Induction of IgG in young nude mice by lipid A or thymus grafts.

Postnatal serum concentrations of IgG2a of paternal allotype, measured in congenitally thymusless nude mice, increase with kinetics and titers comparable to their normal congeneic counterparts. Lipid A, the mitogenic part of LPS, stimulates IgG synthesis in nude mice when it is given 7 days after birth. IgG concentrations at 15 days of age are 6- to 8-fold higher than in untreated control nudes; this is considerably lower, however, than in normal mice, which show up to 45-fold higher IgG2ab levels after lipid A treatment. A thymus graft from nearly congeneic donors of the same age, transplanted at 4 days after birth, also stimulates long-lasting IgG synthesis in the nude recipients. If the grafted nudes are injected with lipid A 3 days later, IgG synthesis is further stimulated 8- to 16-fold. The data are discussed in relation to the thymus dependency of IgG production and the conditions for lipid A stimulation.

Induction of IgG by lipid A in the newborn mouse.

IgG of paternal allotype first becomes detectable in the serum of (BALB/c x C57BL/6)F(1) mice between day 12 and 14 after birth and reaches adult levels at an age of 5 wk. Since in mice there is a transfer of maternal IgG molecules through the placenta and via milk, F(1) heterozygous at the allotype locus were used and the concentrations of IgG with paternal allotype were measured. This was done by a sensitive method capable of detecting IgG concentrations as low as 5 x 10(-4) of normal adult serum levels. It is based on the quantitative inhibition of allotype-specific facilitation of hemolysis. When lipid A or Salmonella bacteria were injected into neonatal mice, a stimulation of IgG synthesis was observed. Thus IgG levels were enhanced 10-30-fold compared to the nontreated mice. No increase in IgG levels was obtained in adult mice after treatment with lipid A. Whether the newborns were injected at birth, on day 2, 4, or 7, IgG was first demonstrable in the treated mice at an age of 6-11 days. The increase in IgG levels was not paralleled by a demonstrable antibody activity against lipid A, SRBC, and LPS. Thus the bulk of newly induced IgG is probably a statistical distribution of different specificities.

Many roads lead to "auxin": of nitrilases, synthases, and amidases.

Recent progress in understanding the biosynthesis of the auxin, indole-3-acetic acid (IAA) in Arabidopsis thaliana is reviewed. The current situation is characterized by considerable progress in identifying, at the molecular level and in functional terms, individual reactions of several possible pathways. It is still too early to piece together a complete picture, but it becomes obvious that A. thaliana has multiple pathways of IAA biosynthesis, not all of which may operate at the same time and some only in particular physiological situations. There is growing evidence for the presence of an indoleacetamide pathway to IAA in A. thaliana, hitherto known only from certain plant-associated bacteria, among them the phytopathogen Agrobacterium tumefaciens.

Domains of yeast plasma membrane and ATPase-associated glycoprotein.

In yeast homogenates the plasma membrane H(+)-ATPase and a major surface glycoprotein of about 115 kDa are present in two membrane fractions with peak densities in sucrose gradients of 1.17 and 1.22. Immunogold electron microscopy of frozen yeast sections indicates that the ATPase is exclusively (greater than 95%) present at the surface membrane. Therefore the two ATPase-containing fractions appear to correspond to different domains of the plasma membrane. The 115 kDa glycoprotein is tightly associated with the ATPase during solubilization and purification of the enzyme. However, in a mutant lacking the glycoprotein the activity of the plasma membrane H(+)-ATPase is similar to wild type, suggesting that this association is fortuitous. The ATPase and the glycoprotein are difficult to separate by electrophoresis and therefore binding of concanavalin A to the ATPase cannot be unambiguously demonstrated in wild-type yeast. By utilizing the mutant without glycoprotein it was shown that the ATPase band of 105 kDa binds concanavalin A.

Structure of the gene encoding nitrilase 1 from Arabidopsis thaliana.

The nitrilases of Arabidopsis thaliana (At) catalyze the conversion of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), thus controlling the last step of auxin biosynthesis. A full-length genomic clone encoding the complete cluster of the At nitrilases 1 to 3 (NIT1-3), including the respective promoter regions, has been isolated and the NIT1 isoform has been sequenced. The coding region (nit1) spans about 2.3 kb and is composed of five exons separated by four introns. The exon-intron splice junctions agree with the consensus sequences typical for plant genes. In agreement with the known cDNA sequence, the exons encode a protein of 346 amino acids (aa) with a deduced molecular mass of 38.2 kDa. The transcription start point (tsp) of nit1 was determined by primer extension experiments. This tsp defines a 5' untranslated region of 36 bp and is located 32 bp downstream from a TATA box. The promoter region of nit1 is located within the approx. 1.5-kb intergenic part that separates the nit2 and nit1 coding sections.

