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Oxygen-linked equilibrium CuB-CO species in cytochrome ba3 oxidase from thermus thermophilus. Implications for an oxygen channel ar the CuB site.

We report the first study of O(2) migration in the putative O(2) channel of cytochrome ba(3) and its effect to the properties of the binuclear heme a(3)-Cu(B) center of cytochrome ba(3) from Thermus thermophilus. The Fourier transform infrared spectra of the ba(3)-CO complex demonstrate that in the presence of 60-80 micro m O(2), the nu(C-O) of Cu(B)1+-C-O at 2053 cm(-1) (complex A) shifts to 2045 cm(-1) and remains unchanged in H(2)O/D(2)O exchanges and in the pH 6.5-9.0 range. The frequencies but not the intensities of the C-O stretching modes of heme a(3)-CO (complex B), however, remain unchanged. The change in the nu(C-O) of complex A results in an increase of k(-2), and thus in a higher affinity of Cu(B) for exogenous ligands. The time-resolved step-scan Fourier transform infrared difference spectra indicate that the rate of decay of the transient Cu(B)1+-CO complex at pH 6.5 is 30.4 s(-1) and 28.3 s(-1) in the presence of O(2). Similarly, the rebinding to heme a(3) is slightly affected and occurs with k(2) = 26.3 s(-1) and 24.6 s(-1) in the presence of O(2). These results provide solid evidence that in cytochrome ba(3), the ligand delivery channel is located at the Cu(B) site, which is the ligand entry to the heme a(3) pocket. We suggest that the properties of the O(2) channel are not limited to facilitating ligand diffusion to the active site but are extended in controlling the dynamics and reactivity of the reactions of ba(3) with O(2) and NO.

Binding Sites↗

Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism.

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA overflow into the synaptic cleft. Facilitated exchange diffusion is the classical model used to describe AMPH-induced DA efflux. This model hypothesizes that AMPH-induced DA efflux is mediated by DAT and results from the transport of AMPH into the cell followed by a counter movement of DA out to the extracellular compartment. To further characterize the action of AMPH, we used the patch clamp technique in the whole-cell configuration combined with amperometry on human embryonic kidney HEK-293 cells stably transfected with the human DAT (DAT cells). In DAT cells, AMPH-induced DAT-mediated currents were blocked by cocaine. We demonstrate that DA efflux mediated by DAT is voltage-dependent, electrogenic, and dependent on intracellular Na(+) concentration in the recording electrode. Intracellular Na(+) fluorescence, as measured by confocal microscopy using a Na(+)-sensitive dye, was enhanced by AMPH application. Furthermore, the ability of AMPH to induce DA efflux was regulated by intracellular Na(+) concentration and correlated with the size of the DAT-mediated, AMPH-induced ion flux across the plasma membrane. In the absence of intracellular Na(+) but the presence of high intracellular Cl(-), AMPH-induced inward currents elicited DA efflux proportionally to their dimension and duration. Thus, we propose that AMPH-induced DA efflux depends on two correlated transporter processes. First, AMPH binds to the DAT and is transported, thereby causing an inward current. Second, because of this AMPH-induced inward current, Na(+) becomes more available intracellularly to the DAT, thereby enhancing DAT-mediated reverse transport of DA.

Amphetamine↗

Zebrafish reveals different and conserved features of vertebrate neuroglobin gene structure, expression pattern, and ligand binding.

Neuroglobin has been identified as a respiratory protein that is primarily expressed in the mammalian nervous system. Here we present the first detailed analysis of neuroglobin from a non-mammalian vertebrate, the zebrafish Danio rerio. The zebrafish neuroglobin gene reveals a mammalian-type exon-intron pattern in the coding region (B12.2, E11.0, and G7.0), plus an additional 5'-non-coding exon. Similar to the mammalian neuroglobin, the zebrafish protein displays a hexacoordinate deoxy-binding scheme. Flash photolysis kinetics show the competitive binding on the millisecond timescale of external ligands and the distal histidine, resulting in an oxygen affinity of 1 torr. Western blotting, immune staining, and mRNA in situ hybridization demonstrate neuroglobin expression in the fish central nervous system and the retina but also in the gills. Neurons containing neuroglobin have a widespread distribution in the brain but are also present in the olfactory system. In the fish retina, neuroglobin is mainly present in the inner segments of the photoreceptor cells. In the gills, the chloride cells were identified to express neuroglobin. Neuroglobin appears to be associated with mitochondria-rich cell types and thus oxygen consumption rates, suggesting a myoglobin-like function of this protein in facilitated oxygen diffusion.

