Sex-specific control and tuning of the pattern generator for courtship song in Drosophila.

The differentially spliced transcription factors encoded by the fruitless (fru) gene are key determinants of sexual behavior in Drosophila. They are expressed in a minority of neurons with limited dimorphisms and regulate neural processes that remain largely unknown. Here, we use light-activated ion channels to stimulate fru-expressing neurons in the thoracic-abdominal ganglia, enabling direct functional comparisons of homologous circuitry between sexes. Optical stimulation of males or females initiates the unilateral wing vibrations that normally generate the male courtship song. The pattern-generating circuit operates differently in the two sexes, producing wing movement and sound in both but authentic songs only in males and in females expressing male fru product. A song-like motor program is thus present in females but lies dormant because the neural commands required for song initiation are absent. Supplying such commands artificially reveals fru-specific differences in the internal dynamics of the song generator and sets the stage for exploring their physiological basis.

Mutational analysis of the conserved cysteines of the rat P2X2 purinoceptor.

P2X receptors are ATP-gated cation channels that are widely expressed in the brain. The extracellular domains of all seven P2X receptors contain 10 conserved cysteines, which could form disulfide bonds or binding sites for transition metals that modulate P2X receptors. To test whether these cysteines are critical for receptor function, we studied wild-type rat P2X(2) receptors and 10 mutant P2X(2) receptors, each containing an alanine substituted for a cysteine. Nine mutants were functional but had reduced maximum currents compared with wild-type P2X(2) expressed in either Xenopus oocytes or human embryonic kidney (HEK) 293 cells. The 10th mutant (C224A) did not respond to ATP when expressed in oocytes and gave very small currents in HEK 293 cells. Seven mutants (C113A, C124A, C130A, C147A, C158A, C164A, and C214A) showed rightward shifts (9- to 30-fold) in their ATP concentration-response relationships and very little potentiation by zinc. In contrast, C258A and C267A had EC(50) values similar to those of wild-type P2X(2) and were potentiated by zinc. Acidic pH potentiated wild-type and all mutant receptor currents. Despite the loss of zinc potentiation in seven mutants, these cysteines are unlikely to be exposed in the zinc-binding site, because [2-(trimethylammonium)ethyl] methanethiosulfonate bromide did not prevent zinc potentiation of wild-type receptor currents. On the basis of correlations in the maximum current, EC(50), zinc potentiation, and pH potentiation, we suggest that the following cysteine pairs form disulfide bonds: C113-C164, C214-C224, and C258-C267. We also suggest that C124, C130, C147, and C158 form two disulfide bonds, but we are unable to assign specific cysteine pairs to these two bonds.

A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum.

The detection of a little as 0.2 pg (60,000 molecules) of hepatitis B viral (HBV) DNA in human serum samples in 4 h has been demonstrated using a solution-hybridization and bead-capture method. An amplification method based on chemically crosslinked oligodeoxyribonucleotides was coupled with a horseradish peroxidase-labeling scheme for the ultimate detection of the analyte. Two sets of HBV complementary synthetic oligodeoxyribonucleotide probes containing one of two types of single-stranded (ss) overhangs were employed. These ss overhangs were used to capture the probe-analyte complex onto a bead and subsequently to label it. Detection was achieved with either a chemiluminescent or colorimetric output substrate for the enzyme. Only in the presence of the virus was label specifically bound to the support. The assay was relatively unaffected by either sample composition or by the presence of heterologous nucleic acids.

Expression level dependent changes in the properties of P2X2 receptors.

