jurisprudence over the past two decades has focused on the outer limits of federal power, suggesting a mutually exclusive division of jurisdiction between the states and the federal government, where subjects are regulated by one sovereign or the other but not both. This is not an accurate picture of American governance as it has operated over the past half century — most important areas of American life are regulated concurrently by both the federal government and the states. The Supreme Court's June 2012 decision clearing the way for the Patient Protection and Affordable Care Act (PPACA) to move forward thus should not be regarded as an affront to state sovereignty but as a realistic embrace of state power in its active, modern form. The PPACA is infused with multiple major roles for the states, and as the statute goes into operation over the next few years, states retain, and are already exercising, substantial policy discretion.
Wearing the Crown of Solomon? Chief Justice Roberts and the Affordable Care Act “Tax”
Authors: Muise Robert J. , Yerushalmi David
Published: December 2012

Attempting to play the role of King Solomon in his PPACA decision, Chief Justice John Roberts split the baby perversely by ruling it was not a tax under the Anti-Injunction Act, which would have likely deprived the Court of jurisdiction to hear this pre-enforcement challenge to the individual mandate, but it was a tax for taxing and spending purposes even though Congress said it was a “penalty” and not a tax. And the Chief Justice had to twist further his “wisdom” to hold that it was not an unconstitutional direct tax, even though that is exactly what it is, if it is a tax in the first instance.
Behavioral abnormalities and Parkinson's-like changes resulting from Id2 inactivation in mice
Authors: Havrda Matthew C. , Paolella Brenton R. , Ward Nora M. , Holroyd Kathryn B.
Published: December 2012

Characterizing dopaminergic neuronal development and function in novel genetic animal models might uncover strategies for researchers to develop disease-modifying treatments for neurologic disorders. Id2 is a transcription factor expressed in the developing central nervous system. Id2-/- mice have fewer dopaminergic neurons in the olfactory bulb and reduced olfactory discrimination, a pre-clinical marker of Parkinson's disease. Here, we summarize behavioral, histological, and in vitro molecular biological analyses to determine whether midbrain dopaminergic neurons are affected by Id2 loss. Id2-/- mice were hyperactive at 1 and 3 months of age, but by 6 months showed reduced activity. Id2-/- mice showed age-dependent histological alterations in dopaminergic neurons of the substantia nigra pars compacta (SNpC) associated with changes in locomotor activity. Reduced dopamine transporter (DAT) expression was observed at early ages in Id2-/- mice and DAT expression was dependent on Id2 expression in an in vitro dopaminergic differentiation model. Evidence of neurodegeneration, including activated caspase-3 and glial infiltration, were noted in the SNpC of older Id2-/- mice. These findings document a novel role for Id2 in the maintenance of midbrain dopamine neurons. The Id2-/- mouse should provide unique opportunities to study the progression of neurodegenerative disorders involving the dopamine system.
A new and clinically relevant murine model of solid-organ transplant aspergillosis
Authors: Herbst Susanne , Shah Anand , Carby Martin , Chusney Gary , Kikkeri Naresh , Dorling Anthony , Bignell Elaine , Shaunak Sunil , Armstrong-James Darius
Published: December 2012

