Effects of carbon dioxide exposure on early brain development in rats

The developing brain is vulnerable to environmental factors. We investigated the effects of air that contained 0.05, 0.1 and 0.3% CO₂ on the hippocampus, prefrontal cortex (PFC) and amygdala. We focused on the circuitry involved in the neurobiology of anxiety, spatial learning, memory, and on insulin-like growth factor-1 (IGF-1), which is known to play a role in early brain development in rats. Spatial learning and memory were impaired by exposure to 0.3% CO₂ air, while exposure to 0.1 and 0.3% CO₂ air elevated blood corticosterone levels, intensified anxiety behavior, increased superoxide dismutase (SOD) enzyme activity and MDA levels in hippocampus and PFC; glutathione peroxidase (GPx) enzyme activity decreased in the PFC with no associated change in the hippocampus. IGF-1 levels were decreased in the blood, PFC and hippocampus by exposure to both 0.1 and 0.3% CO₂. In addition, apoptosis was increased, while cell numbers were decreased in the CA1 regions of hippocampus and PFC after 0.3% CO₂ air exposure in adolescent rats. A positive correlation was found between the blood IGF-1 level and apoptosis in the PFC. We found that chronic exposure to 0.3% CO₂ air decreased IGF-1 levels in the serum, hippocampus and PFC, and increased oxidative stress. These findings were associated with increased anxiety behavior, and impaired memory and learning.     

Sorting of MHC Class II Molecules into Exosomes through a Ubiquitin-Independent Pathway

Major histocompatibility complex class II (MHC-II) molecules accumulate in exocytic vesicles, called exosomes, which are secreted by antigen presenting cells. These vesicles are released following the fusion of multivesicular bodies (MVBs) with the plasma membrane. The molecular mechanisms regulating cargo selection remain to be fully characterized. As ubiquitination of the MHC-II β-chain cytoplasmic tail has recently been demonstrated in various cell types, we sought to determine if this post-translational modification is required for the incorporation of MHC-II molecules into exosomes. First, we stably transfected HeLa cells with a chimeric HLA-DR molecule in which the β-chain cytoplasmic tail is replaced by ubiquitin. Western blot analysis did not indicate preferential shedding of these chimeric molecules into exosomes. Next, we forced the ubiquitination of MHC-II in class II transactivator (CIITA)-expressing HeLa and HEK293 cells by transfecting the MARCH8 E3 ubiquitin ligase. Despite the almost complete downregulation of MHC-II from the plasma membrane, these molecules were not enriched in exosomes. Finally, site-directed mutagenesis of all cytoplasmic lysine residues on HLA-DR did not prevent inclusion into these vesicles. Taken together, these results demonstrate that ubiquitination of MHC-II is not a prerequisite for incorporation into exosomes.     

Stimulation of tPA-dependent provisional extracellular fibrin matrix degradation by human recombinant soluble melanotransferrin

Tissue-type plasminogen activator (tPA) and its substrate plasminogen (Plg) are key components in the fibrinolytic system. We have recently demonstrated, that truncated human recombinant soluble melanotransferrin (sMTf) could stimulate the activation of Plg by urokinase plasminogen activator and inhibit angiogenesis. Since various angiogenesis inhibitors were shown to stimulate tPA-mediated plasminogen activation, we examined the effects of sMTf on tPA-dependent fibrinolysis. This study demonstrated that sMTf enhanced tPA-activation of Plg by 6-fold. sMTf also increased the release of [125I]-fibrin fragments by tPA-activated plasmin. Moreover, we observed that the interaction of sMTf with Plg provoked a change in the fibrin clot structure by cleaving the fibrin alpha and beta chains. Overall, the present study shows that sMTf modulates tPA-dependent fibrinolysis by modifying the clot structure. These results also suggest that sMTf properties could involve enhanced dissolution of the provisional extracellular fibrin matrix.     

An in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants

Proteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a 'mouse trap' against the host proteases during extraction. After confirming the importance of trypsin- and chymotrypsin-like activities in crude protein extracts of potato (Solanum tuberosum L.) leaves, transgenic lines of potato expressing either tomato cathepsin D inhibitor (CDI) or bovine aprotinin, both active against trypsin and chymotrypsin, were generated by Agrobacterium tumefaciens-mediated genetic transformation. Leaf crude protein extracts from CDI-expressing lines, showing decreased levels of cathepsin D-like and ribulose 1,5-bisphosphate carboxylase/oxygenase hydrolysing activities in vitro, conducted decreased turnover rates of the selection marker protein neomycin phosphotransferase II (NPTII) relative to the turnover rates measured for transgenic lines expressing only the marker protein. A similar stabilizing effect on NPTII was observed in leaf protein extracts from plant lines coexpressing bovine aprotinin, confirming the ability of ectopically expressed broad-spectrum serine proteinase inhibitors to reproduce the protein-stabilizing effect of low-molecular-weight proteinase inhibitors generally added to protein extraction media.     