The currents of P2X(2) receptors expressed in Xenopus oocytes or HEK293 cells show significant cell-to-cell variation in many properties including the rate of desensitization and the magnitude of potentiation by zinc or acidic pH. In this study, we examined whether differences in expression levels underlie this variability. We injected Xenopus oocytes with different concentrations of RNA encoding rat P2X(2) to give a wide range of maximum current amplitudes, and then measured the potentiation of responses to 10 micro M adenosine 5'-triphosphate (ATP) by zinc or acidic pH. Individual oocytes showed potentiation ratios that ranged from 1.4- to 25-fold. Oocytes with small amplitude responses to a saturating concentration of ATP tended to have larger potentiation ratios than oocytes with large amplitude responses. This phenomenon was explained by an inverse correlation between the EC(50) for ATP and the maximum current amplitude, with the EC(50) decreasing from about 37 to 7 micro M as expression level increased. In contrast, the Hill coefficient was not correlated with the maximum current amplitude. Truncated receptors lacking the last 76 amino acids also showed an inverse correlation between the EC(50) and the maximum current amplitude. Thus, the interactions that cause expression-dependent changes in P2X(2) receptor properties must involve domains proximal to position H397.

Storage of platelet concentrates--an in vitro study of four types of plastic packs.

Four types of plastic blood collection packs were studied for their ability to preserve platelet function during a 5 d storage period. The platelet concentrates were stored in polyvinyl chloride (Tuta Laboratories), PL 1240 and PL 732 (Fenwal Laboratories) and CLX (Cutter Laboratories) packs, on a Fenwal elliptical rotator at 20 degrees-24 degrees C. Plasma pH, lactate concentration, hypotonic shock response (HSR), platelet aggregation in response to ADP, collagen and ristocetin and levels of the plasticisers, di-2-ethylhexyl phthalate (DEHP) and tri-ethylhexyl trimellitate (TEHTM), were measured. Morphological changes were assessed by electron microscopy. No significant fall in pH occurred in any type of pack but in vitro function and platelet morphology was generally better preserved in Tuta and CLX packs than in PL 732 and PL 1240. Very little TEHTM leached out of the PL 1240 and CLX packs whereas the mean concentration of DEHP in the platelet concentrates stored in Tuta packs was 27.4 mg/100 ml plasma after 5 d of storage. The results indicate that it is possible to prepare and store platelet concentrates in polyvinyl chloride plastic packs for a period of 5 d and maintain their function and viability.

When "both sides" are not enough: the restricted debate over health care reform.

In covering the health care reform debate, the major news outlets go out of their way to avoid mentioning a Canadian-style single-payer reform as a progressive alternative to the Clinton plan.

Postcoital finesse.

Mating changes female reproductive behavior in profound ways. In Drosophila, the trigger for this behavioral switch is a small peptide called sex peptide (SP), which is transferred with the male seminal fluid during insemination. Two papers in this issue of Neuron (Häsemayer et al. and Yang et al.) show that SP inhibits a small set of internal sensory neurons in the female genital tract. These neurons project to the CNS to control the female's reproductive behavior.

Significance of algal excretory products for growth of epilimnetic bacteria.

Light-stimulated uptake of CO(2) and differential filtration through Nucleopore filters were used to estimate the significance of phytoplankton excretion as a source of bacterial carbon in water samples collected at different seasons of the year in Lake Mendota, Wis. On an annual basis, about 14% of the estimated bacterial production was accounted for by algal excretion, although at certain times of year the fraction of bacterial carbon derived from algal excretion was considerably higher. About 20% of the annual primary production was estimated to pass through the bacterial component.

The role of histidine residues in modulation of the rat P2X(2) purinoceptor by zinc and pH.

P2X(2) receptor currents are potentiated by acidic pH and zinc. To identify residues necessary for proton and zinc modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X(2) receptor. Wild-type and mutant receptors were expressed in Xenopus oocytes and analysed with two-electrode voltage clamp. All mutations caused less than a 2-fold change in the EC(50) of the ATP concentration-response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 microM ATP of wild-type P2X(2) and eight mutant receptors more than 4-fold, but the response of the mutant receptor H319A was potentiated only 1.4-fold. The H319A mutation greatly attenuated the maximal potentiation that could be produced by a drop in pH, shifted the pK(a) (-log of dissociation constant) of the potentiation to a more basic pH as compared with P2X(2) and revealed a substantial pH-dependent decrease in the maximum response with a pK(a) near 6.0. Substituting a lysine for H319 reduced the EC(50) for ATP 40-fold. Zinc (20 microM) potentiated the responses to 10 microM ATP of wild-type P2X(2) and seven histidine mutants by approximately 8-fold but had virtually no effect on the responses of two mutants, H120A and H213A. Neither H120A nor H213A removed the voltage-independent inhibition caused by high concentrations of zinc. The observation that different mutations selectively eliminated pH or zinc potentiation implies that there are two independent sites of action, even though the mechanisms of pH and zinc potentiation appear similar.

