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Biomedical subjects

K Kaiser

Publications and source records attributed to K Kaiser.

At least 19 recordsLinked to original sources
PubMed2001

Production of refractory dissolved organic matter by bacteria.

Most of the oceanic reservoir of dissolved organic matter (DOM) is of marine origin and is resistant to microbial oxidation, but little is known about the mechanisms of its formation. In a laboratory study, natural assemblages of marine bacteria rapidly (in <48 hours) utilized labile compounds (glucose, glutamate) and produced refractory DOM that persisted for more than a year. Only 10 to 15% of the bacterially derived DOM was identified as hydrolyzable amino acids and sugars, a feature consistent with marine DOM. These results suggest that microbial processes alter the molecular structure of DOM, making it resistant to further degradation and thereby preserving fixed carbon in the ocean.

Amino Acids↗
PubMed2001

Real time quantitative PCR and RT--PCR for analysis of Pneumocystis carinii hominis.

Pneumocystis carinii hominis is a common cause of pneumonia in immunocompromised patients and particularly in those infected by HIV. Giemsa- and Gomori--Grocott-stained smears are widely used for detection and quantification of this opportunistic fungus obtained from biological samples or from in vitro culture. But these methods are fastidious and time-consuming. Thus, instead of performing a count of organisms, we focused our attention on the level of specific DNA by a quantitative PCR technique. This procedure has the advantage of greater precision and more objectivity. To verify the presence of organisms, quantitative RT--PCR based on DHFR and a cell cycle mRNA have been developed. In this current study, we present a detailed description of these methods and their applications for analysis of P. carinii hominis.

Animals↗
PubMed2001

Synchronized neural activity in the Drosophila memory centers and its modulation by amnesiac.

The mushroom bodies are key features of the brain circuitry for insect associative learning, especially when evoked by olfactory cues. Mushroom bodies are also notable for the close-packed parallel architecture of their many intrinsic neuronal elements, known as Kenyon cells. Here, we report that Kenyon cells of adult Drosophila exhibit synchronous oscillation of intracellular calcium concentration, with a mean period of approximately 4 min. Robust oscillation within a dissected brain persists for hours in insect saline and is strongly modulated in amplitude by the product(s) of the memory consolidation gene, amnesiac. It is also sensitive to pharmacological agents specific for several classes of ion channel and for acetylcholine and GABA receptors. A role in memory consolidation involving transcriptionally mediated synaptic strengthening is proposed.

Aequorin↗
PubMed2000

The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory.

Mutations in the amnesiac gene in Drosophila affect both memory retention and ethanol sensitivity. The predicted amnesiac gene product, AMN, is an apparent preproneuropeptide, and previous studies suggest that it stimulates cAMP synthesis. Here we show that, unlike other learning-related Drosophila proteins, AMN is not preferentially expressed in mushroom bodies. Instead, it is strongly expressed in two large neurons that project over all the lobes of the mushroom bodies, a finding that suggests a modulatory role for AMN in memory formation. Genetically engineered blockade of vesicle recycling in these cells abbreviates memory as in the amnesiac mutant. Moreover, restoration of amn gene expression to these cells reestablishes normal olfactory memory in an amn deletion background. These results indicate that AMN neuropeptide release onto the mushroom bodies is critical for normal olfactory memory.

Animals↗
PubMed2000

Determination of amino sugars in environmental samples with high salt content by high-performance anion-exchange chromatography and pulsed amperometric detection.

Amino sugars were determined in natural samples, including seawater, using high-performance anion-exchange chromatography with pulsed amperometric detection and a new-off-line sample cleanup procedure. Samples were hydrolyzed with 3 M HCl for 5 h (100 degrees C) and neutralized with anion retardation resin. Before injection, salts and organic contaminants were removed with a strong cation exchanger in the Na+ form. Detection limits for amino sugars were between 1 and 4 nM (signal-to-noise ratio 3), allowing for the first time quantification of amino sugars in seawater without preconcentration. Precision was 2-11% at the 20 nM level. The relatively simple and rapid sample preparation makes it suitable for routine analyses.