Invasive fungal infections (IFIs) are a major cause of death in organ transplant patients. The murine hydrocortisone-mediated immunosuppression model of pulmonary aspergillosis is commonly used to characterise IFIs in these patients. However, this model does not take into account the effects of calcineurin inhibitors on transplant immunity to IFIs or the fungal calcineurin pathway, which is required for both virulence and antifungal drug resistance. To address these two issues, a new and clinically relevant transplant immunosuppression model of tacrolimus (FK506) and hydrocortisone-associated pulmonary aspergillosis was developed. We first characterised IFIs in 406 patients with a lung transplant. This showed that all of the patients with pulmonary aspergillosis were immunosuppressed with calcineurin inhibitors and steroids. Murine pharmacokinetic studies demonstrated that an ideal dose of 1 mg/kg/day of FK506 intraperitoneally produced blood trough levels in the human therapeutic range (5-12 ng/ml). There was increased mortality from pulmonary aspergillosis in a transplant-relevant immunosuppression model using both FK506 and hydrocortisone as compared with immunosuppression using hydrocortisone only. Lung histopathology showed neutrophil invasion and tracheobronchitis that was associated with reduced lung tumour necrosis factor-α (TNFα), JE (homologue of human MCP-1) and KC (homologue of human IL-8) at 24 hours, but increased lung TNFα, JE and KC at 48 hours when fungal burden was high. Furthermore, FK506 directly impaired fungal killing in alveolar macrophages in vitro, with FK506-mediated inhibition of the radial growth of Aspergillus fumigatus in vitro occurring at the low concentration of 5 ng/ml. Taken together, these findings show that the immunosuppressive activity of FK506 outweighs its antifungal activity in vivo. These observations demonstrate that FK506 impairs innate immune responses and leads to an incremental increase in susceptibility to IFIs when it is combined with steroids. This new and clinically relevant mouse model of invasive aspergillosis is a valuable addition to the further study of both fungal immunity and antifungal therapy in organ transplantation.
Bile-acid-mediated decrease in endoplasmic reticulum stress: a potential contributor to the metabolic benefits of ileal interposition surgery in UCD-T2DM rats
Authors: Cummings Bethany P. , Bettaieb Ahmed , Graham James L. , Kim Jaehyoung , Ma Fangrui , Shibata Noreene , Stanhope Kimber L. , Giulivi Cecilia , Hansen Frederik , Jelsing Jacob , Vrang Niels , Kowala Mark , Chouinard Michael L. , Haj Fawaz G. , Havel Peter J.
Published: December 2012

Post-operative increases in circulating bile acids have been suggested to contribute to the metabolic benefits of bariatric surgery; however, their mechanistic contributions remain undefined. We have previously reported that ileal interposition (IT) surgery delays the onset of type 2 diabetes in UCD-T2DM rats and increases circulating bile acids, independently of effects on energy intake or body weight. Therefore, we investigated potential mechanisms by which post-operative increases in circulating bile acids improve glucose homeostasis after IT surgery. IT, sham or no surgery was performed on 2-month-old weight-matched male UCD-T2DM rats. Animals underwent an oral fat tolerance test (OFTT) and serial oral glucose tolerance tests (OGTT). Tissues were collected at 1.5 and 4.5 months after surgery. Cell culture models were used to investigate interactions between bile acids and ER stress. IT-operated animals exhibited marked improvements in glucose and lipid metabolism, with concurrent increases in postprandial glucagon-like peptide-1 (GLP-1) secretion during the OFTT and OGTTs, independently of food intake and body weight. Measurement of circulating bile acid profiles revealed increases in circulating total bile acids in IT-operated animals, with a preferential increase in circulating cholic acid concentrations. Gut microbial populations were assessed as potential contributors to the increases in circulating bile acid concentrations, which revealed proportional increases in Gammaproteobacteria in IT-operated animals. Furthermore, IT surgery decreased all three sub-arms of ER stress signaling in liver, adipose and pancreas tissues. Amelioration of ER stress coincided with improved insulin signaling and preservation of β-cell mass in IT-operated animals. Incubation of hepatocyte, adipocyte and β-cell lines with cholic acid decreased ER stress. These results suggest that post-operative increases in circulating cholic acid concentration contribute to improvements in glucose homeostasis after IT surgery by ameliorating ER stress.
Olfactomedin-1 activity identifies a cell invasion checkpoint during epithelial-mesenchymal transition in the embryonic heart
Authors: Lencinas Alejandro , Chhun Danny C. , Dan Kelvin P. , Ross Kristen D. , Hoover Elizabeth A. , Antin Parker B. , Runyan Raymond
Published: December 2012

