An ecdysone-inducible putative "DEAD box" RNA helicase in the spruce budworm (Choristoneura fumiferana).

RNA helicases are a family of enzymes that unwind nucleic acid duplexes, such as RNA/RNA and RNA/DNA, in a 3' to 5' direction into single-stranded polynucleotides. A putative RNA helicase cDNA (CfrHlc64) was isolated from the spruce budworm, Choristoneura fumiferana. CfrHlc64 was 1998 nucleotides in length, and the deduced protein had 565 amino acids with a predicted molecular mass of 64 kDa. It contained eight functional motifs conserved in the "DEAD box" family of RNA helicases. The deduced amino acid sequence showed 10-50% identities to homologues of other species from bacteria to human. In vitro expression of the cDNA resulted in recombinant proteins of 64 kDa as expected from the deduced amino acid sequence. Northern blotting and RT-PCR analyses revealed the presence of CfrHlc64 mRNA in all developmental stages from embryo to adult. Higher levels of CfrHlc64 mRNA were detected in the fat body and midgut than in the epidermis of sixth instar larvae. The CfrHlc64 protein was distributed mainly in the fat body. Female adults expressed CfrHlc64 mRNA at higher levels than male adults. The nonsteroidal ecdysone agonist, tebufenozide, enhanced the expression of CfrHlc64 in a dose-dependent manner.

The modes of action of juvenile hormones: some questions we ought to ask.

This paper argues that the current dogma that juvenile hormones are structurally unique and constitute a family of derivatives of farnesoic acid which are produced by the corpus allatum (CA), secreted into the hemolymph, frequently transported by binding proteins, enter cells by diffusion across the cell membrane and there the products of the CA interact in some way with the genome, probably via nuclear receptors of the steroid superfamily, may not be tenable. It does so by examining the following questions. How many JHs are there? Are there other sources of JH in insects? Are there non-farnesoids with JH activity in insects? How does JH get into cells? Is the product of the CA the effective hormone? How many modes of action are there? How many receptors are there?

Molecular cloning of a female-specific cDNA with unique repeat sequences from the fat body of the adult locust, Locusta migratoria.

A cDNA clone encoding a 25-kDa protein (25K) was isolated from a cDNA library made from RNA isolated from the adult fat body and ovaries of the locust, Locusta migratoria. The longest open reading frame of this cDNA clone encodes a 225-amino acid polypeptide, the N-terminal end of which was similar to the 21-kDa and 19-kDa juvenile hormone induced proteins identified in the locust hemolymph, but the C-terminal end was different. The C-terminal end of the 25K cDNA contained seven unique repeat elements of 10 amino acids each, most of which are polar residues. Expression of the 25K mRNA was tissue-, development- and sex-specific. A 1.2-kb mRNA was detected using the 25K cDNA as a probe only in the fat body of adult females. The mRNA started to appear at day 4 after the insect molted to the adult and rapidly increased by day 6. The mRNA was absent in the ovarian follicle cells and fat body of adult males. In vitro transcription and translation of the 25K cDNA produced a protein that migrated around 32 kDa on sodium dodecyl sulfate polyacrylamide gels. The 25K cDNA was expressed in a baculovirus expression system and the protein produced also migrated around 32 kDa.

Do thyroid hormones function in insects?

Earlier work demonstrated that phenoxy-phenyl compounds such as fenoxycarb and thyroxine mimicked the effects of JH III in causing a reduction in volume of the follicle cells of Locusta migratoria. While these compounds were only moderately effective, a derivative of thyroxine, 3,3',5-triiodothyronine (T3) was as effective as JH III, and T3 has been shown to bind to the same membrane receptor and activate the same pathway as JH III. The current paper shows that other thyroxine derivatives vary in activity. 3,3', 5'-Triiodothyronine (reverse T3) is inactive. 3,5-Diiodothyronine (T2) is more active than JH III, while its relatives (iodines at 3', 5' or at 3,3') are inactive. When follicles are exposed in vitro to rhodamine conjugated T3, the fluorescent compound can be seen to enter the cells and accumulate there: this process is inhibited by cycloheximide or by a temperature of 0 degrees C. The accumulation is antagonised by JH III but not JH I (which does not bind to the JH III membrane receptor) and by an antiserum raised against the putative membrane receptor protein. The action of T3, but not T2, is inhibited by 6-n-propyl-2-thiouracil or by aurothioglucose, both known to inhibit deiodinases. The activity of T3, but not of T2, increases with time of exposure to the follicle cells. These facts suggest that T3 enters the cells by receptor mediated endocytosis and is converted to a more active compound. Immunoreactivity to T3, but not thyroxine, can be detected in the haemolymph of locusts, and the titre varies slightly with the gonotrophic cycle. The food shows immunoreactivity for both thyroxine and T3. These findings suggest that thyroid hormones are ingested by locusts and have the potential to be used as hormonal signals in the control of egg production.