Amino Sugars↗
PubMed2000

A novel Drosophila alkaline phosphatase specific to the ellipsoid body of the adult brain and the lower Malpighian (renal) tubule.

Two independent Drosophila melanogaster P(GAL4) enhancer-trap lines revealed identical GAL4-directed expression patterns in the ellipsoid body of the brain and in the Malpighian (renal) tubules in the abdomen. Both P-element insertions mapped to the same chromosomal site (100B2). The genomic locus, as characterized by plasmid rescue of flanking DNA, restriction mapping, and DNA sequencing, revealed the two P(GAL4) elements to be inserted in opposite orientations, only 46 bp apart. Three genes flanking the insertions have been identified. Calcineurin A1 (previously mapped to 21E-F) lies to one side, and two very closely linked genes lie to the other. The nearer encodes Aph-4, the first Drosophila alkaline phosphatase gene to be identified; the more distant gene [l(3)96601] is novel, with a head-elevated expression, and with distant similarity to transcription regulatory elements. Both in situ hybridization with Aph-4 probes and direct histochemical determination of alkaline phosphatase activity precisely matches the enhancer-trap pattern reported by the original lines. Although the P-element insertions are not recessive lethals, they display tubule phenotypes in both heterozygotes and homozygotes. Rates of fluid secretion in tubules from c507 homozygotes are reduced, both basally, and after stimulation by CAP(2b), cAMP, or Drosophila leucokinin. The P-element insertions also disrupt the expression of Aph-4, causing misexpression in the tubule main segment. This disruption extends to tubule pigmentation, with c507 homozygotes displaying white-like transparent main segments. These results suggest that Aph-4, while possessing a very narrow range of expression, nonetheless plays an important role in epithelial function.

Alkaline Phosphatase↗
PubMed2000

The effects of ectopic white and transformer expression on Drosophila courtship behavior.

The sex determining genes of Drosophila males and females function to establish the potential for sex-specific behaviors. Previous studies suggest that ectopic GAL4-directed misexpression of the female-specific isoform of the sex-determining gene transformer (tra) in specific sub-domains of an otherwise male brain can lead to bisexual courtship behavior, thus identifying brain domains that may mediate sex-specific behavior. However, expression of mini-white, the marker gene used in both P[GAL4] and P[UAS(G)] constructs, also induces males to court other males, questioning whether GAL4-mediated tra expression alone can induce bisexual behavior. Here we demonstrate the consequences of inducing mutations in the mini-white genes within P[GAL4] and P[UAS(G)] constructs to generate flies in which a white mutant phenotype is revealed. In these mini-white mutant strains, P[GAL4]-mediated transformer expression alone is both sufficient and necessary to generate bisexual behavior. In addition, using RT-PCR, we reveal the presence of female transcripts of doublesex and fruitless in the brains of otherwise male (XY) flies exhibiting P[GAL4]-directed tra-expression, demonstrating that P[GAL4]-directed tra is functional at the molecular level. We conclude that P[GAL4]-directed misexpression of tra is responsible for the bisexual behavior previously described and that this is mediated via sex-specific splicing of dsx and fru. Our results support the validity of such strategies for identifying regions of the fly brain that underlie sex-specific behaviors.

ATP-Binding Cassette Transporters↗
PubMed1999

Genetic analysis of the Drosophila ellipsoid body neuropil: organization and development of the central complex.

The central complex is an important center for higher-order brain function in insects. It is an intricate neuropil composed of four substructures. Each substructure contains repeated neuronal elements which are connected by processes such that topography is maintained. Although the neuronal architecture has been described in several insects and the behavioral role investigated in various experiments, the exact function of this neuropil has proven elusive. To describe the architecture of the central complex, we study 15 enhancer-trap lines that label various ellipsoid body neuron types. We find evidence for restriction of gene expression that is correlated with specific neuronal types: such correlations suggest functional classifications as well. We show that some enhancer-trap patterns reveal a single ellipsoid body neuron type, while others label multiple types. We describe the development of the ellipsoid body neuropil in wild-type animals and propose developmental mechanisms based on animals displaying structural mutations of this neuropil. The experiments performed here demonstrate the degree of resolution possible from the analysis of enhancer-trap lines and form a useful library of tools for future structure/function studies of the ellipsoid body.