Pore-forming properties of elicitors of plant defense reactions and cellulolytic enzymes.

Using the planar lipid bilayer technique, it is shown that a yeast elicitor as well as several cellulolytic enzymes used in protoplasting plant cells contain components which strongly interact with the bilayers. This results in the appearance of transmembrane ion fluxes which may pass through membrane defect structures and even large conductance pores with unitary conductances above 400 pS. Since membrane depolarization is an immediate response in the process of defense elicitation in plant cells, elicitors may act directly with the lipid phase of cell membranes, causing depolarizations and thus initiating the process of elicitation. When using enzymatically prepared protoplasts in electrophysiological work, contributions to electrical activity by membrane active constituents originating from the enzymes used must be expected.

Modulation of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica by divalent cations, protons and H2O2.

Electrical properties of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica were analyzed after incorporation of BCC1 into black lipid bilayers. Single channel current fluctuations were modulated by divalent cations, protons and H2O2. Whereas the channel is permeable for Ca2+, Ba2+ and Sr2+, its conductance is strongly reduced in solutions containing MgCl2. Cu2+ and Zn2+ are potent inhibitors of BCC1 in micromolar concentrations. The open channel conductance of BCC1 increases with acidification of the electrolyte solution. H2O2 shows strong inhibitory effects on BCC1. The channel is almost completely closed at submillimolar concentrations of H2O2. The effects of pH and H2O2 on channel properties are directional and affect BCC1 at the Ca exit side, but not on the entry site. Thus, cytosolic pH and H2O2 levels may play an important role in the modulation of the cytoplasmic free calcium concentration through BCC1.

The fusicoccin receptor of plants is a member of the 14-3-3 superfamily of eukaryotic regulatory proteins.

The receptor for the wilt-inducing phytotoxin fusicoccin was purified to homogeneity from plasma membranes of Commelina communis as a complex with the radioligand [3H]9'-nor-8'-hydroxyfusicoccin. The preparation consisted of two polypeptides with apparent molecular masses of 30.5 kDa and 31.5 kDa and with isoelectric points of around pH 5.2 and 5.3, respectively. The proteins were N-terminally blocked. Internal amino acid sequences were obtained for both polypeptides of the fusicoccin-binding complex. Sequence information, as well as subsequent immunological analysis, proved that both polypeptides are members of the eukaryotic 14-3-3 family, which comprises structurally conserved regulatory proteins of widespread occurrence and a wide range of functions. 14-3-3 isoform(s) constituting the fusicoccin receptor are distinguishable from other cellular 14-3-3 proteins by their tight association with the plasma membrane. Applying temperature-induced Triton X-114 phase separation experiments, they, as well as the target enzyme of fusicoccin action, the H(+)-ATPase, partitioned into the phospholipid-rich fraction which contains the most hydrophobic proteins. The results discussed herein provide a basis for the elucidation of the molecular mechanism of fusicoccin action.

The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants.

The phytotoxic principle, coronatine, which is present in several pathovars of the plant pathogen, Pseudomonas syringae was shown to be highly active in completely different, jasmonate-selective bioassays. At nanomolar to micromolar concentrations, coronatine induced the accumulation of defense-related secondary metabolites in several plant cell cultures, induced transcript accumulation of the elicitor-responsive gene encoding the berberine bridge enzyme of Eschscholtzia californica, as well as the coiling response of Bryonia dioica tendrils. Biological activity critically depended upon the structure of coronatine, and slight modifications, such as methylation of the carboxyl moiety or reduction of the carbonyl group, rendered the molecules almost inactive. Coronafacic acid, obtained by hydrolysis of coronatine, was also nearly inactive. Coronatine did not elicit the accumulation of endogenous jasmonic acid in the systems analyzed. While coronafacic acid is similar in structure to jasmonic acid, we found coronatine to be a close structural analogue of the cyclic C18-precursor of jasmonic acid, 12-oxo-phytodienoic acid. The phytotoxic symptoms produced by coronatine can now be understood on the basis of the toxin's action as a mimic of the octadecanoid signalling molecules of higher plants.

