Peripapillary fluorescence lifetime reveals age-dependent changes using fluorescence lifetime imaging ophthalmoscopy in rats.

Many fundus diseases accompany fundus autofluorescence change. Fluorescence lifetime imaging ophthalmoscope (FLIO) is a latest technique in imaging fundus autofluorescence. With FLIO, the fundus fluorescence lifetime (FLT) is recorded topographically, assisting to diagnose and monitor multiple fundus diseases. The purpose of this study was to evaluate the repeatability of FLT using FLIO on adult rats and to analyze the age-dependency of the peripapillary FLT of the fundus in a short spectral channel (498-560 nm) and a long spectral channel (560-720 nm). Sprague Dawley rats (n of eyes = 10) were used for repeatability experiments. Age-dependent changes were investigated in young (two months old, n of eyes = 20) and old (eight months old, n of eyes = 10) rats. Repeatability experiments showed highly corresponding data for all segments in both spectral channel, with higher repeatability in the short spectral channel. FLT decreased significantly in all areas in the short (young: 991 ±â€¯29 ps; old: 547 ±â€¯42 ps) and long (young: 382 ±â€¯28 ps; old: 261 ±â€¯16 ps) spectral channels, indicating an overall metabolic change of the fundus in old animals. FLT of veins increased in the short spectral channel (young: 385 ±â€¯43 ps; old: 424 ±â€¯25 ps) and no change was observed in the long spectral channel (young: 274 ±â€¯9 ps; old: 269 ±â€¯24 ps). FLIO represents a highly repeatable and sensitive method to detect changes of the FLT in aged eyes for monitoring the degeneration of the rodent retinae.

Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

Proteomics of Orbital Tissue in Thyroid-Associated Orbitopathy.

CONTEXT: A potentially altered protein expression profile in orbital tissue from patients with thyroid-associated orbitopathy (TAO) is suspected. OBJECTIVE: To detect for the first time changes in proteomic patterns of orbital connective tissue in TAO and compare these with control tissue using mass spectrometry. DESIGN: Proteomics cross-sectional, comparative study. SETTING: Two academic endocrine institutions. SAMPLES: A total of 64 orbital and peripheral adipose tissue samples were collected from 39 patients with TAO and 25 control subjects. METHODS: Samples were analyzed and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology. MAIN OUTCOME MEASURES: Mean intensity values of all identified peptides per protein. RESULTS: Thirty-one proteins were identified, of which 16 differentiated between controls and patients with TAO. Different protein patterns between orbital and peripheral adipose tissue were observed. Compared to controls, 10 proteins were markedly up-regulated (≥ 2-fold) in the orbital tissue of untreated patients: beta IV spectrin (6.2-fold), GTP binding G protein 2 (5.6-fold), POTE ankyrin domain family member F (5.4-fold), xylulokinase (4.1-fold), kinesin family member 1A and lipocalin 1 (both 3.6-fold), semicarbazide-sensitive metalloproteinase amine oxidase 3 and polymerase I transcript release factor (both 3.4-fold), cell-cycle protein elongin A binding protein 1 (3.3-fold), annexin A2 and cavin (both 3-fold), protein pointing to cell proliferation histone H4 (2.8-fold), and ADAM metallopeptidase with thrombospondin type 1 motif 14 (2.7-fold). The highest protein up-regulations were noted in the orbital tissue of medically untreated patients. Steroid therapy markedly reduced up-regulation of these proteins, foremost in nonsmokers. CONCLUSIONS: Proteins involved in tissue inflammation, adipose tissue differentiation, lipid metabolism, and tissue remodeling were up-regulated in orbital tissue of untreated patients with TAO. Steroids decreased the expression of these proteins, whereas smoking attenuated such effect.

Existence and learning of oscillations in recurrent neural networks.

In this paper we study a particular class of -node recurrent neural networks (RNN's). In the 3-node case we use monotone dynamical systems theory to show, for a well-defined set of parameters, that, generically, every orbit of the RNN is asymptotic to a periodic orbit. Then we investigate whether RNN's of this class can adapt their internal parameters so as to "learn" and then replicate autonomously (in feedback) certain external periodic signals. Our learning algorithm is similar to identification algorithms in adaptive control theory. The main feature of the algorithm is that global exponential convergence of parameters is guaranteed. We also obtain partial convergence results in the -node case.

The Hippo pathway is controlled by Angiotensin II signaling and its reactivation induces apoptosis in podocytes.

