Changes in brain cell shape create residual extracellular space volume and explain tortuosity behavior during osmotic challenge

Diffusion of molecules in brain extracellular space is constrained by two macroscopic parameters, tortuosity factor λ and volume fraction α. Recent studies in brain slices show that when osmolarity is reduced, λ increases while α decreases. In contrast, with increased osmolarity, α increases, but λ attains a plateau. Using homogenization theory and a variety of lattice models, we found that the plateau behavior of λ can be explained if the shape of brain cells changes nonuniformly during the shrinking or swelling induced by osmotic challenge. The nonuniform cellular shrinkage creates residual extracellular space that temporarily traps diffusing molecules, thus impeding the macroscopic diffusion. The paper also discusses the definition of tortuosity and its independence of the measurement frame of reference.

The biotic crisis and the future of evolution

The biotic crisis overtaking our planet is likely to precipitate a major extinction of species. That much is well known. Not so well known but probably more significant in the long term is that the crisis will surely disrupt and deplete certain basic processes of evolution, with consequences likely to persist for millions of years. Distinctive features of future evolution could include a homogenization of biotas, a proliferation of opportunistic species, a pest-and-weed ecology, an outburst of speciation among taxa that prosper in human-dominated ecosystems, a decline of biodisparity, an end to the speciation of large vertebrates, the depletion of “evolutionary powerhouses” in the tropics, and unpredictable emergent novelties. Despite this likelihood, we have only a rudimentary understanding of how we are altering the evolutionary future. As a result of our ignorance, conservation policies fail to reflect long-term evolutionary aspects of biodiversity loss.

Lessons from the past: Evolutionary impacts of mass extinctions

Mass extinctions have played many evolutionary roles, involving differential survivorship or selectivity of taxa and traits, the disruption or preservation of evolutionary trends and ecosystem organization, and the promotion of taxonomic and morphological diversifications—often along unexpected trajectories—after the destruction or marginalization of once-dominant clades. The fossil record suggests that survivorship during mass extinctions is not strictly random, but it often fails to coincide with factors promoting survival during times of low extinction intensity. Although of very serious concern, present-day extinctions have not yet achieved the intensities seen in the Big Five mass extinctions of the geologic past, which each removed ≥50% of the subset of relatively abundant marine invertebrate genera. The best comparisons for predictive purposes therefore will involve factors such as differential extinction intensities among regions, clades, and functional groups, rules governing postextinction biotic interchanges and evolutionary dynamics, and analyses of the factors that cause taxa and evolutionary trends to continue unabated, to suffer setbacks but resume along the same trajectory, to survive only to fall into a marginal role or disappear (“dead clade walking”), or to undergo a burst of diversification. These issues need to be addressed in a spatially explicit framework, because the fossil record suggests regional differences in postextinction diversification dynamics and biotic interchanges. Postextinction diversifications lag far behind the initial taxonomic and morphological impoverishment and homogenization; they do not simply reoccupy vacated adaptive peaks, but explore opportunities as opened and constrained by intrinsic biotic factors and the ecological and evolutionary context of the radiation.

Human-modified ecosystems and future evolution

Our global impact is finally receiving the scientific attention it deserves. The outcome will largely determine the future course of evolution. Human-modified ecosystems are shaped by our activities and their side effects. They share a common set of traits including simplified food webs, landscape homogenization, and high nutrient and energy inputs. Ecosystem simplification is the ecological hallmark of humanity and the reason for our evolutionary success. However, the side effects of our profligacy and poor resource practices are now so pervasive as to threaten our future no less than that of biological diversity itself. This article looks at human impact on ecosystems and the consequences for evolution. It concludes that future evolution will be shaped by our awareness of the global threats, our willingness to take action, and our ability to do so. Our ability is presently hampered by several factors, including the poor state of ecosystem and planetary knowledge, ignorance of human impact, lack of guidelines for sustainability, and a paucity of good policies, practices, and incentives for adopting those guidelines in daily life. Conservation philosophy, science, and practice must be framed against the reality of human-dominated ecosystems, rather than the separation of humanity and nature underlying the modern conservation movement. The steps scientists can take to imbed science in conservation and conservation in the societal process affecting the future of ecosystems and human well-being are discussed.

