Heterologous transposon tagging of the DRL1 locus in Arabidopsis.

The development of heterologous transposon tagging systems has been an important objective for many laboratories. Here, we demonstrate the use of a Dissociation (Ds) derivative of the maize transposable element Activator (Ac) to tag the DRL1 locus of Arabidopsis. The drl1 mutant shows highly abnormal development with stunted roots, few root hairs, lanceolate leaves, and a highly enlarged, disorganized shoot apex that does not produce an inflorescence. The mutation was shown to be tightly linked to a transposed Ds, and somatic instability was observed in the presence of the transposase source. Some plants showing somatic reversion flowered and produced large numbers of wild-type progeny. These revertant progeny always inherited a DRL1 allele from which Ds had excised. Analysis of the changes in DNA sequence induced by the insertion and excision of the Ds element showed that they were typical of those induced by Ac and Ds in maize.     

An assay to measure the simultaneous uptake of [3H]dopamine and [14C]serotonin by the human platelet reveals an imipramine-insensitive [3H]dopamine uptake mechanism.

To determine whether a specific dopamine uptake mechanism is present on the human platelet the simultaneous uptake of [3H]dopamine and [14C]serotonin by platelets was measured. Utilising a dual radiolabel uptake technique, platelets have been shown to take up serotonin more rapidly and to a greater extent than they take up dopamine. Furthermore, at high concentrations serotonin was able to reduce dopamine uptake by platelets by 60% whereas dopamine had no effect on serotonin uptake. Similarly, imipramine and reserpine reduced (97% and 74% respectively) serotonin uptake by platelets in a dose-dependent manner, but did not affect the uptake of dopamine. Our data show that platelets take up dopamine by a mechanism independent of the imipramine-sensitive serotonin uptake mechanism. Furthermore, the increased capacity of platelets to store serotonin is because serotonin, unlike dopamine, is transported into the dense granules of the platelet.     

EFFECTS OF SUBSTRATE RELIEF ON THE DISTRIBUTION OF PERIPHYTON IN LABORATORY STREAMS. II. INTERACTIONS WITH IRRADIANCE1

Laboratory streams were used in a 42-day experiment designed to investigate how the spatial and temporal distribution of lotic periphyton created by current flow over cobble-size substrates is a affected by irradiance. The streams contained 22.5 × 22.5 × 4 cm substrate blocks and were exposed to either 385, 90 or 20 μE·m^?2·s^?1. We monitored periphyton succession in fast current regimes on top of blocks and in slower current regimes on surfaces recessed between blocks. The absolute differences in AFDW algal biomass between top and recessed substrates were significantly affected by irradiance and time. At the end of the experiment, biomass in streams exposed to 385 μE·m^?2·s^?1. was approximately 2 and 8 times greater than in streams exposed to 90 and 20 μE·m^?2·s^?1, respectively. Differences in biomass were greater between irradiance levels than between top and recessed substrates within an irradiance level. Irradiance also had a greater effect than current regime on the taxonomic composition of assemblages. Oscillatoria agardhii Gomont and Navicula minima Grun. characterized assemblages at 20 μE·m^?2·s^?1, whereas Fragilaria vaucheriae (Kütz.), Nitzschia oregona Sov., Navicula arvensis Hust. and Stigeoclonium tenue (Ag.) Kütz. were more abundant at the two higher irradiances. Detrended correspondence analysis indicated that the rate of succession was relatively high for assemblages at high irradiance and in the slow current regimes between blocks. The results suggested that in natural streams, periphyton patches produced by large differences in irradiance should have a greater effect on periphyton heterogeneity than substrate-induced patches. Moreover, the heterogeneity of algal patches produced by hydrologic differences over a substrate is constrained by irradiance level.     

Functional and biochemical disposition of 7,12-dimethylbenz[a]anthracene in murine lymphoid cells

