Multifunctional activities of green tea catechins in neuroprotection. Modulation of cell survival genes, iron-dependent oxidative stress and PKC signaling pathway

Many lines of evidence suggest that oxidative stress resulting in reactive oxygen species (ROS) generation and inflammation play a pivotal role in the age-associated cognitive decline and neuronal loss in neurodegenerative diseases including Alzheimer's (AD), Parkinson's (PD) and Huntington's diseases. One cardinal chemical pathology observed in these disorders is the accumulation of iron at sites where the neurons die. The buildup of an iron gradient in conjunction with ROS (superoxide, hydroxyl radical and nitric oxide) are thought to constitute a major trigger in neuronal toxicity and demise in all these diseases. Thus, promising future treatment of neurodegenerative diseases and aging depends on availability of effective brain permeable, iron-chelatable/radical scavenger neuroprotective drugs that would prevent the progression of neurodegeneration. Tea flavonoids (catechins) have been reported to possess potent iron-chelating, radical-scavenging and anti-inflammatory activities and to protect neuronal death in a wide array of cellular and animal models of neurological diseases. Recent studies have indicated that in addition to the known antioxidant activity of catechins, other mechanisms such as modulation of signal transduction pathways, cell survival/death genes and mitochondrial function, contribute significantly to the induction of cell viability. This review will focus on the multifunctional properties of green tea and its major component (-)-epigallocatechin-3-gallate (EGCG) and their ability to induce neuroprotection and neurorescue in vitro and in vivo. In particular, their transitional metal (iron and copper) chelating property and inhibition of oxidative stress.     

Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

Prefractionations of proteins prior to their proteolysis, chromatography, and MS/MS analyses help reduce complexity and increase the yield of protein identifications. A number of methods were evaluated here for prefractionating serum samples distributed to the participating laboratories as part of the human Plasma Proteome Project. These methods include strong cation exchange (SCX) chromatography, slicing of SDS-PAGE gel bands, and liquid-phase IEF of the proteins. The fractionated proteins were trypsinized and the resulting peptides were resolved and analyzed by multidimensional protein identification technology coupled to IT MS/MS. The MS/MS spectra were clustered, combined, and searched against the IPI protein databank using Pep-Miner. The identification results were evaluated for the efficacy of the different prefractionation methodologies to identify larger numbers of proteins at higher confidence and to achieve the best coverage of the proteins with the identified peptides. Prefractionation based on SCX resulted in the largest number of identified proteins, followed by gel slices and then the liquid-phase IEF. An important observation was that each of the methods revealed a set of unique proteins, some identified with high confidence. Therefore, for comprehensive identification of the serum proteins, several different prefractionation approaches should be used in parallel.     

In vivo debonding strength and enamel damage in two orthodontic debonding methods

Bracket debonding strength related to diverse debonding methods and enamel damage has not been assessed in vivo. The study hypothetized a direct relationship between these three parameters. Debonding strength was measured clinically in the wings method and base method on 50 patients in a split mouth method using a calibrated debonding plier. Brackets from 30 of these patients were scanned in SEM and EDAX for adhesive remnant index and enamel calcium remnants. Base method debonding force was significantly greater than wings method (163.5+/-68.7 N, 106.1+/-66.2 N, respectively, p < 0.001). A positive adhesive remnant index score was found in both methods (68.7%, 66.7%, respectively). Debonding strength vs. adhesive remnant index or calcium index scores were not correlated. However, the latter two were significantly correlated (0.524 < R < 0.895, p < 0.031). Half of the debonding failures developed at the adhesive enamel interface. The results warnts the potential of enamel damage during debonding.     

The tumor suppressor protein p16(INK4a) and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system. Direct evidence for ubiquitination at the N-terminal residue

Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16(INK4a). For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16(INK4a) is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes.     

Body size of insular carnivores: little support for the island rule

Large mammals are thought to evolve to be smaller on islands, whereas small mammals grow larger. A negative correlation between relative size of island individuals and body mass is termed the "island rule." Several mechanisms--mainly competitive release, resource limitation, dispersal ability, and lighter predation pressure on islands, as well as a general physiological advantage of modal size--have been advanced to explain this pattern. We measured skulls and teeth of terrestrial members of the order Carnivora in order to analyze patterns of body size evolution between insular populations and their near mainland conspecifics. No correlations were found between the size ratios of insular/mainland carnivore species and body mass. Only little support for the island rule is found when individual populations rather than species are considered. Our data are at odds with those advanced in support of theories of optimal body size. Carnivore size is subjected to a host of selective pressures that do not vary uniformly from place to place. Mass alone cannot account for the patterns in body size of insular carnivores.     

The scaffolding A/Tpd3 subunit and high phosphatase activity are dispensable for Cdc55 function in the Saccharomyces cerevisiae spindle checkpoint and in cytokinesis

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional enzyme whose trimeric form consists of a scaffolding A subunit, a catalytic C subunit, and one of several regulatory B subunits (B, B', and B'). The adenovirus E4orf4 protein associates with PP2A by directly binding the B or B' subunits. An interaction with an active PP2A containing the B subunit, or its homologue in yeast, Cdc55, is required for E4orf4-induced apoptosis in mammalian cells and for induction of growth arrest in Saccharomyces cerevisiae. In this work, Cdc55 was randomly mutagenized by low-fidelity PCR amplification, and Cdc55 mutants that lost the ability to transduce the E4orf4 toxic signal in yeast were selected. The mutations obtained by this protocol inhibited the association of Cdc55 with E4orf4, or with the PP2A-AC subunits, or both. Functional analysis revealed that a mutant that does not bind Tpd3, the yeast A subunit, as well as wild type Cdc55 in a tpd3Delta background, can form a heterodimer with the catalytic subunit. This association requires C subunit carboxyl methylation. The residual phosphatase activity associated with Cdc55 in the absence of Tpd3 is sufficient to maintain a partially active spindle checkpoint and to prevent cytokinesis defects.