Simulation of Brugada syndrome using cellular and three-dimensional whole-heart modeling approaches

Brugada syndrome (BS) is a genetic disease identified by an abnormal electrocardiogram ( ECG) ( mainly abnormal ECGs associated with right bundle branch block and ST-elevation in right precordial leads). BS can lead to increased risk of sudden cardiac death. Experimental studies on human ventricular myocardium with BS have been limited due to difficulties in obtaining data. Thus, the use of computer simulation is an important alternative. Most previous BS simulations were based on animal heart cell models. However, due to species differences, the use of human heart cell models, especially a model with three-dimensional whole-heart anatomical structure, is needed. In this study, we developed a model of the human ventricular action potential (AP) based on refining the ten Tusscher et al (2004 Am. J. Physiol. Heart Circ. Physiol. 286 H1573 - 89) model to incorporate newly available experimental data of some major ionic currents of human ventricular myocytes. These modified channels include the L-type calcium current (ICaL), fast sodium current (I-Na), transient outward potassium current (I-to), rapidly and slowly delayed rectifier potassium currents (I-Kr and I-Ks) and inward rectifier potassium current (I-Ki). Transmural heterogeneity of APs for epicardial, endocardial and mid-myocardial (M) cells was simulated by varying the maximum conductance of IKs and Ito. The modified AP models were then used to simulate the effects of BS on cellular AP and body surface potentials using a three-dimensional dynamic heart - torso model. Our main findings are as follows. (1) BS has little effect on the AP of endocardial or mid-myocardial cells, but has a large impact on the AP of epicardial cells. (2) A likely region of BS with abnormal cell AP is near the right ventricular outflow track, and the resulting ST-segment elevation is located in the median precordium area. These simulation results are consistent with experimental findings reported in the literature. The model can reproduce a variety of electrophysiological behaviors and provides a good basis for understanding the genesis of abnormal ECG under the condition of BS disease.

Steric stabilization and dissolution characteristics of aqueous suspensions of pharmaceutical interest

The adsorption of two qroups of nonionic surface active agents and a series of hiqh molecular weiqht hydrophilic polymer fractions onto a polystyrene latex and a drug substance diloxanide furoate B.P. has been investigated. The presence of pores within the drug surface has been demonstrated and this is shown to increase the adsorption of low molecular weight polymer species. Differences in the maximum amount of polymer adsorbed at both solid-solution interfaces have been ascribed to the different hydrophobicities of the surface as determined by contact angle measurements. Adsorbed layer thicknesses of polymer on polystyrene latex have been determined by three techniques: microelectrophoresis, intensity fluctuation spectroscopy and by viscometric means. These results, in combination with adsorption data, were used to interpret the configuration of the adsorbed polymer molecules at the interface. The type of druq suspension produced on adsorbing the different polymers in the absence of electrostatic stabilization was correlated with theoretical prediuctions of suspension characteristics deduced from potential energy diagrams, The agreement was good for the adsorption of short chain length surfactants, but for the polyvinylalcohols, discrepancies were found between experiment and theory. This was attributed to the inappropriate use of a mean segment density approximation within the adsorbed layer to calculate attractive potentials between particles. A maximum in the redispersibility values for suspensions coated with adsorbed nonylphenylethoxylates was attributed to "partial static stabilization" of the particles in conjunction with the attractive forces operating in the sediment between bare surface patches on neighbouring particles. No significant change in the dissolution of the drug was observed when nonylphenylethoxylates were adsorbed due to desorption upon contact with the dissolution medium. Pluronic F68 and all the polyvinylalcohol fractions caused a reduction in the dissolution rate which is explained by the decreased diffusion of drug' through the adsorbed polymer layer.

