The triple gene block protein 1 (TGBp1) encoded by open reading frame 2 of bamboo mosaic potexvirus (BaMV) was overexpressed in Escherichia coli and purified in order to test its RNAbinding activity. UV crosslinking assays revealed that the RNA-binding activity was present mainly in the soluble fraction of the refolded TGBp1. The binding activity was nonspecific and salt concentration-dependent : activity was present at 0-50 mM NaCl but was almost abolished at 200 mM. The RNA-binding domain was located by deletion mutagenesis to the N-terminal 3-24 amino acids of TGBp1. Sequence alignment analysis of the N-terminal 25 amino acids of the TGBp1 homologues of potexviruses identified three arginine residues. Arg-to-Ala substitution at any one of the three arginines eliminated most of the RNA-binding activity, indicating that they were all critical to the RNA-binding activity of the TGBp1 of BaMV.

Open reading frame 2 of the bamboo mosaic potexvirus (BaMV) genome encodes a 28 kDa protein, the first of the ' triple-gene-block ' of BaMV which is believed to play a role in cell-to-cell movement of the virus in host plants. The 28 kDa protein was expressed in Escherichia coli and polyclonal antiserum was raised in a rabbit. Western blot analyses showed that the 28 kDa protein was associated mainly with components in the cell wall and 30 000 g pellet fractions of a BaMV-infected leaf homogenate. Immunogold electron microscopy of infected leaf tissues revealed that the 28 kDa protein was associated with electron-dense crystalline bodies (EDCBs) in the cytoplasm and nuclei. Nuclear EDCBs were found closely associated with nucleoli. Gold-labelled EDCB-like structures were also detected in the cytoplasm, but not within nuclei, in protoplasts up to 48 h post-inoculation. No specific labelling of the 28 kDa protein was found within any cytoplasmic structures or within cell walls.

The TGBp1 of bamboo mosaic potexvirus (BaMV) is encoded by the first overlapping gene of the triple-gene-block (TGB), whose products are thought to play roles in virus movement between plant cells. This protein forms cytoplasmic inclusions associated with virus particles in the BaMV-infected tissues. It has been proposed that the inclusion is one of the active forms of TGBp1. To prove this idea, we purified the TGBp1 inclusions from both the BaMV-infected Chenopodium quinoa and Escherichia coli cells overexpressing this protein to test some of their biochemical activities. We found that the TGBp1 inclusions isolated from the infected plant leaves, but not from E. coli, possess the NTP-binding and NTPase activities. However, they lack the RNA-binding activity possessed by the soluble TGBp1. These results indicate that the TGBp1 proteins in the BaMV-infected tissues assume two different functional forms. Mutational analyses and competition experiments show that the two arginine residues, Arg-16 and Arg-21, essential to RNA binding, are also required for the ATP-utilizing activity of the soluble TGBp1. This indicates that a same-structure motif is required for the two functions of the soluble TGBp1. The location of the two arginine residues outside the seven conserved motifs of the NTP-utilizing superfamily I RNA helicases, to which TGBp1 belongs, suggests that an extra-structure motif, besides the seven conserved ones, is required for the NTP-utilizing activity of the TGBp1 protein of BaMV.

This work examines whether microglia-conditioned medium (MCM) is beneficial in stressed spinal cord cells or tissues. MCM was separated into two fractions by 50 kDa molecular cut-off centrifugation. MCM not only promoted survival of neuronal and oligodendroglial cells but effectively reduced LPS stimulation in spinal cord cultures. We further utilized the NYU weight-drop device to induce contusive spinal cord injury (SCI) in rats. Immediately after dropping the impactor from a height of 25 mm onto thoracic spinal segment, MCM was intrathecally administered. At 6 weeks post-injury, SCI rats receiving MCM > 50 kDa treatment showed significant hind-limb improvement over MCM < 50 kDa- or vehicle-treated SCI rats. Consistently, more preserved nerve fibers and fewer activated microglia were found in the injured epicenter of MCM-treated SCI rats. Taken together, secreted substances, mainly > 50 kDa, of microglia was neuroprotective against spinal cord injury

Traumatic spinal cord injury (SCI) initiates a series of cellular and molecular events that include both primary and secondary injury cascades. This secondary cascade provides opportunities for the delivery of therapeutic intervention. Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-β (TGF-β) superfamily, regulates various biological processes in mammals. The effects of GDF11 in the nervous system were not fully elucidated. Here, we perform extensive in vitro and in vivo studies to unravel the effects of GDF11 on spinal cord after injury. In vitro culture studies showed that GDF11 increased the survival of both neuronal and oligodendroglial cells but decreased microglial cells. In stressed cultures, GDF11 effectively inhibited LPS stimulation and also protected neurons from ischemic damage. Intravenous GDF11 administration to rat after eliciting SCI significantly improved hindlimb functional restoration of SCI rats. Reduced neuronal connectivity was evident at 6 weeks post-injury and these deficits were markedly attenuated by GDF11 treatment. Furthermore, SCI-associated oligodendroglial alteration were more preserved by GDF11 treatment. Taken together, GDF11 infusion via intravenous route to SCI rats is beneficial, facilitating its therapeutic application in the future.