Dissociation of the insulin receptor from caveolae during TNFα-induced insulin resistance and its recovery by D-PDMP

Previously, we demonstrated that an inhibitor of ganglioside biosynthesis, d-PDMP, could restore impaired insulin signaling in tumor necrosis factor α (TNFα)-treated adipocytes by blocking the increase of GM3 ganglioside. Here, we analyzed the interaction between insulin receptor (IR) and GM3 in the plasma membranes using immunoelectron microscopy. In normal adipocytes, most GM3 molecules localized at planar and non-caveolar regions. Approximately 19% of IR molecules were detected in caveolar regions. The relative ratio of IRs associated with caveolae in TNFα-treated adipocytes was decreased to one-fifth of that in normal adipocytes, but this decrease was restored by d-PDMP. Thus, we could obtain direct evidence that insulin resistance is a membrane microdomain disorder caused by aberrant expression of ganglioside.     

Infrared thermal imaging of rat somatosensory cortex with whisker stimulation

The present study aims to validate the applicability of infrared (IR) thermal imaging for the study of brain function through experiments on the rat barrel cortex. Regional changes in neural activity within the brain produce alterations in local thermal equilibrium via increases in metabolic activity and blood flow. We studied the relationship between temperature change and neural activity in anesthetized rats using IR imaging to visualize stimulus-induced changes in the somatosensory cortex of the brain. Sensory stimulation of the vibrissae (whiskers) was given for 10 s using an oscillating whisker vibrator (5-mm deflection at 10, 5, and 1 Hz). The brain temperature in the observational region continued to increase significantly with whisker stimulation. The mean peak recorded temperature changes were 0.048 ± 0.028, 0.054 ± 0.036, and 0.097 ± 0.015°C at 10, 5, and 1 Hz, respectively. We also observed that the temperature increase occurred in a focal spot, radiating to encompass a larger region within the contralateral barrel cortex region during single-whisker stimulation. Whisker stimulation also produced ipsilateral cortex temperature increases, which were localized in the same region as the pial arterioles. Temperature increase in the barrel cortex was also observed in rats treated with a calcium channel blocker (nimodipine), which acts to suppress the hemodynamic response to neural activity. Thus the location and area of temperature increase were found to change in accordance with the region of neural activation. These results indicate that IR thermal imaging is viable as a functional quantitative neuroimaging technique.     

Suppression of Rac1 activity at the apical membrane of MDCK cells is essential for cyst structure maintenance

Using MDCK cells that constitutively express a Förster resonance energy transfer biosensor, we found that Rac1 activity is homogenous at the entire plasma membrane in early stages of cystogenesis, whereas in later stages Rac1 activity is higher at the lateral membrane than at the apical plasma membrane. If Rac1 is activated at the apical membrane in later stages, however, the monolayer cells move into the luminal space. In these cells, tight junctions are disrupted, accompanied by mislocalization of polarization markers and disorientation of cell division. These observations indicate that Rac1 suppression at the apical membrane is essential for the maintenance of cyst structure.     

Localization of mRNAs encoding human mitochondrial oxidative phosphorylation proteins

The mitochondrial oxidative phosphorylation (OXPHOS) proteins are encoded by both nuclear and mitochondrial DNA. The nuclear-encoded OXPHOS mRNAs have specific subcellular localizations, but little is known about which localize near mitochondria. Here, we compared mRNAs in mitochondria-bound polysome fractions with those in cytosolic, free polysome fractions. mRNAs encoding hydrophobic OXPHOS proteins, which insert into the inner membrane, were localized near mitochondria. Conversely, OXPHOS gene which mRNAs were predominantly localized in cytosol had less than one transmembrane domain. The RNA-binding protein Y-box binding protein-1 is localized at the mitochondrial outer membrane and bound to the OXPHOS mRNAs. Our findings offer new insight into mitochondrial co-translational import in human cells.     

Phytochemistry and antiglycation activity of Aloe sinkatana Reynolds

A new anthraquinone along with 10 known compounds were isolated from the leaves of Aloe sinkatana Reynolds (Aloaceae), and their structures were elucidated as the new compound 2,8-dihydroxy-6-(hydroxymethyl)-1-methoxyanthracene-9,10-dione (1) and the known compounds Aloe-emodin (2), feralolide (3), 1-hydroxy-5-methoxy-3-methyl-9,10 dihydroanthracene 9,10-dione (4), β-sitosterol (5), β-sitosterol with glycosidic bond (6), microdontin (7), homoaloins A (8) and B (9) and aloins A (10) and B (11). Characterization of compounds 1–9 was based on spectral analyses and comparison with reported data, particularly the new compound 1 was identified by 1D- and 2D NMR, mass spectroscopic and X-ray crystallography analyses. Antiglycation activity of the extracts and isolated compounds were carried out using the hemoglobin-δ-gluconolactone and glucose–bovine serum albumin assays. The results obtained showed that MeOH and EtOAc extracts as well as compound 1 showed an inhibitory effect on early stage protein glycation. Compound 1 also showed significant inhibitory effects against glucose-induced advanced glycation end-products.     