Role of the transgenic human thyrotropin receptor A-subunit in thyroiditis induced by A-subunit immunization and regulatory T cell depletion

Transgenic BALB/c mice that express intrathyroidal human thyroid stimulating hormone receptor (TSHR) A-subunit, unlike wild-type (WT) littermates, develop thyroid lymphocytic infiltration and spreading to other thyroid autoantigens after T regulatory cell (Treg) depletion and immunization with human thyrotropin receptor (hTSHR) adenovirus. To determine if this process involves intramolecular epitope spreading, we studied antibody and T cell recognition of TSHR ectodomain peptides (A–Z). In transgenic and WT mice, regardless of Treg depletion, TSHR antibodies bound predominantly to N-terminal peptide A and much less to a few downstream peptides. After Treg depletion, splenocytes from WT mice responded to peptides C, D and J (all in the A-subunit), but transgenic splenocytes recognized only peptide D. Because CD4+ T cells are critical for thyroid lymphocytic infiltration, amino acid sequences of these peptides were examined for in silico binding to BALB/c major histocompatibility complex class II (IA–d). High affinity subsequences (inhibitory concentration of 50% < 50 nm) are present in peptides C and D (not J) of the hTSHR and mouse TSHR equivalents. These data probably explain why transgenic splenocytes do not recognize peptide J. Mouse TSHR mRNA levels are comparable in transgenic and WT thyroids, but only transgenics have human A-subunit mRNA. Transgenic mice can present mouse TSHR and human A-subunit-derived peptides. However, WT mice can present only mouse TSHR, and two to four amino acid species differences may preclude recognition by CD4+ T cells activated by hTSHR-adenovirus. Overall, thyroid lymphocytic infiltration in the transgenic mice is unrelated to epitopic spreading but involves human A-subunit peptides for recognition by T cells activated using the hTSHR.

Understanding anuran community dynamics in temporary wetlands: The interaction and importance of landscape and biotic processes

The major objective of this study was to determine the relative importance of landscape factors, local abiotic factors, and biotic interactions in influencing tadpole community structure in temporary wetlands. I also examined the influence of agricultural activities in South-central Florida by comparing tadpole communities in native prairie wetlands (a relatively unmodified habitat) at the Kissimmee Prairie Sanctuary (KPS) to tadpole communities in three agriculturally modified habitats found at MacArthur Agro-Ecology Research Center (MAERC). Environmental characteristics were measured in 24 isolated wetlands, and tadpoles were sampled using throw-traps and dipnets during the 1999 wet season (June–October). Landscape characteristics were expected to predominately influence all aspects of community structure because anurans associated with temporary wetland systems are likely to exist as metapopulations. Both landscape characteristics (wetland proximity to nearest woodland and the amount of woodland surrounding the wetland) and biotic interactions (fish predation) had the largest influence on tadpole community structure. Predatory fish influenced tadpole communities more than expected due to the ubiquity of wetlands, lack of topographic relief, and dispersal abilities of several fish species. Differences in tadpole community structure among habitat types were attributed to differences in woodland attributes and susceptibility to fish colonization. Furthermore, agricultural modification of prairie habitats in South-central Florida may benefit amphibian communities, particularly woodland-dwelling species that are unable to coexist with predatory fish. From a conservation standpoint, temporary wetlands proximal to woodland areas and isolated from permanent water sources appear to be most important to amphibians. In addition, the high tadpole densities attained in these wetlands suggest that these wetlands serve as biological hotspots within the landscape, and their benefits extend into the adjacent terrestrial matrix. Further research efforts are needed to quantify the biological productivity of these systems and determine spatial dynamics of anurans in surrounding terrestrial habitats. ^

Responses of neotropical mangrove seedlings grown in monoculture and mixed culture under treatments of hydroperiod and salinity