Animals↗

Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation.

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.

Animals↗

Fine structure of the cilia of rotifers.

The fine structure of the coronal cilia of the rotifer Philodina citrina has been studied in detail. Specimens were fixed with OsO(4) and embedded in butyl-methyl methacrylate, Epon 812, or Vestopal and sectioned with a Porter-Blum microtome. The details of structure of the rootlets, basal bodies, basal plates, and free cilia are described. The general structure of the rotifer ciliary apparatus conforms well to that established for other species. One of the main observations is the difference in structure of the peripheral filaments in the opposing halves of a cross-section of the free cilium. Also, in longitudinal sections evidence is offered for the existence of a helical structure in the peripheral filaments.

Basal Bodies↗

Molecular basis of pH and Ca2+ regulation of aquaporin water permeability.

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca(2+) increase the water permeability of bovine AQP0 expressed in Xenopus oocytes. We now show that external histidines in loops A and C mediate the pH dependence. Furthermore, the position of histidines in different members of the aquaporin family can "tune" the pH sensitivity toward alkaline or acid pH ranges. In bovine AQP0, replacement of His40 in loop A by Cys, while keeping His122 in loop C, shifted the pH sensitivity from acid to alkaline. In the killifish AQP0 homologue, MIPfun, with His at position 39 in loop A, alkaline rather than acid pH increased water permeability. Moving His39 to His40 in MIPfun, to mimic bovine AQP0 loop A, shifted the pH sensitivity back to the acid range. pH regulation was also found in two other members of the aquaporin family. Alkaline pH increased the water permeability of AQP4 that contains His at position 129 in loop C. Acid and alkaline pH sensitivity was induced in AQP1 by adding histidines 48 (in loop A) and 130 (in loop C). We conclude that external histidines in loops A and C that span the outer vestibule contribute to pH sensitivity. In addition, we show that when AQP0 (bovine or killifish) and a crippled calmodulin mutant were coexpressed, Ca(2+) sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca(2+) and pH modulation are separable and arise from processes on opposite sides of the membrane.

Amino Acid Sequence↗

The permeability of thin lipid membranes to bromide and bromine.

Thin lipid (optically black) membranes were made from sheep red cell lipids dissolved in n-decane. The flux of Br across these membranes was measured by the use of tracer (82)Br. The unidirectional flux of Br (in 50-100 mM NaBr) was 1-3 x 10(-12) mole/cm(2)sec. This flux is more than 1000 times the flux predicted from the membrane electrical resistance (>10(8) ohm-cm(2)) and the transference number for Br(-) (0.2-0.3), which was estimated from measurements of the zero current potential difference. The Br flux was not affected by changes in the potential difference imposed across the membrane (+/-60 mv) or by the ionic strength of the bathing solutions. However, the addition of a reducing agent, sodium thiosulfate (10(-3)M), to the NaBr solution bathing the membrane caused a 90% reduction in the Br flux. The inhibiting effect of S(2)O(3) (=) suggests that the Br flux is due chiefly to traces of Br(2) in NaBr solutions. As expected, the addition of Br(2) to the NaBr solutions greatly stimulated the Br flux. However, at constant Br(2) concentration, the Br flux was also stimulated by increasing the Br(-) concentration, in spite of the fact that the membrane was virtually impermeable to Br(-). Finally, the Br flux appeared to saturate at high Br(2) concentrations, and the saturation value was roughly proportional to the Br(-) concentration. These results can be explained by a model which assumes that Br crosses the membrane only as Br(2) but that rapid equilibration of Br between Br(2) and Br(-) occurs in the unstirred layers of aqueous solution bathing the two sides of the membrane. A consequence of the model is that Br(-) "facilitates" the diffusion of Br across the unstirred layers.

