Event-related brain potentials in the study of visual selective attention

Event-related brain potentials (ERPs) provide high-resolution measures of the time course of neuronal activity patterns associated with perceptual and cognitive processes. New techniques for ERP source analysis and comparisons with data from blood-flow neuroimaging studies enable improved localization of cortical activity during visual selective attention. ERP modulations during spatial attention point toward a mechanism of gain control over information flow in extrastriate visual cortical pathways, starting about 80 ms after stimulus onset. Paying attention to nonspatial features such as color, motion, or shape is manifested by qualitatively different ERP patterns in multiple cortical areas that begin with latencies of 100–150 ms. The processing of nonspatial features seems to be contingent upon the prior selection of location, consistent with early selection theories of attention and with the hypothesis that spatial attention is “special.”

Somatic mosaicism in Wiskott–Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism

Somatic mosaicism caused by in vivo reversion of inherited mutations has been described in several human genetic disorders. Back mutations resulting in restoration of wild-type sequences and second-site mutations leading to compensatory changes have been shown in mosaic individuals. In most cases, however, the precise genetic mechanisms underlying the reversion events have remained unclear, except for the few instances where crossing over or gene conversion have been demonstrated. Here, we report a patient affected with Wiskott–Aldrich syndrome (WAS) caused by a 6-bp insertion (ACGAGG) in the WAS protein gene, which abrogates protein expression. Somatic mosaicism was documented in this patient whose majority of T lymphocytes expressed nearly normal levels of WAS protein. These lymphocytes were found to lack the deleterious mutation and showed a selective growth advantage in vivo. Analysis of the sequence surrounding the mutation site showed that the 6-bp insertion followed a tandem repeat of the same six nucleotides. These findings strongly suggest that DNA polymerase slippage was the cause of the original germ-line insertion mutation in this family and that the same mechanism was responsible for its deletion in one of the propositus T cell progenitors, thus leading to reversion mosaicism.

Selective constraints on the activation domain of transcription factor Pit-1.

The POU transcription factor Pit-1 activates members of the prolactin/growth hormone gene family in specific endocrine cell types of the pituitary gland. Although Pit-1 is structurally conserved among vertebrate species, evolutionary changes in the pattern of Pit-1 RNA splicing have led to a notable "contraction" of the transactivation domain in the mammalian lineage, relative to Pit-1 in salmonid fish. By site-directed mutagenesis we demonstrate that two splice insertions in salmon Pit-1, called beta (29 aa) and gamma (33 aa), are critical for cooperative activation of the salmon prolactin gene. Paradoxically, Pit-1-dependent activation of the prolactin gene in rat is enhanced in the absence of the homologous beta-insert sequence. This apparent divergence in the mechanism of activation of prolactin genes by Pit-1 is target gene specific, as activation of rat and salmon growth hormone genes by Pit-1 splice variants is entirely conserved. Our data suggest that efficient activation of the prolactin gene in the vertebrate pituitary has significantly constrained the pattern of splicing within the Pit-1 transactivation domain. Rapid evolutionary divergence of prolactin gene function may have demanded changes in Pit-1/protein interactions to accommodate new patterns of transcriptional control by developmental or physiological factors.

4-Hydroxylation of estradiol by human uterine myometrium and myoma microsomes: implications for the mechanism of uterine tumorigenesis.

Estradiol is converted to catechol estrogens via 2- and 4-hydroxylation by cytochrome P450 enzymes. 4-Hydroxyestradiol elicits biological activities distinct from estradiol, most notably an oxidant stress response induced by free radicals generated by metabolic redox cycling reactions. In this study, we have examined 2- and 4-hydroxylation of estradiol by microsomes of human uterine myometrium and of associated myomata. In all eight cases studied, estradiol 4-hydroxylation by myoma has been substantially elevated relative to surrounding myometrial tissue (minimum, 2-fold; mean, 5-fold). Estradiol 2-hydroxylation in myomata occurs at much lower rates than 4-hydroxylation (ratio of 4-hydroxyestradiol/2-hydroxyestradiol, 7.9 +/- 1.4) and does not significantly differ from rates in surrounding myometrial tissue. Rates of myometrial 2-hydroxylation of estradiol were also not significantly different from values in patients without myomata. We have used various inhibitors to establish that 4-hydroxylation is catalyzed by a completely different cytochrome P450 than 2-hydroxylation. In myoma, alpha-naphthoflavone and a set of ethynyl polycyclic hydrocarbon inhibitors (5 microM) each inhibited 4-hydroxylation more efficiently (up to 90%) than 2-hydroxylation (up to 40%), indicating > 10-fold differences in Ki (<0.5 microM vs. > 5 microM). These activities were clearly distinguished from the selective 2-hydroxylation of estradiol in placenta by aromatase reported previously (low Km, inhibition by Fadrozole hydrochloride or ICI D1033). 4-Hydroxylation was also selectively inhibited relative to 2-hydroxylation by antibodies raised against cytochrome P450 IB1 (rat) (53 vs. 17%). These data indicate that specific 4-hydroxylation of estradiol in human uterine tissues is catalyzed by a form(s) of cytochrome P450 related to P450 IB1, which contribute(s) little to 2-hydroxylation. This enzyme(s) is therefore a marker for uterine myomata and may play a role in the etiology of the tumor.

Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates.

Although the gene defect responsible for Huntington disease (HD) has recently been identified, the pathogenesis of the disease remains obscure. One potential mechanism is that the gene defect may lead to an impairment of energy metabolism followed by slow excitotoxic neuronal injury. In the present study we examined whether chronic administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, can replicate the neuropathologic and clinical features of HD in nonhuman primates. After 3-6 weeks of 3-NP administration, apomorphine treatment induced a significant increase in motor activity as compared with saline-treated controls. Animals showed both choreiform movements, as well as foot and limb dystonia, which are characteristic of HD. More prolonged 3-NP treatment in two additional primates resulted in spontaneous dystonia and dyskinesia accompanied by lesions in the caudate and putamen seen by magnetic resonance imaging. Histologic evaluation showed that there was a depletion of calbindin neurons, astrogliosis, sparing of NADPH-diaphorase neurons, and growth-related proliferative changes in dendrites of spiny neurons similar to changes in HD. The striosomal organization of the striatum and the nucleus accumbens were spared. These findings show that chronic administration of 3-NP to nonhuman primates can replicate many of the characteristic motor and histologic features of HD, further strengthening the possibility that a subtle impairment of energy metabolism may play a role in its pathogenesis.

Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.

An evaluation of potential mechanism-based inactivation of human drug metabolizing cytochromes P450 by monoamine oxidase inhibitors, including isoniazid

To characterize potential mechanism-based inactivation (MBI) of major human drug-metabolizing cytochromes P450 (CYP) by monoamine oxidase (MAO) inhibitors, including the antitubercular drug isoniazid. Human liver microsomal CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities were investigated following co- and preincubation with MAO inhibitors. Inactivation kinetic constants (K-I and k(inact)) were determined where a significant preincubation effect was observed. Spectral studies were conducted to elucidate the mechanisms of inactivation. Hydrazine MAO inhibitors generally exhibited greater inhibition of CYP following preincubation, whereas this was less frequent for the propargylamines, and tranylcypromine and moclobemide. Phenelzine and isoniazid inactivated all CYP but were most potent toward CYP3A and CYP2C19. Respective inactivation kinetic constants (K-I and k(inact)) for isoniazid were 48.6 mu M and 0.042 min(-1) and 79.3 mu M and 0.039 min(-1). Clorgyline was a selective inactivator of CYP1A2 (6.8 mu M and 0.15 min(-1)). Inactivation of CYP was irreversible, consistent with metabolite-intermediate complexation for isoniazid and clorgyline, and haeme destruction for phenelzine. With the exception of phenelzine-mediated CYP3A inactivation, glutathione and superoxide dismutase failed to protect CYP from inactivation by isoniazid and phenelzine. Glutathione partially slowed (17%) the inactivation of CYP1A2 by clorgyline. Alternate substrates or inhibitors generally protected against CYP inactivation. These data are consistent with mechanism-based inactivation of human drug-metabolizing CYP enzymes and suggest that impaired metabolic clearance may contribute to clinical drug-drug interactions with some MAO inhibitors.