Declines of biomes and biotas and the future of evolution

Although panel discussants disagreed whether the biodiversity crisis constitutes a mass extinction event, all agreed that current extinction rates are 50–500 times background and are increasing and that the consequences for the future evolution of life are serious. In response to the on-going rapid decline of biomes and homogenization of biotas, the panelists predicted changes in species geographic ranges, genetic risks of extinction, genetic assimilation, natural selection, mutation rates, the shortening of food chains, the increase in nutrient-enriched niches permitting the ascendancy of microbes, and the differential survival of ecological generalists. Rates of evolutionary processes will change in different groups, and speciation in the larger vertebrates is essentially over. Action taken over the next few decades will determine how impoverished the biosphere will be in 1,000 years when many species will suffer reduced evolvability and require interventionist genetic and ecological management. Whether the biota will continue to provide the dependable ecological services humans take for granted is less clear. The discussants offered recommendations, including two of paramount importance (concerning human populations and education), seven identifying specific scientific activities to better equip us for stewardship of the processes of evolution, and one suggesting that such stewardship is now our responsibility. The ultimate test of evolutionary biology as a science is not whether it solves the riddles of the past but rather whether it enables us to manage the future of the biosphere. Our inability to make clearer predictions about the future of evolution has serious consequences for both biodiversity and humanity.

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet1

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387–392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.

NADH-Monodehydroascorbate Oxidoreductase Is One of the Redox Enzymes in Spinach Leaf Plasma Membranes1

Amino acid analysis of internal sequences of purified NADH-hexacyanoferrate(III) oxidoreductase (NFORase), obtained from highly purified plasma membranes (PM) of spinach (Spinacia oleracea L.) leaves, showed 90 to 100% homology to internal amino acid sequences of monodehydroascorbate (MDA) reductases (EC 1.6.5.4) from three different plant species. Specificity, kinetics, inhibitor sensitivity, and cross-reactivity with anti-MDA reductase antibodies were all consistent with this identification. The right-side-out PM vesicles were subjected to consecutive salt washing and detergent (polyoxyethylene 20 dodecylether and 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane sulfonate [CHAPS]) treatments, and the fractions were analyzed for NFORase and MDA reductase activities. Similar results were obtained when the 300 mm sucrose in the homogenization buffer and in all steps of the salt-washing and detergent treatments had been replaced by 150 mm KCl to mimic the conditions in the cytoplasm. We conclude that (a) MDA reductase is strongly associated with the inner (cytoplasmic) surface of the PM under in vivo conditions and requires washing with 1.0 m KCl or CHAPS treatment for removal, (b) the PM-bound MDA reductase activity is responsible for the majority of PM NFORase activity, and (c) there is another redox enzyme(s) in the spinach leaf PM that cannot be released from the PM by salt-washing and/or CHAPS treatment. The PM-associated MDA reductase may have a role in reduction of ascorbate in both the cytosol and the apoplast.

Dopamine inhibits mammalian photoreceptor Na+,K+-ATPase activity via a selective effect on the alpha3 isozyme.

