Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene.

Indole-3-acetonitrile (IAN) is a candidate precursor of the plant growth hormone indole-3-acetic acid (IAA). We demonstrated that IAN has auxinlike effects on Arabidopsis seedlings and that exogenous IAN is converted to IAA in vivo. We isolated mutants with reduced sensitivity to IAN that remained sensitive to IAA. These mutants were recessive and fell into a single complementation group that mapped to chromosome 3, within 0.5 centimorgans of a cluster of three nitrilase-encoding genes, NIT1, NIT2, and NIT3. Each of the three mutants contained a single base change in the coding region of the NIT1 gene, and the expression pattern of NIT1 is consistent with the IAN insensitivity observed in the nit1 mutant alleles. The half-life of IAN and levels of IAA and IAN were unchanged in the nit1 mutant, confirming that Arabidopsis has other functional nitrilases. Overexpressing NIT2 in transgenic Arabidopsis caused increased sensitivity to IAN and faster turnover of exogenous IAN in vivo.     

Physical interactions between members of the DnaK chaperone machinery: characterization of the DnaK.GrpE complex

Many of the functions of the Escherichia coli Hsp 70, DnaK, require two cofactors, DnaJ and GrpE. GrpE acts as a nucleotide exchange factor in the DnaK reaction cycle but the details of its mechanism remain unclear. GrpE has high affinity for monomeric native DnaK, with a K_d estimated at ≤50 nM. GrpE is a very asymmetric molecule and exists as either a dimer or trimer in its native state. The stoichiometry of GrpE to DnaK in the isolated complex was 3:1, suggesting a trimer. Formation of the complex is quite fast (k_on >1 S⁻¹, whereas the off-rate is very slow on the HPLC timescale (k_off ≤ 10⁻⁴ S⁻¹). GrpE has no affinity for ATP or ADP, nor the oligomeric and moltn globule states of DnaK. The complex is much more thermally stable than either GrpE or DnaK alone, and prevents the formation of the molten globule-like state of DnaK at physiologically relevant temperatures. Formation of the complex does not cause any change in secondary structure, as determined by the lack of change in the circular dichroism spectrum. However, binding of GrpE induces a similar tertiary strcutral change in DnaK to that induced by binding of ATP₁ based on the blue shift in λ_max from the fluroscence of the single tryptophan in DnaK. The nucleotide exchange properties of GrpE can be explained by the conformational change which may represent the opening of the nucleotide cleft on DnaK, subsequently inducing a low affinity state for ADP.     

Violaxanthin accessibility and temperature dependency for de-epoxidation in spinach thylakoid membranes

Using DTT and iodoacetamide as a novel irreversible method to inhibit endogenous violaxanthin de-epoxidase, we found that violaxanthin could be converted into zeaxanthin from both sides of the thylakoid membrane provided that purified violaxanthin de-epoxidase was added. The maximum conversion was the same from both sides of the membrane. Temperature was found to have a strong influence both on the rate and degree of maximal violaxanthin to zeaxanthin conversion. Thus only 50% conversion of violaxanthin was detected at 4 °C, whereas at 25 °C and 37 °C the degree of conversion was 70% and 80%, respectively. These results were obtained with isolated thylakoids from non-cold acclimated leafs. Pigment analysis of sub-thylakoid membrane domains showed that violaxanthin was evenly distributed between stroma lamellae and grana partitions. This was in contrast to chlorophyll a and -carotene which were enriched in stroma lamellae fractions while chlorophyll b, lutein and neoxanthin were enriched in the grana membranes. In combination with added violaxanthin de-epoxidase we found almost the same degree of conversion of violaxanthin to zeaxanthin (73–78%) for different domains of the thylakoid membrane. We conclude that violaxanthin de-epoxidase converts violaxanthin in the lipid matrix and not at the proteins, that violaxanthin does not prefer one particular membrane region or one particular chlorophyll protein complex, and that the xanthophyll cycle pigments are oriented in a vertical manner in order to be accessible from both sides of the membrane when located in the lipid matrix.     

Sponge Carrying by Dolphins (Delphinidae,Tursiops sp.): A Foraging Specialization Involving Tool Use?

During long-term research on bottlenose dolphins (Tursiops sp.) in Shark Bay, Western Australia, several individuals were observed carrying sponges, Echinodictyum mesenterinum, on their rostra. Over multiple years, five regularly sighted individuals were usually carrying sponges when encountered (67–100% of encounters). Four additional regularly sighted individuals were observed with sponges just one time each. All five individuals that routinely carried sponges were female. Two of the anomalous, one-time carriers were female, one was likely female, and one was male. Most observations of sponge carrying occurred within a restricted area, a relatively deep water channel (8–10 m deep). Surface observations of sponge carrying, including focal animal observations, revealed a stereotyped surfacing and diving pattern, and occasional indications of prey consumption. Three hypotheses are considered regarding the function of sponge carrying: 1. dolphins were playing with the sponges; 2. the sponges contain some compound of use to the dolphins (e.g. for medicinal purposes); and 3. the sponges were used as a tool to aid in foraging. The foraging tool hypothesis is best supported, but the exact manner in which sponges are used remains to be discovered. Sponge carrying is a behavioural specialization, probably involving foraging, and regularly engaged in by only a small proportion of female dolphins in Shark Bay.     

Modulation of Phosphorylation of Neuronal Cytoskeletal Proteins by Neuronal Depolarization

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5.2. In aggregating neuronal–glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM.3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.     