The metabolism of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) was studied in murine lymphocytes. This carcinogen has previously been shown to be immunosuppressive to lymphocytes regardless of their ability to be induced via the Ah locus and receptor. Experiments were designed to quantify the generation of metabolites of DMBA by lymphocytes incubated with [14C]DMBA and to ascertain whether radioactivity was covalently bound to cellular macromolecules in DMBA-exposed lymphocytes. No significant metabolism of DMBA was detected in culture supernatants, except when cultures were incubated in the presence of Arochlor-induced rat liver 9000 x g supernatants (S9). Covalent binding of 14C to cellular macromolecules was enhanced approximately eightfold in the presence of S9. Inhibition of monooxygenase activity by alpha-naphthoflavone did not modulate the immunosuppressive character of DMBA. Furthermore, addition of S9 did not amplify or ablate DMBA-mediated suppression of lymphocyte proliferation to the mitogen concanavalin A (Con A). Selected metabolites of DMBA were evaluated for immunosuppressive effects in cultures stimulated with mitogens and cellular alloantigens. 7-Hydroxymethyl-12-methylbenz[a]anthracene (OHMe) and 5,6-dihydro-5,6-dihydroxybenz[a]anthracene (Diol) were found to cause only slightly greater suppression of lymphocyte responses than DMBA. Thus, it appears that metabolites of DMBA were not responsible for the immunosuppression observed in lymphocyte cultures and that lymphocytes were not equipped to metabolize any significant amount of DMBA. These data lend support to the hypothesis that parent compound alone is responsible for the immunosuppressive effects observed in murine lymphocyte culture.     

Ape-like endocast of “ape-man” Taung

I have identified and illustrated a spherical “dimple” or “depression” on the Taung endocast as indicating the most likely position of the medial end of the lunate sulcus but have not drawn an actual lunate sulcus on Taung because one is not visible. In a recent paper, R.L. Holloway (Am. J. Phys. Anthropol. 77:27–33, 1988) drew a lunate sulcus on his copy of the Taung endocast, incorrectly attributed this sulcus to me, and used it to obtain a ratio of 0.254 to describe “Falk's” position of the lunate sulcus. My published ratio of 0.242 for Taung (Falk: Am. J. Phys. Anthropol. 67:313–315, 1985a) was not considered, although the focus of Holloway's paper was my assessment of the position of the lunate sulcus. Holloway also excluded published ratios for a chimpanzee in my collection from his statistical analysis but, even so, my published ratio for Taung is still only 1.5 standard deviations from his chimpanzee mean. If my chimpanzee brain is included in the sample, the ratio for Taung is 1.2 standard deviations from the mean. Furthermore, one of Holloway's own chimpanzees (B60–7) has a ratio of 0.241, just 0.001 below my ratio for Taung. There is no sulcus where Holloway has drawn one on Taung, his “F(LS)” is not mine, his 2 mm error is not mine, and the correct ratio for my measurement of Tuang is the one that I published, not the one that Holloway attributes to me. Assessment of Holloway's chimpanzee data supports my claim that the dimple on the Taung endocast is within the chimpanzee range for the medial end of the lunate sulcus.     

Intermittent altitude exposures improve muscular performance at 4,300 m.

Chronic altitude residence improves muscular performance at altitude, but the effect of intermittent altitude exposures (IAE) on muscular performance at altitude has not been defined. The purpose of this study was to determine the effects of 3 wk of IAE, in combination with rest and cycle training, on muscular performance at altitude. Six lowlanders (23 +/- 2 yr, 77 +/- 6 kg; means +/- SE) completed a cycle time trial and adductor pollicis endurance test at sea level and during a 30-h acute exposure to 4,300 m altitude equivalent (barometric pressure = 446 mmHg) once before (pre-IAE) and once after (post-IAE) a 3-wk period of IAE (4 h/day, 5 days/wk, 4,300 m). During each IAE, three subjects cycled for 45-60 min/day at 60%-70% of maximal O2 uptake and three subjects rested. Cycle training during each IAE did not appear to affect muscular performance at altitude. Thus data from all six subjects were combined. Three weeks of IAE resulted in 1) a 21 +/- 6% improvement (P < 0.05) in cycle time-trial performance (min) from pre-IAE (32.8 +/- 3.7) to post-IAE (24.8 +/- 1.2), 2) a 63 +/- 26% improvement (P < 0.05) in adductor pollicis endurance (min) from pre-IAE (9.2 +/- 2.8) to post-IAE (14.8 +/- 4.2), and 3) a 10 +/- 4% increase (P < 0.05) in resting arterial O2 saturation (%) from pre-IAE (82 +/- 2) to post-IAE (90 +/- 1). These improvements in muscular performance after IAE correlated strongly with increases in resting arterial O2 saturation and were comparable to those reported previously after chronic altitude residence. IAE may therefore be used as an alternative to chronic altitude residence to facilitate improvements in muscular performance in athletes, soldiers, mountaineers, shift workers, and others that are deployed to altitude.     

