Directed evolution of a single-chain class II MHC product by yeast display

Many autoimmune diseases have been linked to the class II region of the major histocompatibility complex (MHC). The linkage is thought to be a result of autoreactive T cells that recognize self-peptides bound to a product of this locus. For example, T cells from non-obese diabetic mice recognize specific 'diabetogenic' peptides bound to a class II MHC allele called I-A(g7). The I-A(g7) molecule is noted for being unstable and difficult to work with, especially in soluble form. In this work, yeast surface display combined with fluorescence-activated cell sorting was used as a means of directed evolution to engineer stabilized variants of a single-chain form of I-A(g7). A library containing mutations at two residues (positions 56 and 57 of the I-A(g7) beta-chain) that are important in the class II disease associations yielded stabilized mutants with preferences for a glutamic acid at residue 56 and a leucine at residue 57. Random mutation of I-A(g7) followed by selection with an anti-I-A(g7) antibody also yielded stabilized variants with mutations in other residues. The methods described here allow the discovery of novel MHC complexes that could facilitate structural studies and provide new opportunities in the development of diagnostics or antagonists of class II MHC-associated diseases.     

Enhancement of antidepressant potency by a potentiator of AMPA receptors

1. AMPA receptor potentiators (ARPs) exhibit antidepressant-like activity in preclinical tests (for example, the forced swim test) that are highly predictive of efficacy in humans. Unlike most currently used antidepressants, ARPs do not elevate extracellular levels of biogenic amines (e.g., 5HT, NE) in prefrontal cortex at doses that are active in the forced swim test.2. The present series of experiments examined the effects of combining the ARP, LY 392098, with biogenic amine-based antidepressants in the forced swim test. Male, NIH Swiss mice were placed in a cylinder of water and observed for attempted escape behaviors and immobility.3. LY 392098 dose-dependently decreased immobility as did a range of classical antidepressants. At doses of LY 392098 below those that decreased immobility, this compound significantly increased the potency with which fluoxetine and citalopram (SSRI antidepressants), imipramine (tricyclic antidepressant), duoxetine (norepinephrine/serotonin uptake blocker), nisoxetine (norepinephrine uptake inhibitor), and rolipram (PDE4 inhibitor) decreased immobility in the forced swim test with potency shifts upward of 5-fold (fluoxetine, imipramine, and rolipram). Likewise, ineffective doses of the traditional antidepressants potentiated the effects LY 392098 with shifts in the dose-effect functions that were 10-fold or more for citalopram, fluoxetine, imipramine, and duloxetine.4. Combined with other evidence for a role of AMPA receptors in the efficacy of antidepressants, the current data suggest that the addition of an ARP may augment the activity and perhaps the onset of the therapeutic effects of biogenic amine and second messenger-based antidepressants.     

Functional organization of adult motor cortex is dependent upon continued protein synthesis

The functional organization of adult cerebral cortex is characterized by the presence of highly ordered sensory and motor maps. Despite their archetypical organization, the maps maintain the capacity to rapidly reorganize, suggesting that the neural circuitry underlying cortical representations is inherently plastic. Here we show that the circuitry supporting motor maps is dependent upon continued protein synthesis. Injections of two different protein synthesis inhibitors into adult rat forelimb motor cortex caused an immediate and enduring loss of movement representations. The disappearance of the motor map was accompanied by a significant reduction in synapse number, synapse size, and cortical field potentials and caused skilled forelimb movement impairments. Further, motor skill training led to a reappearance of movement representations. We propose that the circuitry of adult motor cortex is perpetually labile and requires continued protein synthesis in order to maintain its functional organization.     

All-optical histology using ultrashort laser pulses

As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 microJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100 microm or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.     

Plasma 3-nitrotyrosine and outcome in neonates with severe bronchopulmonary dysplasia after inhaled nitric oxide

Plasma protein levels of 3-nitrotyrosine and 3-chlorotyrosine were measured by LC-MS/MS at 0 and 72 h after the initiation of inhaled nitric oxide (INO) at 20 ppm in 22 prematurely born infants with clinically documented bronchopulmonary dysplasia. Infants were classified at the time of hospital discharge as either "off mechanical ventilation," "on mechanical ventilation," or "expired/organ failure." These outcomes were tested for association with changes in plasma levels of 3-nitrotyrosine and 3-chlorotyrosine and selected clinical risk factors. Infants whose 3-nitrotyrosine levels decreased over the 72 h period were more likely to wean off of mechanical ventilation (p =.03). There was no significant association between changes in 3-chlorotyrosne levels and outcome. After controlling for other variables, an odds ratio of 8.3 (95% CI: 1.3-54.4) for improved outcomes was observed if the 3-nitrotyrosine levels decreased. These data suggest that nitrative and oxidative stress may be related to the severity of lung disease and, consequentially, the overall outcome in this select group of infants with severe bronchopulmonary dysplasia.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.     

Identification of lipophilic flavones and flavonols by comparative HPLC,TLC and UV spectral analysis

The identification of lipophilic flavones and flavonols using a combination of high performance liquid chromatography, thin layer chromatography and UV spectral analysis is discussed. Data are provided for the flavones, apigenin, luteolin and tricetin and twelve of their methyl ethers, 8-hydroxyluteolin, 6-hydroxyluteolin and scutellarein and fourteen of their methyl ethers, and some 6,8-dihydroxyapigenin and 6,8-dihydroxyluteolin derivatives. Data for some forty two flavonols with extra 6- and/or 8-hydroxylation, mostly 6-hydroxykaempferol and quercetagetin derivatives, are also presented. The remaining compounds analysed include fourteen 5-deoxyflavones, four 5-methoxyflavones and five 5-deoxyflavonols plus further 5-hydroxylated flavones and flavonols without B-ring oxidation or with 2'-, 5'- or 6'-hydroxylation.