Endothelia in the atrioventricular (AV) canal of the developing heart undergo a prototypical epithelial mesenchymal transition (EMT) to begin heart valve formation. Using an in vitro invasion assay, an extracellular matrix protein found in the heart, Olfactomedin-1 (OLFM1), increases mesenchymal cell numbers. Both anti-OLFM1 antibody and OLFM1 siRNA treatment inhibit mesenchymal cell formation. OLFM1 does not alter cell proliferation, migration or apoptosis. Dispersion, but lack of invasion in the presence of inhibiting antibody, identifies a specific role for OLFM1 in cell invasion during EMT. This role is conserved in other epithelia, as OLFM1 similarly enhances invasion by MDCK epithelial cells in a trans-well assay. OLFM-1 activity is cooperative with TGFβ, as synergy is observed when TGFβ2 and OLFM1 are added to MDCK cell cultures. Inhibition of both OLFM1 and TGFβ in heart invasion assays shows a similar cooperative role during development. To explore OLFM1 activity during EMT, representative EMT markers were examined. Effects of OLFM1 protein and anti-OLFM1 on transcripts of cell-cell adhesion molecules and the transcription factors, Snail-1, Snail-2, Twist1, and Sox-9, argue that OLFM1 does not initiate EMT. Rather, regulation of transcripts of Zeb1 and Zeb2, secreted proteases and mesenchymal cell markers by both OLFM1 and anti-OLFM1 is consistent with regulation of the cell invasion step of EMT. We conclude that OLFM1 is present and necessary during EMT in the embryonic heart. Its role in cell invasion and mesenchymal cell gene regulation suggests an invasion checkpoint in EMT where OLFM1 acts to promote cell invasion into the three-dimensional matrix.
Pharmacological preconditioning with erythropoietin attenuates the organ injury and dysfunction induced in a rat model of hemorrhagic shock
Authors: Nandra Kiran K. , Collino Massimo , Rogazzo Mara , Fantozzi Roberto , Patel Nimesh S. A. , Thiemermann Christoph
Published: December 2012

Pre-treatment with erythropoietin (EPO) has been demonstrated to exert tissue-protective effects against 'ischemia-reperfusion'-type injuries. This protection might be mediated by mobilization of bone marrow endothelial progenitor cells (EPCs), which are thought to secrete paracrine factors. These effects could be exploited to protect against tissue injury induced in cases where hemorrhage is foreseeable, for example, prior to major surgery. Here, we investigate the effects of EPO pre-treatment on the organ injury and dysfunction induced by hemorrhagic shock (HS). Recombinant human EPO (1000 IU/kg/day i.p.) was administered to rats for 3 days. Rats were subjected to HS on day 4 (pre-treatment protocol). Mean arterial pressure was reduced to 35±5 mmHg for 90 minutes, followed by resuscitation with 20 ml/kg Ringer’s lactate for 10 minutes and 50% of the shed blood for 50 minutes. Rats were sacrificed 4 hours after the onset of resuscitation. EPC (CD34+/flk-1+ cell) mobilization was measured following the 3-day pre-treatment with EPO and was significantly increased compared with rats pre-treated with phosphate-buffered saline. EPO pre-treatment significantly attenuated organ injury and dysfunction (renal, hepatic and neuromuscular) caused by HS. In livers from rats subjected to HS, EPO enhanced the phosphorylation of Akt (activation), glycogen synthase kinase-3β (GSK-3β; inhibition) and endothelial nitric oxide synthase (eNOS; activation). In the liver, HS also caused an increase in nuclear translocation of p65 (activation of NF-κB), which was attenuated by EPO. This data suggests that repetitive dosing with EPO prior to injury might protect against the organ injury and dysfunction induced by HS, by a mechanism that might involve mobilization of CD34+/flk-1+ cells, resulting in the activation of the Akt-eNOS survival pathway and inhibition of activation of GSK-3β and NF-κB.
Critical role for αvβ6 integrin in enamel biomineralization
Authors: Mohazab L. , Koivisto L. , Jiang G. , Kytömäki L. , Haapasalo M. , Owen G.R. , Wiebe C. , Xie Y. , Heikinheimo K. , Yoshida T. , Smith C.E. , Heino J. , Häkkinen L. , McKee M.D. , Larjava H.
Published: December 2012

Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with ameloblast plasma membrane. Although it is known that integrins mediate cell-matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. Thus, we hypothesized that αvβ6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both β6 integrin mRNA and protein. The maxillary incisors of Itgb6−/− mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6−/− mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6−/− enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6−/− mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, which did not use αvβ6 integrin as an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin αvβ6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition/turnover and subsequent enamel biomineralization.
Akt1 promotes focal adhesion disassembly and cell motility through phosphorylation of FAK in growth factor-stimulated cells
Authors: Higuchi Maiko , Kihara Rina , Okazaki Tomohiko , Aoki Ichiro , Suetsugu Shiro , Gotoh Yukiko
Published: December 2012