We investigated the combined effects of salinity and hydroperiod on seedlings of Rhizophora mangle and Laguncularia racemosa grown under experimental conditions of monoculture and mixed culture by using a simulated tidal system. The objective was to test hypotheses relative to species interactions to either tidal or permanent flooding at salinities of 10 or 40 g/l. Four-month-old seedlings were experimentally manipulated under these environmental conditions in two types of species interactions: (1) seedlings of the same species were grown separately in containers from September 2000 to August 2001 to evaluate intraspecific response and (2) seedlings of each species were mixed in containers to evaluate interspecific, competitive responses from August 2002 to April 2003. Overall, L. racemosa was strongly sensitive to treatment combinations while R. mangle showed little effect. Most plant responses of L. racemosa were affected by both salinity and hydroperiod, with hydroperiod inducing more effects than salinity. Compared to R. mangle, L. racemosa in all treatment combinations had higher relative growth rate, leaf area ratio, specific leaf area, stem elongation, total length of branches, net primary production, and stem height. Rhizophora mangle had higher biomass allocation to roots. Species growth differentiation was more pronounced at low salinity, with few species differences at high salinity under permanent flooding. These results suggest that under low to mild stress by hydroperiod and salinity, L. racemosa exhibits responses that favor its competitive dominance over R. mangle. This advantage, however, is strongly reduced as stress from salinity and hydroperiod increase.

Water Uptake Patterns of an Invasive Exotic Plant in Coastal Saline Habitats

Schinus terebinthifolius Raddi (Schinus) is one of the most widely found woody exotic species in South Florida. This exotic is distributed across environments with different hydrologic regimes, from upland pine forests to the edges of sawgrass marshes and into saline mangrove forests. To determine if this invasive exotic had different physiological attributes compared to native species in a coastal habitat, we measured predawn xylem water potentials (Ψ), oxygen stable isotope signatures (δ18O), and sodium (Na+) and potassium (K+) contents of sap water from plants within: (1) a transition zone (between a mangrove forest and upland pineland) and (2) an upland pineland in Southwest Florida. Under dynamic salinity and hydrologic conditions, Ψ of Schinus appeared less subject to fluctuations caused by seasonality when compared with native species. Although stem water δ18O values could not be used to distinguish the depth of Schinus and native species' water uptake in the transition zone, Ψ and sap Na+/K+ patterns showed that Schinus was less of a salt excluder relative to the native upland species during the dry season. This exotic also exhibited Na+/K+ ratios similar to the mangrove species, indicating some salinity tolerance. In the upland pineland, Schinus water uptake patterns were not significantly different from those of native species. Differences between Schinus and native upland species, however, may provide this exotic an advantage over native species within mangrove transition zones.

Isolation and characterization of the U1 and U2 snRNAs in the silk gland of the 5th instar larval stage silk moth Bombyx mori

In isolation and characterization studies, expression level U1 and U2 snRNA isoforms were obtained from the 5th instar larval stage silk gland (SG). The DNA content of the SG cells is approximately 200,000-fold higher compared to the usual (2N) somatic cells of B. mori due to endoreduplication. In this study, the existence of U1 and U2 snRNA isoforms in the SG of the organism is investigated. Bombyx mori U1 and U2-specific RT-PCR libraries from the silk gland were generated. Five U1 and eight U2 isoforms were isolated and characterized. Nucleotide differences, structural alterations, as well as protein and RNA interaction sites were analyzed in these variants. For the U1 snRNA variants, they were compared to the previously reported BmN isoforms. In all these U-snRNA variants, polymorphic sites do not predominate at the core of known functional sequences, which were interspecifically conserved. Variant sites and inter-species differences are located in moderately conserved regions. Free energy (ΔG) values for the entire U1 and U2 snRNA secondary structures and for the individual stem/loops domains of the isoforms were generated and compared to determine their structural stability. This will be the first time that U1 and U2 variants are shown specific for a development stage (larval) other than embryonic or adult. ^ Using phylogenetic analysis, evolutionary trees were generated for the U1 and U2 snRNAs using animal, plant, protista and fungal species. The resulting trees were boostrapped for robustness and rooted with the self-splicing RNA group II intron sequence from the cyanobacterium Calothrix. Using phylogenetic analyses, possible structural and functional evolutionary interdependence between the U1 and U2 snRNAs was investigated. ^