The rat retina contains dopaminergic interplexiform cells that send processes to the outer plexiform layer where dopamine is released in a light-dependent manner. We report herein that physiologically relevant concentrations of dopamine inhibited ouabain-sensitive photoreceptor oxygen consumption in dark- and light-adapted rat retinas and inhibited Na+,K+-ATPase specific activity (EC 3.6.1.37) in a rat rod outer-inner segment preparation. Experiments with the selective D1 agonist fenoldopam or D2 agonist quinpirole and experiments with dopamine plus either the D1 antagonist SCH23390 or D2/D4 antagonist clozapine showed that the inhibition of oxygen consumption and enzyme activity were mediated by D2/D4-like receptors. The amphetamine-induced release of dopamine, monitored by the inhibition of oxygen consumption, was blocked by L-2-amino-4-phosphonobutyric acid and kynurenic acid. Pharmacological and biochemical experiments determined that the IC50 values of ouabain for the alpha1-low and alpha3-high ouabain affinity isozymes of photoreceptor Na+,K+-ATPase were approximately 10(-5) and approximately 10(-7) M, respectively, and that the D2/D4-like mediated inhibition of Na+,K+-ATPase was exclusively selective for the alpha3 isozyme. The dopamine-mediated inhibition of alpha3 first occurred at 5 nM, was maximal at 100 microM (-47%), had an IC50 value of 382 +/- 23 nM, and exhibited negative cooperativity (Hill coefficient, 0.27). Prior homogenization of the rod outer-inner segment completely prevented the long-lasting inhibition, suggesting that the effect was coupled to a second messenger. Although the physiological significance of our findings to photoreceptor function is unknown, we hypothesize that these results may have relevance for the temporal tuning properties of rods.

Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L.

An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combined use of an improved isolation medium and jasmonic acid-induced etiolated seedlings of Sinapis alba L. An 8-fold induction of de novo biosynthesis of the L-tyrosine-derived p-hydroxybenzylglucosinolate was obtained in etiolated S. alba seedlings upon treatment with jasmonic acid. Formation of inhibitory glucosinolate degradation products upon tissue homogenization was prevented by inactivation of myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. The biosynthetically active microsomal enzyme system converted L-tyrosine into p-hydroxyphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The Km and Vmax values of the enzyme system were 346 microM and 538 pmol per mg of protein per h, respectively. The nature of the enzyme catalyzing the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 by photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous cytochrome P450 inhibitors confirms the involvement of cytochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glycosides.

Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes.

Signals for endocytosis and for basolateral and lysosomal sorting are closely related in a number of membrane proteins, suggesting similar sorting mechanisms at the plasma membrane and in the trans-Golgi network (TGN). We tested the hypothesis that basolateral membrane proteins are transported to the cell surface via endosomes for the asialoglycoprotein receptor H1. This protein was tagged with a tyrosine sulfation site (H1TS) to allow specific labeling with [35S]sulfate in the TGN. Madin-Darby canine kidney cells expressing H1TS were pulse-labeled and chased for a period of time insufficient for labeled H1TS to reach the cell surface. Upon homogenization and gradient centrifugation, fractions devoid of TGN were subjected to immunoisolation of compartments containing mannose 6-phosphate receptor, which served as an endosomal marker. H1TS in transit to the cell surface was efficiently coisolated, whereas a labeled secretory protein and free glycosaminoglycan chains were not. This indicates an indirect pathway for the asialoglycoprotein receptor to the plasma membrane via endosomes and has important implications for protein sorting in the TGN and endosomes.

Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution.

The internal transcribed spacers (ITS) of nuclear ribosomal DNA of 33 species of genus Paeonia (Paeoniaceae) were sequenced. In section Paeonia, different patterns of nucleotide additivity were detected in 14 diploid and tetraploid species at sites that are variable in the other 12 species of the section, suggesting that reticulate evolution has occurred. Phylogenetic relationships of species that do not show additivity, and thus ostensibly were not derived through hybridization, were reconstructed by parsimony analysis. The taxa presumably derived through reticulate evolution were then added to the phylogenetic tree according to additivity from putative parents. The study provides an example of successfully using ITS sequences to reconstruct reticulate evolution in plants and further demonstrates that the sequence data could be highly informative and accurate for detecting hybridization. Maintenance of parental sequences in the species of hybrid origin is likely due to slowing of concerted evolution caused by the long generation time of peonies. The partial and uneven homogenization of parental sequences displayed in nine species of putative hybrid origin may have resulted from gradients of gene conversion. The documented hybridizations may have occurred since the Pleistocene glaciations. The species of hybrid origin and their putative parents are now distantly allopatric. Reconstruction of reticulate evolution with sequence data, therefore, provides gene records for distributional histories of some of the parental species.