A nonantisense sequence-selective effect of a phosphorothioate oligodeoxynucleotide directed against the epidermal growth factor receptor in A431 cells

The overexpression of epidermal growth factor receptor (EGFr) has been implicated as a causative factor and a poor prognostic marker in a number of carcinomas. Therefore, strategies that down-regulate EGFr expression may be therapeutically useful. We designed antisense ODNs complementary to the initiation codon region of the EGFr mRNA and evaluated their efficacy in several tumor-derived cells, including the A431 cell line that express amplified levels of EGFr. A 15-mer phosphorothioate (PS) antisense ODN (erbB1AS15) induced a concentration-dependent reduction in proliferation that was accompanied by a change in the morphology of A431 cells into more tightly clustered and discrete colonies. A 15-mer sense (PS) control oligodeoxynucleotide (ODN) and a phosphodiester (PO) version of erbB1AS15 had little or no effect on cell number of morphology, and erbB1AS15 (PS) did not induce these effects in control cell lines expressing lower levels of EGFr. The effects of erbB1AS15 (PS) on A431 cells were not mediated by a true antisense mechanism in that there was no reduction in the level of EGFr mRNA or protein over a 24-hr period, as determined by Northern and Western blotting, respectively. However, autophosphorylation of the receptor was significantly reduced by erbB1AS15 (PS) and not by control ODNs. The results of further studies suggested that this effect was mediated by a direct, dose-dependent inhibition of the EGFr tyrosine kinase enzyme and was not due to impairment of either ligand-binding or receptor dimerization. These data suggest that erbB1AS15 (PS) can inhibit proliferation and alter the morphology of A431 cells by a sequence-selective, but nonantisense mechanism affecting receptor tyrosine kinase activity.

Mechanism of attenuation of skeletal muscle protein catabolism in cancer cachexia by eicosapentaenoic acid

Cancer cachexia is characterized by selective depletion of skeletal muscle protein reserves. Soleus muscles from mice bearing a cachexia-inducing tumor (MAC16) showed an increased protein degradation in vitro, as measured by tyrosine release, when compared with muscles from nontumor-bearing animals. After incubation under conditions that modify different proteolytic systems, lysosomal, calcium-dependent, and ATP-dependent proteolysis were found to contribute to the elevated protein catabolism. Treatment of mice bearing the MAC16 tumor with the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), attenuated loss of body weight and significantly suppressed protein catabolism in soleus muscles through an inhibition of an ATP-dependent proteolytic pathway. The ATP-ubiquitin-dependent proteolytic pathway is considered to play a major role in muscle catabolism in cachexia, and functional proteasome activity, as determined by “chymotrypsin-like” enzyme activity, was significantly elevated in gastrocnemius muscle of mice bearing the MAC16 tumor as weight loss progressed. When animals bearing the MAC16 tumor were treated with EPA, functional proteasome activity was completely suppressed, together with attenuation of the expression of 20S proteasome a-subunits and the p42 regulator, whereas there was no effect on the expression of the ubiquitin-conjugating enzyme (E214k). These results suggest that EPA induces an attenuation of the up-regulation of proteasome expression in cachectic mice, and this was correlated with an increase in myosin expression, confirming retention of contractile proteins. EPA also inhibited growth of the MAC16 tumor in a dose-dependent manner, and this correlated with suppression of the expression of the 20S proteasome a-subunits in tumor cells, suggesting that this may be the mechanism of tumor growth inhibition. Thus EPA antagonizes loss of skeletal muscle proteins in cancer cachexia by down-regulation of proteasome expression, and this may also be the mechanism for inhibition of tumor growth.

