Avoiding viral contamination in biotechnological and pharmaceutical processes.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
K Kaiser
Publications and source records attributed to K Kaiser.
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↗
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↗
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↗
Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos.
The protein serine/threonine phosphatase 4 (PP4), which localises to centrosomes/spindle pole bodies in human cells, is shown to exhibit a similar localisation in Drosophila cells and embryos and possess a highly conserved (91% identical) amino acid sequence from humans to invertebrates. A homozygous Drosophila melanogaster strain mutant in the PP4 gene at 19C1-2 has been produced using P element mutagenesis. This strain, termed centrosomes minus microtubules (cmm), has reduced amounts of PP4 mRNA, approximately 25% of normal PP4 protein in early embryos and exhibits a semi-lethal phenotype with only 10% viability in certain conditions. Reversion mutagenesis shows that the phenotype is due to the presence of the P element in the PP4 mRNA. In early cmm embryos, nuclear divisions become asynchronous and large regions containing centrosomes with no well defined radiating microtubules are visible. In such areas, most nuclei arrest during mitosis with condensed DNA, and mitotic spindle microtubules are either absent, or aberrant and unconnected to the centrosome. A reduction in the staining of gamma-tubulin at centrosomes in cmm embryos suggests a conformational change or relocation of this protein, which is known to be essential for initiation of microtubule growth. These findings indicate that PP4 is required for nucleation, growth and/or stabilisation of microtubules at centrosomes/spindle pole bodies.
Animals↗
13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for ecotoxicity.
There is growing public pressure to minimize the use of vertebrates in ecotoxicity testing; therefore, effective alternatives to toxicity tests causing suffering are being sought. This report discusses alternatives and differs in some respects from the reports of the other three groups because the primary concern is with harmful effects of chemicals at the level of population and above rather than with harmful effects upon individuals. It is concluded that progress toward the objective of minimizing testing that causes suffering would be served by the following initiatives--a clearer definition of goals and strategies when undertaking testing procedures; development of alternative assays, including in vitro test systems, that are based on new technology; development of nondestructive assays for vertebrates (e.g., biomarkers) that do not cause suffering; selection of most appropriate species, strains, and developmental stages for testing procedures (but no additional species for basic testing); better integrated and more flexible testing procedures incorporating biomarker responses, ecophysiological concepts, and ecological end points (progress in this direction depends upon expert judgment). In general, testing procedures could be made more realistic, taking into account problems with mixtures, and with volatile or insoluble chemicals.
Animal Testing Alternatives↗
Defective learning in mutants of the Drosophila gene for a regulatory subunit of cAMP-dependent protein kinase.
Disruptions of a Drosophila gene encoding a regulatory subunit of cAMP-dependent protein kinase homologous to mammalian RIbeta (dPKA-RI) were targeted to the first (noncoding) exon of dPKA-RI via site-selected P element mutagenesis. Flies homozygous for either of two mutant alleles showed specific defects in olfactory learning but not in subsequent memory decay. In contrast, olfactory acuity and shock reactivity, component behaviors required for normal odor avoidance learning, were normal in these mutants. Northern and Western blot analyses of mRNA and protein extracted from adult heads have revealed a complex lesion of the PKA-RI locus, including expression of a novel product and over- or underexpression of wild-type products in mutants. Western blot analysis revealed reductions in RI protein in mutants. PKA activity in the absence of exogenous cAMP also was significantly higher than normal in homogenates from mutant adult heads. These two mutant alleles failed to complement each other for each of these phenotypic defects, eliminating second-site mutations as a possible explanation. These results establish a role for an RI regulatory subunit of PKA in Pavlovian olfactory conditioning.
Animal Structures↗
KLP38B: a mitotic kinesin-related protein that binds PP1.
We have identified a new member of the kinesin superfamily in Drosophila, KLP38B (kinesin-like protein at 38B). KLP38B was isolated through its two-hybrid interaction with the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1). We demonstrate that recombinant KLP38B and PP1 associate in vitro. This is the first demonstration of direct binding of a kinesin-related protein to a regulatory enzyme. Though most closely related to the Unc-104 subfamily of kinesin-related proteins, KLP38B is expressed only in proliferating cells. KLP38B mutants show cell proliferation defects in many tissues. KLP38B is required for normal chromatin condensation as embryos from KLP38B mutant mothers have undercondensed chromatin at metaphase and anaphase. This is the first time that a kinesin-related protein has been shown to have such a role. Incomplete lethality of a strong KLP38B allele suggests partial redundancy with one or more additional kinesin-related proteins.
Amino Acid Sequence↗
Sexual behaviour: secrets and flies.
Recent studies of Drosophila courtship mutants provide a molecular foundation for sexual orientation and behaviour.
Animals↗
Functional domains are specified to single-cell resolution in a Drosophila epithelium.
Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P[GAL4] enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary ("stellate") cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an "identified cell" system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.
Animals↗
Ectopic expression of sex-peptide in a variety of tissues in Drosophila females using the P[GAL4] enhancer-trap system.
Sex-peptide (SP), which is secreted by the accessory gland of Drosophila males, is transferred to the female during copulation, thereby reducing her sexual appetite (receptivity to males) and stimulating ovulation/oviposition. SP is known to be taken up into the hemolymph of mated females, but it is not clear whether there are two separate target tissues, for behavioral changes and ovulation or only one target for both responses. We have employed the GAL4-UAS system to express SP transgene constructs, both in different tissues and in different cellular components of virgin females. A cytoplasmic form of SP lacking a signal sequence did not evoke any responses, even when expressed ubiquitously. In contrast, a membrane-bound form of SP induced typical post-mating behavior, indicating that SP must be outside the cell in order to exert its biological effects. A total of 204 randomly selected P[GAL4] enhancer-trap lines were screened for their ability to induce SP responses in combination with the membrane-bound SP expressed under GAL4 control. Thirty-three lines were associated with both behavioral change and stimulated ovulation. No line was associated with only one of the two responses, implying that the SP target(s) mediating the two responses are either identical, very closely located, or present in two distinct tissues with a common set of genetic determinants. Western blot analysis of head, thorax, and abdominal extracts revealed that the biological activity was correlated with expression in the head fraction.