The crosstalk between spatial adhesion signals and temporal soluble signals is key in regulating cellular responses such as cell migration. Here we show that soluble growth factors (GFs) enhance integrin signaling through Akt phosphorylation of FAK at Ser695 and Thr700. PDGF treatment or overexpression of active Akt1 in fibroblasts increased autophosphorylation of FAK at Tyr397, an essential event for integrin turnover and cell migration. Phosphorylation-defective mutants of FAK (S695A and T700A) underwent autophosphorylation at Tyr397 and promoted cell migration in response to the integrin ligand fibronectin (FN), but importantly, not in response to PDGF. This study has unveiled a novel function of Akt as an “ignition kinase” of FAK in GF signaling and may shed light on the mechanism by which GFs regulate integrin signaling.
Roles of chemokine receptor CX3CR1 in maintaining murine bone homeostasis through the regulation of both osteoblasts and osteoclasts
Authors: Hoshino Akiyoshi , Ueha Satoshi , Hanada Sanshiro , Imai Toshio , Ito Masako , Yamamoto Kenji , Matsushima Kouji , Yamaguchi Akira , Iimura Tadahiro
Published: December 2012

Chemokines have recently been reported to be involved in pathological bone destruction. However, the physiological roles of chemokines in bone metabolism in vivo have not been well documented. We analyzed the bone phenotypes in Cx3cr1-deficient mice. The mice exhibited slight but significant increases in trabecular and cortical thickness, reduced numbers of osteoclasts and increased rates of osteoid formation. Although the morphometric parameters showed marginal differences, the Cx3cr1-deficient bones showed an elevated expression of Osterix/SP7, an essential transcriptional factor for osteoblasts, while a late marker, Osteocalcin/Bglap, was downregulated. The levels of various osteoclastic markers, such as receptor activator of NF-κB (Rank)/Tnfrsf11a, receptor activator of NF-κB ligand (RANKL)/Tnfsf11, tartrate-resistant acid phosphatase 5b (Trap5b)/Acp5b, Cathepsin K/Ctsk, Mmp3 and Mmp13, were significantly decreased in the Cx3cr1-deficient bones. Cultured Cx3cr1-deficient osteoblastic cells showed inverse temporal patterns of osteoblastic marker expression and reduced calcium deposition. Further in vitro studies and immunofluorescence staining against CX3CR1 and CX3CL1 suggested a role for the CX3CR1-CX3CL1axis in an early stage of osteoblast differentiation, possibly through their trans- and cis-interactions. Cultured Cx3cr1-deficient pre-osteoclasts showed impaired differentiation, mainly due to a deficiency of the CD115+CD11blo osteoclastogenic population of myeloid-lineage precursors. The treatment of bone marrow-derived osteoclastic cultures with recombinant CX3CL1 at different time points suggested that the CX3CR1-CX3CL1 axis favors the maintenance of osteoclastic precursors, but not differentiated osteoclasts. The current observations uncovered novel roles of the CX3CR1-CX3CL1 axis in the differentiation of both osteoblasts and osteoclasts.
Dictyostelium ACAP-A is an ArfGAP involved in cytokinesis, cell migration and actin cytoskeleton dynamics
Authors: Dias Marco , Blanc Cédric , Thazar-Poulot Nelcy , Ben Larbi Sabrina , Cosson Pierre , Letourneur François
Published: December 2012

ACAPs and ASAPs are Arf-GTPase-activating proteins with BAR, PH, GAP and ankyrin repeat domains and are known to regulate vesicular traffic and actin cytoskeleton dynamics in mammalian cells. The amoeba Dictyostelium has only two proteins with this domain organization instead of six in human, enabling a more precise functional analysis. Genetic invalidation of acapA, resulted in multinucleated cells with cytokinesis defects. Mutant acapA− cells were hardly motile and their multicellular development was significantly delayed. In addition, formation of filopodial protrusions was deficient in these cells. Conversely, re-expression of ACAP-A-GFP resulted in numerous and long filopodia-like protrusions. Mutagenesis studies showed that ACAP-A actin remodeling function was dependent on its ability to activate its substrate, the small GTPase ArfA. Likewise, the expression of a constitutively active ArfA•GTP mutant in wild-type cells led to a significant reduction of filopodia length. Together our data support a role for ACAP-A in the control of the actin cytoskeleton organization and dynamics through an ArfA-dependent mechanism.
The Drosophila inner-membrane protein PMI controls cristae biogenesis and mitochondrial diameter
Authors: Macchi Marc , El Fissi Najla , Tufi Roberta , Bentobji Mélanie , Liévens Jean-Charles , Martins L. Miguel , Royet Julien , Rival Thomas
Published: December 2012

