Tsukamurella strandjordae sp. nov., a proposed new species causing sepsis.

We have isolated a gram-positive, weakly acid-alcohol-fast, irregular rod-shaped bacterium from cultures of blood from a 5-year-old girl with acute myelogenous leukemia. This isolate was compared with 14 other strains including reference strains of Tsukamurella species by a polyphasic approach based on physiological and biochemical properties, whole-cell short-chain fatty acid and mycolic acid analyses, DNA-DNA hybridization, and sequencing of the 16S rRNA gene. This isolate represents a new taxon within the genus Tsukamurella for which we propose the name Tsukamurella strandjordae sp. nov. Our study also revealed that Tsukamurella paurometabola ATCC 25938 represents a misnamed Tsukamurella inchonensis isolate and confirms that Tsukamurella wratislaviensis belongs to the genus Rhodococcus.

Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes.

Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by /=99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.

Application of 16S rRNA gene sequencing to identify Bordetella hinzii as the causative agent of fatal septicemia.

We report on the first case of fatal septicemia caused by Bordetella hinzii. The causative organism exhibited a biochemical profile identical to that of Bordetella avium with three commercial identification systems (API 20E, API 20 NE, and Vitek GNI+ card). However, its cellular fatty acid profile was not typical for either B. avium or previously reported strains of B. hinzii. Presumptive identification of the patient's isolate was accomplished by traditional biochemical testing, and definitive identification was achieved by 16S rRNA gene sequence analysis. Phenotypic features useful in distinguishing B. hinzii from B. avium were production of alkali from malonate and resistance to several antimicrobial agents.

A library-based bioinformatics services program.

Support for molecular biology researchers has been limited to traditional library resources and services in most academic health sciences libraries. The University of Washington Health Sciences Libraries have been providing specialized services to this user community since 1995. The library recruited a Ph.D. biologist to assess the molecular biological information needs of researchers and design strategies to enhance library resources and services. A survey of laboratory research groups identified areas of greatest need and led to the development of a three-pronged program: consultation, education, and resource development. Outcomes of this program include bioinformatics consultation services, library-based and graduate level courses, networking of sequence analysis tools, and a biological research Web site. Bioinformatics clients are drawn from diverse departments and include clinical researchers in need of tools that are not readily available outside of basic sciences laboratories. Evaluation and usage statistics indicate that researchers, regardless of departmental affiliation or position, require support to access molecular biology and genetics resources. Centralizing such services in the library is a natural synergy of interests and enhances the provision of traditional library resources. Successful implementation of a library-based bioinformatics program requires both subject-specific and library and information technology expertise.

G protein control of Drosophila photoreceptor phospholipase C.

Light stimulates phosphatidylinositol bisphosphate phospholipase C (PLC) activity in Drosophila photoreceptors. We have investigated the mechanism of this reaction by assaying PLC activity in Drosophila head membranes using exogenous phospholipid substrates. PLC activation depends on the photoconversion of rhodopsin to metarhodopsin and is reduced in norpAEE5 PLC and ninaEP332 rhodopsin mutants. NorpA PLC is stimulated by light at free Ca2+ concentrations between 10 nM and 1 microM. This finding is consistent with a Ca(2+)-mediated positive feedback mechanism that contributes to the rapid temporal response of invertebrate photoreceptor cells. The guanyl nucleotide dependence of light-stimulated PLC activity indicates that a G protein regulates NorpA. This was confirmed by the observation that light stimulation of PLC activity is deficient in mutants that lack the eye-specific G protein beta subunit G beta e. These results indicate that G beta e functions as the beta subunit of the G protein coupling rhodopsin to NorpA PLC.

In situ assay of light-stimulated G protein activity in Drosophila photoreceptor G protein beta mutants.

An in situ 35S-labeled guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding procedure was developed to assay light-stimulated G protein activity in Drosophila compound eyes. We found that Drosophila with mutations in G beta e, an abundant photoreceptor-specific G protein beta subunit essential for photoexcitation, are defective in light-stimulated [35S]GTP gamma S binding. We confirmed that G beta e interacts with a GTP-binding protein by demonstrating that immunoprecipitation of G beta e is sensitive to GTP gamma S. These results suggest that G beta e functions as the beta subunit of a heterotrimeric G protein that couples photoactivation of rhodopsin to downstream components in the Drosophila phototransduction cascade.

An eye-specific G beta subunit essential for termination of the phototransduction cascade.

Heterotrimeric G proteins couple various receptors to intracellular effector molecules. Although the role of the G alpha subunit in effector activation, guanine nucleotide exchange and GTP hydrolysis has been well studied, the cellular functions of the G beta subunits are less well understood. G beta gamma dimers bind G alpha subunits and anchor them to the membrane for presentation to the receptor. In specific systems, the G beta subunits have also been implicated in direct coupling to ion channels and to effector molecules. We have isolated Drosophila melanogaster mutants defective in an eye-specific G-protein beta-subunit (G beta e), and show here that the beta-subunit is essential for G-protein-receptor coupling in vivo. Remarkably, G beta mutants are also severely defective in the deactivation of the light response, demonstrating an essential role for the G beta subunit in terminating the active state of this signalling cascade.

