Short hairpin RNAs against eotaxin or interleukin‐5 decrease airway eosinophilia and hyper‐responsiveness in a murine model of asthma

Background

Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up‐regulation of eotaxin, which, together with interleukin (IL)‐5, is important for differentiation, chemo‐attraction, degranulation, and survival of eosinophils in local tissue. In a previous study, we found that administration of lentivirus‐delivered short hairpin RNA (shRNA) to suppress the expression of IL‐5 inhibited airway inflammation. The present study aimed to investigate the role of eotaxin shRNA and the synergistic effect of eotaxin and IL‐5 shRNAs on airway inflammation in an ovalbumin (OVA)‐induced murine model of asthma.

Methods

Lentivirus‐delivered shRNAs were used to suppress the expression of eotaxin and/or IL‐5 in local tissue in an OVA‐induced murine asthma model.

Results

Intra‐tracheal administration of lentivirus containing eotaxin shRNA expressing cassette (eoSEC3.3) efficiently moderated the characteristics of asthma, including airway hyper‐responsiveness, cellular infiltration of lung tissues, and eotaxin and IL‐5 levels in bronchio‐alveolar lavage fluid. Administration of lentiviruses expressing IL‐5 or eotaxin shRNAs (IL5SEC4 + eoSEC3.3) also moderated the symptoms of asthma in a mouse model.

Conclusions

Local delivery of lentiviruses expressing IL‐5 and eotaxin shRNAs provides a potential tool in moderating airway inflammation and also has the potential for developing clinical therapy based on the application of shRNAs of chemokines and cytokines involved in T helper 2 cell inflammation and eosinophilia. Copyright © 2008 John Wiley & Sons, Ltd.     

The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.     

Is unconventional secretion inhibited during cell division by Cdk1 activity?

A process of unconventional secretion that is dependent on the Golgi stacking protein GRASP and multiple components of the autophagy machinery has recently been documented for several cytoplasmic and membrane protein. Classical secretion via the exocytic pathway is inhibited during cell division in animal cells, as key membrane compartments, particularly the Golgi, are disassembled and fragmented. The question as to whether unconventional secretion is likewise inhibited during mitosis has not been explored. This mode of secretion supposedly bypasses the Golgi. However, GRASP and Vps34 (a key autophagy protein) are both substrates of the cell cycle regulating cyclin‐dependent kinase 1 (Cdk1), and their activities are apparently inhibited by Cdk1 phosphorylation. Is unconventional secretion therefore similarly inhibited during cell division like conventional secretion? The story may yet turn out to be more complicated. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Glucose,glycolysis, and neurodegenerative diseases

Prolonged survival of a typical postmitotic neuron hinges on a balance between multiple processes, among these are a sustenance of ATP production and protection against reactive oxygen species. In neuropathological conditions, mitochondrial defects often lead to both a drop in ATP levels, as well as increase reactive oxygen species production from inefficient electron transport processes and NADPH-oxidases activities. The former often resulted in the phenomenon of compensatory aerobic glycolysis. The latter stretches the capacity of the cell's redox buffering capacity, and may lead to damages of key enzymes involved in energy metabolism. Several recent reports have indicated that enhancing glucose availability and uptake, as well as increasing glycolytic flux via pharmacological or genetic manipulation of glycolytic enzymes, could be protective in animal models of several major neurodegenerative diseases, including Parkinson's disease, Huntington's disease, and Amyotrophic lateral sclerosis. Activation of canonical Wnt signaling, which improves disease symptoms in mouse models of Alzheimer's disease also appears to work via an elevation of glycolytic enzymes and enhance glucose metabolism. Here, I discuss these findings and the possible underlying mechanisms of how an increase in glucose uptake and glycolysis could be neuroprotective. Increased glycolytic production of ATP would help alleviate energy deficiency, and ATP's hydrotropic effect may enhance solubility and clearance of toxic aggregates prevalent in many neurodegenerative diseases. Furthermore, channeling of glucose into the Pentose Phosphate Pathway would increase the redox buffering capacity of the cell.     

Nogo/RTN4 isoforms and RTN3 expression protect SH-SY5Y cells against multiple death insults

Among the members of the reticulon (RTN) family, Nogo-A/RTN4A, a prominent myelin-associated neurite growth inhibitory protein, and RTN3 are highly expressed in neurons. However, neuronal cell-autonomous functions of Nogo-A, as well as other members of the RTN family, are unclear. We show here that SH-SY5Y neuroblastoma cells stably over-expressing either two of the three major isoforms of Nogo/RTN4 (Nogo-A and Nogo-B) or a major isoform of RTN3 were protected against cell death induced by a battery of apoptosis-inducing agents (including serum deprivation, staurosporine, etoposide, and H₂O₂) compared to vector-transfected control cells. Nogo-A, -B, and RTN3 are particularly effective in terms of protection against H₂O₂-induced increase in intracellular reactive oxygen species levels and ensuing apoptotic and autophagic cell death. Expression of these RTNs upregulated basal levels of Bax, activated Bax, and activated caspase 3, but did not exhibit an enhanced ER stress response. The protective effect of RTNs is also not dependent on classical survival-promoting signaling pathways such as Akt and Erk kinase pathways. Neuron-enriched Nogo-A/Rtn4A and RTN3 may, therefore, exert a protective effect on neuronal cells against death stimuli, and elevation of their levels during injury may have a cell-autonomous survival-promoting function.     