Animal Structures↗
PubMed1999

Rapid estimation of avidin and streptavidin by fluorescence quenching or fluorescence polarization.

A new biotin-carboxyfluorescein conjugate has been presented in the accompanying study (G. Kada et al., Biochim. Biophys. Acta 000 (1999) 000-000) which contains ethylene diamine as a 4-atom spacer. This so-called biotin-4-fluorescein showed exceptionally fast and tight binding to avidin and streptavidin, and binding was accompanied by strong quenching. In the present study the specific quenching of 'biotin-4-fluorescein' was utilized to measure (strept)avidin concentrations (0.2-2 nM) by the extent of fluorescence quenching at 8 nM ligand concentration. Adsorption of (strept)avidin to the assay tubes was suppressed by inclusion of bovine serum albumin (0.1 mg/ml). Virtually the same specific response to avidin and streptavidin was also observed with commercial 'fluorescein-biotin', except that >10 h incubation times were required. The slow association of 'fluorescein-biotin' was attributed to the anti-cooperative binding which is due to the much longer spacer as compared to 'biotin-4-fluorescein'. The third ligand tested in this study was 'biotin-4-FITC' which was analogous to 'biotin-4-fluorescein' except that carboxyfluorescein was replaced by the fluorescein isothiocyanate residue. Surprisingly, this probe was much less quenched by avidin but this was compensated by an exceptionally high fluorescence polarization in the avidin-bound state. In conclusion, the new ligand 'biotin-4-fluorescein' appeared to be the most general and convenient probe: quenching was most pronounced and linearly dependent on (strept)avidin concentrations, the dose response for streptavidin was almost the same as for avidin, and the association kinetics were fast enough to reach equilibrium within 30 min incubation time.

Avidin↗
PubMed1999

Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR.

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp. hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.

AIDS-Related Opportunistic Infections↗
PubMed1999

Abnormal courtship conditioning in males mutant for the RI regulatory subunit of Drosophila protein kinase A.

The previously described site-selected P-element mutagenesis of a Drosophila gene encoding the regulatory subunit of cAMP-dependent protein kinase generates mutants that have defective behavior in the olfactory learning test. Here we describe the effect of the same mutations in a courtship conditioning assay. Wild-type males can distinguish between virgin females (which they court vigorously), and fertilized females (which they court less vigorously). After exposure to fertilized females, wild-type males modify their behavior by decreasing courtship to subsequent target virgins, an effect that may last for many hours. Like wild-type males, PKA-RI mutant males are also able to distinguish between virgin and fertilized females. PKA-RI males also modify their behavior towards virgin females after prior exposure to a fertilized female, but such an effect is short-lived, suggesting a defect in memory rather than learning. We also show that under these conditions the behavior of PKA-RI males is similar to that of amnesiac, dunce and rutabaga males.

Animals↗
PubMed1998

Accurate titration of avidin and streptavidin with biotin-fluorophore conjugates in complex, colored biofluids.

A new fluorimetric assay is presented for the specific and reliable quantitation of >/=2 nM avidin and streptavidin. The assay is based on pronounced changes in the fluorescence properties of commercial fluorescein-biotin, or of a newly synthesized biotin-poly(ethylene glycol)-pyrene conjugate, which occur upon binding to avidin and streptavidin. Accurate measurement of (strept)avidin in complex, colored biofluids, such as crude egg white or serum relies on a simple titration protocol. Only occasional recalibration of the reagent solution is required. Due to these merits the proposed assay is particularly suited for rapid measurement of few samples on short notice, for functional control of (strept)avidin-containing reagents after storage, and for the monitoring of (strept)avidin concentrations in large scale processes.

Animals↗
PubMed1998

Isolation and characterisation of smallminded, a Drosophila gene encoding a new member of the Cdc48p/VCP subfamily of AAA proteins.