Pertussis toxin expression in Drosophila alters the visual response and blocks eating behaviour.

Pertussis toxin inactivates certain G-proteins by introducing an ADP-ribose group near the carboxyl-terminus of the alpha-subunit. The major pertussis toxin substrate in Drosophila tissues is Go alpha. We introduced a pertussis toxin gene under control of the hsp70 heat-shock promoter into the Drosophila genome. When heat-shocked, transformed flies produce active pertussis toxin which ADP-ribosylates endogenous Go alpha. Pertussis toxin is expressed in photoreceptors, in the lamina of the eye and in epithelial cells lining the gut. As expected from the absence of Go alpha in photoreceptors, pertussis toxin does not affect the photoreceptor component of the Drosophila visual response. However, it abolishes light on- and off-transients in the electroretinogram. These transients normally arise from the lamina, a tissue where Go alpha transcripts have been detected. Pertussis toxin expression also blocks embryonic development and shortens the lifetime of adult Drosophila. Following heat-shock, transformed adults are active, but they fail to take up nutrients because they stop eating. High energy metabolites are significantly depleted shortly after pertussis toxin expression is induced and the flies die within 48 h.

A G beta protein in the Drosophila compound eye is different from that in the brain.

A G protein beta subunit gene (Gbe) is expressed only in the eyes of adult D. melanogaster. This gene was identified by probing a Drosophila head cDNA expression library with monoclonal antibodies to a previously characterized Drosophila G protein beta subunit (Gbb). Immunoblot and Northern analyses demonstrate that Gbe protein and mRNA is not present in Drosophila mutants that lack eyes. Immunocytochemical and in situ hybridization analyses further demonstrate that Gbe is expressed in the eyes but not in the brain, whereas Gbb is abundantly expressed in the brain. The Gbe product is approximately 45% identical to previously identified G beta subunits and defines a new G beta class. Its localization suggests a possible role in phototransduction.

The Drosophila Go alpha-like G protein gene produces multiple transcripts and is expressed in the nervous system and in ovaries.

Drosophila melanogaster cDNA clones coding for the alpha subunit of a Go-like G protein have been isolated. The sequence of two cDNA clones shows there is alternative splicing in the 5'-coding region which, on conceptual translation, would give rise to two proteins with slightly different amino termini. A partial genomic clone indicates there are four introns in the carboxyl-terminal half of the clone. Two transcripts, 3.8 and 5.3 kilobases long, are expressed at a high level in the heads of adult flies and also in larvae, pupae, and embryos. Hybridization of the cDNA probes to sections of adult flies indicates the RNA is present predominantly in nervous tissue (in the cortex of the brain and the thoracic ganglion); it is also expressed in the ovaries. Transcripts corresponding to both cDNAs are present in the central nervous system, but only one of them is found in detectable levels in the ovaries. The gene maps to 47A on the Drosophila second chromosome.

Cloning of a Drosophila melanogaster guanine nucleotide regulatory protein beta-subunit gene and characterization of its expression during development.

A Drosophila melanogaster gene encoding a protein with greater than 80% sequence identity to the beta subunits of mammalian guanine nucleotide-binding regulatory proteins (G proteins) has been cloned. The gene, which was mapped to 13F on the X chromosome by in situ hybridization, was cloned from a Drosophila genomic library by using a bovine transducin beta-subunit cDNA probe. Genomic DNA blot hybridization analysis indicated that there is a single Drosophila G-protein beta-subunit gene. Multiple transcripts were detected throughout development; in adult flies the mRNA is expressed at higher levels in heads than in bodies. The proposed coding region is uninterrupted by introns, but there is evidence for differential mRNA splicing in the 5' nontranslated region.

Urinary glucuronidase and arylsulfatases in identical twins of bladder cancer patients.

Studies showing that bladder cancer patients have unusually high levels of urinary beta-glucuronidase and arylsulfatases A and B led to the suggestion that these urinary enzymes may participate in bladder cancer etiology. An alternative explanation of the high levels of these urinary enzymes in bladder cancer patients is that the disease itself causes the elevation. Since the levels of these enzymes are genetically determined, measuring these enzymes in healthy identical twins of bladder cancer patients can test whether high enzyme levels occurred prior to bladder cancer. Five healthy identical cotwins of bladder cancer patients, together with matched controls, were measured for urinary beta-glucuronidase, arylsulfatases A and B, and two other lysosomal enzymes as controls, alpha- and beta-galactosidases. The mean levels of all five enzymes were not very different in the cotwins and controls, suggesting that high levels of urinary enzymes observed in bladder cancer patients are a consequence of disease rather than occurring prior to disease and contributing to its etiology.