Sodium-dependent vitamin C transporter SVCT2: Expression and function in bone marrow stromal cells and in osteogenesis

Ascorbic acid (Vitamin C) has a critical role in bone formation and osteoblast differentiation, but very little is known about the molecular mechanisms of ascorbic acid entry into bone marrow stromal cells (BMSCs). To address this gap in knowledge, we investigated the identity of the transport system that is responsible for the uptake of ascorbic acid into bone marrow stromal cells (BMSCs). First, we examined the expression of the two known isoforms of the sodium-coupled ascorbic acid transporter, namely SVCT1 and SVCT2, in BMSCs (Lin ? ve Sca1 + ve) and bone at the mRNA level. Only SVCT2 mRNA was detected in BMSCs and bone. Uptake of ascorbic acid in BMSCs was Na⁺-dependent and saturable. In order to define the role of SVCT2 in BMSC differentiation into osteoblasts, BMSCs were stimulated with osteogenic media for different time intervals, and the activity of SVCT2 was monitored by ascorbic acid uptake. SVCT2 expression was up-regulated during the osteogenic differentiation of BMSCs; the expression was maximal at the earliest phase of differentiation. Subsequently, osteogenesis was inhibited in BMSCs upon knock-down of SVCT2 by lentivirus shRNA. We also found that the expression of the SVCT2 could be negatively or positively modulated by the presence of oxidant (Sin-1) or antioxidant (Ascorbic acid) compounds, respectively, in BMSCs. Furthermore, we found that this transporter is also regulated with age in mouse bone. These data show that SVCT2 plays a vital role in the osteogenic differentiation of BMSCs and that its expression is altered under conditions associated with redox reaction. Our findings could be relevant to bone tissue engineering and bone related diseases such as osteoporosis in which oxidative stress and aging plays important role.     

Combination of neurofilament heavy chain and complement C3 as CSF biomarkers for ALS

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease with an average survival of 3 years from symptom onset. Rapid and conclusive early diagnosis is essential if interventions with disease-modifying therapies are to be successful. Cytoskeletal modification and inflammation are known to occur during the pathogenesis of ALS. We measured levels of cytoskeletal proteins and inflammatory markers in the CSF of ALS, disease controls and healthy subjects. We determined threshold values for each protein that provided the optimal sensitivity and specificity for ALS within a training set, as determined by receiver operating characteristic analysis. Interestingly, the optimal assay was a ratio of the levels for phosphorylated neurofilament heavy chain and complement C3 (pNFH/C3). We next applied this assay to a separate test set of CSF samples to verify our results. Overall, the predictive pNFH/C3 ratio identified ALS with 87.3% sensitivity and 94.6% specificity in a total of 71 ALS subjects, 52 disease control subjects and 40 healthy subjects. In addition, the level of CSF pNFH correlated with survival of ALS patients. We also detected increased pNFH in the plasma of ALS patients and observed a correlation between CSF and plasma pNFH levels within the same subjects. These findings support large-scale prospective biomarker studies to determine the clinical utility of diagnostic and prognostic signatures in ALS.     

Vesicular ATP is the predominant cause of intercellular calcium waves in astrocytes

Brain astrocytes signal to each other and neurons. They use changes in their intracellular calcium levels to trigger release of transmitters into the extracellular space. These can then activate receptors on other nearby astrocytes and trigger a propagated calcium wave that can travel several hundred micrometers over a timescale of seconds. A role for endogenous ATP in calcium wave propagation in hippocampal astrocytes has been suggested, but the mechanisms remain incompletely understood. Here we explored how calcium waves arise and directly tested whether endogenously released ATP contributes to astrocyte calcium wave propagation in hippocampal astrocytes. We find that vesicular ATP is the major, if not the sole, determinant of astrocyte calcium wave propagation over distances between approximately 100 and 250 microm, and approximately 15 s from the point of wave initiation. These actions of ATP are mediated by P2Y1 receptors. In contrast, metabotropic glutamate receptors and gap junctions do not contribute significantly to calcium wave propagation. Our data suggest that endogenous extracellular astrocytic ATP can signal over broad spatiotemporal scales.     

Swimbladder Leiomyosarcoma in Atlantic salmon (Salmo salar) in North America

Leiomyosarcoma with associated retrovirus were found in North America for the first time in adult Atlantic salmon (Salmo salar) held in a quarantine facility at the North Attleboro National Fish Hatchery (NANFH), Massachusetts, USA. The fish had been collected as age 1-2 yr animals from the Pleasant River, Maine, and were to be used as brood stock in a population augmentation program for that river. Neoplastic disease was observed at NANFH initially in older (age 4 yr) fish, followed by age 3 yr fish. Disease was not observed in age 2 yr fish. The mortality pattern was chronic.     

Truncation of the GABAA-Receptor γ2 Subunit in a Family with Generalized Epilepsy with Febrile Seizures Plus

Recent findings from studies of two families have shown that mutations in the GABA(A)-receptor gamma2 subunit are associated with generalized epilepsies and febrile seizures. Here we describe a family that has generalized epilepsy with febrile seizures plus (GEFS(+)), including an individual with severe myoclonic epilepsy of infancy, in whom a third GABA(A)-receptor gamma2-subunit mutation was found. This mutation lies in the intracellular loop between the third and fourth transmembrane domains of the GABA(A)-receptor gamma2 subunit and introduces a premature stop codon at Q351 in the mature protein. GABA sensitivity in Xenopus laevis oocytes expressing the mutant gamma2(Q351X) subunit is completely abolished, and fluorescent-microscopy studies have shown that receptors containing GFP-labeled gamma2(Q351X) protein are retained in the lumen of the endoplasmic reticulum. This finding reinforces the involvement of GABA(A) receptors in epilepsy.