Degradação enzimática de clorofenol em microrreator.

O microrreator faz parte de conjunto de dispositivos de uma nova e promissora tecnologia, que podem ser chamados de micro fabricados, atuante em campos como a da química, biológica, farmacêutica, engenharia química e biotecnologia. Trata-se de um dispositivo que possibilita reação química, tais como os reatores convencionais, mas com dimensões menores, com canais na escala micrométrica. A tecnologia de miniaturização de dispositivos para reações químicas vem se expandindo promovendo uma importante evolução, com microssistemas que abrange dispositivos mais eficazes, com configuração e geometrias específicas e menor consumo de energia, onde reações com elevadas taxas de transporte podem ser usadas para muitas finalidades diferentes, tais como, reações rápidas, mistura, reações sensíveis à temperatura, temperatura de homogeneização, ou até mesmo precipitação de nano partículas. Devido sua escala ser extremamente reduzida em relação à escala macro, oferecem um sistema que permite uma investigação do processo em um curto espaço de tempo, sendo muito útil para o rastreio de substratos, enzimas, condições de reação, bem como a determinação de parâmetros cinéticos. O presente trabalho teve por objetivo estudar a biodegradação enzimática de 2,4,6-Triclorofenol, com a utilização das enzimas Lacase e Soybean Peroxidase em microrreator da Syrris com volume de 250 ?l, que permite o estudo de cinéticas muito rápidas. Para as análises de degradação utilizou-se duas enzimas, a Lacase em concentrações de 0,05; 0,1 e 0,2 mg/ml; e a Soybean Peroxidase em concentrações de 0,0005; 0,001 e 0,002 mg/ml com a adição de Peróxido de Hidrogênio. Através dos ensaios realizados obteve-se dados experimentais da reação enzimática, possibilitando a verificação da taxa inicial de reação e sua cinética. Posteriormente, realizou-se as análises em simulação utilizando os dados experimentais, que através de um sistema de EDOs estimando inicialmente as constantes cinéticas k1, k2 e k3 usando a ferramenta ESTIMA, onde apresentaram duas respostas, uma resposta típica de mínimos quadrados, e a outra resposta que a velocidade inicial, que foi melhor representada pelos parâmetros obtidos. O método empregado na degradação do substrato, o microrreator mostrou-se eficiente, permitindo a detecção de baixo consumo de substrato para a determinação da taxa inicial, em curto tempo de residência. Perante os ensaios realizados com Lacase e Soybean Peroxidase, o microrreator é também um equipamento eficaz na repetitividade e na reprodutibilidade dos dados obtidos em diferentes concentrações.

Mineral and chemical composition of hydrothermally altered rocks and sediments from the Middle Valley, ODP Leg 139 data

