Translation of soybean mosaic virus RNA in vitro: evidence of protein processing

The genomic RNA of soybean mosaic virus (SMV), a member of the potyvirus group of plant viruses, was translated in both the wheat germ and reticulocyte cell free systems to identify some viral encoded proteins and as an approach to determining the translational strategy of the virus. The RNA was translated into the same specific set of 10 to 12 polypeptides in both in vitro systems. Immunological tests and peptide analyses indicate that six translation products are related to SMV coat protein, and one of these comigrated with coat protein during electrophoresis. Two other antigenically distinct classes of polypeptides were identified by their specific immunoprecipitation with antibody against the SMV cytoplasmic inclusion body protein or tobacco etch virus nuclear inclusion body protein. To determine if any products of the in vitro translation reactions resulted from proteolytic processing of a precursor molecule, translation reactions were carried out with amino terminal label N-formyl[35S]methionyl-tRNA(Met)i (f-Met), or with [35S]methionine, and the resultant products were compared. The putative SMV coat protein and a translation product related to the nuclear inclusion body protein were not labeled with f-Met indicating that they were generated by proteolytic processing at their amino termini. Consistent with this finding is the accumulation of new polypeptides of greater apparent molecular weight when amino acid analogs were present during translation.     

Osteoblasts produce monocyte chemoattractant protein-1 in a murine model of Staphylococcus aureus osteomyelitis and infected human bone tissue

Incidences of osteomyelitis caused by Staphylococcus aureus have increased dramatically in recent years, in part, due to the appearance of community-acquired antibiotic-resistant strains. Therefore, understanding the pathogenesis of this organism has become imperative. Recently, we have described the surprising ability of bone-forming osteoblasts to secrete a number of important immune mediators when exposed to S. aureus in vitro. In the present study, we provide the first evidence for the in vivo production of the pivotal inflammatory chemokine, monocyte chemoattractant protein-1 (MCP-1), by osteoblasts during S. aureus-associated bone infection. Quantitative real-time PCR was employed to determine levels of mRNA encoding MCP-1 in vivo using a mouse model that closely resembles the pathology of trauma-induced staphylococcal osteomyelitis. Expression of this inflammatory chemokine and osteoblast-specific markers was investigated by confocal laser scanning microscopy in bone tissue from organ cultures of neonatal mouse calvaria and from the in vivo mouse model. Furthermore, the clinical relevancy of these findings was investigated by performing similar studies on infected human bone tissue from patients with S. aureus-associated osteomyelitis. Here, we confirm that expression of mRNA encoding MCP-1 is elevated in bacterially infected murine bone tissue. Importantly, we show that these increases translate into marked elevations in the expression of MCP-1 protein that co-localizes with osteoblast markers in infected bone tissue. Such increases could not be attributed solely to mechanical damage as a similar response was observed in infected but otherwise undamaged organ cultures. Finally, we have demonstrated the in vivo production of MCP-1 by osteoblasts in bone specimens from patients with S. aureus-associated osteomyelitis. As such, these studies demonstrate that bacterial challenge of osteoblasts during bone diseases such as staphylococcal osteomyelitis induces cells to produce a key inflammatory chemokine that can direct appropriate host responses or may contribute to progressive inflammatory damage.     

The baromodulatory effect of gamma knife irradiation of the hypothalamus in the obese Zucker rat

OBJECTIVE: The aim of this study was to evaluate the effect on body weight set point over time of focused, subnecrotic doses of radiation via gamma knife (GK) to the hypothalamus of the genetically obese Zucker rat. METHODS: A total of 36 adolescent animals were used in this experiment and placed in 6 groups of 6. The genetically obese homozygous Zucker rat was used in 4 groups (n = 24) and received GK, subcutaneous cobalt protoporphyrin (CoPP), both treatments combined or sham treatment. The heterozygous lean Zucker rat was used in 2 control groups (n = 12) and received either GK or sham treatment. All animals were weighed at the beginning of the experiment and at weekly intervals for 34 weeks. GK irradiation was accomplished using a specially designed stereotactic frame and a total dose of 40 Gy was given to 2 nearby targets in the medial hypothalamus. The drug subgroups received weekly subcutaneous injections. All animals were housed in the same environment with unlimited access to food. RESULTS: There were no significant differences in weight between the lean GK and sham groups. For the obese cohort, beginning at week 7 and throughout the remainder of the experiment, there were significant and sustained reductions in weight set point for animals that received GK (p < 0.05) and CoPP (p < 0.05) compared to sham-treated animals. Curiously, there was no statistical difference between the combined treatment and sham subgroups, though there was a trend toward weight reduction (p < 0.10). With the exception of one animal in the obese GK cohort in which there was a small area of necrosis lateral to the target area, histopathological analysis failed to reveal any abnormalities. There were no gross behavioral abnormalities noted. CONCLUSION: Our experimental results suggest that a single dose of GK irradiation to the hypothalamus can produce sustained reduction in the weight set point without emaciation in adolescent animals. The effect of this treatment is comparable to a well-studied drug therapy with a metalloporphyrin. We hypothesize that this involves a resetting of the hypothalamic set point for body weight through an as yet uncharacterized neuromodulatory effect.     