Expression of a cloned Lepore globin gene.

Lepore globin is synthesized in markedly diminished amounts (approximately 10% to 15% of normal beta-globin) in human erythroid cells. To study the molecular mechanisms responsible for the diminished biosynthesis of Lepore globin, the Lepore-Boston gene was cloned from a charon phage DNA library and expressed in HeLa cells. Northern blotting and S1 nuclease analyses indicated that the Lepore gene produced less globin mRNA than a beta-gene and more than a delta-gene. The results indicate that expression of the Lepore-Boston gene in HeLa cells is reduced to an extent comparable to that seen in erythroid precursors in vivo. This indicates that the decrease in Lepore globin gene transcription is due to the delta-nucleotide sequences either in the 5' flanking region or within this gene.

Application of a rapid non-radioisotopic nucleic acid analysis system to the detection of sexually transmitted disease-causing organisms and their associated antimicrobial resistances.

We devised a versatile method for detecting nucleic acids in crude lysates of biological samples. A controlled network of nucleic acid hybrids composed of the target fragment, several oligonucleotide probes, branched DNA amplifiers, and labeled oligonucleotides is produced on a solid phase to ultimately incorporate 60 to 300 molecules of alkaline phosphatase, which are detected with a chemiluminescent substrate. The visible light output can be recorded on a luminometer or on instant black-and-white film. Assays have been developed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and for genes conferring penicillin and tetracycline resistance. Conducted much like ELISAS, the assays are performed in about 4 h (for 96 samples) in microliter dishes. The molecular detection limit of approximately 50,000 molecules of double-stranded DNA has permitted us to detect 1 to 10 x 10(3) of C. trachomatis and N. gonorrhoeae with specific probe sequences. Both plasmid and genomic target sequences can be detected by the same procedure. All of the assay components, except for a set of unmodified oligonucleotide probes, are universally applicable for all targets.

A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes.

N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxy trityl -5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.

A rapid chemiluminescent DNA hybridization assay for the detection of Chlamydia trachomatis.

With an estimated 3-4 million new cases per year, human infections from Chlamydia trachomatis are probably the most prevalent sexually transmitted disease (STD) in the United States. Diagnosis of Chlamydia is usually conducted by tissue culture methods. Direct immunofluorescence and ELISA tests have become available, but there remains a need for a test with better specificity and sensitivity. In response to this need, we have developed a rapid DNA hybridization assay using synthetic oligonucleotide probes to detect the presence of the Chlamydia trachomatis specific 7.4 kb plasmid. The assay involves solution phase hybridization of unlabelled probes, rapid capture of the probe-target duplex onto a microtitre dish surface, a new signal amplification technique that employs chemically cross-linked oligonucleotides, and an alkaline phosphatase labelled probe. Signal is obtained by reacting the labelled probe-target complex with an enzyme triggerable dioxetane substrate. Detection of the chemiluminescent output is performed either with a luminometer or by exposure to instant film. All 15 serovars of Chlamydia trachomatis react positively, while organisms known to co-inhabit the human urogenital tract react negatively.

Structure and expression of two beta genes in a beta thalassemia homozygote.

Two beta globin gene alleles have been cloned and characterized from a patient with beta + thalassemia. Both beta genes have single base mutations in the small intervening sequence (IVS 1); one 6 nucleotides and the other 110 nucleotides from the 5' end of IVS 1. Both genes lead to abnormal splicing of beta globin mRNA precursors when expressed in HeLa cells. Despite the fact that both alleles produce some normal beta globin mRNA transcripts, the patient has clinically severe beta + thalassemia (Cooley's anemia).