Functional binding of a vertebrate hormone, L-3,5,3'-triiodothyronine (T3), on insect follicle cell membranes.

A vertebrate hormone, L-3,5,3'-triiodothyronine (T3), induces volume reduction in the follicle cells of Locusta migratoria and Rhodnius prolixus. The effect of T3 on locust follicle cells is inhibited by ouabain and by antibodies raised against a membrane binding protein for juvenile hormone (JH). [125I]-T3 binds to membrane preparations of vitellogenic follicles in a specific and saturable fashion, with a KD in the low nanomolar range. T3 and JH III exhibited equivalent abilities to compete for the T3 binding site. These findings strongly suggest that T3 and JH act via the same receptor in follicle cells.

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.

Spruce budworm (Choristoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning.

We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.

Comparison of the API Candida system with the AUXACOLOR system for identification of common yeast pathogens.

Two commercial systems for the identification of yeasts were evaluated by using 159 clinical isolates that had also been identified by conventional biochemical and morphological methods. The API Candida system correctly identified 146 isolates (91.8%), and the AUXACOLOR system correctly identified 145 isolates (91.2%). However, of the 146 isolates identified by the API Candida system, 23 required supplemental biochemical tests or morphological assessment to obtain the correct identification. The AUXACOLOR system gave no identification in 13 cases (8.2%), while the API Candida system gave an unreadable profile in only one case. Incorrect identifications were more common with the API Candida system (12 isolates; 7.5%) than with the AUXACOLOR system (1 isolate; 0.6%).

The correlation of the visual field with scanning laser ophthalmoscope measurements in glaucoma.

PURPOSE: To assess the relationship between Humphrey visual field data and optic disc topographical data collected by the Heidelberg Retina Tomograph (HRT) scanning laser ophthalmoscope in chronic glaucoma patients. METHODS: The mean deviation (MD) and corrected pattern standard deviation (CPSD) from Humphrey visual fields of 106 eyes of 106 patients with glaucoma were analysed for correlation with the multiple topographical measures calculated by the HRT. RESULTS: Significant correlations were found between MD of the visual field and several optic disc measurements. These included neuroretinal rim volume, mean nerve fibre layer thickness and cross-sectional area, and the cup shape measure. CPSD correlated significantly only with mean nerve fibre layer cross-sectional area. This pattern was common to the whole circumference of the disc with the exception of the directly temporal segment. CONCLUSION: Optic disc topography performed by HRT reflects the optic disc pathology in correlation with perimetry.

Identification and developmental expression of the mitochondrial phosphate transport protein gene from the spruce budworm, Choristoneura fumiferana.

Phosphate transport protein (PTP) is a mitochondrial inner membrane protein responsible for the translocation of inorganic phosphate into the mitochondrial matrix. A full length cDNA clone encoding the PTP was isolated from the spruce budworm, Choristoneura fumiferana. The deduced amino acid sequence of the longest ORF of CfPTP cDNA showed high similarity with the amino acid sequences of PTPs cloned from several species. Phylogenetic tree analysis indicated that CfPTP occupied an intermediate position between vertebrates on the one side and yeast and nematodes on the other side. Studies on the developmental expression of CfPTP mRNA showed that higher levels of mRNA were present during the feeding and growing stages than during molting periods.

Comparison of a new commercial colorimetric microdilution method with a standard method for in-vitro susceptibility testing of Candida spp. and Cryptococcus neoformans.

The Sensititre Yeast One method (AccuMed International Ltd, East Grinstead, UK) is a microplate-based procedure that incorporates an oxidation-reduction indicator, Alamar Blue, for the in-vitro testing of five antifungal agents (amphotericin B, fluconazole, itraconazole, ketoconazole and flucytosine). We compared this colorimetric method with a standard broth microdilution test, performed according to US National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in-vitro susceptibilities of 180 isolates of Candida spp. (50 Candida albicans, 50 Candida glabrata, ten Candida kefyr, 20 Candida krusei, ten Candida lusitaniae, 20 Candida parapsilosis and 20 Candida tropicalis) and 20 isolates of Cryptococcus neoformans. The overall agreement between the results of the two methods (colorimetric MICs within +/- two log2 dilutions of standard MICs) were 99% for amphotericin B, 96.5% for flucytosine, 93% for itraconazole, 91.5% for fluconazole and 85.5% for ketoconazole. The overall levels of agreement between the two methods were > or = 94% for six of the eight species tested, the exceptions being C. neoformans and C. tropicalis where the overall agreement was 89% and 80% respectively. The poorest agreement between the results for individual agents was seen with C. tropicalis and the three azole agents (60-75% of colorimetric MICs within +/- two log2 dilutions of standard MICs), and C. neoformans for ketoconazole (50%). The Yeast One method appears to be a suitable alternative procedure for routine antifungal susceptibility testing of Candida spp. and C. neoformans.

