Coxsackievirus preferentially replicates and induces cytopathic effects in undifferentiated neural progenitor cells

Enteroviruses, including coxsackieviruses, exhibit significant tropism for the central nervous system, and these viruses are commonly associated with viral meningitis and encephalitis. Previously, we described the ability of coxsackievirus B3 (CVB3) to infect proliferating neuronal progenitor cells located in the neonatal subventricular zone and persist in the adult murine central nervous system (CNS). Here, we demonstrate that cultured murine neurospheres, which comprise neural stem cells and their progeny at different stages of development, were highly susceptible to CVB3 infection. Neurospheres, or neural progenitor and stem cells (NPSCs), isolated from neonatal C57BL/6 mice, supported high levels of infectious virus production and high viral protein expression levels following infection with a recombinant CVB3 expressing enhanced green fluorescent protein (eGFP) protein. Similarly, NPSCs isolated from neonatal actin-promoter-GFP transgenic mice (actin-GFP NPSCs) were highly susceptible to infection with a recombinant CVB3 expressing DsRed (Discosoma sp. red fluorescent protein). Both nestin-positive and NG2(+) progenitor cells within neurospheres were shown to preferentially express high levels of viral protein as soon as 24 h postinfection (p.i.). By day 3 p.i., viral protein expression and viral titers increased dramatically in NPSCs with resultant cytopathic effects (CPE) and eventual cell death. In contrast, reduced viral replication, lower levels of CPE, and diminished viral protein expression levels were observed in NPSCs differentiated for 5 or 16 days in the presence of fetal bovine serum (FBS). Despite the presence of CPE and high levels of cell death following early CVB3 infection, surviving neurospheres were readily observed and continued to express detectable levels of viral protein as long as 37 days after initial infection. Also, CVB3 infection of actin-GFP NPSCs increased the percentage of cells expressing neuronal class III β-tubulin following their differentiation in the presence of FBS. These results suggest that neural stem cells may be preferentially targeted by CVB3 and that neurogenic regions of the CNS may support persistent viral replication in the surviving host. In addition, normal progenitor cell differentiation may be altered in the host following infection.     

Isolation and expression analysis of partial sequences of heavy metal transporters from <Emphasis Type="Italic">Brassica juncea</Emphasis> by coupling high throughput cloning with a molecular fingerprinting technique

Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. These are important candidate genes to study in metal tolerant and accumulator plants for their potential use in environmental clean up. We coupled a degenerate primer-based RT-PCR approach with a molecular fingerprinting technique based on amplified rDNA restriction analysis (ARDRA) to identify novel ESTs corresponding to heavy metal transporters from metal accumulator Brassica juncea. We utilized this technique to clone several family members of natural resistance-associated macrophage proteins (NRAMP) and yellow stripe-like proteins (YSL) in a high throughput manner to distinguish between closely related isoforms and/or allelic variants from the allopolyploid B. juncea. Partial clones of 23 Brassica juncea NRAMPs and 27 YSLs were obtained with similarity to known Arabidopsis thaliana and Noccaea (Thlaspi) caerulescens NRAMP and YSL genes. The cloned transporters showed Brassica-specific changes in domains, which can have important functional consequences. Semi-quantitative RT-PCR-based expression analysis of chosen members indicated that even closely related isoforms/allelic variants of BjNRAMP and BjYSL have distinct tissue-specific and metal-dependent expressions which might be essential for adaptive fitness and heavy metal tolerance. Consistent to this, BjYSL6.1 and BjYSL5.8 were found to show elevated expressions specifically in cadmium-treated shoots and lead-treated roots of B. juncea, respectively.     