Animals↗

The effect of calcium ionophores on fragmented sarcoplasmic reticulum.

X-537 A and A 23187, two antibiotics which form liphophilic complexes with divalent cations, function as ionophores in vesicular fragments of sarcoplasmic reticulum (SR). Addition of either ionophore to SR preloaded with calcium in the presence of adenosine triphosphate (ATP), causes rapid release of calcium. Furthermore, net calcium accumulation by SR is prevented, when the ionophores are added to the reaction mixture before ATP. On the contrary, ATP-independent calcium binding to SR is not inhibited. This effect is specific for the two antibiotics and could not be reproduced, either by inactive derivatives, or by other known ionophores. Neither ionophore produces alterations of the electron microscopic appearance of SR membranes or inhibition of the calcium-dependent ATPase. In fact, the burst of ATP hydrolysis obtained on addition of calcium, is prolonged in the presence of the ionophores. Lanthanum inhibits ATP-independent calcium binding to SR, ATP-dependent calcium accumulation and calcium-dependent ATPase. However, addition of lanthanum to SR preloaded in the presence of ATP, does not cause calcium release. The reported experiments indicated that: (a) ATP-dependent calcium accumulation by SR results in primary formation of calcium ion gradients across the membrane. (b) Most of the accumulated calcium is not available for displacement by lanthanum on the outer surface of the membrane. (c) Calcium ionophores induce rapid equilibration of the gradients, by facilitating cation diffusion across the membrane.

Adenosine Triphosphatases↗

Differences in pH between interstitial fluid and arterial blood in water-breathing and air-breathing vertebrates.

Most cells are bathed by interstitial fluid, but extracellular pH measurements are mostly for arterial plasma. Whole-body mean pH differences between the two fluids have been estimated in terms of a simple model. This relates to the diffusive exchange of carbon dioxide and oxygen and utilizes literature data, for 22 vertebrate species, on arterial and mixed-venous tensions of both gases. Uncertainties arise because the carbon dioxide reaction in blood may sometimes be in disequilibrium and because carbon dioxide diffusion is facilitated to unknown degrees in the presence of buffers. Nevertheless, the model suggests that the pH difference should tend to vary inversely with arterial carbon dioxide tension. In some species, this may aid interstitial pH homeostasis, but a clearer implication is that the difference should be generally greater in water breathers than in air breathers. It has previously been found that arterial pH in water-breathing teleosts also tends to be higher than in air-breathing tetrapods (when allowance is made for temperature and plasma sodium concentration) and to a comparable extent. Thus, mean interstitial pH may be more nearly similar in the two groups than is arterial pH. Direct measurements of interstitial pH do not yet suffice to test the model.

Air↗

Regulation of submergence-induced enhanced shoot elongation in Oryza sativa L.

Rice (Oryza sativa L.) is the only cereal that can be cultivated in the frequently flooded river deltas of South-East and South Asia. The survival strategies used by rice have been studied quite extensively and the role of several phytohormones in the elongation response has been established. Deep-water rice cultivars can diminish flooding stress by rapid elongation of their submerged tissues to keep up with the rising waters. Other rice cultivars may react by mechanisms of submergence tolerance. Aerenchyma and aerenchymatous adventitious roots are formed that facilitate oxygen diffusion to prevent anaerobic conditions in the submerged tissues. This paper discusses the molecular aspects of the mechanism that leads to shoot elongation (leaves of seedlings and internodes), the regulation of which involves metabolism of, and interactions between, ethylene, gibberellins and abscisic acid. Finally, the importance of new techniques in future research is assessed. Current molecular technology can reveal subtle differences in gene activity between tolerant and non-tolerant cultivars, and identify genes that are involved in the regulation of submergence avoidance and tolerance.

Abscisic Acid↗

Structure of the developing pea seed coat and the post-phloem transport pathway of nutrients.