Animals↗
GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of Drosophila.
We have screened a collection of approximately 400 GAL4 enhancer trap lines for useful patterns of expression in the embryo, larval brain, imaginal discs, and ovary using a UAS-lacZ reporter construct. Although similar patterns of expression have previously been reported in the original P[lacZ] enhancer trap screens, these lines are useful for directing ectopic expression of genes in discrete patterns during these stages. In addition, we have identified some unique patterns of expression that have not been previously reported.
Animals↗
Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.
Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.
Amino Acid Sequence↗
Comparing a computer-based childhood vaccination registry with parental vaccination cards: a population-based study of Delaware children.
We conducted a population-based study in Delaware to examine the reliability of childhood vaccination data in a comprehensive computer-based record system versus parental vaccination cards. We sampled 1,005 children born between January, 1991, and September, 1993. We oversampled for children whose mothers received Medicaid or were uninsured at the time of delivery. Of the survey responders, 276 (56%) had access to written records, and 409 (83%) records were located in the Delaware immunization computer database. The kappa coefficient was 0.18. The observed agreement was 59.8%. When the two databases were combined, the up-to-date rate for 2-year-olds was 58.4%, an increase of 12.7% and 24.2% from the computer database and the parental records, respectively. The computer database was 78.1% sensitive and the parental records were 54.9% sensitive. These results indicate that a comprehensive computer-based record system, with adequate provider participation and proper data management, can be more reliable than parental vaccination cards.
Child↗
Cell-type specific calcium signalling in a Drosophila epithelium.
Calcium is a ubiquitous second messenger that plays a critical role in both excitable and non-excitable cells. Calcium mobilisation in identified cell types within an intact renal epithelium, the Drosophila melanogaster Malpighian tubule, was studied by GAL4-directed expression of an aequorin transgene. CAP2b, a cardioactive neuropeptide that stimulates fluid secretion by a mechanism involving nitric oxide, causes a rapid, dose-dependent rise in cytosolic calcium in only a single, genetically-defined, set of 77 principal cells in the main (secretory) segment of the tubule. In the absence of external calcium, the CAP2b-induced calcium response is abolished. In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, elevates [Ca2+]i only in the smaller stellate cells, suggesting that principal cells do not contain a thapsigargin-sensitive intracellular pool. Assays for epithelial function confirm that calcium entry is essential for CAP2b to induce a physiological response in the whole organ. Furthermore, the data suggest a role for calcium signalling in the modulation of the nitric oxide signalling pathway in this epithelium. The GAL4-targeting system allows general application to studies of cell-signalling and pharmacology that does not rely on invasive or cytotoxic techniques.
Adenosine Triphosphatases↗
Molecular genetic analysis of V-ATPase function in Drosophila melanogaster.
V-ATPases are phylogenetically widespread, highly conserved, multisubunit proton pumps. Originally characterised in endomembranes, they have been found to energise transport across plasma membranes in a range of animal cells and particularly in certain epithelia. While yeast is the model of choice for the rapid generation and identification of V-ATPase mutants, it does not allow their analysis in a plasma membrane context. For such purposes, Drosophila melanogaster is a uniquely suitable model. Accordingly, we have cloned and characterised genes encoding several V-ATPase subunits in D. melanogaster and, using P-element technology, we have succeeded in generating multiple new alleles. Reporter gene constructs reveal ubiquitous expression, but at particularly high levels in those epithelial thought to be energised by V-ATPases, and several of the alleles have lethal recessive phenotypes characterised by epithelial dysfunction. These results, while providing the first gene knockouts of V-ATPases in animals, also illustrate the general utility of D. melanogaster as a model for the genetic analysis of ion transport and its control in epithelia.
Alleles↗
Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies.
Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3',5' monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein alpha subunit (Galphas*) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when Galphas* was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of Galphas* did not affect gross MB morphology, and wild-type Galphas expression did not affect learning. Thus, olfactory learning depends on regulated Gs signaling in Drosophila MBs.
Adenylyl Cyclases↗
Analysis and inactivation of vha55, the gene encoding the vacuolar ATPase B-subunit in Drosophila melanogaster reveals a larval lethal phenotype.
Vacuolar ATPases play major roles in endomembrane and plasma membrane proton transport in eukaryotes. A Drosophila melanogaster cDNA encoding vha55, the 55-kDa vacuolar ATPase (V-ATPase) regulatory B-subunit, was characterized and mapped to 87C2-4 on chromosome 3R. A fly line was identified that carried a single lethal P-element insertion within the coding portion of gene, and its LacZ reporter gene revealed elevated expression in Malpighian tubules, rectum, antennal palps, and oviduct, regions where V-ATPases are believed to play a plasma membrane, rather than an endomembrane, role. The P-element vha55 insertion was shown to be allelic to a known lethal complementation group l(3)SzA (= l(3)87Ca) at 87C, for which many alleles have been described previously. Deletions of the locus have been shown to be larval lethal, whereas point mutations show a range of phenotypes from subvital to embryonic lethal, implying that severe alleles confer a partial dominant negative phenotype. The P-element null allele of vha55 was shown also to suppress ectopic sex combs in Polycomb males, suggesting that transcriptional silencing may be modulated by genes other than those with known homeotic or DNA binding functions.
Alleles↗