Understanding anuran community dynamics in temporary wetlands: the interaction and importance of landscape and biotic processes

The major objective of this study was to determine the relative importance of landscape factors, local abiotic factors, and biotic interactions in influencing tadpole community structure in temporary wetlands. I also examined the influence of agricultural activities in South-central Florida by comparing tadpole communities in native prairie wetlands (a relatively unmodified habitat) at the Kissimmee Prairie Sanctuary (KPS) to tadpole communities in three agriculturally modified habitats found at MacArthur Agro- Ecology Research Center (MAERC). Environmental characteristics were measured in 24 isolated wetlands, and tadpoles were sampled using throw-traps and dipnets during the 1999 wet season (June - October). Landscape characteristics were expected to predominately influence all aspects of community structure because anurans associated with temporary wetland systems are likely to exist as metapopulations. Both landscape characteristics (wetland proximity to nearest woodland and the amount of woodland surrounding the wetland) and biotic interactions (fish predation) had the largest influence on tadpole community structure. Predatory fish influenced tadpole communities more than expected due to the ubiquity of wetlands, lack of topographic relief, and dispersal abilities of several fish species. Differences in tadpole community structure among habitat types were attributed to differences in woodland attributes and susceptibility to fish colonization. Furthermore, agricultural modification of prairie habitats in South-central Florida may benefit amphibian communities, particularly woodland-dwelling species that are unable to coexist with predatory fish. From a conservation standpoint, temporary wetlands proximal to woodland areas and isolated from permanent water sources appear to be most important to amphibians. In addition, the high tadpole densities attained in these wetlands suggest that these wetlands serve as biological hotspots within the landscape, and their benefits extend into the adjacent terrestrial matrix. Further research efforts are needed to quantify the biological productivity of these systems and determine spatial dynamics of anurans in surrounding terrestrial habitats.

Behavioural profiles of captive capuchin monkeys (Sapajus spp.): analyses at group and individual levels

The use of behavioural indicators of suffering and welfare in captive animals has produced ambiguous results. In comparisons between groups, those in worse condition tend to exhibit increased overall rate of Behaviours Potentially Indicative of Stress (BPIS), but when comparing within groups, individuals differ in their stress coping strategies. This dissertation presents analyses to unravel the Behavioural Profile of a sample of 26 captive capuchin monkeys, of three different species (Sapajus libidinosus, S. flavius and S. xanthosternos), kept in different enclosure types. In total, 147,17 hours of data were collected. We explored four type of analysis: Activity Budgets, Diversity indexes, Markov chains and Sequence analyses, and Social Network Analyses, resulting in nine indexes of behavioural occurrence and organization. In chapter One we explore group differences. Results support predictions of minor sex and species differences and major differences in behavioural profile due to enclosure type: i. individuals in less enriched enclosures exhibited a more diverse BPIS repertoire and a decreased probability of a sequence with six Genus Normative Behaviour; ii. number of most probable behavioural transitions including at least one BPIS was higher in less enriched enclosures; iii. proeminence indexes indicate that BPIS function as dead ends of behavioural sequences, and proeminence of three BPIS (pacing, self-direct, active I) were higher in less enriched enclosures. Overall, these data are not supportive of BPIS as a repetitive pattern, with a mantra-like calming effect. Rather, the picture that emerges is more supportive of BPIS as activities that disrupt organization of behaviours, introducing “noise” that compromises optimal activity budget. In chapter Two we explored individual differences in stress coping strategies. We classified individuals along six axes of exploratory behaviour. These were only weakly correlated indicating low correlation among behavioural indicators of syndromes. Nevertheless, the results are suggestive of two broad stress coping strategies, similar to the bold/proactive and shy/reactive pattern: more exploratory capuchin monkeys exhibited increased values of proeminence in Pacing, aberrant sexual display and Active 1 BPIS, while less active animals exhibited increased probability in significant sequences involving at least one BPIS, and increased prominence in own stereotypy. Capuchin monkeys are known for their cognitive capacities and behavioural flexibility, therefore, the search for a consistent set of behavioural indictors of welfare and individual differences requires further studies and larger data sets. With this work we aim contributing to design scientifically grounded and statistically correct protocols for collection of behavioural data that permits comparability of results and meta-analyses, from whatever theoretical perspective interpretation it may receive.

