Functional expression and characterisation of human cytochrome P45017alpha in Pichia pastoris

Human cytochrome P45017alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initially hampered due to low level in vivo expression of CYP17. Heterologous expression systems have contributed greatly to our current knowledge of CYP17's dual catalytic activity. However, due to the hydrophobic nature of this membrane-bound protein, primarily truncated and modified forms of CYP17 are currently being expressed heterologously. Although the N-terminally modified enzyme has been well characterised, protein structure and function studies still necessitate the expression of unmodified, wild-type CYP17. We report here the expression of a catalytically active, unmodified human CYP17 in the industrial methylotrophic yeast, Pichia pastoris. A typical P450 carbon monoxide difference spectrum, with an absorption maximum at 448nm and a substrate-induced type I spectrum were recorded using a detergent-solubilised cellular fraction containing CYP17. The expressed enzyme catalysed the conversion of progesterone to 17-hydroxyprogesterone as well as 16-hydroxyprogesterone, a product unique to human and chimpanzee CYP17. This is the first report showing the heterologous expression of a fully functional human steroidogenic cytochrome P450 enzyme in P. pastoris.     

Culture and PCR analysis of joint fluid in the diagnosis of prosthetic joint infection

This prospective study compared PCR and culture techniques in the diagnosis of prosthetic joint infection (PJI). We obtained joint fluid samples (JFS; n=115) from patients who had failed total joint arthroplasty between January 2003 and June 2005; 49 were positive for PJI according to established strict criteria. JFS were analyzed by PCR (n=35; control n=66) or culture (n=46, control n=48). PCR was positive in 71% of PJI cases, resulting in sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and likelihood ratio for positive results as follows: 0.71; 0.97; 0.88; 0.93; 0.87 and 23.6, respectively. Culture was positive in 44% of PJI samples. Corresponding statistics were 0.44; 0.94; 0.69; 0.87; 0.63 and 7.0, respectively. Significantly higher sensitivity, accuracy and negative predictive values were calculated for PCR versus culture, and there was 83% concordance between the results of intraoperative culture and PCR detection of causative bacteria. Therefore, we conclude that PCR analysis of synovial fluid increases the utility of pre-operative aspiration for patients who require revision total joint surgery.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Daphnid morphology deters fish predators

Spine and helmet production in zooplankton are thought to provide protection from invertebrate rather than vertebrate predators. We examined selectivity for Daphnia lumholtzi, a species that exhibits extreme cyclomorphosis with a large helmet and long tail spine (total length can exceed 5 mm), by juvenile bluegill (15–80 mm) in the laboratory and field. Bluegill consumed more D. pulex than D. lumholtzi when the species were presented alone. When the daphnids were offered together in equal numbers, bluegill selected against D. lumholtzi. Bluegill foraging behavior helped explain the observed nonrandom feeding. Bluegill capture efficiency foraging on D. pulex was high (85–100%) and handling times were low (usually too short to detect), whereas efficiencies were lower (40–96%) and handling times were longer (1–3 s) when foraging on D. lumholtzi, particularly for fish <50 mm. As they gained experience, bluegill <50 mm that oriented towards D. lumholtzi rejected them more often than striking. In addition, more D. lumholtzi were rejected and expelled than were D. pulex. From these experiments, we conclude that larger bluegill (>50 mm) are able to forage more successfully on D. lumholtzi than smaller fish. Selectivity by bluegill collected from a reservoir infested with D. lumholtzi verified our laboratory conclusions. Smaller bluegill selected against D. lumholtzi, whereas it was a preferred diet item for bluegill >50 mm. These results show that the morphology of D. lumholtzi interferes with predation by small planktivorous fish, posing foraging constraints for these fish more similar to those of piscivores, where handling time is important, than to those of planktivores, where prey density is of primary importance. Received: 13 August 1998 / Accepted 21 August 1998     

Semisynthetic epsilon-isorhodomycins: their synthesis using glycals and their structure-activity relationship

Syntheses and structure-activity relationships of 7-O-(3-amino-2,3,6-trideoxy-a-L-lyxo- (18), -L-arabino- (20) and -L-ribo- hexopyranosyl)-epsilon-isorhodomycins (25) and their 3'-dimethylamino derivatives 22, 23 and 26 are described. Condensation (trimethylsilyl triflate, molecular sieves 4 A, 10:1 dichloromethane-acetone, -15 degrees) of epsilon-isorhodomycinone (epsilon-isoRMN, 6) with 1,5-anhydro-4-O-p-nitrobenzoyl-3-trifluoroacetamido-L-lyxo- (5) -L-arabino- (9) or -L-ribo-hex-l-enitols (10) afforded mainly the 7-O-a-glycosyl-epsilon-isoRMNs 7, 11, and 12. Similar glycosylation of 6 with 1,5-anhydro-3-azido-4-O-p-nitrobenzoyl-2,3,6-trideoxy-L-arabino-hex-1-++ +enitol (15) yielded a-glycoside 16. Removal (M NaOH) of the p-nitrobenzoyl and trifluoroacetyl groups from 7, 11, and 12 gave the 7-O-(3-amino-2,3,6-trideoxy-a-L-hexopyranosyl)-epsilon-isoRMNs 18, 20, and 25. Reductive alkylation (CH2O, NaCNBH3) of these products afforded the 3'-N,N-dimethyl analogues 22, 23, and 26. The cytotoxic effect (IC50) of the semisynthetic epsilon-isorhodomycins was tested in vitro in leukemia cell line L1210.     

