High resolution spatio-temporal analysis of aquatic chemical signals using microelectrochemical electrodes

Detailed understanding of chemoreceptor cell transduction andfiltering depends on precise control and thus measurement ofthe chemical stimulus. In contrast to vision and hearing, accuratestimulus measurement in chemoreception has not been possibleat biologically relevant spatial and temporal scales. In thispaper we introduce a new high-speed (200 hz) electrochemicalmethod for the direct measurement of odor signals at biologicallyrelevant space scales (10-100 µm). We tested this systemin three applications: (i) temporal and spatial features ofodor plumes, (ii) stimulus calibrations in physiological recordingchambers and (iii) boundary layer diffusion measurements withinreceptor structures.     

Kinetics of basic fibroblast growth factor binding to its receptor and heparan sulfate proteoglycan: a mechanism for cooperactivity.

Basic fibroblast growth factor (bFGF) binds to cell surface receptor (CSR) proteins and to heparan sulfate proteoglycans (HSPG). On the basis of equilibrium dissociation constants (Kd), the CSR has been considered a "high-affinity" binding site and HSPG a "low-affinity" site. We measured the apparent individual on and off rate constants (kon and koff) for bFGF binding to these two sites on intact cells and to each class of binding site in the absence of the other. While the kon's for CSR and HSPG on intact cells were not statistically different (konC = 2.27 x 10(8) M-1 min-1; konH = 0.90 x 10(8) M-1 min-1), the koff for the HSPG was 22.7-fold greater than that for the CSR (koffC = 0.003 min-1; koffH = 0.68 min-1). Thus, the difference in Kd's appears to result from the faster rate at which bFGF is released from the HSPG sites compared to the CSR. The kon's for isolated CSR and HSPG, and the koff for isolated HSPG, did not differ significantly from those for intact cells konC = 2.50 x 10(8) M-1 min-1; konH = 0.92 x 10(8) M-1 min-1; koffH = 0.095 min-1). However, the off rate for isolated CSR (koffC = 0.048 min-1) was statistically indistinguishable from the off rate for HSPG and 16-fold greater than the off rate for CSR on intact cells. The "high-affinity" binding of bFGF to intact cells probably refers only to a complex of bFGF with both CSR and HSPG, and not to the CSR alone.     

Antisense oligonucleotides to crabp I and II alter the expression of TGF-β3, RAR-β, and tenascin in primary cultures of embryonic palate cells

Summary The cellular retinoic acid-binding proteins (CRABPs) are thought to modulate the responsiveness of cells to retinoic acid (RA). We have previously shown that primary cultures of murine embryonic palate mesenchymal (MEPM) cells express both CRABP-I and CRABP-II genes and that this expression is regulated by RA and transforming growth factor β (TGF-β). These cells also express high levels of TGF-β3, which is also regulated by RA and TGF-β. We have used an antisense strategy to investigate the role of the CRABPs in retinoid-induced gene expression. Subconfluent cultures of MEPM cells were treated for several days with phosphorothioate modified 18-mer oligonucleotides antisense to CRABP-I or CRABP-II and then with all-trans-retinoic acid at a concentration of 3.3 μM or 0.33 μM for 5 or 22 h. Total RNA was then extracted and the expression of TGF-β3, retinoic acid receptor β (RAR-β), and tenascin was assessed by northern blot analysis. Antisense oligonucleotides to CRABP-I partially inhibited the RA-induced TGF-β3, RAR-β, and tenascin mRNA expression. The corresponding mis-sense oligonucleotides were without effect. Antisense oligonucleotides to CRABP-II also partially inhibited RA-induced expression of these genes. As with the CRABP-I antisense, mis-sense oligonucleotides to CRABP-II had no effect. These data suggest that both CRABPs modulate the responsiveness of MEPM cells to retinoic acid. Inhibition of endogenous CRABP expression renders MEPM cells less responsive to RA with respect to induction of TGF-β3, RAR-β, and tenascin gene expression. These results have important implications for our understanding of the role of the CRABPs in retinoid teratology.     

Building the State, Making the Nation: The Bases and Limits of State Centralization in "Modern" Peru

Most contemporary discussions of the nature of the state, state-building, and national consciousness formation are based on an oppositional model of state-society relations. In such models, state-building is depicted as a process in which the state must impose its central institutions and cultural/moral values on the recalcitrant local populations found within its territorial boundaries who, in resisting the state, cling to a myriad of local, oppositional identities. The present article critiques the oppositional model, proposes an alternative conceptualization that encompasses dimensions of cooperation and conflict in state-society relations, and thus points toward the forces that underlie the fragmentation and consolidation of states and national cultures.     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait low brood-viability resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.     

