Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.     

Microhabitat,water relations,and photosynthesis of a desert fern,Notholaena parryi

Summary Interrelationships between morphology, microhabitat, water relations, and photosynthesis of a xeric fern, Notholaena parryi D.C. Eat. (Pteridaceae), were examined in the western Colorado desert. In its typical microhabitat rock outcroppings protected N. parryi from direct sunlight and moderated the diurnal variations in air temperature. For example, frond temperature at noon in late winter was 15.3° C, which was 7.3° C cooler than an energy budget simulation predicting frond temperature at an exposed site. The lowest soil water potential leading to daytime stomatal opening was about-1.5 MPa (-15 bars). Rainfall runoff that was channeled to the periphery of the rocks caused ^soil near the fern roots to rise above-1.5 MPa even after light rainfalls, and it remained above-1.5 MPa longer after rainfall than in non-rocky sites.The water potential gradient along the stipe necessary to support the observed rates of transpiration was about-10 MPa m^-1; such a large gradient reflected the small conducting area in the xylem. The water vapor conductance decreased as the frond temperature was raised, an effect that became proportionally greater as the soil dried out. The daytime water-use efficiency (mass CO₂ fixed/mass water transpired) was 0.0058 for a spring day. Individual fronds reached 90% of light saturation for photosynthesis at only 100 Einsteins m^-2 s^-1, a photosynthetically active radiation similar to that from the diffuse sunlight incident on the generally north-facing microhabitat of the fern. Below 50 E m^-2 s^-1 the quantum requirement was 13 Einsteins absorbed/mole CO₂ fixed. The ratio of chlorophyll to P700 was 552, indicating a fairly large photosynthetic unit that is characteristic of plants adapted to shaded habitats. The temperature optimum for net photosynthesis shifted from 13° C in midwinter (mean daily air temperature of 11° C) to 19° C in early fall (air temperature of 23° C).     

ORIENTATION,PAR INTERCEPTION,AND NOCTURNAL ACIDITY INCREASES FOR TERMINAL CLADODES OF A WIDELY CULTIVATED CACTUS,OPUNTIA FICUS-INDICA

Terminal vertical cladodes (flattened stems) of Opuntia ficus-indica growing in widely separated locations were nonrandomly oriented. On plantations at 33°S latitude in Chile individual cladodes tended to orient in the same direction as the planted cladodes on which they developed. However, after 2 years unshaded new cladodes tended to face east-west. Terminal cladodes also tended to face east-west for irrigated O. ficus-indica in California (at 34°N) and in Israel (32 to 33°N), but cladodes developing in the winter tended to face north-south. Except for the residual effect of initial planting direction, the observed patterns tended to maximize the interception of photosynthetically active radiation (PAR). Specifically, east-west cladode orientation would maximize PAR interception, except for cladodes developing near the winter solstice at latitudes more than 27° from the equator. Nocturnal acidity increases and hence productivity would generally be light-limited, since the nocturnal increase in acidity was 90% saturated for a total daytime PAR of 24 mol m^–2 day^–1 and the PAR received on vertical surfaces is usually less than this. Topographical features can modify the orientation patterns, since at a site where PAR was considerably blocked by surrounding mountains the maximal nocturnal acidity increases and peak in cladode orientation occurred 20° from facing east–west. Laboratory studies showed that developing cladodes oriented toward a horizontal light and were rotated an average of 16° in a direction that increased PAR interception compared to the cladodes on which they developed. Such phototropic responses, the higher productivity of favorably oriented cladodes, and the tendency to orient similarly to the underlying cladode presumably accounts for the overall orientation patterns observed, where up to four times more cladodes may face in a particular direction than at right angles to it.     

Carbon and water relations for developing fruits of Opuntia ficus-indica (L.) Miller, including effects of drought and gibberellic acid

Growth, gas exchange rates, and carbohydrate content were studied for developing fruits of the cultivated cactus Opuntia ficus-indica (L.) Miller, including effects of drought and exogenous gibberellic acid (GA3). Fruit development required 110 d from the time of bud differentiation to ripening at 80 d after anthesis, when the fruit mass averaged 67 g. Stomatal conductance and net CO2 uptake rates for fruits were higher during the night; they were maximal at 7 d before anthesis and decreased as development progressed. Fruits undergoing drought, imposed by detaching terminal stems bearing fruits, were 50% smaller than the control at 80 d after anthesis and did not ripen. Fruits injected with 2 ml of 500 ppm GA3 were 30% smaller than the control at 80 d after anthesis; they contained a large proportion of aborted seeds that produced a weak sink signal for dry mass accumulation. Gas exchange was higher at 21 d after anthesis for fruits treated with GA3. Total soluble sugars represented 40% of the fruit's dry mass until 45 d after anthesis, when the sugar content rapidly increased, reaching 90% at 73 d after anthesis. Such an increase was not observed for fruits treated with GA3, and the sugar content for fruits undergoing drought remained low throughout development. Starch content increased for developing fruits of O. ficus-indica until 14 d after anthesis and, except for the fruits undergoing drought, decreased thereafter. Fruit development for O. ficus-indica is apparently regulated by water availability as well as hormonal signals originating both within and outside the fruit.     