An important function of the seed coat is to deliver nutrients to the embryo. To relate this function to anatomical characteristics, the developing seed coat of pea (Pisum sativum L.) was examined by light- and cryo-scanning electron microscopy (cryo-SEM) from the late pre-storage phase until the end of seed filling. During this time the apparently undifferentiated seed coat tissues evolve into the epidermal macrosclereids, the hypodermal hourglass cells, chlorenchyma, ground parenchyma and branched parenchyma. Using the fluorescent symplast tracer 8-hydroxypyrene-1,3,6-trisulfonic acid, it could be demonstrated that solutes imported by the phloem move into the chlorenchyma and ground parenchyma, but not into the branched parenchyma. From a comparison with literature data of common bean (Phaseolus vulgaris L.) and broad bean (Vicia faba L.), it is concluded that in the three species different parenchyma layers, but not the branched parenchyma, may be involved in the post-phloem symplasmic transport of nutrients in the seed coat. In pea, the branched parenchyma dies during the storage phase, and its cell wall remnants then form the boundary layer between the living seed coat parenchyma cells and the cotyledons. Using cryo-SEM, clear images were obtained of this boundary layer which showed that many intracellular spaces in the seed coat parenchyma are filled with an aqueous solution. This is suggested to facilitate the diffusion of nutrients from the site of unloading towards the cotyledons.

Arylsulfonates↗

Aerenchyma formation and recovery from hypoxia of the flooded root system of nodulated soybean.

BACKGROUND AND AIMS: Flooding results in hypoxia of the root system to which N2 fixation of nodulated roots can be especially sensitive. Morphological adaptions, such as aerenchyma formation, can facilitate the diffusion of oxygen to the hypoxic tissues. Using soybean, the aim of the study was to characterize the morphological response of the nodulated root system to flooding and obtain evidence for the recovery of N metabolism. METHODS: Sections from submerged tissues were observed by light microscopy, while sap bleeding from the xylem was analysed for nitrogenous components. KEY RESULTS: Flooding resulted in the rapid formation of adventitious roots and aerenchyma between the stem (immediately above the water line), roots and nodules. In the submerged stem, taproot, lateral roots and adventitious roots, lysigenous aerenchyma arose initially in the cortex and was gradually substituted by secondary aerenchyma arising from cells derived from the pericycle. Nodules developed aerenchyma from cells originating in the phellogen but nodules situated at depths greater than 7-8 cm showed little or no aerenchyma formation. As a result of aerenchyma formation, porosity of the taproot increased substantially between the 4th and 7th days of flooding, coinciding with the recovery of certain nitrogenous products of N metabolism of roots and nodules transported in the xylem. Thus, on the first day of flooding there was a sharp decline in xylem ureides and glutamine (products of N2 fixation), together with a sharp rise in alanine (product of anaerobic metabolism). Between days 7 and 10, recovery of ureides and glutamine to near initial levels was recorded while recovery of alanine was partial. CONCLUSIONS: N metabolism of the nodulated soybean root system can recover at least partially during a prolonged period of flooding, a process associated with aerenchyma formation.

Nitrogen Fixation↗

Innovation in nursing homes: which facilities are the early adopters?

PURPOSE: This study examined organizational and market factors associated with nursing homes that are most likely to be early adopters of innovations. Early adopter institutions, defined as the first 20% of facilities to adopt an innovation, are important because they subsequently facilitate the diffusion of innovations to others in the industry. DESIGN AND METHODS: Two groups of innovations were examined, special care units and subacute care services. I used discrete-time logistic regression analysis and nationally representative data from 13,162 facilities at risk of being early adopters of innovations during twelve 6-month intervals from 1992 to 1997. RESULTS: Organizational factors that increase the likelihood of early innovation adoption are larger bed size, chain membership, and high levels of private-pay residents. Four market factors that increase the likelihood of early innovation adoption are: a retrospective Medicaid reimbursement methodology, a more competitive environment, higher average income in the county, and a higher number of hospital beds in the county. IMPLICATIONS: This analysis shows that organizational and market characteristics of nursing homes affect their propensity toward early adoption of innovations. Some of the results may be useful for nursing home administrators and policy makers attempting to promote innovation.

Aged↗

Carbon metabolism in developing soybean root nodules: the role of carbonic anhydrase.

