rBAT is an Amino Acid Exchanger with Variable Stoichiometry

The rBAT protein, when expressed in Xenopus oocytes, was previously shown to reproduce the selectivity of the Na⁺-independent neutral and basic amino acid transport system called b^o,+. More recently, the capacity of rBAT to generate a transmembrane current was demonstrated when addition of neutral amino acids stimulated the efflux of cations (presumably basic amino acids) in rBAT-injected oocytes. In the present paper, aminoisobutyric acid (AIB), a neutral amino acid analogue, was shown to induce outward currents (efflux of basic amino acids) through rBAT similar to those caused by alanine in terms of affinity, maximal currents and I-V curves. Despite generating similar currents, the AIB transport rate was more than 30 times lower than that of alanine, thus challenging the assumption that rBAT functions as a classical exchanger. Experiments using a cut-open oocyte voltage clamp demonstrated that AIB was capable of stimulating rBAT-mediated currents from either side of the membrane. AIB, like alanine, was able to stimulate the efflux of radiolabeled alanine and arginine while no rBAT-mediated efflux was measurable in the absence of external rBAT substrates. These results demonstrate that (i) the presence of amino acids is required on both sides of the membrane for rBAT to mediate amino acid flux and thus rBAT must be some type of exchanger but (ii) rBAT-mediated amino acid influx is not stoichiometrically related to the efflux. A model of a ``double gated pore' is proposed to account for these properties of rBAT, which contravene standard models of exchangers and other transporters. Received: 15 June 1995/Revised: 21 September 1995     

Synthesis of an inhibitor of glutamyl-tRNA synthetase

A specific inhibitor of glutamyl-tRNA synthetase, analog of glutamyl-adenylate, the N⁶-benzoyl-L-glutamol AMP, has been synthesized. This compound does not inhibit glutaminyl-tRNA synthetase.     

Large scale sequencing projects using rapidly prepared double-stranded plasmid DNA.

We have developed a simple rapid plasmid DNA mini-preparation method which yields DNA of sufficient quality to be used in large scale sequencing projects. The method, which is a modification of the alkaline method of Birnboim and Doly (1979), requires less than two hours. We have eliminated the use of organic extractions, RNase digestion and alkaline denaturation of the DNA for annealing of the primer. The proportion of supercoiled plasmid DNA obtained is close to 100%. Greater than 80% of the clones yield at least 500 bp of sequence information per primer. The sequencing reactions from these double-stranded templates can be done on both strands using the universal and reverse sequence primers with the usual two reactions per primer, one to read close to the primer and one to read far from it. Thus, each clone yields at least 1 kb of sequence information. The preparation of the templates and the sequencing reactions can be done in less than three hours so that the sequencing gel can be run the same day.     

THE EFFECTS OF LIGHT AND NITROGEN ON GROWTH,PIGMENT CONTENT,AND BIOCHEMICAL COMPOSITION OF GRACILARIA FOLIIFERA V. ANGUSTISSIMA (GIGARTINALES,RHODOPHYTA)1

The combined effects of light intensity and nitrogen (NO₃^?) on growth rate, pigment content, and biochemical composition of Gracilaria foliifera v. angustissima (Harvey) Taylor was investigated using outdoor continuous cultures. Growth of Gracilaria increased linearly with increasing light to 0.43 doublings d^?1 at high light levels (383 ly d^?1 of in situ light), suggesting that light may often limit growth of this plant in nature. Chlorophyll a and phycoerythrin contents were inversely proportional to light level and growth rate. However, pigment content did not affect the growth capacity of Gracilaria. There was no increase in growth or pigment content with increasing additions of nitrogen. The low nitrogen treatment was unenriched seawater that had higher NO₃^? levels than most coastal waters (influent = 8.61 μM; residual = 0.94 μM). When growing near its maximum rate under high light intensities, Gracilaria had a significantly (P < 0.001) lower phycoerythrin: chlorophyll a ratio (phyco: Chl a) than did Gracilaria growing more slowly under lower light (Phyco:Chl a of 2.8 ± 0.2 vs. 3.8 ± 0.3). Faster growing plants also had C:N ratios above 10, indicating N- limitation. In addition to harvesting light the phycobiliproteins of Gracilaria may store nitrogen. Growth rates of Gracilaria correlated negatively with ash (r =–0.85) and positively with the carbon: phycoerythrin ratio (r = 0.85), suggesting that these two indices can be used to estimate growth in the field.     

Pathogens in children with severe combined immune deficiency disease or AIDS.

We evaluated the frequency and severity of illnesses caused by various microbial pathogens in 15 children with severe combined immune deficiency disease (SCID) and 8 with acquired immune deficiency syndrome (AIDS). There were 35 viral, 23 bacterial, 19 mycotic and 13 parasitic infections. Nineteen of the 23 patients died of infection; Pneumocystis carinii pneumonia, giant-cell pneumonia due to paramyxoviruses and various disseminated viral infections were responsible for most deaths in both groups. The emerging role of paramyxoviruses was illustrated by the fact that they were responsible for giant-cell pneumonia in seven patients. Viral enteric infections were frequent in both groups. The variety of infectious microorganisms and the severity of resulting illnesses in the patients with AIDS were similar to those in the patients with SCID.     

Discovery and characterization of chemical signals for citrus root weevil, Diaprepes abbreviatus

The tropical root weevil Diaprepes abbreviatus (L. 1758) (Coleoptera: Curculionidae) is a polyphagous insect from the Caribbean Islands and an invasive insect in the southern part of the United States where it is pest of citrus crops and ornamental trees. Adults feed upon foliage where aggregation, mating and oviposition take place. Here, the headspace volatiles from Citrus macrophylla Wester (Rutaceae), D. abbreviatus adults feeding on this plant, adults alone and adult feces, were collected by aeration and solid-phase microextraction (SPME) for analysis by gas chromatography-linked mass spectrometry (GC-MS). Electrophysiological responses of weevil antennal receptors to volatile headspace extracts and synthetic analogues were recorded by gas chromatography-linked electroantennographic detection (GC-EAD) and electroantennograms (EAGs). Antennal responses were recorded to the monoterpenes (R)-(−)-linalool, citronellal, nerol, citral, and geraniol; all present in the headspace of C. macrophylla. Antennal responses were also recorded to carvacrol, present in the headspace of adults and adult feces. The green leaf volatiles cis-3-hexen-1-ol and trans-2-hexen-1-ol, produced by other host plants, elicited reliable responses on the D. abbreviatus antenna. When comparing EAGs between (±)-linalool and (R)-(−)-linalool, no significant difference was found; responses to (R)-(+)-citronellal were larger than for (S)-(−)-citronellal. Among the individual compounds and blends tested using an open T-track dual choice olfactometer, only the blend of (±)-linalool, cis-3-hexen-1-ol and carvacrol (source dose 25:25:2.5 μg) elicited significant attraction of females, the same blend was repellent for males. The biologically active compounds found here likely play a role in host finding by D. abbreviatus and other interactions of the insect with its hostplant. Handling editor: Sam Cook