Social Decision-Making in Bonobos and Chimpanzees

Humans are natural politicians. We obsessively collect social information that is both observable (e.g., about third-party relationships) and unobservable (e.g., about others’ psychological states), and we strategically employ that information to manage our cooperative and competitive relationships. To what extent are these abilities unique to our species, and how did they evolve? The present dissertation seeks to contribute to these two questions. To do so, I take a comparative perspective, investigating social decision-making in humans’ closest living relatives, bonobos and chimpanzees. In Chapter 1, I review existing literature on theory of mind—or the ability to understand others’ psychological states—in these species. I also present a theoretical framework to guide further investigation of social cognition in bonobos and chimpanzees based on hypotheses about the proximate and ultimate origins of their species differences. In Chapter 2, I experimentally investigate differences in the prosocial behavior of bonobos and chimpanzees, revealing species-specific prosocial motivations that appear to be less flexible than those exhibited by humans. In Chapter 3, I explore through decision-making experiments bonobos’ ability to evaluate others based on their prosocial or antisocial behavior during third-party interactions. Bonobos do track the interactions of third-parties and evaluate actors based on these interactions. However, they do not exhibit the human preference for those who are prosocial towards others, instead consistently favoring an antisocial individual. The motivation to prefer those who demonstrate a prosocial disposition may be a unique feature of human psychology that contributes to our ultra-cooperative nature. In Chapter 4, I investigate the adaptive value of social cognition in wild primates. I show that the recruitment behavior of wild chimpanzees at Gombe National Park, Tanzania is consistent with the use of third-party knowledge, and that those who appear to use third-party knowledge receive immediate proximate benefits. They escape further aggression from their opponents. These findings directly support the social intelligence hypothesis that social cognition has evolved in response to the demands of competing with one’s own group-mates. Thus, the studies presented here help to better characterize the features of social decision-making that are unique to humans, and how these abilities evolved.

Stable isotope record of planktonic foraminifera of sediment core TR163-19

Stable isotope data from eastern equatorial Pacific (EEP) core TR163-19 (2°15'N, 90°57'W, 2348 m) are presented for the surface-dwelling foraminifers Globigerinoides ruber and G. sacculifer and thermocline-dwelling Globorotalia menardii and Neogloboquadrina dutertrei. Using species-specific normalization factors derived from experimental and plankton tow data, we reconstruct a 360 kyr record of water column hydrography across the past three glacial cycles. We demonstrate that G. ruber maintains a mixed layer habitat throughout the entire record, while G. sacculifer records a mixture of thermocline and mixed layer conditions and G. menardii and N. dutertrei record thermocline properties. We conclude that G. sacculifer is not appropriate for paleoceanographic applications in regions with steep vertical hydrographic gradients. Results suggest that this region of the EEP had a thicker mixed layer and deeper d13CDIC boundary between the surface and equatorial undercurrent during the last two glacial periods. A shift in N. dutertrei and G. sacculifer geochemistry prior to ~185 kyr suggests water column structure and chemocline gradients changed, possibly due to a shift in the position of the undercurrent relative to this site. The timing and magnitude of glacial-interglacial d13C variations between species indicates that near-surface carbon chemistry is controlled by changes in productivity, atmospheric circulation, and advected intermediate water sources north of the Antarctic polar front. These results demonstrate that when properly calibrated for species differences, multispecies geochemical data sets can be invaluable for reconstructing water column structure and properties in the past.