Backcrossing of nematode-resistant sugar beet: a second nematode resistance gene at the locus containing Hs1pro-1?

Beet cyst nematode-resistant sugar beet plants, containing the Hs1^pro-1 locus from Beta procumbens, show a female transmission frequency of the resistance of ca. 90%. Such plants often suffer from tumour formation on leaves and root systems, and from the occurrence of a so-called multi-top phenotype. With the aim of obtaining resistant sugar beet material lacking these negative traits, nematode-resistant plants with a reduced size of the chromosome segment of the wild beet that carries the Hs1^pro-1 gene were selected from backcrosses between the resistant stocks B883 or AN1-65-2 and susceptible sugar beet (Beta vulgaris). Analysis of such plants, referred to as Sat-minus plants, showed that the transmission frequency of the resistance to subsequent generations had dropped dramatically to ca. 0.5%. The multi-top phenotype was still present in the newly selected material, indicating that improvement of the resistant sugar beet material by further backcrossing will be hard to achieve. Two of the selected resistant offspring plants were analysed at the molecular level. With the aid of AFLP markers it was found that the size of the alien chromosome segment had decreased to 35% and 17% of the original size, respectively. Surprisingly, both plants had lost the Hs1^pro-1 nematode resistance gene that recently was isolated from the original introgression material. This shows that more than one gene conferring resistance must be present in the locus in B883 and AN1-65-2 carrying the resistance gene Hs1^pro-1.     

Cerebral areas associated with motor control of speech in humans

Murphy, K., D. R. Corfield, A. Guz, G. R. Fink, R. J. S. Wise, J. Harrison, and L. Adams. Cerebral areas associated withmotor control of speech in humans. J. Appl.Physiol. 83(5): 1438-1447, 1997.We have definedareas in the brain activated during speaking, utilizing positronemission tomography. Six normal subjects continuously repeated thephrase "Buy Bobby a poppy" (requiring minimal languageprocessing) in four ways: A) spoken aloud, B) mouthed silently,C) without articulation, andD) thought silently. Statisticalcomparison of images from conditions Awith C andB withD highlighted areas associated witharticulation alone, because control of breathing for speech wascontrolled for; we found bilateral activations in sensorimotor cortexand cerebellum with right-sided activation in the thalamus/caudate nucleus. Contrasting images from conditionsA with B andC with D highlighted areas associated withthe control of breathing for speech, vocalization, and hearing, becausearticulation was controlled for; we found bilateral activations insensorimotor and motor cortex, close to but distinct from theactivations in the preceding contrast, together with activations inthalamus, cerebellum, and supplementary motor area. In neithersubtraction was there activation in Broca's area. These resultsemphasize the bilaterality of the cerebral control of "speaking"without language processing.

     

Steroid metabolism in the hormone dependent MCF-7 human breast carcinoma cell line and its two hormone resistant subpopulations MCF-7/LCC1 and MCF-7/LCC2

Androgen and estrogen metabolism was investigated in the hormone-dependent human breast cancer cell line MCF-7 and its two hormone-resistant sublines MCF-7/LCC1 and MCF-7/LCC2. Using the product isolation method, the activity of aromatase, 5-reductase, 3/β-hydroxysteroid oxidoreductase and 17β-hydroxysteroid oxidoreductase were investigated isolating the following steroids: estriol (E₃), estradiol (E₂), estrone (E₁), 3/β-androstanediol (A-diol), testosterone (T), dihydrotestosterone (DHT), androsterone (AND), androstenedion (4-AD) and androstanedione (A-dion). For all experiments, cells were preincubated with cortisol and subsequently incubated with [¹⁴C]T or [¹⁴C]4-AD as the substrate in medium without phenol red and with serum charcoal stripped of steroids. The results showed no aromatase activity in any of the cell lines under the experimental conditions used, and preincubation with cortisol had no effect on the enzyme activity. With [¹⁴C]T as the substrate, the metabolized level of DHT was very similar in the three cell lines, though MCF-7/LCC1 and MCF-7/LCC2 utilized the substrate to a much lesser extent. The amount of DHT and 4-AD produced were comparable in the two hormone-resistant cell lines, while the amount of 4-AD was significantly higher in MCF-7 cells. No differences in enzyme activity were found in the three cell lines when [¹⁴C]4-AD was used as the substrate. This study showed an altered androgen metabolism in the MCF-7/LCC1 and MCF-7/LCC2 sublines compared to the parent MCF-7. However, since treatment with DHT and T inhibited cell growth equally well in all three tumor cell lines, it is unlikely that the found differences in steroid metabolism was involved in the acquisition of the endocrine resistance of the two MCF-7 sublines.     

Precision and accuracy of radioimmunoassay in the analysis of endogenous 3-indoleacetic acid from needles of scots pine

The precision and accuracy of a radioimmunoassay (RIA) for 3-indoleacetic acid (IAA) were assessed when applied to the analysis of extracts from Pinus sylvestris seedlings. It was found that extracts contained contaminants that adversely affected both the precision and accuracy of quantitative estimates of IAA. When samples were subjected to purification procedures prior to RIA, accurate and more precise estimates were obtained. Parallel analysis with high performance liquid chromatography using a fluorescence detector (HPLC-FL) indicated that HPLC-FL provided accurate estimates of the IAA content of Pinus extracts at the same stage of purification as RIA. The precision of HPLC-FL estimates was similar to that obtained with RIA.