Nitrogen and oxygen isotopes in nitrate in the groundwater and surface water discharge from two rural catchments: implications for nitrogen loading to coastal waters

In Prince Edward Island, Canada, widespread intensive potato production has contributed to elevated nitrate concentrations in groundwater and streams, and eutrophic or anoxic conditions occur regularly in several estuarine systems. In this research, the stable isotopes of nitrogen and oxygen in nitrate in intertidal groundwater discharge and stream water were used, in conjunction with water quality and quantity data and land use information, to better understand the characteristics of nitrate delivered to two small estuaries with contrasting land use in their contributory catchments. Most of the water samples collected during the two-year study had isotopic signatures that fell in the range expected for nitrate derived from ammonium-based fertilizers (26.5 % of the samples) or in the overlapping range formed between ammonium-based fertilizers and nitrate derived from soil (64 % of the samples). Overall, isotopic signatures spanned over relatively narrow ranges, and correlations with other water quality parameters, or catchment characteristics, were weak. Nitrate in groundwater discharge and surface water in the Trout River catchment exhibited significantly different isotopic signatures only for the nitrogen isotope, while in the McIntyre Creek catchment groundwater discharge and surface water had similar isotopic signatures. When the isotopic results for the waters from the two catchments were compared, the surface waters were found to be similar, while the isotopic signatures of nitrate in groundwater were distinct only for the nitrogen isotope. Denitrification in the two study catchments was not evident based on the isotopic results for nitrate; however, in the case of the Trout River catchment, where a small freshwater pond exists, an average nitrate load reduction of 14 % was inferred based on a comparison of nitrate loads entering and leaving the pond. Overall, it appears that natural attenuation processes, occurring either in the streams or groundwater flow systems, do not significantly reduce nitrate loading to these estuaries.     

Classification of established atopic dermatitis in children with the in vivo imaging methods

Atopic dermatitis (AD) is a cutaneous disease resulting from a defective barrier and dysregulated immune response. The severity scoring of atopic dermatitis (SCORAD) is used to classify AD. Noninvasive imaging approaches supplementary to SCORAD were investigated. Cr:forsterite laser‐based microscopy was employed to analyze endogenous third‐harmonic generation (THG) and second‐harmonic generation (SHG) signals from skin. Imaging parameters were compared between different AD severities. Three‐dimensional reconstruction of imaged skin layers was performed. Finally, statistic models from quantitative imaging parameters were developed for predicting disease severity. Our data demonstrate that THG signal intensity of lesional skin in AD were significantly increased and was positively correlated with AD severity. Characteristic gray level co‐occurrence matrix (GLCM) values were observed in more severe AD. In the 3D reconstruction video, individual dermal papilla and obvious fibrosis in the upper papillary dermis were easily identified. Our estimation models could predict the disease severity of AD patients with an accuracy of nearly 85%. The THG signal intensity and characteristic GLCM patterns are associated with AD severity and can serve as quantitative predictive parameters. Our imaging approach can be used to identify the histopathological changes of AD objectively, and to complement the SCORAD index, thus improving the accuracy of classifying AD severity.      

Neuritogenesis: polarization of constitutive exocytosis by effectors of Rho-family GTPases?

The sprouting of neurites from a neuron represents a highly specialized form of cellular morphogenesis that must involve coordinated changes in two major cellular processes at two membrane locations: reorganization of the cytoskeleton and redirection of membrane traffic from the trans-Golgi network to the plasma membrane of the growth tip. How exactly are these two processes linked and how is spatial and temporal coordination achieved at the first instance of neurite sprouting? Recent advances may have already revealed some, if not most of the pieces in the puzzle. We discuss below, with some extrapolations, of what has recently come to light, and what more is needed to construct a coherent picture.     

The action mode of the ribosome-inactivating protein α-sarcin

Based on the tertiary structure of the ribosome-inactivating protein alpha-sarcin, domains that are responsible for hydrolyzing ribosomes and naked RNA have been dissected. In this study, we found that the head-to-tail interaction between the first amino beta-strand and the last carboxyl beta-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids. Instead, a four-strand pleated beta-sheet is indispensable for catalyzing both substrates, suggesting that alpha-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center. The integrity of an amino beta-hairpin and that of the loop L3 in alpha-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo. However, a mutant protein without the beta-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids. The functional involvement of the beta-hairpin and the loop L3 in the sarcin stem/loop RNA of ribosomes is demonstrated by a docking model, suggesting that the two structures are in essence naturally designed to distinguish ribosome-inactivating proteins from RNase T1 to inactivate ribosomes.