Cristae are mitochondrial inner-membrane structures which concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling cristae morphogenesis are poorly understood and few cristae determinants have been identified. Among them are the Mitofilins that are required to establish cristae junctions and ATP-synthase subunits that bend membrane at cristae tips. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondria ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI over-expression is sufficient to reduce cristae length in vivo. Finally, those cristae defaults are associated with impaired respiratory chain activity and increased level of reactive oxygen species. Since PMI and its human ortholog TMEM11 are regulators of mitochondria morphology, our data suggest that, by controlling cristae length, PMI influences mitochondria diameter and tubular shape.
MTR120/KIAA1383, a novel microtubule-associated protein, promotes microtubule stability and ensures cytokinesis
Authors: Fong Ka-wing , Leung Justin Wai-chung , Li Yujing , Wang Wenqi , Feng Lin , Ma Wenbin , Liu Dan , Songyang Zhou , Chen Junjie
Published: December 2012

Microtubules (MTs) are the major constituent of the mitotic apparatus. Deregulation of MT dynamics leads to chromosome missegregation, cytokinesis failure and improper inheritance of genetic materials. Here, we describe the identification and characterization of KIAA1383/MTR120 (microtubule regulator 120 kDa) as a novel MT-associated protein. We found that MTR120 localizes to stabilized MTs during interphase and to the mitotic apparatus during mitosis. MTR120 overexpression results in MT bundling and acetylation. In vitro, purified MTR120 protein binds to and bundles preassembled MTs. Moreover, depletion of MTR120 by RNA interference leads to cytokinesis failure and polyploidy. These phenotypes can be rescued by wild-type MTR120 but not by the MT non-binding mutant of MTR120. Together, these data suggest that MTR120 is a novel MT-associated protein that directly stabilizes MTs and hence ensures the fidelity of cell division.
MicroRNA-17∼92a upregulation by estrogen leads to Bim targeting and inhibition of osteoblasts apoptosis
Authors: Guo Lei , Xu Jiping , Qi Jin , Zhang Lianfang , Wang Jinsheng , Liang Jing , Qian Niandong , Zhou Hanbing , Wei Li , Deng Lianfu
Published: December 2012

Anti-apoptotic effects of estrogen on osteoblasts are very important in the etiology of estrogen protecting the adult skeleton against bone loss. The mechanisms of this process are still not fully understood. Recent studies implicated an important role of microRNAs in estrogen-mediated responses in various cellular processes, including cell apoptosis and proliferation. Therefore, we hypothesized that these regulatory molecules might be implicated in the process of estrogen protecting osteoblasts from apoptosis. Western blot, quantitative real-time PCR, flow cytometry, and luciferase assay were employed to investigate the role of microRNA in estrogen protecting osteoblasts against apoptosis. The microRNA cluster miR-17∼92a, a post-transcriptional regulator, was significantly reduced during dexamethasone, etoposide and TNF-a induced osteoblasts apoptosis. The repression of microRNA cluster miR-17∼92a was significantly attenuated by estrogen. To delineate the role of miR-17∼92a in apoptosis, we respectively silenced and overexpressed miR-17∼92a in osteoblasts. We found that miR-17∼92a depletion significantly enhanced dexamethasone-induced apoptosis and over-expressing miR-17∼92a remarkably increased anti-apoptotic effects of estrogen on osteoblasts. Mechanistic studies showed that microRNA-17∼92a inhibited Bim expression through a microRNA-17∼92a-binding site within the 3′- untranslational region of Bim. The post-transcriptional repression of Bim was further confirmed by luciferase reporter assay. These results showed that microRNA cluster miR-17∼92a, an important protecting factor, plays a significant role in the process of estrogen protecting osteoblasts against apoptosis, by regulating Bim expression.
Identification of a novel motif that affects the conformation and activity of the MARCH1 E3 ubiquitin ligase
Authors: Bourgeois-Daigneault Marie-Claude , Thibodeau Jacques
Published: December 2012