Determinants of performance in the isocitrate dehydrogenase of Escherichia coli.

The substrate specificity of the NADP-dependent isocitrate dehydrogenase of Escherichia coli was investigated by combining site-directed mutagenesis and utilization of alternative substrates. A comparison of the kinetics of the wild-type enzyme with 2R-malate reveals that the gamma-carboxylate of 2R,3S-isocitrate contributes a factor of 12,000,000 to enzyme performance. Analysis of kinetic data compiled for 10 enzymes and nine different substrates reveals that a factor of 1,650 can be ascribed to the hydrogen bond formed between S113 and the gamma-carboxylate of bound isocitrate, a factor of 150 to the negative charge of the gamma-carboxylate, and a factor of 50 for the gamma-methyl. These results are entirely consistent with X-ray structures of Michaelis complexes that show a hydrogen bond positions the gamma-carboxylate of isocitrate so that a salt bridge can form to the nicotinamide ring of NADP.     

Extracellular addition of a domain of HIV-1 Vpr containing the amino acid sequence motif H(S/F)RIG causes cell membrane permeabilization and death

Vpr is a virion-associated protein of human immuno-deficiency virus type 1 (HIV-1) whose function in acquired immune deficiency syndrome (AIDS) has been uncertain. We previously employed yeast as a model to examine the effects of Vpr on basic cellular functions; intracellular Vpr was shown to cause cell-growth arrest and structural defects, and these effects were caused by a region of Vpr containing the sequence HFRIGCRHSRIG. Here we show that peptides containing the H(S/F)RIG amino acid sequence motif cause death when added externally to a variety of yeast including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Candida albicans and Schizosaccharomyces pombe. Such peptides rapldly entered the cell from the time of addition, resulting in cell death. Elevated levels of ions, particularly magnesium and calcium ions, abrogated the cytotoxic effect by preventing the Vpr peptides from entering the cells. Extracellular Vpr found in the serum, or breakdown products of extracellular Vpr, may have similar effects to the Vpr peptides described here and could explain the death of uninfected by-stander cells during AIDS.     

Poplar (Populus nigra L.) plants transformed with aBacillus thuringiensis toxin gene: insecticidal activity and genomic analysis

Insect-resistant poplar (Populus nigra L.) plants have been produced by infecting leaves withAgrobacterium tumefaciens strains carrying a binary vector containing different truncated forms of aBacillus thuringiensis (B.t.) toxin gene under a duplicated CaMV 35S promoter. Putative transgenic plants were propagated by cuttings at two experimental farms (in Beijing and Xinjiang, China). At 2–3 years after transformation, 17 of them were selected on the bases of insect-tolerance and good silvicultural traits, and evaluated for insect resistance, for the presence of theB.t. toxin DNA fragment (Southern blots and PCR) and for the expression of the transgene (western and northern blots). Somaclonal variation, as suggested by the appearance of permanent changes in the shape of the leaves, was also investigated with molecular tools (RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA) and microsatellite DNA).Bioassays withApochemia cineraius andLymantria dispar on the leaves of the selected clones showed different and, in some cases, high levels of insecticidal activity. The molecular analysis demonstrated integration and expression of the foreign gene. Somatic changes were correlated to extensive genomic changes and were quantified in dendrograms, in terms of genomic similarity. The analysis of control plants suggested that genomic changes were correlated to thein vitro culture step necessary forA. tumefaciens-mediated gene transfer, rather than to the integration of the foreign genes.Three transgenic clones (12, 153 and 192), selected for insect resistance, reduced morphological changes and promising silvicultural traits, are now under large-scale field evaluation in six different provinces in China.     

Expression, Purification, and Initial Characterization of the Recombinant Storage Protein Precursor ofTheobroma cacao

The gene encoding the 67-kDa cocoa storage protein precursor has been cloned fromTheobroma cacaoand expressed inEscherichia coliusing the pET expression system. The recombinant storage protein has been renatured from inclusion bodies at 30°C using 20 m glycine–NaOH buffer, pH 10.0, containing 1 m oxidized glutathione and 0.1% Brij. The renatured protein was purified and demonstrated to adopt a stable native conformation by optical spectroscopy. Secondary structure analysis from circular dichroism indicated the protein to be 23% α-helix and 38% β-sheet, in close agreement with values obtained using a secondary structure prediction program.