A study of the electrical basis for the inhibition and excitation in autonomically innervated smooth muscle

The aim of this thesis was to investigate the electrical and mechanical responses to inhibitory non-adrenergic noncholinergic (NANC) nerve stimulation in the bovine retractor penis muscle (BRP) and compare them with those to an inhibitory extract made from this muscle. The extract may contain the NANC inhibitory transmitter of the BRP and possibly of other smooth muscles. Because of species differences in the electrical response to NANC nerves in the rat and rabbit anococcygeus the effects of the extract on these tissues was also investigated. Prior to the investigation of the extract, both the excitatory and inhibitory responses to field stimulation in the BRP, and the effects of passive membrane potential displacement were studied using conventional intra- or extracellular (sucrose gap) recording techniques. The majority of cells in the BRP were electrically quiescent independent of the resting tone. The most frequent (in approximately 25% of preparations) form of spontaneous activity, oscillations in membrane potential and tone, may represent a pacemaker activity. The BRP had cable properties; the time constant and space constant indicated a high membrane resistance. In the absence of tone, field stimulation of the BRP evoked excitatory junction potentials (ejps) in every cell impaled and contractions, graded with the strength, frequency and number of pulses; spikes were not observed. Guanethidine (1-3 x 10-5M) abolished the ejps and contractions, confirming their adrenergic origin. Noradrenaline added exogenously depolarised and contracted the muscle. These effects were blocked by the a-adrenoceptor antagonists, phentolamine and prazosin. However, phentolamine (2.5x 10-6M) inhibited the contraction without reducing the ejp significantly. These effects may be independent of adrenoceptor blockade or the ejp may be mediated by a substance other than noradrenaline (e.g. ATP) released from adrenergic nerves. Prazosin (1.4 x lO-6M) failed to block either the ejp or contraction, indicating the possible existence of two types of adrenoceptor in the BRP; one activated by neuronally-released and the other by exogenously-added noradrenaline. ATP, a contaminant in the extract, also depolarised and contracted the BRP. Physostigmine reduced whilst atropine enhanced the ejps and contractions without similarly affecting the response to exogenous noradrenaline. This confirmed the presence of a cholinergic inhibitory innervation acting on the excitatory adrenergic fibres (Klinge and Sjostrand, 1977). TEA (1 x lO-4M) enhanced the ejp and contraction. Higher concentrations (0.5 to 10 x 10-3M) depolarised, increased the tone and evoked electrical and mechanical oscillations but no spikes. The depolarisation and contraction to exogenous noradrenaline were not enhanced, indicating that TEA acts on the adrenergic nerves. Some post-synaptic effect to block K+ channels also seems likely. The relationship between ejp amplitude and membrane potential in the double sucrose gap was linear and indicated a reversal potential more positive than -30mV. Electrotonic pulse amplitude decreased during the ejp, indicating an increased membrane conductance. Ejps and contractions were reduced following the replacement of the NaCl of the Krebs solution with sodium glutamate. This may be due to the effects of glutamate itself (e.g. Ca2+ chelation) rather than reduction in the membrane Cl- gradient. Tone usually developed spontaneously and was accompanied by membrane depolarisation (from -53 to -45mV) which may open voltage-dependent channels, causing Ca2+ entry and/or its release from intracellular binding sites. Field stimulation produced inhibitory potentials (ijps) and relaxations graded with the strength and number of pulses but showing little frequency dependence. Rebound depolarisation and contraction often followed the ijp and relaxation. Tetrodotoxin (3 x IO-6M), but not adrenergic or cholinergic antagonists, abolished the ijp and relaxation, confirming their non-adrenergic non-cholinergic neurogenic nature. The extract, prepared and acid-activated as described by Gillespie, Hunter and Martin (1981), hyperpolarised and relaxed the BRP, as did sodium nitroprusside and adenosine triphosphate (ATP). Unlike the activated extract or sodium nitroprusside, desensitisation to ATP occurred rapidly and without any change in the inhibitory electrical or mechanical responses to field stimulation. The ijp and relaxation in the BRP were insensitive to apamin but abolished by oxyhaemoglobin (4-8 x 10-6M), as were the responses to extract and sodium nitroprusside. In TEA (10-2M), field stimulation evoked relaxations with no accompanying electrical change. The ijp may be unconnected with or additional to another mechanism producing relaxation. The relationship between membrane potential and ijp in the BRP was non-linear. Ijp amplitude was initially increased during membrane potential displacement from -45mV to approximately -60mV. Thereafter (-60 to -l03mV) the ijp was reduced. Ijps were abolished at -27 and -103mV; reversal was not observed. The hyperpolarisation to extract was also enhanced during passive displacement of the membrane potential to more negative values (-57mV). Membrane resistance increased during the ijp. The extract produced inconsistent changes in membrane resistance, possibly because of the presence of more than one active component. K+ withdrawal failed to enhance the ijp or hyperpolarisation to extract and 20mM K+ did not abolish the the ijp at membrane potentials exceeding EK (-49mV). Thus, the ijp or hyperpolarisation to extract are unlikely to be mediated by an increased K+ conductance. Reducing the Cl- abolished the hyperpolarisation to field stimulation and extract. This occurred more quickly than the anticipated reduction in the Cl- gradient and may be due to Ca2+ chelation by the anion substitute (glutamate or benzenesulphonate) or blockade of the resting conductance which is normally inactivated by the transmitter. Ouabain (1-5x 10-5M), which reduces both the Na+ and Cl- gradients, abolished the ijp, implicating either of these ions as the ionic species involved. In the rat and rabbit anococcygeus, field stimulation and extract each reduced guanethidine-induced tone. This was unaccompanied in the majority of cells in the rat by any significant electrical response. In the remaining cells, inhibition of the membrane potential oscillations occurred. The rabbit anococcygeus differed in that inhibition of the electrical oscillations was observed in every cell exhibiting this behaviour. However, the majority of cells in the rabbit were electrically quiescent and showed only small hyperpolarisations to field stimulation and no electrical response to extract. Apamin (1 x 10-7M) failed to block the electrical and mechanical response to field stimulation in the rabbit but did inhibit transiently that to extract. The latter effect may be due to the initial excitatory effects of apamin. The similarities between the electrical effects of the extract and those of inhibitory nerve stimulation in the BRP, rat and rabbit anococcygeus muscles are generally consistent with their being mediated by the same active component. Moreover, the ijp in the BRP shows properties which have not been reported in other non-adrenergic noncholinergically innervated smooth muscles.