MARCH1, a member of the membrane-associated RING-CH family of E3 ubiquitin ligases, regulates antigen presentation by down-regulating the cell surface expression of MHC class II and CD86 molecules. MARCH1 is a transmembrane protein that exposes both its N- and C-terminus to the cytoplasm. Here, we have conducted a structure-function analysis of its two cytoplasmic tails to gain insights into the trafficking of MARCH1 in the endocytic pathway. Fusion of the N-terminal portion of MARCH1 to a type II transmembrane reporter molecule revealed that this cytoplasmic tail contains endosomal sorting motifs. The C-terminal domain also appears to contain intracellular sorting signals as it reduced surface expression of a type I transmembrane reporter molecule. Mutation of the two putative C-terminal tyrosine-based sorting signals did not affect the activity of human MARCH1. However, it reduced its incorporation into exosomes. Moreover, site-directed mutagenesis pointed to a functional C-terminal 221VQNC224 sequence that affects the spatial organization of the two cytoplasmic regions. This motif is also found in other RING-type E3 ubiquitin ligases, such as parkin. Altogether, these findings highlight the complex regulation of MARCH1 trafficking in the endocytic pathway as well as the intricate interactions between its cytoplasmic tails.
PtdIns(3,4,5)P3 is constitutively synthesized and required for spindle translocation during meiosis in mouse oocytes
Authors: Zheng Ping , Baibakov Boris , Wang Xi-hong , Dean Jurrien
Published: December 2012

Prior to ovulation, mammalian oocytes complete their first meiotic division and arrest at metaphase II. During this marked asymmetric cell division, the meiotic spindle moves dramatically from the center of the oocyte to the cortex to facilitate segregation of half of its chromosomal content into the diminutive first polar body. Recent investigations have documented critical roles for filamentous actin (F-actin) in meiotic spindle translocation. However, the identity of the upstream regulators responsible for these carefully orchestrated movements has remained elusive. Utilizing fluorescence-tagged probes and time-lapse confocal microscopy, we document that phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is constitutively synthesized with spatial and temporal dynamics similar to that of F-actin and Formin 2 (Fmn2). Blockage of PtdIns(3,4,5)P3 synthesis by LY294002, a specific inhibitor of phosphoinositide 3-kinases (PI3K), disrupts cytoplasmic F-actin organization and meiotic spindle migration to the cortex. F-actin nucleator Fmn2 and Rho GTPase Cdc42 play roles in mediating the effect of PtdIns(3,4,5)P3 on F-actin assembly. Moreover, the spatial and temporal dynamics of PtdIns(3,4,5)P3 is impaired by depletion of MATER or Filia, two oocyte proteins encoded by maternal effect genes. Thus, PtdIns(3,4,5)P3 is synthesized during meiotic maturation and acts upstream of Cdc42 and Fmn2, but downstream of MATER/Filia proteins to regulate the F-actin organization and spindle translocation to the cortex during mouse oocyte meiosis.
The sugar meal of the African malaria mosquito Anopheles gambiae (Giles) and how deterrent compounds interfere with it: a behavioural and neurophysiological study
Authors: Kessler Sébastien , Vlimant Michèle , Guerin Patrick M.
Published: December 2012