Effects of lead and natriuretic hormone on kinetics of sodium-potassium-activated adenosine triphosphatase: possible relevance to hypertension.

Inhibition of vascular smooth muscle sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) has been postulated as a central mechanism in enhancing vascular contractility. In the present study, kinetics of inhibition of Na-K-ATPase by lead, ouabain, and natriuretic hormone (NH) was studied in a purified hog cerebral cortex enzyme preparation. Determination of I50 values for lead, ouabain, and NH revealed that NH is the most potent inhibitor of the enzyme system (0.8 x 10(-6) M ouabain equivalents). Kinetic analyses indicated that lead and NH exhibited different inhibitory mechanisms. The inhibition by lead was noncompetitive with respect to potassium and competitive with respect to sodium and MgATP. Natriuretic hormone was noncompetitive with respect to potassium, uncompetitive with respect to MgATP, and exhibited no inhibitory effect with respect to sodium. Synergism between lead and NH in the inhibition of Na-K-ATPase raises the possibility that lead may be a contributory factor in hypertension via this mechanism.

Apparent trans-chromosomal antibody class switch in mice bearing an Igh(a) mu-chain transgene on an Igh(b) genetic background.

Native high molecular weight dextran induces a thymus-independent response in BALB/c mice. When the dextran epitope is linked to a protein carrier the response becomes thymus-dependent. IgG antibodies produced after secondary immunization had epitope specificity and idiotope of myeloma M104E. The antibody of M104E (mu, lambda1) is representative for antibodies produced by mice with immunoglobulin haplotype Igh(a) in response to immunization with dextran B1355S. Myeloma product and physiological antibodies share specificity for the alpha(1-3) glucosidic linkage and have idiotopes in common. Mice with haplotypes other than Igh(a) (e.g. Igh(b)) are unable to yield this type of response. A complete rearranged immunoglobulin mu-chain gene with a VDJ-region from BALB/c (Igh(a)) myeloma protein M104E had been introduced into the genome of BALB/c congenic mice having the haplotype Igh(b). As was shown previously in our laboratory the M104E mu-chain transgene confers Igh(a)-type reactivity to Igh(b) mice. In experiments described in this report we used the thymus-dependent form of the antigen to immunize mice bearing the M104E mu-chain, either alone or together with the lambda1-chain, as a transgene on an Igh(b) genetic background. Serological analysis revealed a class switch to IgG very similar to that seen in BALB/c mice with respect to magnitude, kinetics, epitope and idiotope specificity. The pattern of IgG subclass expression was indistinguishable in mu-chain transgenic Igh(b) and normal BALB/c mice. The class switch occurred even though, as is shown here, the transgene had become incorporated in a site not linked to the Igh locus on chromosome 12. We propose a model for this apparent trans-chromosomal class switch recombination which is based on mechanisms known for conventional switch recombination.

Octadecanoid and hexadecanoid signalling in plant defence.

Plants respond to situations requiring the initiation of inducible defence reactions with a complex array of signalling events that ultimately result in the activation of sets of defence genes. Among the chemical signals involved in the induction of defence reactions are cyclic oxylipins derived from C18- or C16-unsaturated fatty acids, the octadecanoids and the hexadecanoids. Key to understanding octadecanoid biology are the C18-metabolite 12-oxophytodienoic acid (OPDA) and the C12-compound jasmonic acid which is biosynthetically derived from 12-oxophytodienoic acid. Different octadecanoids likely have different biological functions. The bouquet of signalling compounds, rather than any single compound, is probably decisive for the biological response that results. This means that the processes regulating the pool sizes of different octadecanoids and their distribution within the plant are key to understanding octadecanoid biology. Recent results, including the cloning of several enzymes of the octadecanoid biosynthetic pathway, have provided first insights into these processes and into how the octadecanoid system is linked to other defence-related signalling pathways of the plant cell.

Predominance of high molecular weight plasma Na(+)-K(+)-ATPase inhibitor in essential hypertension.