Naphthalene carboxamides as inhibitors of human cytomegalovirus DNA polymerase

ortho-Hydroxynaphthalene carboxamides have been identified as inhibitors of HCMV DNA polymerase. SAR investigations have demonstrated that both the amide and hydroxy functionalities are required for activity. Substitution on the naphthalene ring has led to inhibitors with submicromolar IC50s against HCMV polymerase. These compounds have been found to be >100-fold selective for inhibition of HCMV polymerase versus human alpha polymerase and display antiviral activity in a cell-based plaque reduction assay.     

Clustered protocadherin family

The brain is a complex system composed of enormous numbers of differentiated neurons, and brain structure and function differs among vertebrates. To examine the molecular mechanisms underlying brain structure and function, it is important to identify the molecules involved in generating neural diversity and organization. The clustered protocadherin (Pcdh) family is the largest subgroup of the diverse cadherin superfamily. The clustered Pcdh proteins are predominantly expressed in the brain and their gene structures in vertebrates are diversified. In mammals, the clustered Pcdh family consists of three gene clusters: Pcdh -α, Pcdh -β, and Pcdh -γ. During brain development, this family is upregulated by neuronal differentiation, and Pcdh-α is then dramatically downregulated by myelination. Clustered Pcdh expression continues in the olfactory bulb, hippocampus, and cerebellum until adulthood. Structural analysis of the first cadherin domain of the Pcdh-α protein revealed it lacks the features that classical cadherins require for homophilic adhesiveness, but it contains Pcdh-specific loop structures. In Pcdh-α, an RGD motif on a specific loop structure binds β1-integrin. For gene expression, the gene clusters are regulated by multiple promoters and alternative cis splicing. At the single-cell level, several dozen Pcdh -α and -γ mRNA are regulated monoallelically, resulting in the combinatorial expression of distinct variable exons. The Pcdh-α and Pcdh-γ proteins also form oligomers, further increasing the molecular diversity at the cell surface. Thus, the unique features of the clustered Pcdh family may provide the molecular basis for generating individual cellular diversity and the complex neural circuitry of the brain.     

Rottlerin activates AMPK possibly through LKB1 in vascular cells and tissues

AMP-activated protein kinase (AMPK) is a cellular energy sensor involved in multiple cell signaling pathways that has become an attractive therapeutic target for vascular diseases. It is not clear whether rottlerin, an inhibitor of protein kinase Cδ, activates AMPK in vascular cells and tissues. In the present study, we have examined the effect of rottlerin on AMPK in vascular smooth muscle cells (VSMCs) and isolated rabbit aorta. Rottlerin reduced cellular ATP and activated AMPK in VSMCs and rabbit aorta; however, inhibition of PKCδ by three different methods did not activate AMPK. Both VSMCs and rabbit aorta expressed the upstream AMPK kinase LKB1 protein, and rottlerin-induced AMPK activation was decreased in VSMCs by overexpression of dominant-negative LKB1, suggesting that LKB1 is involved in the upstream regulation of AMPK stimulated by rottlerin. These data suggest for the first time that LKB1 mediates rottlerin-induced activation of AMPK in vascular cells and tissues.     

Disulfide-linked bovine beta-lactoglobulin dimers fold slowly, navigating a glassy folding landscape

To gain insight into the folding of large proteins, we constructed a bovine beta-lactoglobulin (beta-lg) dimeric mutant, A34C/C121A beta-lg. In the mutant, a free thiol group of wild-type beta-lg at Cys121 was removed and two beta-lg molecules were linked by a disulfide bridge through Cys34 created at the dimer's interface. Under strongly native conditions at low concentrations of urea, the refolding yield of A34C/C121A beta-lg was low when monitored by heteronuclear NMR spectroscopy. However, under marginally native conditions, the yield improved notably, although the refolding was still slow. H-D exchange pulse labeling monitored using heteronuclear NMR spectroscopy indicated that A34C/C121A beta-lg forms a folding intermediate similar to monomeric C121A beta-lg in spite of its slow folding. These results indicate that the rapid formation of folding intermediates driven by local interactions occurs in a manner independent of the molecular size and that, if the non-native interactions are too strong, the kinetic trap is set, leading to a glasslike misfolded state. The results suggest the important roles of marginal stability and pathways in making the folding of large proteins possible.