In this study, we show that female African malaria mosquitoes Anopheles gambiae (Giles) starved for 3-5 hours start to engorge on sucrose at concentrations between 50 to 75 mM. Half of the feeding response (ED50) is reached at 111 mM and the maximum response (0.4 mg) occurs from 146 mM (5% m/v). Two receptor cells in a trichoid sensillum of the labellum, called the 'sucrose' and the 'water' neurones, are activated by sucrose and water, respectively. The electrophysiological response of the sucrose receptor cell starts well below the level of sugar necessary to induce feeding. An. gambiae is most sensitive to small increments in sucrose concentration up to 10 mM with a response plateau at a maximum frequency of 53 spikes per 2 s from 50 mM, the concentration at which female An. gambiae start to engorge on sucrose. Fructose has a mild phagostimulatory effect on An. gambiae, whereas no significant differences in meal sizes between water and glucose were found. However, when 146 mM fructose plus glucose are mixed, the same engorgement as on 146 mM sucrose is observed. Likewise, even though the sucrose receptor cell is not activated by either fructose or glucose alone, equimolar solutions of fructose plus glucose activate the neurone. We conclude that there is a behavioural and a neurophysiological synergism between fructose and glucose, the two hexose sugars of sucrose. We show that bitter tasting products for humans have a deterrent effect on feeding in An. gambiae. When 1 mM quinidine, quinine or denatonium benzoate is added to 146 mM sucrose, feeding is almost totally inhibited. The effect of berberine is lower and no significant inhibition on engorgement occurs for caffeine. The deterrent effect depends on concentration for both quinine and quinidine. Capillary feeding experiments show that contact chemosensilla on the mouthparts are sufficient for the detection of sucrose and bitter products. The feeding assay findings with deterrents correlate with the neurophysiological responses of the sucrose and the water labellar neurones which are both inhibited by the bitter compounds denatonium benzoate, quinine and berberine between 0.01 and 1 mM, but not by the same concentrations of caffeine which has no effect on feeding. In conclusion, sucrose which stimulates feeding activates the labellar sucrose neurone whereas feeding deterrents inhibit both the sucrose and the water neurones. This study provides an initial understanding of the physiological mechanisms involved in sugar feeding in An. gambiae and shows how some bitter products interfere with it.
The relative contribution of calcium, zinc and oxidation-based cross-links to the stiffness ofArion subfuscusglue
Authors: Braun Melanie , Menges Meghan , Opoku Freda , Smith Andrew M.
Published: December 2012

Metal ions are present in many different biological materials, and are capable of forming strong cross-links in aqueous environments. The relative contribution of different metal-based cross-links was measured in the defensive glue produced by the terrestrial slug Arion subfuscus. This glue contains calcium, zinc, manganese, iron and copper. These metals are essential to the integrity of the glue and to gel stiffening. Removal of all metals caused at least a fifteen-fold decrease in the storage modulus of the glue. Selectively disrupting cross-links involving hard Lewis acids such as calcium weakened the glue, while disrupting cross-links involving borderline Lewis acids such as zinc did not. Calcium is the most common cation bound to the glue (40 mmol L-1), and its charge is balanced primarily by sulfate at 82 to 84 mmol L-1. Thus, these ions likely play a primary role in bringing polymers together directly. Imine bonds formed as a result of protein oxidation also contribute substantially to the strength of the glue. Disrupting these bonds with hydroxylamine caused a 33% decrease in storage modulus of the glue, while stabilizing them by reduction with sodium borohydride increased the storage modulus by 40%. Thus, a combination of metal-based bonds operates in this glue. Most likely, cross-links directly involving calcium play a primary role in bringing together and stabilizing the polymer network, followed by imine bond formation and possible iron coordination.
The metabolic cost of incline locomotion in the Svalbard rock ptarmigan (Lagopus muta hyperborea): the effects of incline grade and seasonal fluctuations in body mass
Authors: Lees John , Folkow Lars , Stokkan Karl-Arne , Codd Jonathan
Published: December 2012

In a terrestrial environment animals must locomote over different terrain; despite this, the majority of studies focus on level locomotion. The influence moving up an inclined surface has on the metabolic cost of locomotion and the efficiency with which animals perform positive work against gravity is still not well understood. Generally speaking, existing data sets lack consistency in the use of grades, further compounded by differences between species in terms of morphology and locomotor gait. Here we investigated the metabolic cost of locomotion using respirometry in the Svalbard ptarmigan (Lagopus muta hyperborea). The Svalbard ptarmigan provides a unique opportunity to investigate the cost of incline locomotion as it undergoes a seasonal fluctuation in body mass, which doubles in winter, meaning the requirement for positive mechanical work also fluctuates with season. We demonstrate that at the same degree of incline, the cost of lifting 1 kg by 1 vertical metre remains relatively constant between seasons despite the large differences in body mass from summer to winter. These findings are consistent with the notion that positive mechanical work alone dictates the cost of lifting above a certain body mass. However, our data indicate that this cost may vary according to the degree of incline and gait.