A full-length cDNA clone encoding carbonic anhydrase (CA) was isolated from a soybean nodule cDNA library. In situ hybridization and immunolocalization were performed in order to assess the location of CA transcripts and protein in developing soybean nodules. CA transcripts and protein were present at high levels in all cell types of young nodules, whereas in mature nodules they were absent from the central tissue and were concentrated in cortical cells. The results suggested that, in the earlier stages of nodule development, CA might facilitate the recycling of CO2 while at later stages it may facilitate the diffusion of CO2 out of the nodule system. In parallel, sucrose metabolism was investigated by examination of the temporal and spatial transcript accumulation of sucrose synthase (SS) and phosphoenolpyruvate carboxylase (PEPC) genes, with in situ hybridization. In young nodules, high levels of SS gene transcripts were found in the central tissue as well as in the parenchymateous cells and the vascular bundles, while in mature nodules the levels of SS gene transcripts were much lower, with the majority of the transcripts located in the parenchyma and the pericycle cells of the vascular bundles. High levels of expression of PEPC gene transcripts were found in mature nodules, in almost all cell types, while in young nodules lower levels of transcripts were detected, with the majority of them located in parenchymateous cells as well as in the vascular bundles. These data suggest that breakdown of sucrose may take place in different sites during nodule development.

Amino Acid Sequence↗

Glucose transporter expression in rat embryo and uterus during decidualization, implantation, and early postimplantation.

Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryo's production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.

Animals↗

Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses.

We used silver-enhanced immunogold electron microscopy to reveal synaptic localization of the glutamate transporter EAAT4 in mouse cerebellar Purkinje cells (PCs). Gold-silver particles representing the EAAT4 were densely localized on extra-junctional membrane, but not on junctional membrane of PC spines in contact with parallel fiber or climbing fiber terminals. No particle accumulations were observed at inhibitory synapses formed on cell body and dendritic shafts of PCs. Therefore, the EAAT4 is selectively targeted to the extra-junctional site of excitatory PC synapses. The finding suggests that the EAAT4 transports glutamate or its related amino acids from outside the synaptic cleft, which would facilitate glutamate diffusion from the synaptic cleft to the extrasynaptic space and restrict glutamate spillover to adjacent synapses.

Amino Acid Transport System X-AG↗

Individual, contextual, and social network factors affecting exposure to HIV/AIDS risk among older residents living in low-income senior housing complexes.

This study examines the influence of individual, contextual (building location and characteristics), and social network characteristics on HIV prevalence and risk behavior among people older than 50 years of age living in low-income senior housing in two cities, Hartford, Connecticut and Chicago, Illinois. The authors' study focuses on older residents of six buildings located in impoverished neighborhoods with high rates of HIV transmission through injection drug use and unprotected sexual activity, including the exchange of sex for drugs and money. The article is organized into three sections. First, the authors explore the HIV prevalence, distribution, and risk behaviors that may contribute to new infections among the buildings' older residents in general. These practices include unprotected sexual intercourse, sex with commercial sex workers, casual or multiple partners, regular use of alcohol and/or illicit drugs, and other high-risk activities that expose older residents to HIV, sexually transmitted diseases, and other negative health consequences. Next, the authors examine the relationship between building and neighborhood characteristics, the internal social organization of buildings, and the risk behaviors of individual residents by building residency. Finally, the authors use a social network analysis to identify possible entry points and transmission routes for HIV infection through drug and sexual exchanges between and among building residents and visitors. This approach also reveals variations in building structures that may facilitate the diffusion of HIV prevention efforts.

Acquired Immunodeficiency Syndrome↗

Formation and splitting of paired hydroxyl groups on reduced TiO2(110).

A combination of high-resolution scanning tunneling microscopy and density functional theory is utilized to study the interaction of water with the reduced TiO2(110)-(1 x 1) surface. As the direct product of water dissociation in oxygen vacancies, paired hydroxyl groups are formed. These pairs are immobile and stable unless they interact with adsorbed water molecules. As a result of these interactions, protons are transferred to adjacent oxygen rows, thereby forming single hydroxyl groups. Additionally, we show that hydroxyl groups facilitate the diffusion of water molecules over the oxygen rows.

Journal Article↗