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD+ from NADH. The production of NADH stimulated by D-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K-m) values determined for D-glyceraldehyde-3-phosphate and NAD(+) were K-m = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.

Analysis of the Nicotiana tabacum Stigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species

The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process.

Morphometric differences in two calanoid sibling species, Boeckella gracilipes and B. titicacae (Crustacea, Copepoda)

Calanoid copepods are abundant in South American inland waters and include widespread species, such as Boeckella gracilipes (Daday, 1902), which occurs from the Ecuador to Tierra del Fuego Island. This species occurs under various environmental conditions, and is found in oligotrophic lakes in Patagonia (39-54°S) and in shallow mountain lakes north of 39°S. The aim of the present study is to conduct a morphometric comparison of male specimens of B. titicacae collected in Titicaca and B. gracilipes collected in Riñihue lakes, with a third population of B. gracilipes collected in shallow ponds in Salar de Surire. Titicaca and Riñihue lakes are stable environments, whereas Salar de Surire is an extreme environment. These ponds present an extreme environment due to high exposure to solar radiation and high salinity levels. The results of the study revealed differences among the three populations. These results agree well with systematic descriptions in the literature on differences between the populations of Titicaca and Riñihue lakes, and population of Salar de Surire differs slightly from the other two populations. It is probable that the differences between the population of Salar de Surire and the other two populations result from the extreme environment in Salar de Surire. High exposure to solar radiation, high salinity and extreme variations in temperature enhance genetic variations that are consequently expressed in morphology.