Identification of a crotoxin-binding protein in membranes from guinea pig brain by photoaffinity labeling

Crotoxin is a neurotoxic phospholipase A₂ capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin withN-hydroxysuccinimidyl-4-azidobenzoate and with Na[¹²⁵I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin,Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC₅₀ of about 10^–8 M. Mojave toxin, caudoxin, notexin,Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while -bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.On leave from Department of Biochemistry and Biophysics, University of Hawaii School of Medicine, Honolulu, Hawaii.     

Effect of cystine loading and cystine dimethylester on renal brushborder membrane transport

The effect of loading renal tubule cells with cystine was studied by incubating them with cystine dimethylester. Proline uptake into brushborder membrane vesicles isolated from the cystine loaded cells was not different from that observed into brushborder vesicles isolated from tubules incubated in buffer alone. Incubating brushborder membranes with 2 mM cystine dimethylester for 10 minutes reduced the uptake of proline by 27% after 15 seconds of incubation and by 21% after 60 seconds of incubation. There was no effect after 20 minutes of incubation. Pre-incubating brushborder membrane vesicles with cystine dimethylester had no statistically significant effect on the affinity of priline for the carrier, but did reduce the maximal rate of proline uptake by 49%.     

Receptor expression and oxidase activity in human neutrophils: Regulation by granulocyte-macrophage colony-stimulating factor and dependence upon protein biosynthesis

Incubation of human bloodstream neutrophils with 50 u/ml recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) primed the respiratory burst (as assessed by fMet-Leu-Phe stimulated luminol-dependent chemiluminescence) and resulted in a rapid (within 15 min) upregulation of expression of CD11b and CD18 (as measured by FACS analysis). This rapid priming and modulation of receptor expression was not inhibited by cycloheximide and hence appeared to be independent ofde novo protein biosynthesis. When neutrophils were incubated for up to 5 h in culture, the fluorescence distributions of CD11b and CD18 declined indicating the loss of expression of these receptors as the neutrophils aged, but in rGM-CSF treated suspensions receptor expression was maintained. When neutrophils were incubated in the presence of cycloheximide, they progressively lost their ability to generate reactive oxidants in response to fMet-Leu-Phe so that by 5 h incubation with this inhibitor they could only generate about 25% of the oxidative response stimulated in untreated cells, and the expression of CD16 and CD18 was grossly impaired. Similar effects were observed in rGM-CSF treated suspensions except that cycloheximide required longer incubation times (typically 4–5 h) before impairment of function or receptor expression occurred. These data show thatde novo protein biosynthesis is required for both the maintenance of neutrophil function and also for the continued expression of some plasma membrane receptors.Abbreviations fMet-Leu-Phe N-formylmethionyl-Leucyl-Phenylalanine - rGM-CSF recombinant granulocyte-macrophage colony-stimulating factor - FITC fluorescein isothiocyanate conjugate - Luminol 5-amino-2,3-dihydrophthalazine-1,4-dione     

Functional properties of the anaerobic responsive element of the maize Adh1 gene

The functional properties of the anaerobic responsive element (ARE) of the maize Adh1 gene have been analysed using a transient expression assay in electroporated maize protoplasts. The ARE functions in both orientations although inversion of the ARE sequence relative to the TATA box element produces slightly weaker promoter activity under anaerobic conditions and elevated expression under aerobic conditions. Promoter activity under anaerobic conditions is proportional to the number of complete ARE sequences in the Adh1 promotor. The ARE contains two sub-regions and dimers of sub-region II are as efficient as the wild-type sequence in activating gene expression under anaerobic conditions. However, sub-region I dimers do not appear capable of inducing gene expression in response to anaerobic stress. We conclude that sub-region II is essential for anaerobic induction of gene expression. Reporter gene expression remains constant when the spacing between sub-regions of the ARE is increased up to at least 64 bp, but increased spacing of 136 bp or greater abolishes expression in both aerobic and anaerobic conditions, indicating that a close association of the two sub-regions is required both for anaerobic responsiveness and for maximal levels of aerobic gene expression. When the ARE is placed upstream of position –90 of the CaMV 35S promoter, the ARE produces a high level of expression in both aerobic and anaerobic conditions. The general enhancement of gene expression driven by the hybrid ARE/35S promoter in aerobic conditions requires an intact sub-region II motif since mutation or deletion of sub-region II from the hybrid promoter reduces the level of expression to that observed for the truncated 35S promoter alone. In addition, mutation of the sub-region I sequences in the ARE/35S hybrid promoter does not significantly reduce expression in aerobic conditions, relative to pARE/35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.     