Mechanism of protein catobolism in skeletal muscle during cancer cachexia

Cancer cachexia is characterised by selective depletion of skeletal muscle protein reserves. The ubiquitin-proteasome proteolytic pathway has been shown to be responsible for muscle wasting in a range of cachectic conditions including cancer cachexia. To establish the importance of this pathway in muscle wasting during cancer (and sepsis), a quantitative competitive RT-PCR (QcRT-PCR) method was developed to measure the mRNA levels of the proteasome sub units C2a and C5ß and the ubiquitin-conjugating enzyme E214k. Western blotting was also used to measure the 20S proteasome and E214k protein expression. In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma (MAC16) demonstrated the effect of progressive weight loss on the mRNA and protein expression for 20S proteasome subunits, as well as the ubiquitin-conjugating enzyme, E214k, in gastrocnemius and pectoral muscles. QcRT-PCR measurements showed a good correlation between expression of the proteasome subunits (C2 and CS) and the E214k enzyme mRNA and weight loss in gastrocnemius muscle, where expression increased with increasing weight loss followed by a decrease in expression at higher weight losses (25-27%). Similar results were obtained in pectoral muscles, but with the expression being several fold lower in comparison to that in gastrocnemius muscle, reflecting the different degrees of protein degradation in the two muscles during the process of cancer cachexia. Western blot analysis of 20S and E214k protein expression followed a similar pattern with respect to weight loss as that found with mRNA. In addition, mRNA and protein expression of the 20S proteasome subunits and E214k enzyme was measured in biopsies from cachectic cancer patients, which also showed a good correlation between weight loss and proteasome expression, demonstrating a progressive increase in expression of the proteasome subunits and E214k mRNA and protein in cachectic patients with progressively increasing weight loss.The effect of the cachexia-inducing tumour product PIF (proteolysis inducing factor) and 15-hydroxyeicosatetraenoic acid (15-HETE), the arachidoinic acid metabolite (thought to be the intracellular transducer of PIF action) has also been determined. Using a surrogate model system for skeletal muscle, C2C12 myotubes in vitro, it was shown that both PIF and 15-HETE increased proteasome subunit expression (C2a and C5ß) as well as the E214k enzyme. This increase gene expression was attenuated by preincubation with EPA or the 15-lipoxygenase inhibitor CV-6504; immunoblotting also confirmed these findings. Similarly, in sepsis-induced cachexia in NMRI mice there was increased mRNA and protein expression of the 20S proteasome subunits and the E214k enzyme, which was inhibited by EPA treatment. These results suggest that 15-HETE is the intracellular mediator for PIF induced protein degradation in skeletal muscle, and that elevated muscle catabolism is accomplished through upregulation of the ubiquitin-proteasome-proteolytic pathway. Furthermore, both EPA and CV -6504 have shown anti-cachectic properties, which could be used in the future for the treatment of cancer cachexia and other similar catabolic conditions.

Rapid aquaporin translocation regulates cellular water flow:the mechanism of hypotonicity-induced sub-cellular localization of the aquaporin 1 water channel

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The family's structural features and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this has only been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations, through transient receptor potential channels, that trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly-changing local cellular water availability. Moreover, since calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.

The Selective Advantages of Anthocyanins in Developing Leaves of Mango and Cacao

Althought he developingl eaves of mango and cacao containa ppreciablec oncentrationosf anthocyaninns ot presenti n mature leaves,t hesec ompoundsa re a smallp roportiono f the totalp henolicc oncentrationF.o r thisa nd otherr easonsa nthocyanindso not seem to be importantin the developingl eaves as: (1) a screena gainstu ltravioletr adiation;( 2) a mechanismf ore levatingl eaf temperature(;3 ) a means of defenseo r aposematicc olorationa gainsth erbivorya;n d (4) a part of any postulatedp hysiological mechanism,a s photosynthesiAs.n thocyanico lorationm ay be a by-producto f the metabolismo f otherf lavonoidc ompoundsi n theser apidlyg rowingo rgans.

Nuclear magnetic resonance spectroscopic and computational investigations of chloroperoxidase catalyzed regio- and enantio-selective transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. ^ To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. ^ To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O 2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.^

Selective Preparation of Semiconducting Single-Walled Carbon Nanotubes: From Fundamentals to Applications

Aligned single-walled carbon nanotubes (SWNTs) synthesized by the chemical vapor deposition (CVD) method have exceptional potential for next-generation nanoelectronics. However, there are considerable challenges in the preparation of semiconducting (s-) SWNTs with controlled properties (e.g., density, selectivity, and diameter) for their application in solving real-world problems. This dissertation describes research that aims to overcome the limitations by novel synthesis strategies and post-growth treatment. The application of as-prepared SWNTs as functional devices is also demonstrated. The dissertation includes the following parts: 1) decoupling the conflict between density and selectivity of s-SWNTs in CVD growth; 2) investigating the importance of diameter control for the selective synthesis of s-SWNTs; 3) synthesizing highly conductive SWNT thin film by thiophene-assisted CVD method; 4) eliminating metallic pathways in SWNT crossbars by gate-free electrical breakdown method; 5) enhancing the density of SWNT arrays by strain-release method; 6) studying the sensing mechanism of SWNT crossbar chemical sensors.

Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns.

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.