A SELDI‐TOF MS procedure for the detection,quantitation, and preliminary characterization of low‐molecular‐weight recombinant proteins expressed in transgenic plants

We describe a SELDI‐TOF MS procedure for the rapid detection and quantitation of low‐molecular‐weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens‐mediated transformation, and then used as test material for the analyses. Real‐time RT‐PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI‐TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.     

Using knowledge brokers to facilitate the uptake of pediatric measurement tools into clinical practice: a before-after intervention study

Background

There is a large quality of care gap for patients with osteoporosis. As a fragility fracture is a strong indicator of underlying osteoporosis, it offers an ideal opportunity to initiate investigation and treatment. However, studies of post-fracture populations document screening and treatment rates below 20% in most settings. This is despite the fact that bone mineral density (BMD) scans are effective at identifying patients at high risk of fracture, and effective drug treatments are widely available. Effective interventions are required to remedy this incongruity in current practice.

Methods

This study reviewed randomised controlled trials (RCT) involving fully qualified healthcare professionals caring for patients with a fragility fracture in all healthcare settings. Any intervention designed to modify the behaviour of healthcare professionals or implement a service delivery change was considered. The main outcomes were BMD scanning and osteoporosis treatment with anti-resorptive therapy. The electronic databases Medline and Embase were searched from 1994 to June 2010 to identify relevant articles in English. Post-intervention risk differences (RDs) were calculated for the main outcomes and any additional study primary outcomes; the trials were meta-analysed.

Results

A total of 2814 potentially relevant articles were sifted; 18 were assessed in full text. Nine RCTs evaluating ten interventions met the inclusion criteria for the review. All were from North America. Four studies focused on patients with a hip fracture, three on fractures of the wrist/distal forearm, and two included several fracture sites consistent with a fragility fracture. All studies reported positive effects of the intervention for the main study outcomes of BMD scanning and osteoporosis treatment. For BMD scanning the overall risk ratio (95% CI) was 2.8 (2.16 to 3.64); the RD was 36% (21% to 50%). For treatment with anti-resorptive therapy the overall risk ratio (95% CI) was 2.48 (1.92 to 3.2); the RD was 20% (10% to 30%).

Conclusions

All interventions produced positive effects on BMD scanning and osteoporosis treatment rates post-fracture. Despite sizeable increases, investigation and treatment rates remain sub-optimal. Long-term compliance with osteoporosis medications needs to be addressed, as the majority of studies reported treatment rates at six-month follow up only. Studies would be more informative if treatment criteria were defined a priori to facilitate understanding of whether patients were being treated appropriately and integrated economic analyses would be helpful for informing policy implementation decisions.     

Overexpression of type 1 angiotensin II receptors impairs excitation-contraction coupling in the mouse heart

Transgenic mice that overexpress human type 1 angiotensin II receptor (AT(1)R) in the heart develop cardiac hypertrophy. Previously, we have shown that in 6-mo AT(1)R mice, which exhibit significant cardiac remodeling, fractional shortening is decreased. However, it is not clear whether altered contractility is attributable to AT(1)R overexpression or is secondary to cardiac hypertrophy/remodeling. Thus the present study characterized the effects of AT(1)R overexpression on ventricular L-type Ca(2+) currents (I(CaL)), cell shortening, and Ca(2+) handling in 50-day and 6-mo-old male AT(1)R mice. Echocardiography showed there was no evidence of cardiac hypertrophy in 50-day AT(1)R mice but that fractional shortening was decreased. Cellular experiments showed that cell shortening, I(CaL), and Ca(v)1.2 mRNA expression were significantly reduced in 50-day and 6-mo-old AT(1)R mice compared with controls. In addition, Ca(2+) transients and caffeine-induced Ca(2+) transients were reduced whereas the time to 90% Ca(2+) transient decay was prolonged in both age groups of AT(1)R mice. Western blot analysis revealed that sarcoplasmic reticulum Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger protein expression was significantly decreased in 50-day and 6-mo AT(1)R mice. Overall, the data show that cardiac contractility and the mechanisms that underlie excitation-contraction coupling are altered in AT(1)R mice. Furthermore, since the alterations in contractility occur before the development of cardiac hypertrophy, it is likely that these changes are attributable to the increased activity of the renin-angiotensin system brought about by AT(1)R overexpression. Thus it is possible that AT(1)R blockade may help maintain cardiac contractility in individuals with heart disease.