Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation

In several vascular inflammatory reactions (i.e. immunity and thrombosis) inflammatory mediators lead to the activation of vascular endothelial cells (EC). To date, a number of functional molecules induced on the surface of activated-EC have been identified. We report here that Globotetraosylceramide (Gb4), a glycosphingolipid expressed in EC, is a novel inducible molecule on EC activated by TNF-α. The cell surface expression of Gb4 is increased in a time-dependent manner under TNF-α stimulation, which shows distinct expression kinetics of major proteins induced by TNF-α on EC. MALDI-TOF-MS analysis revealed that the enhanced Gb4 predominantly contains C24:0 fatty acid in the ceramide moiety. Isolated caveolae/lipid raft-enriched detergent insoluble membrane domains in activated-EC predominantly contain this molecular species of Gb4. Gb4 containing C16:0 fatty acid in the ceramide moiety, which is known to constitute the major species of Gb4 in plasma, is also found as a major molecular species in EC. These observations indicate that Gb4, especially with very long fatty acid, is enhanced in EC during its inflammatory reaction, and suggest the potential utility of Gb4 as a biomarker for monitoring inflammation status of EC involving its related diseases.     

Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA

The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.     

Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-L-alanine-D-glutamate-meso-diaminopimelic acid-D-alanyl-D-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal D-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.     

Cell-to-cell movement of the CAPRICE protein in Arabidopsis root epidermal cell differentiation

CAPRICE (CPC), a small, R3-type Myb-like protein, is a positive regulator of root hair development in Arabidopsis. Cell-to-cell movement of CPC is important for the differentiation of epidermal cells into trichoblasts (root hair cells). CPC is transported from atrichoblasts (hairless cells), where it is expressed, to trichoblasts, and generally accumulates in their nuclei. Using truncated versions of CPC fused to GFP, we identified a signal domain that is necessary and sufficient for CPC cell-to-cell movement. This domain includes the N-terminal region and a part of the Myb domain. Amino acid substitution experiments indicated that W76 and M78 in the Myb domain are critical for targeted transport, and that W76 is crucial for the nuclear accumulation of CPC:GFP. To evaluate the tissue-specificity of CPC movement, CPC:GFP was expressed in the stele using the SHR promoter and in trichoblasts using the EGL3 promoter. CPC:GFP was able to move from trichoblasts to atrichoblasts but could not exit from the stele, suggesting the involvement of tissue-specific regulatory factors in the intercellular movement of CPC. Analyses with a secretion inhibitor, Brefeldin A, and with an rhd3 mutant defective in the secretion process in root epidermis suggested that intercellular CPC movement is mediated through plasmodesmata. Furthermore, the fusion of CPC to tandem-GFPs defined the capability of CPC to increase the size exclusion limit of plasmodesmata.     

Airway Wall Area Derived from 3-Dimensional Computed Tomography Analysis Differs among Lung Lobes in Male Smokers

Background

It is time-consuming to obtain the square root of airway wall area of the hypothetical airway with an internal perimeter of 10 mm (√Aaw at Pi10), a comparable index of airway dimensions in chronic obstructive pulmonary disease (COPD), from all airways of the whole lungs using 3-dimensional computed tomography (CT) analysis. We hypothesized that √Aaw at Pi10 differs among the five lung lobes and √Aaw at Pi10 derived from one certain lung lobe has a high level of agreement with that derived from the whole lungs in smokers.

Methods

Pulmonary function tests and chest volumetric CTs were performed in 157 male smokers (102 COPD, 55 non-COPD). All visible bronchial segments from the 3^rd to 5^th generations were segmented and measured using commercially available 3-dimensional CT analysis software. √Aaw at Pi10 of each lung lobe was estimated from all measurable bronchial segments of that lobe.

Results

Using a mixed-effects model, √Aaw at Pi10 differed significantly among the five lung lobes (R² = 0.78, P<0.0001). The Bland-Altman plots show that √Aaw at Pi10 derived from the right or left upper lobe had a high level of agreement with that derived from the whole lungs, while √Aaw at Pi10 derived from the right or left lower lobe did not.