The Presence of Multiple Cellular Defects Associated with a Novel G50E Iron-Sulfur Cluster Scaffold Protein (ISCU) Mutation Leads to Development of Mitochondrial Myopathy

Iron-sulfur (Fe-S) clusters are versatile cofactors involved in regulating multiple physiological activities, including energy generation through cellular respiration. Initially, the Fe-S clusters are assembled on a conserved scaffold protein, iron-sulfur cluster scaffold protein (ISCU), in coordination with iron and sulfur donor proteins in human mitochondria. Loss of ISCU function leads to myopathy, characterized by muscle wasting and cardiac hypertrophy. In addition to the homozygous ISCU mutation (g.7044G→C), compound heterozygous patients with severe myopathy have been identified to carry the c.149G→A missense mutation converting the glycine 50 residue to glutamate. However, the physiological defects and molecular mechanism associated with G50E mutation have not been elucidated. In this report, we uncover mechanistic insights concerning how the G50E ISCU mutation in humans leads to the development of severe ISCU myopathy, using a human cell line and yeast as the model systems. The biochemical results highlight that the G50E mutation results in compromised interaction with the sulfur donor NFS1 and the J-protein HSCB, thus impairing the rate of Fe-S cluster synthesis. As a result, electron transport chain complexes show significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, the G50E mutant mitochondria display enhancement in iron level and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. Thus, our findings provide compelling evidence that the respiration defect due to impaired biogenesis of Fe-S clusters in myopathy patients leads to manifestation of complex clinical symptoms.     

Tight,cell type-specific control of LNX expression in the nervous system,at the level of transcription,translation and protein stability

LNX1 and LNX2 are E3 ubiquitin ligases that can interact with Numb — a key regulator of neurogenesis and neuronal differentiation. LNX1 can target Numb for proteasomal degradation, and Lnx mRNAs are prominently expressed in the nervous system, suggesting that LNX proteins play a role in neural development. This hypothesis remains unproven, however, largely because LNX proteins are present at very low levels in vivo. Here, we demonstrate expression of both LNX1 and LNX2 proteins in the brain for the first time. We clarify the cell-type specific expression of LNX isoforms in both the CNS and PNS, and identify a novel LNX1 isoform. Using luciferase reporter assays, we show that the 5′ untranslated region of the Lnx1_variant 2 mRNA, that generates the LNX1p70 isoform, strongly suppresses protein production. This effect is mediated in part by the presence of upstream open reading frames (uORFs), but also by a sequence element that decreases both mRNA levels and translational efficiency. By contrast, uORFs do not negatively regulate LNX1p80 or LNX2 expression. Instead, we find some evidence that protein turnover via proteasomal degradation may influence LNX1p80 levels in cells. These observations provide plausible explanations for the low levels of LNX1 proteins detected in vivo.     

Solubilization of tricalcium phosphate by P(3HB) accumulating Azotobacter chroococcum MAL-201

Cells of Azotobacter chroococcum MAL-201 (MTCC 3853) are capable of accumulating the intracellular poly(3-hydroxybutyric acid) [P(3HB)], accounting for 65–71 % of its cell dry weight and also capable of synthesizing the enzyme alkaline phosphatase (APase), when grown in glucose and tricalcium phosphate containing nitrogen-free modified Stockdale medium. The concentration of insoluble phosphate in broth medium was optimized as 0.25 % (w/v) for growth and biosynthesis of APase. However, the suboptimal concentration of phosphate (0.1 %, w/v) appeared as the best suited for accumulation of P(3HB) by the strain. The significant differences were observed in biosynthesis of polymer and APase enzyme under variable phosphate concentrations. Glucose, 3.0 % (w/v) was recorded as the optimum concentration for all of the three parameters. The continuation of APase biosynthesis was observed during the period of significant decline in the cellular content of the polymer in the late phase of growth. In order to study the role of P(3HB), the rate of autodigestion of biopolymer and phosphate solubilization rate (k, mineralization constant) were determined in carbon-free medium under batch cultivation process and the parameters were found to be positively correlated. The maximum phosphate solubilization rate (k = 0.0154) by the strain MAL-201 timed at the 10th hour of incubation when the rate of polymer degradation concomitantly attained its peak corresponding to 87 mg/l/h and then declined gradually. Only a negligible amount of residual polymer remained undigested. These data strongly support the functional role of P(3HB) in response to multinutritional stress condition.