Dephosphorylation and internalization of cell adhesion molecule L1 induced by theta burst stimulation in rat hippocampus

The neural cell adhesion molecule L1 may participate in initiating and maintaining synaptic changes during learning in the hippocampus. One prominent form of synaptic change in the hippocampus is long-term potentiation (LTP) that occurs following specific patterns of synaptic activity. We present evidence that Y1176 of the YRSL motif within L1 cytoplasmic domain is dephosphorylated in LTP-induced hippocampus. The dephosphorylated L1 is associated with AP-2 and AP180 that are required for clathrin-mediated internalization of L1. These data suggest that clathrin-mediated recycling of L1 at presynaptic sites is enhanced by certain kinds of neural activity, and that maintenance of LTP-induced synaptic changes is regulated by L1 recycling.     

Disrupted Schwann cell-axon interactions in peripheral nerves of mice with altered L1-integrin interactions

The cell adhesion molecule L1 is important for peripheral nerve development. Mice lacking the 6th Ig domain of L1 (L1-6D mice) lose L1 homophilic binding and RGD dependent L1-integrin binding [Itoh, K., Cheng, L., Kamei, Y., Fushiki, S., Kamiguchi, H., Gutwein, P., Stoeck, A., Arnold, B., Altevogt, P., Lemmon, V., 2004. Brain development in mice lacking L1-L1 homophilic adhesion. J. Cell Biol. 165, 145-154]. We examined the ultrastructure of sciatic nerves from L1-6D at postnatal day 7 and 8 weeks. Unmyelinated axons frequently detached at the edge of Schwann cells, and naked axons were observed. Myelin was thinner in L1-6D and abnormal, multiple axons wrapped in a single myelin sheath were routinely observed. Previous work has shown that L1 on axons interacts with a heterophilic binding partner on Schwann cells to facilitate normal peripheral nerve formation. Taken together, it is likely that L1 on axons binds integrins on Schwann cells, resulting in interactions between axons and Schwann cells that are essential for ensheathment and myelination.     

Reliability and validity of useful field of view test scores as administered by personal computer

The Useful Field of View test (UFOV(1)) is a measure of processing speed that predicts driving performance and other functional abilities in older adults. In comparison to a number of other visual and cognitive measures, the UFOV measure has consistently been found to be the strongest predictor of motor vehicle crashes of older adults. This measure has valuable applications in that computerized, performance-based measures that are predictive of crashes in the elderly population can provide an objective criterion for determining the need for driver restriction or rehabilitation. Administration of the UFOV test has evolved from the standard version (administered via touch-screen with the Visual Attention Analyzer) to two briefer versions, which are administered on a personal desktop computer (PC) using either a touch screen or mouse response option. These new versions of the test are briefer and require less specialized equipment, making the test more portable and practical for use in clinical settings. This study examined the reliability and validity of the scores from these two new versions. Results indicate that test-retest reliabilities of the scores from the UFOV PC versions are high (r's= 0 .884 for mouse and 0.735 for touch), and performance on both PC versions correlates well with performance on the standard version (r's = 0.658 for mouse and 0.746 for touch). Furthermore, scores were highly correlated (r = 0.916) when participants used either a touch screen or a mouse to input responses. In conclusion, the reliability and validity coefficients are of sufficient magnitude to make the touch and mouse PC versions of the UFOV practical for use in clinical evaluations.     

The reliability of ultrasound measurements of carotid stenosis compared to MRA and DSA

OBJECTIVE: Carotid ultrasound (US) is a screening test for patients with transient ischemic attacks (TIAs) or stroke who then undergo Digital Subtraction Angiogram (DSA) or Magnetic Resonance Angiography (MRA). Gold standard DSA is invasive with inherent risks and costs. MRA is an evolving technology. This study compares reliability of MRA and US modes with DSA in determining degree of internal carotid artery stenosis. METHODS: A five year retrospective analysis of 140 carotid arteries from patients who had carotid US and DSA, and possibly Magnetic Resonance Angiography was undertaken. Recorded US parameters were peak systolic velocity (PSV), end diastolic velocity (EDV), and ICA/CCA peak systolic velocity ratio. The MRA and DSA parameters used NASCET technique for measuring stenosis. Statistical analysis included ROC curves and Kappa computation. RESULTS: US grading of carotid stenosis can be made more reliable by choosing appropriate parameters. The best combination of sensitivity and specificity for stenosis > 70% in our hospital was seen at PSV > 173 cm/s (sensitivity 0.87, specificity 0.8, Positive Predictive Value (PPV) 0.70, Negative Predictive Value (NPV) 0.93, kappa 0.64 and weighted kappa 0.71). MRA kappa was 0.78, (sensitivity 0.75, specificity 1.0, PPV 1.0, NPV 0.85). CONCLUSIONS: US parameters should be validated in each centre. At best, US can only approximate the accuracy of DSA, probably due to inherent limitations of this modality. Magnetic Resonance Angiography has a perfect specificity and PPV but this technique needs to be standardized. Simultaneous use of MRA and US for screening increases sensitivity to over 0.9 without compromising specificity in > 70% stenosis.