Conclusion

In male smokers, CT-derived airway wall area differs among the five lung lobes, and airway wall area derived from the right or left upper lobe is representative of the whole lungs.     

Cell cycle arrest by monochloramine through the oxidation of retinoblastoma protein

Impairment of cell cycle control has serious effects on inflammation, tissue repair, and carcinogenesis. We report here the G1 cell cycle arrest by monochloramine (NH2Cl), a physiological oxidant derived from activated neutrophils, and its mechanism. When Jurkat cells were treated with NH2Cl (70 microM, 10 min) and incubated for 24 h, the S phase population decreased significantly with a slight increase in the hypodiploid cell population. The G0/ G1 phase and G2/M phase populations did not show marked changes. Three hours after NH2Cl treatment, the retinoblastoma protein (pRB) was dephosphorylated especially at Ser780 and Ser795, both of which are important phosphorylation sites for the G1 checkpoint function. The phosphorylation at Ser807/811 showed no apparent change. The expression of cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors showed no apparent change. Moreover, the kinase activity that phosphorylates pRB remained constant even after NH2Cl treatment. The protein phosphatase activity that dephosphorylates pRB showed a marginal increase. Notably, when the recombinant pRB was oxidized by NH2Cl in vitro, the oxidized pRB became difficult to be phosphorylated by kinases, especially at Ser780 and Ser795, but not at Ser807/811. Amino acid analysis of oxidized pRB showed methionine oxidation to methionine sulfoxide. The NH2Cl-treated Jurkat cell proteins also showed a decrease in methionine. These observations suggested that direct pRB oxidation was the major cause of NH2Cl-induced cell cycle arrest. In the presence of 2 mM NH4+, NaOCl (200 microM) or activated neutrophils also induced a G1 cell cycle arrest. As protein methionine oxidation has been reported in inflammation and aging, cell cycle modulation by pRB oxidation may occur in various pathological conditions.     

Developmental expression profiles of axon guidance signaling and the immune system in the marmoset cortex: Potential molecular mechanisms of pruning of dendritic spines during primate synapse formation in late infancy and prepuberty (I)

The synapse number and the related dendritic spine number in the cerebral cortex of primates shows a rapid increase after birth. Depending on the brain region and species, the number of synapses reaches a peak before adulthood, and pruning takes place after this peak (overshoot-type synaptic formation). Human mental disorders, such as autism and schizophrenia, are hypothesized to be a result of either too weak or excessive pruning after the peak is reached. Thus, it is important to study the molecular mechanisms underlying overshoot-type synaptic formation, particularly the pruning phase.     

Prosaposin Overexpression following Kainic Acid-Induced Neurotoxicity

Because excessive glutamate release is believed to play a pivotal role in numerous neuropathological disorders, such as ischemia or seizure, we aimed to investigate whether intrinsic prosaposin (PS), a neuroprotective factor when supplied exogenously in vivo or in vitro, is up-regulated after the excitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, PS immunoreactivity and its mRNA expression in the hippocampal and cortical neurons showed significant increases on day 3 after KA injection, and high PS levels were maintained even after 3 weeks. The increase in PS, but not saposins, detected by immunoblot analysis suggests that the increase in PS-like immunoreactivity after KA injection was not due to an increase in saposins as lysosomal enzymes after neuronal damage, but rather to an increase in PS as a neurotrophic factor to improve neuronal survival. Furthermore, several neurons with slender nuclei inside/outside of the pyramidal layer showed more intense PS mRNA expression than other pyramidal neurons. Based on the results from double immunostaining using anti-PS and anti-GABA antibodies, these neurons were shown to be GABAergic interneurons in the extra- and intra-pyramidal layers. In the cerebral cortex, several large neurons in the V layer showed very intense PS mRNA expression 3 days after KA injection. The choroid plexus showed intense PS mRNA expression even in the normal rat, and the intensity increased significantly after KA injection. The present study indicates that inhibitory interneurons as well as stimulated hippocampal pyramidal and cortical neurons synthesize PS for neuronal survival, and the choroid plexus is highly activated to synthesize PS, which may prevent neurons from excitotoxic neuronal damage. To the best of our knowledge, this is the first study that demonstrates axonal transport and increased production of neurotrophic factor PS after KA injection.