Circulating inhibitors of the transport enzyme, sodium-potassium-activated adenosine triphosphatase (Na(+)-K(+)-ATPase), have been shown to be of possible pathogenetic importance in the mechanism of essential hypertension. Although previous studies have demonstrated the presence of both high molecular weight (HMW) and low molecular weight (LMW) natriuretic plasma Na(+)-K(+)-ATPase inhibitors, no previous attempts have been made to ascertain whether HMW or LMW forms predominate in hypertension. In this study, plasma samples obtained from 26 patients with essential hypertension, 12 normotensive controls, and six normotensives with a family history of hypertension, were separated into HMW and LMW moieties by passage through a 1 kDa Amicon membrane. The LMW moiety was separated on C18 Sep-Pak cartridges, applying a 10% step-wise acetonitrile trifluoroacetic acid gradient. The HMW moiety was further separated on Sephadex G-75. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the fraction with inhibitory activity contained a distinct 12 kDa protein band, with staining intensity depending on the presence or absence of hypertension. Na(+)-K(+)-ATPase inhibitory activity was found in several LMW fractions, but differences between hypertensives and normotensives were observed in only one fraction (0.29 +/- 0.12 SD v 0.11 +/- .12 mumol/L ouabain equivalents, P < .01). Na(+)-K(+)-ATPase inhibitory activity in the HMW fraction was 38 x the inhibitory activity in the LMW fraction and was significantly increased in hypertensives as compared to normotensive controls (10.9 +/- 8.9 v 1.3 +/- 0.8 mumol/L ouabain equivalents, P < .01). Inhibitory activity in both HMW and LMW fractions correlated positively with mean blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a human-derived sodium transport inhibitor on in vitro vascular reactivity.

Concentrations of sodium-transport inhibitors (STI) which block the sodium-potassium-ATPase pump are increased in the plasma and urine of volume-expanded and low-renin hypertensive humans and animals. To evaluate the physiologic relevance of STI to blood-pressure-raising mechanisms, we have examined the in vitro properties of STI extracted from the urine of human subjects. STI produced a significant (by ANOVA: P less than .01) dose-contraction response in the isolated rabbit femoral artery. Moreover, small noncontractile doses of STI in this in vitro preparation produced a fivefold leftward shift in the contraction dose-response curve of norepinephrine (P less than .01) and a threefold shift for angiotensin II (P less than .01); at lower, physiologic, concentrations of these vasoconstrictor hormones the amplifications in contraction caused by STI ranged from 100% to 500%. In studies in calcium-free tissue bath solutions, the direct contractile action of STI was abolished; however, its amplification of responses to norepinephrine remained, suggesting that this latter effect of STI is not entirely dependent upon calcium influx into vascular smooth muscle cells. The glycoside ouabain produced effects identical to those of STI in arteries, but its actions on a rabbit atrium preparation were different: ouabain stimulated powerful inotropic effects, whereas human STI failed to cause myocardial contractions even though the amounts of STI administered had the same sodium-transport inhibitory capacity as the ouabain. Thus, the actions of human STI are primarily in the peripheral circulation, where they directly produce arterial contractions and also enhance contractile responses to other pressor hormones, suggesting that they may have a role in regulating systemic blood pressure.

Lead-induced hypertension: possible role of endothelial factors.

The results of this study confirm that low lead (0.01%) but not high lead (0.5%) administration results in increased blood pressure in rats treated for up to 12 months. This effect appeared to be related to an imbalance of endothelially-derived vasoconstrictor and vasodilator compounds in low lead-treated animals but not in high lead-treated animals. In low lead-treated rats, measurement of plasma endothelins 1 and 3 (ET-1 and ET-3) revealed that ET-3 concentration increased significantly after both 3 months (Experimental, 92.1 +/- 9.7 v Control, 46.7 +/- 12.0 pmol/mL; P < .001) and 12 months (Experimental, 105.0 +/- 9.3 v Control, 94.1 +/- 5.0 pmol/mL; P < .01) while ET-1 was unaffected. Plasma and urinary cGMP concentrations (as a reflection of endothelium-derived relaxing factor (EDRF)) decreased significantly at 3 months (plasma, Experimental, 1.8 +/- 0.9 v Control, 4.2 +/- 1.6 pmol/mL; P < .001) and 12 months (plasma, Experimental, 2.2 +/- 0.7 v Control, 4.2 +/- 0.9 pmol/mL; P < .001). Thus, the path to development of hypertension in low lead rats may be through an increase in the concentration of the vasoconstrictor hormone, ET-3, and a decrease in the vasodilator hormone, EDRF. High levels of lead exposure did not result in hypertension, perhaps because plasma concentrations of ET-1, ET-3 and cGMP were unaltered at 3 months, while ET-1, ET-3 and cGMP concentrations were coordinately and significantly decreased at 12 months.

Na-K-ATPase inhibitor dissociated from hypertension-associated plasma protein.