Differential recovery of polymorphonuclear neutrophils,B and T cell subpopulations in the thymus,bone marrow,spleen and blood of mice following split-dose polychemotherapy

In these studies, we examined the effect of a maximum-tolerated, split-dose chemotherapy protocol of cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea carmustine on neutrophil and lymphocyte subpopulations in the peripheral blood (PBL), thymus, bone marrow and spleen. It was found that this protocol of polychemotherapy, modeled after the induction protocol used with autologous bone marrow transplantation for breast cancer, suppressed both B and T cell populations and T cell function at times when the absolute neutrophil count had returned to normal or supernormal numbers. In the peripheral blood, 7 days following initiation of chemotherapy, there was a twofold increase in the percentage of granulocytes as compared to the level in control animals on the basis of a differential count. The polymorphonuclear neutrophil (PMN) frequency in the bone marrow was increased on day 14 and statistically identical to that in control mice on all other days analyzed. In contrast to the bone marrow cells and PBL on day 7, the frequency of PMN in the spleen and thymus was depressed. B cells (B220+) were depressed in the PBL, spleen and bone marrow and took 18–32 days to return to their normal frequency, while the frequency of B cells in the thymus was increased owing to a loss of immature T cells. The percentage of CD3+ cells in the thymus, spleen and bone marrow was significantly increased and required 10–18 days to return to normal levels, while the absolute number of CD3+ cells in the blood varied around the normal value. The ratio of CD4+ to CD8+ cells in all the organs studied varied only slightly owing to a similar reconstitution of CD4+ and CD8+ cells. In contrast to the phenotypic recovery of the CD3+, CD4+ and CD8+ cells, the ability of the splenic lymphocytes to respond to concanavalin-A was depressed and remained depressed, despite the phenotypic reconstitution of the T cell subsets, on the basis of both percentage and absolute cell number. These results show a selective T and B cell depression following multi-drug, split-dose chemotherapy in tissue and blood leukocyte populations and a chronic depression in T cell function.     

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg²⁺-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase -dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergenta availability and cation sensitivity on the apparent distribution of PAPH in neutrophil sub-cellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.     

Spectroscopic studies of the manganese complex of Photosystem II

Our recent EPR and EXAFS experiments investigating the structure of the oxygen-evolving complex of PS II are discussed. PS II treatments which affect the cofactors calcium and chloride have been used to poise samples in modified forms of the S-states, S₁, S₂ and S₃. X-ray absorption studies indicate a similar overall structure for the manganese complex between treated and native samples although the influence of the treatments and cofactors is observed. Manganese oxidation (or oxidation of a ligand to the manganese cluster) is indicated to occur on each of the transitions S₁ S₂ and S₂ S₃ in these modified samples. The cluster appears to contain at least two inequivalent Mn-Mn pairs. In the native samples the Mn-Mn distance is 2.7 Å, but in samples where the calcium site is affected, one of the pairs has a 3.0 Å Mn-Mn distance. The intensity of the 3.3/3.6 Å interaction is reduced on sodium chloride treatment (calcium depletion) perhaps indicating calcium binding close to the manganese cluster. From EPR data we also propose that treatments which affect calcium and chloride binding cause a modification of the native S₂ state, slow the reduction of Y_z and allow an S3 EPR signal to be observed following illumination. The origin of the S3 EPR signal, a modified S₃ or S₂ X where X is an organic radical of unknown charge, is discussed in relation to the results from the EXAFS studies.Abbreviations EPR electron paramagnetic resonance spectroscopy - EXAFS extended X-ray absorption fine structure - HTG n-heptyl -d-thioglucoside - MES 2(N-morpholino)ethanesulfonic acid - OEC oxygen evolving complex - PPBQ phenyl-1,4-benzoquinone - PS II Photosystem II - Y_z redox active tyrosine     

Clearance of Exogenous Dopamine in Rat Dorsal Striatum and Nucleus Accumbens: Role of Metabolism and Effects of Locally Applied Uptake Inhibitors

In vivo electrochemistry was used to investigate the mechanisms contributing to the clearance of locally applied dopamine in the dorsal striatum and nucleus accumbens of urethane-anesthetized rats. Chronoamperometric recordings were continuously made at 5 Hz using Nafion-coated carbon fiber electrodes. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible dopamine signals were detected. Substitution of L-a-methyldopamine, a substrate for the dopamine transporter but not for monoamine oxidase, for dopamine in the micropipette did not substantially alter the time course of the resulting signals. This indicates that metabolism of locally applied dopamine to 3,4-dihydroxyphenylacetic acid is not responsible for the decline in the dopamine signal. Similarly, changing the applied oxidation potential from ±0.45 to ±0.80 V, which allows for detection of 3-methoxytyramine formed from dopamine via catechol-O-methyltransferase, had little effect on signal amplitude or time course. In contrast, lesioning the dopamine terminals with 6-hydroxydopamine, or locally applying the dopamine uptake inhibitors cocaine or nomifensine before pressure ejection of dopamine, significantly increased the amplitude and time course of the dopamine signals in both regions. The effects of cocaine and nomifensine were greater in the nucleus accumbens than in the dorsal striatum. Local application of lidocaine and procaine had no effect on the dopamine signals. Initial attempts at modeling resulted in curves that were in qualitative agreement with our experimental findings. Taken together, these data indicate that (1) uptake of dopamine by the neuronal dopamine transporter, rather than metabolism or diffusion, is the major mechanism for clearing locally applied dopamine from the extracellular milieu of the dorsal striatum and nucleus accumbens, and (2) the nucleus accumbens is more sensitive to the effects of inhibitors of dopamine uptake than is the dorsal striatum.