The Middle Valley segment at the northern end of the Juan de Fuca Ridge is a deep extensional rift blanketed with 200-500 m of Pleistocene turbiditic sediment. Sites 857 and 858 were drilled during Ocean Drilling Program Leg 139 to determine whether these two sites were hydrologically linked end members of an active hydrothermal circulation system. Site 858 was placed in an area of active hydrothermal discharge with fluids up to 270°C venting through anhydrite-bearing mounds on top of altered sediment. The shallow basement of fine-grained basalt that underlies the vents at Site 858 is interpreted as a seamount that was subsequently buried by turbidites. Site 857 was placed 1.6 km south of the Site 858 vents in a zone of high heat flow and numerous seismically imaged ridge-parallel faults. Drilling at Site 857 encountered sediments that are increasingly altered with depth and that overlie a series of mafic sills at depths of 460-940 m below sea floor. Sill margins and adjacent baked sediment are highly altered to magnesian chlorite and crosscut with veins filled with quartz, chlorite, sulfides, epidote, and wairakite. The sill interiors vary from slightly altered, with unaltered plagioclase and clinopyroxene in a mesostasis replaced by chlorite, to local zones of intense alteration and brecciation. In these latter zones, the sill interiors are pervasively replaced by chlorite, epidote, quartz, pyrite, titanite, and rare actinolite. The most complete replacement is associated with brecciated horizons with low recovery and slickensides on fracture surfaces, which we interpret as intersections between faults and the sills. Geochemically, the alteration of the sill complex is reflected in significant whole-rock depletions in Ca, Sr, and Na with corresponding enrichments in Mg, Al, and most metals. The latter results from the formation of conspicuous sulfide poikiloblasts. In contrast, metamorphism of the Site 858 seamount includes incomplete albitization of plagioclase phenocrysts and replacement of sparse mafic phenocrysts. Much of the basement alteration at Site 858 is confined to crosscutting veins except for a highly altered and veined horizon at the contact between basaltic basement and the overlying sediment. The sill complex at Site 857 is more highly depleted in 18O (d18O = 2.4 per mil - 4.7 per mil) and more pervasively replaced by secondary minerals relative to the extrusives at Site 858 (d18O = 4.5 per mil - 5.5 per mil). There is no evidence of significant albitization of the plagioclase at Site 857, suggesting high Ca/Na in the pore fluids. Fluid-inclusion data from hydrothermal minerals in altered mafic rocks and veins at Sites 857 and 858 show a consistency of homogenization temperatures, varying from 245 to 270°C, which is within the range of temperatures observed for the fluids venting at Site 858. The consistency of the fluid inclusion temperatures, the lack of albitization within the Site 857 sills, and the apparently low water/rock ratio collectively suggest that the sill complex at Site 857 is in thermal equilibrium and being altered by a highly evolved Ca-rich fluid similar to the fluids now venting at Site 858. The alteration evident in these two deep crustal drillsites is a result of the ongoing hydrothermal circulation and is consistent with downhole logging results, instrumented borehole results, and hydrothermal fluid chemistry. The pervasive alteration of the laterally extensive sill-sediment complex at Site 857 determines the chemistry of the fluids that are venting at Site 858. The limited alteration of the Site 858 lavas suggests that this basement edifice acts as a penetrator or ventilator for the regional hydrothermal reservoir with much of the flow focussed at the highly altered and veined sediment-basalt contact.

Chemistry and fluid inclusions of the sheeted dike comples of ODP Holes 137-504B and 140-504B

Fluid inclusions in variably altered diabase recovered from Ocean Drilling Program Legs 137 and 140 at Hole 504B, Costa Rica Rift, exhibit fluid salinities up to 3.7 times that of seawater values (11.7 wt% NaCl equivalent) and exhibit uncorrected homogenization temperatures of 125°C to 202°C. The liquid-dominated inclusions commonly are entrapped in zones of secondary plagioclase and may be primary in origin. Fluid salinities are similar to compositions of fluids venting on the seafloor (0.4-7.0 wt% NaCl) and overlap with those measured in metabasalt samples recovered from near the Kane Fracture Zone on the Mid-Atlantic Ridge and from the Troodos ophiolite, Cyprus. The salinity variations may reflect hydration reactions involving formation of secondary mineral assemblages under rock-dominated conditions, which modify the ionic strength of hydrothermal fluids by consuming or liberating water and chloride ion. Rare CO2-CH4-bearing inclusions, subjacent to zones where talc after olivine becomes an important secondary mineral phase (1700 mbsf), may have formed due to local interaction of seawater and olivine at low water to rock ratios. Corrected average fluid inclusion homogenization temperatures exhibit a gradient from 159°C at a depth of 1370 mbsf to 183°C at a depth of 1992 mbsf and are in apparent equilibrium with the present conductive downhole temperatures. These data indicate that fluid inclusions may be used to estimate downhole temperatures if logging data are unavailable. The compositional and thermal evolution of the diabase-hosted fluids may reflect late-stage, off-axis circulation and conductive heating of compositionally modified seawater in the sheeted dike complex at Hole 504B.

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric pepticloglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity. (C) 2004 Wiley Periodicals, Inc.