It has been demonstrated that human plasma contains a low molecular weight sodium-potassium-stimulated adenosine triphosphatase (Na-K-ATPase) inhibitor, which can be dissociated from a circulating protein with a molecular weight of approximately 12,000 daltons. The dissociated factor was found to have a molecular weight <500 daltons, and shared many characteristics with ouabain. Similar to ouabain, this factor was found to be a potent inhibitor of both the Na-K-ATPase and potassium-stimulated para-nitrophenyl phosphatase (K-pNPPase) enzyme systems, and to bind to both high- and low-affinity binding sites on Na-K-ATPase, but unlike ouabain did not cross-react with digoxin antibody. The factor was further separated by HPLC and electrochemical detection into two active compounds (p-NKAI-1 and p-NKAI-2). P-NKAI-1 was demonstrated on mass spectroscopy to have a molecular weight of 408 daltons. In a vasoconstrictor assay employing rabbit femoral artery segments, this compound was a direct vasoconstrictor and potentiated the vasoconstriction produced by norepinephrine. It behaved similarly to ouabain in counteracting the relaxing effect on rabbit femoral artery of increasing potassium concentrations in the tissue bath.

Proliferation decrease in the olfactory epithelium during postnatal development.

Olfactory sensory cells are replaced continuously throughout the life of an animal. In postnatal rats proliferation density decreases dramatically, and continues to decrease into adulthood at least up to 11 months of age. This is true in both the basal cell and supporting cell populations. However, correlation analysis revealed there was no correlation in mitotic rate between the two cell types, suggesting that proliferation of the two cell types is regulated differently. With age, the rat body size and the area covered by olfactory epithelium increases. We present evidence that supporting cell proliferation provides only for growth, whereas proliferation of basal cells provides for both growth and replacement. Further, we present evidence that in older animals the sensory cells live longer than they do in younger animals.

Olfactory deprivation enhances normal spine loss in the olfactory bulb of developing ferrets.

Ferrets show a sensitive phase in their postnatal development during which they can become imprinted to food odors. At the same time the number of granule cell spines in the olfactory bulb reaches a maximum, declining significantly thereafter. In ferrets, exposed continuously to saturated levels of geraniol odor in the cage environment, the normal decline in spine number (occurring between day 60 and 90) is significantly enhanced. No such effects were observed during earlier ontogenetic phases. This late postnatal phase is further associated with a marked and significant decrease in total brain weight. The significance of these events to olfactory imprinting and plasticity in the developing brain is discussed.

Quantitation of the octadecanoid 12-oxo-phytodienoic acid, a signalling compound in plant mechanotransduction.

The octadecanoid 12-oxo-phytodienoic acid (OPDA) is an intermediate in biosynthesis of jasmonic acid in plants. A technique for the quantitation of this compound is described which has a limit of detection of 20 pg cis-OPDA corresponding to 4 ng g-1 tissue for the overall procedure and which uses high isotopic abundance [2H5]cis-(+/-)-OPDA, synthesized enzymatically by recombinant allene oxide synthase, as internal standard. The levels of cis-OPDA have been determined in a wide variety of monocotyledonous and dicotyledonous angiosperms and were found to vary considerably among different species. In mechanically stimulated tendrils of Bryonia dioica, the level of cis-OPDA increases several-fold, correlating with the initiation and progression of the free coiling response. In Phaseolus vulgaris internodes undergoing a thigmomorphogenic response, the levels of cis-OPDA were also found to increase several-fold well before the development of thigmomorphogenic symptoms. The thigmomorphogenic reaction could also be triggered by application of the octadecanoid structural analog, coronatine. Coronatine did not induce OPDA accumulation in treated tissues and is thus active per se. In both species, Bryonia dioica and Phaseolus vulgaris, the (+)-enantiomer of cis-OPDA is found and accumulates after mechanical stimulation. Our results establish 12-oxo-phytodienoic acid as a signalling compound in higher plant mechanotransduction.

Direct radioimmunoassay for the determination of 16-acetyl-gitoxin in serum.

A simple and reliable radioimmunoassay procedure for the specific determination of 16-acetyl-gitoxin, the main cardioactive metabolite of penta-acetyl-gitoxin, in serum is described. Antisera raised against 15-acetyl-gitoxin-bovine serum albumin conjugates proved to be highly specific, thus permitting direct analysis of serum. The assay is capable of detecting 0.4 ng of 16-acetyl-gitoxin. This covers the therapeutic and toxic range when 0.1 ml serum is analyzed. A single worker can assay about 100 serum samples per day in triplicate.