The Hippo pathway fulfills a crucial function in controlling the balance between proliferation, differentiation and apoptosis in cells. Recent studies showed that G protein-coupled receptors (GPCRs) serve as upstream regulators of Hippo signaling, that either activate or inactivate the Hippo pathway via the large tumor suppressor kinase (LATS) and its substrate, the co-transcription factor Yes-associated protein (YAP). In this study, we focused on the Angiotensin II type 1 receptor (AT1R), which belongs to the GPCR family and has an essential role in the control of blood pressure and water homeostasis. We found that Angiotensin II (Ang II) inactivates the pathway by decreasing the activity of LATS kinase; therefore, leading to an enhanced nuclear shuttling of unphosphorylated YAP in HEK293T cells. This shuttling of YAP is actin-dependent as disruption of the actin cytoskeleton inhibited dephosphorylation of LATS and YAP. Interestingly, in contrast to HEK293T cells, podocytes, which are a crucial component of the glomerular filtration barrier, display a predominant nuclear YAP localization in vivo and in vitro. Moreover, stimulation with Ang II did not alter Hippo pathway activity in podocytes, which show a deactivated pathway. Reactivation of the LATS kinase activity in podocytes resulted in an increased cytoplasmic YAP localization accompanied by a strong induction of apoptosis. Thus, our work indicates that the control of LATS activation and subsequent YAP localization is important for podocyte homeostasis and survival.

The Apaf-1-binding protein Aven is cleaved by Cathepsin D to unleash its anti-apoptotic potential.

The anti-apoptotic molecule Aven was originally identified in a yeast two-hybrid screen for Bcl-x(L)-interacting proteins and has also been found to bind Apaf-1, thereby interfering with Apaf-1 self-association during apoptosome assembly. Aven is expressed in a wide variety of adult tissues and cell lines, and there is increasing evidence that its overexpression correlates with tumorigenesis, particularly in acute leukemias. The mechanism by which the anti-apoptotic activity of Aven is regulated remains poorly understood. Here we shed light on this issue by demonstrating that proteolytic removal of an inhibitory N-terminal Aven domain is necessary to activate the anti-apoptotic potential of the molecule. Furthermore, we identify Cathepsin D (CathD) as the protease responsible for Aven cleavage. On the basis of our results, we propose a model of Aven activation by which its N-terminal inhibitory domain is removed by CathD-mediated proteolysis, thereby unleashing its cytoprotective function.

An automated multidimensional protein identification technology for shotgun proteomics.

We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.

Prephenate dehydratase of the actinomycete Amycolatopsis methanolica: purification and characterization of wild-type and deregulated mutant proteins.

Prephenate dehydratase (PDT) is a key regulatory enzyme in L-phenylalanine biosynthesis in the Gram-positive bacterium Amycolatopsis methanolica. The PDT protein was purified to homogeneity (1957-fold) from wild-type cells with a final yield of 6.5%. It was characterized as a 150 kDa homotetrameric protein with a subunit size of 34 kDa. The first 35 N-terminal amino acids were identified, revealing highest similarity to the PDT proteins from Corynebacterium glutamicum and Bacillus subtilis. Kinetic studies showed that the A. methanolica PDT is allosterically inhibited by phenylalanine and activated by tyrosine. Phenylalanine caused an increase in the S0.5 for prephenate and a decrease in the Vmax. Tyrosine caused a decrease in the S0.5 for prephenate and an increase in the Vmax. Spontaneous o-fluoro- and p-fluoro-DL-phenylalanine-resistant mutants of A. methanolica were isolated. Kinetic studies with the partially purified PDT proteins of strains pFPhe32 and oFPhe84 showed that these mutant proteins had become (partly) insensitive to both phenylalanine inhibition and tyrosine activation.

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants.

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.

[Pt(dien)]2+ migrates intramolecularly from methionine S to imidazole Nepsilon2 in the peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh.

The pH- and time-dependent reaction of [Pt(dien)(H2O)]2+ with the methionine- and histidine-containing peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh at 313 K has been investigated by HPLC and NMR spectroscopy. For both peptides, initial relatively rapid formation of the kinetically favoured methionine S-bound complex is followed by slow intramolecular migration of the [Pt(dien)]2+ fragment to imidazole Nepsilon2 (or, in the case of H-His-Gly-Met-OH, to a much lesser extent to the competing imidazole Ndelta1) of the histidine side chain over a period of 500 h. Time-dependent studies for the pentapeptide at pH 8.0 demonstrate that this isomerization can take place by either direct S-->Nepsilon2 migration or by a two-step mechanism involving initial Nepsilon2 coordination of a second [Pt(dien)]2+ fragment and subsequent cleavage of the orginal Pt-S bond in the resulting dinuclear complex. The rate of kappaS/kappaNepsilon2 isomerization is markedly reduced on lowering the pH to 5.1.