Transport of groundwater-borne nutrients from watersheds and their effects on coastal waters

Anthropogenic activities on coastal watersheds increase nutrient concentrations of groundwater. As groundwater travels downslope it transports these nutrients toward the adjoining coastal water. The resulting nutrient loading rates can be significant because nutrient concentrations in coastal groundwaters may be several orders of magnitude greater than those of receiving coastal waters. Groundwater-borne nutrients are most subject to active biogeochemical transformations as they course through the upper 1 m or so of bottom sediments. There conditions favor anaerobic processes such as denitrification, as well as other mechanisms that either sequester or release nutrients. The relative importance of advective vs. regenerative pathways of nutrient supply may result in widely different rates of release of nutrients from sediments. The relative activity of denitrifiers also may alter the ratio of N to P released to overlying waters, and hence affect which nutrient limits growth of producers. The consequences of nutrient (particularly nitrate) loading include somewhat elevated nutrient concentrations in the watercolumn, increased growth of macroalgae and phytoplankton, reduction of seagrass beds, and reductions of the associated fauna. The decline in animals occurs because of habitat changes and because of the increased frequency of anoxic events prompted by the characteristically high respiration rates found in enriched waters.     

Hemolysis of rabbit erythrocytes in the presence of copper ions

The hemolysis of red blood cells (RBC) induced by Cu(II) is modified by ceruloplasmin (Cp) and albumin. The time course of hemolysis for rabbit RBC by Cu(II) consisted of two parts, an induction period followed by a catastrophic lysis period. The induction period decreased and the lysis rate increased with increasing Cu(II) concentration. Cp or albumin, modified Cu(II) induced hemolysis, by increasing the duration of the induction period and decreasing the overall rate of hemolysis of RBC. The catastrophic lysis period coincided with a sharp increase in the formation of metHb within the cell and in a rapid uptake of Cu(II). The presence of Cp led to an increase in the induction period prior to the rapid increase in metHb formation and in Cu(II) uptake. Porcine Cp was prepared with either two or three nonprosthetic copper binding sites (sites where Cu(II) is easily removed by passing over Chelex-100). Cp with three nonprosthetic binding sites gave more protection than Cp with two. Likewise, albumin can be prepared with three and five nonprosthetic copper binding sites. The albumin with five sites gave more protection than the albumin with three sites.     

Soluble metals in the atmosphere and their biological implications

Multivariate statistical analysis has been applied to time series measurements of aerosol elemental composition from PIXE analysis of filter samples, and principal components have been resolved that represent distinct particle types in an external mixture in the atmosphere. In this study, it is argued that a combination of chemical and statistical analyses of the data may be more powerful in determining chemical species in atmospheric aerosols than studies that employ mainly direct chemical analysis of chemical species in unresolved mixtures of aerosol particle samples. Sulfur is generally associated with mineral dust elements. It is reasoned that the association may represent sulfuric acid coatings on particles that can lead to mineral dissolution and solubilization of significant amounts of aluminum, iron, and other metals. Upon wet or dry deposition to the surface, the fluxes of these metals in biologically-available form may be sufficient to affect primary productivity in the world ocean and cause ecological damage in lakes. As a consequence, the fluxes of biogenic trace gases to the atmosphere may be changed, possibly leading to changes in the tropospheric concentration of ozone. The inputs to lakes of soluble aluminum, which is toxic to fish, may be partly by deposition directly from the atmosphere, thus not limited to leaching of soils by acid deposition. Human inhalation of soluble aluminum and other solulilized mineral metals may account, in part, for the observed geographic pattern of deaths attributed to chronic obstructive pulmonary disease (COPD) that show high rates in cities of the Western US and the southeast region, but low in most of the midwest and northeast.     

Analysis of an ABA-responsive rice gene promoter in transgenic tobacco

We have analyzed in transgenic tobacco the expression of a chimeric gene containing 5 sequences of the rice rab-16B gene fused to the -glucuronidase (GUS) reporter gene. This construct, a translational fusion (–482 to +184) including 14 amino acids of the RAB-16B protein, is expressed only in zygotic and pollen-derived embryos. In zygotic embryos, GUS activity begins to accumulate 10 days after flowering (daf), and increases until seed maturation at 25 daf. Immunological measurements of endogenous abscisic acid (ABA) accumulation in these seeds showed a close parallel between hormone levels and GUS activity. However, GUS activity could not be reproducibly induced by treatment of immature embryos with ABA (10 M). Neither GUS activity nor GUS mRNA could be detected in leaves of transgenic tobacco even after ABA treatment. In contrast, GUS activity could be induced to high levels in pollen-derived embryos by treatment with ABA. Our results show that 482 bp of 5 sequences of the rice rab-16B promoter can confer in transgenic tobacco developmentally regulated expression in embryos but not ABA-responsive expression in vegetative tissues.     

Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.