Coliform concentration reduction and related performance evaluation of a down-flow anaerobic fixed bed reactor treating low-strength saline wastewater

Low-strength saline wastewater may be generated by tourist facilities, industries and communities located in coastal areas. Sea salts, mostly chlorides, when present in wastewaters at high concentrations, can cause inhibition on biological treatment processes. In this study, a laboratory down-flow anaerobic fixed bed reactor (DFAFBR) was used for treating saline wastewater. This wastewater was simulated by dilution of piggery manure in a synthetic saline water to obtain a final total COD concentration in the range of 1100-2900 mg/l and a salt concentration of 15 g/l. The DFAFBR was operated at hydraulic retention times (HRT) of 96, 48, 24 and 12 h. The results showed that at sea salts concentrations in the range from 5 to 15 g/l, total coliform concentration reduction efficiencies higher than 97% were achieved. A decrease in the total and faecal coliform concentration reduction efficiencies from 99.5% to 90.5% and 92.5%, respectively, was observed when the HRT decreased from 96 to 12 h. Enumeration of coliform bacteria isolated from the biofilm in different zones of the reactor showed that more than 94% of the total amount was removed in the upper zone. A HRT of 24 h was required to obtain total COD, organic-N, total-P and faecal coliform concentration reduction efficiencies higher than 72%, 51%, 39% and 98%, respectively. A concentration of 8.4 g/l for chlorides, 1.25 g/l for sulphates and 4.6 g/l for sodium did not affect the process performance.     

Expression of interleukin 1 alpha and beta, and interleukin 1 receptor antagonist mRNA in the rat central nervous system after peripheral administration of lipopolysaccharides

Interleukin 1alpha (IL-1alpha) and IL-1beta, and the endogenous IL-1 receptor antagonist (IL-1ra) are known members of the IL-1 family. Using in situ hybridization histochemistry we demonstrated that following endotoxin injection (lipopolysaccharides, LPS, 2.0 mg/kg, i.p.) a time dependent expression and partly different expression patterns of the cytokines occurred within the rat brain and pituitary gland. All cytokines were observed in the choroid plexus. In addition, IL-1ra mRNA expressing cells were observed scattered in the brain parenchyma, whereas scattered IL-1beta mRNA expressing cells were restricted to central thalamic nuclei, the dorsal hypothalamus, and cortical regions, such as the parietal and frontal cortex. A strong IL-1beta mRNA expression was found in the circumventricular organs. In the pituitary gland, a low IL-1alpha and a high IL-1beta mRNA expression was observed, with the highest density of cytokine-expressing cells seen in the posterior pituitary. The cell types expressing the mRNA's of IL-1alpha, IL-1beta and IL-1ra were identified as monocytes in the circumventricular organs and the pituitary gland, and as microglia in the brain parenchyma. In conclusion, the present findings revealed that cytokine production in response to a peripheral endotoxin challenge mainly occurs in peripherally derived monocytes in the circumventricular organs and the pituitary gland. IL-1beta is the predominant form expressed, whereas the expression of IL-1alpha mRNA and IL-1ra mRNA is lower. Our observations support the view that peripherally derived IL-1 may play a role in the induction of centrally mediated illness symptoms.     

Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis

To investigate the involvement of K⁺ efflux in apoptotic cell shrinkage, we monitored efflux of the K⁺ congener,⁸⁶ Rb⁺, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in ⁸⁶Rb⁺ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed ⁸⁶Rb⁺ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K⁺ channels could not inhibit CD95-mediated efflux of⁸⁶ Rb⁺, however, the uptake of⁸⁶ Rb⁺ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of⁸⁶ Rb⁺ in Jurkat cells was sensitive to ouabain (a specific Na⁺/K⁺-ATPase inhibitor), demonstrating Na⁺/K⁺-ATPase dependent K⁺ uptake. Ouabain induced significant⁸⁶ Rb⁺ efflux in untreated cells, as well as it seemed to compete with⁸⁶ Rb⁺ efflux induced by the anti-CD95 antibody, supporting a role for Na⁺/K⁺-ATPase in the CD95-mediated⁸⁶ Rb⁺ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na⁺+/K⁺-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.     

Hydraulic conductance and mercury-sensitive water transport for roots of Opuntia acanthocarpa in relation to soil drying and rewetting

Drought-induced changes in root hydraulic conductance (LP) and mercury-sensitive water transport were examined for distal (immature) and mid-root (mature) regions of Opuntia acanthocarpa. During 45 d of soil drying, LP decreased by about 67% for distal and mid-root regions. After 8 d in rewetted soil, LP recovered to 60% of its initial value for both regions. Axial xylem hydraulic conductivity was only a minor limiter of LP. Under wet conditions, HgCl2 (50 microM), which is known to block membrane water-transport channels (aquaporins), decreased LP and the radial hydraulic conductance for the stele (L(R, S)) of the distal root region by 32% and 41%, respectively; both LP and L(R, S) recovered fully after transfer to 2-mercaptoethanol (10 mM). In contrast, HgCl2 did not inhibit LP of the mid-root region under wet conditions, although it reduced L(R, S) by 41%. Under dry conditions, neither LP nor L(R, S) of the two root regions was inhibited by HgCl2. After 8 d of rewetting, HgCl2 decreased LP and L(R, S) of the distal region by 23% and 32%, respectively, but LP and L(R, S) of the mid-root region were unaltered. Changes in putative aquaporin activity accounted for about 38% of the reduction in LP in drying soil and for 61% of its recovery for the distal region 8 d after rewetting. In the stele, changes in aquaporin activity accounted for about 74% of the variable L(R, S) during drought and after rewetting. Thus, aquaporins are important for regulating water movement for roots of O. acanthocarpa.     

Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1

The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.