Preparation and characterization of catalase-positive particles ('microperoxisomes') from Harder's gland of the rat.

Catalase-positive particles (diameter 0.1–0.3 μm) from Harder's gland of the rat were prepared by differential centrifugation. It was demonstrated that these particles do not contain the oxidases thought to be characteristic of peroxisomal systems (i.e. urate oxidase, d-amino acid oxidase, and α-hydroxy acid oxidase). Cytochemical DAB reaction was employed to demonstrate the organelles in the gland tissue and in subcellular fractions by electron microscopy.     

Structure-activity dependence in some novel ring-substituted 3,3-dimethyl-1-phenyltriazenes. Genetic effects in Drosophila melanogaster and in Saccharomyces cerevisiae by a direct and a host-mediated assay

The structure-activity dependence of ten ring-substituted 3,3-dimethyl-1-phenyltriazenes (DMPT), 3,3-dimethyl-1-(3-pyridyl)-triazene (3-PyDMT) and of 3,3-dimethyl-1-(3-pyridyl-N-oxide)-triazene (3-PyODMT) was investigated by the induction of recessive lethal mutations in Drosophila melanogaster and of mitotic gene conversions in Saccharomyces cerevisiae using both direct and host-mediated assays. Significant differences in genetic effectiveness were detected not only between structurally related compounds but also between the responses of each test system to the same mutagen. Triazenes which are easily cleaved at physological conditions showed the highest genetic activity in the direct yeast test whereas stable triazenes, especially those with ortho and para positions blocked by a halogen, were most active in Drosophila. We have concluded that (1) the released arenediazonium cation is most probably responsible for the convertogenic activity in yeast; (2) metabolites, arising from hydroxylation of the methyl group, are essential for the mutagenic activity in Drosophila. A possible molecular basis which could account for the diversity in genetic effectiveness is discussed in terms of reaction mechanisms which can be predicted from the structural features of the tested triazenes.     

Induction of mitotic gene conversion by 3,3-dimethyl-I-phenyltriazene, I-(3-hydroxyphenyl-)-3,3-dimethyltriazene and by I-(4-hydroxyphenyl)-3,3-dimethyltriazene in Saccharomyces cerevisiae

The induction of mitotic gene conversion by 3,3-dimethyl-1-phenyltriazene (DMPT), 1-(3-hydroxyphenyl)-3,3-dimethyltriazene (3-HO-PDMT) and by 1-(4-hydroxyphenyl)-3,3-dimethyltriazene (4-HO-PDMT) in the diploid strain D4 of Saccharomyces cerevisiae was investigated. The frequencies of the non-reciprocal intragenic recombinations at two unlinked loci ade2 (adenine) and trp5 (tryptophan) were determined. Although all three triazenes showed marked convertogenic activities, significant differences in their genetic effectiveness have been observed. Thus both phenolic triazenes were found to be much stronger convertogens than the unhydroxylated parent compound, DMPT. An attempt is made to account for the established differences in convertogenicity by chemical reactivity that could be expected from the structural features of the tested alkaryltriazenes.     

Hydraulic properties of layered soils influence survival of Rhodes grass (<Emphasis Type="Italic">Chloris gayana</Emphasis> Kunth.) during water stress

Survival of vegetation on soil-capped mining wastes is often impaired during dry seasons due to the limited amount of water stored in the shallow soil capping. Growth and survival of Rhodes grass (Chloris gayana) during soil drying on various layered capping sequences constructed of combinations of topsoil, subsoil, seawater-neutralised residue sand and low grade bauxite was determined in a glasshouse. The aim was to describe the survival of Rhodes grass in terms of plant and soil water relationships. The soil water characteristic curve and soil texture analysis was a good predictor of plant survival. The combination of soil with a high water holding capacity and low soil water diffusivity (e.g. subsoil with high clay contents) with soil having a high water holding capacity and high diffusivity (e.g. residue sand) gave best survival during drying down (up to 88 days without water), whereas topsoil and low grade bauxite were unsuitable (plants died within 18–39 days). Clayey soil improved plant survival by triggering a water stress response during peak evaporative water demand once residue sand dried down and its diffusivity fell below a critical range. Thus, for revegetation in seasonally dry climates, soil capping should combine one soil with low diffusivity and one or more soils with high total water holding capacity and high diffusivity.