Smallminded (smid) encodes a new member of the cdc48p/VCP subfamily of AAA proteins in Drosophila. The gene was isolated by plasmid rescue from a GAL4 enhancer trap line which shows reporter gene expression in neuroblasts, imaginal disks and a subset of sensory neurons. Larvae homozygous for the insert arrest development as second instar larvae and die without pupating. The most obvious defect in these larvae is a significantly reduced CNS, hence the naming of the gene as smallminded. The deduced amino acid sequence of smid contains a tandem duplication of the AAA nucleotide binding domain characteristic of the cdc48p/VCP subfamily. Overall, smid shares 33% identical residues with its closest relative, yeast L0919-chrXII and 26-29% with other members of the cdc48p/VCP subfamily. The most highly conserved regions of the predicted protein structure are found in and around the nucleotide binding domains. The gene is expressed at all developmental stages.

Amino Acid Sequence↗
PubMed1998

Fluorogenic detection of viable Toxoplasma gondii.

In order to easily assess growth and destruction of Toxoplasma gondii in vitro, this report describes two double staining assays that both visualize live and dead organisms: acridine orange--ethidium bromide (AO-EB) and bisbenzimide (Hoechst 33258)--propidium iodide (B-PI). EB and PI were chosen for dead organisms staining while AO and B stain viable organisms. Thus, both double staining assays seem more informative than Giemsa staining or indirect immunofluorescence. They offer methods to study internal structure of the parasite as well as information on host-parasite relationships. Moreover, detection in culture are sensitive, easier, and less time consuming than previous methods. So, they should to be useful in strains behaviour analysis.

Acridine Orange↗
PubMed1998

Molecular and behavioral analysis of four period mutants in Drosophila melanogaster encompassing extreme short, novel long, and unorthodox arrhythmic types.

Of the mutationally defined rhythm genes in Drosophila melanogaster, period (per) has been studied the most. We have molecularly characterized three older per mutants-perT, perClk, and per04-along with a novel long-period one (perSLIH). Each mutant is the result of a single nucleotide change. perT, perClk, and perSLIH are accounted for by amino acid substitutions; per04 is altered at a splice site acceptor and causes aberrant splicing. perSLIH exhibits a long period of 27 hr in constant darkness and entrains to light/dark (L/D) cycles with a later-than-normal evening peak of locomotion. perSLIH males are more rhythmic than females. perSLIH's clock runs faster at higher temperatures and slower at lower ones, exhibiting a temperature-compensation defect opposite to that of perLong. The per-encoded protein (PER) in the perT mutant cycles in L/D with an earlier-than-normal peak; this peak in perSLIH is later than normal, and there was a slight difference in the PER timecourse of males vs. females. PER in per04 was undetectable. Two of these mutations, perSLIH and perClk, lie within regions of PER that have not been studied previously and may define important functional domains of this clock protein.

Animals↗
PubMed1998

Hormonally controlled chloride movement across Drosophila tubules is via ion channels in stellate cells.

Anion conductance across the Drosophila melanogaster Malpighian (renal) tubule was investigated by a combination of physiological and transgenic techniques. Patch-clamp recordings identified clusters of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive "maxi-chloride" channels in a small domain of the apical membrane. Fluid secretion assays demonstrated sensitivity to the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, anthracene-9-carboxylic acid, and niflumic acid. Electrophysiological analysis showed that the calcium-mediated increase in anion conductance was blocked by the same agents. Vibrating probe analysis revealed a small number of current density hot spots, coincident with "stellate" cells, that were abolished by low-chloride saline or the same chloride channel blockers. GAL-4-targeted expression of an aequorin transgene revealed that the neurohormone leucokinin elicits a rapid increase in intracellular calcium levels in stellate cells that precedes the fastest demonstrable physiological effect. Taken together, these data show that leucokinins act on stellate cells through intracellular calcium to increase transcellular chloride conductance through channels. As electrogenic cation conductance is confined to principal cells, the two pathways are spatially segregated in this tissue.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