In-vitro susceptibility of Cryptococcus neoformans isolates to fluconazole and itraconazole.

The in-vitro susceptibilities of 143 isolates of Cryptococcus neoformans, collected from British patients between 1994 and 1996, to fluconazole and itraconazole were compared with those of 36 isolates collected between 1971 and 1985, 41 isolates collected between 1986 and 1989, and 43 Ugandan isolates collected in 1996. Testing was done with a broth microdilution method in YNB medium supplemented with glucose, incubation at 30 degrees C for 72 h, and an endpoint of 50% inhibition. The results showed that the MIC ranges, MIC50s and MIC90s of fluconazole and itraconazole for C. neoformans isolates from the UK have remained unchanged despite the recent widespread use of triazoles for long-term maintenance of patients with AIDS-associated cryptococcal meningitis. The MIC ranges, MIC50s and MIC90s of the 43 isolates from untreated Ugandan patients with AIDS were similar to those of the British isolates. Examination of our records for 1994-96 revealed six cases in which a four-fold or greater rise in the MIC of fluconazole was associated with relapsed cryptococcal meningitis.

Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans.

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of Candida spp. (50 C. albicans, 50 C. glabrata, 10 C. kefyr, 20 C. krusei, 10 C. lusitaniae, 20 C. parapsilosis, and 20 C. tropicalis isolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of C. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.

Fenoxycarb and thyroid hormones have JH-like effects on the follicle cells of Locusta migratoria in vitro.

Earlier work had shown that JH acts on the membrane of the follicle cell of Locusta migratoria, bringing about a rapid reduction in volume which can be detected in vitro by measuring the increase in optical path difference using quantitative interference microscopy. The juvenoid fenoxycarb, a phenoxyphenyl derivative, is unrelated in structure to the juvenile hormones (which are derivatives of farnesoic acid), but it also caused a reduction in volume of the cells in vitro as measured by an increase in the optical path difference. The vertebrate hormone thyroxine, and thyronine, the non-iodinated derivative of thyroxine, also phenoxy phenyl compounds, evoked a response like fenoxycarb. The effect of thyroxine was abolished by ouabain, which inhibits Na+/K+ ATPase, the effector molecule for JH, and inhibited by ethoxyzolamide which inhibits the binding of JH to a putative membrane receptor. Triiodothyronine, the effective vertebrate hormone, acted at a lower threshold and optimum concentration, and had a greater magnitude of effect than the other compounds tested. These facts suggest that these phenoxyphenyl compounds are JH agonists and that the membrane receptor for JH may resemble a possible membrane receptor for thyroxine.

Toxic gas generation from plastic mattresses and sudden infant death syndrome.

Microbial generation of toxic gases from antimony, arsenic, or phosphorus in compounds used as fire retardants in cot mattresses has been proposed as a cause of sudden infant death. To test this hypothesis, 23 polyvinyl chloride mattress samples from cot death cases were incubated on malt agar plates until good microbial growth was obtained. Silver nitrate and mercuric chloride test papers were then inserted and the colour reactions recorded. The predominant organism, recovered from all mattresses tested, was not, as claimed in earlier work, the fungus Scopulariopsis brevicaulis, but a mix of common environmental Bacillus spp. Test paper colour changes occurred whenever bacterial growth was present, but these reactions also occurred in control tests in which no mattress material was present on the plates. Chemical and instrumental analyses of exposed test papers showed that the colour reactions were not due to deposits of antimony, arsenic, or phosphorus. Our findings do not support the hypothesis that toxic gases derived from antimony, arsenic, or phosphorus are a cause of sudden infant death. More sulphur was found in test papers exposed in plates containing bacterial growth than in those without such growth. This result suggests that the test paper reactions were due to the generation of sulphur-containing compounds during bacterial growth on the agar medium.

Itraconazole susceptibilities of fluconazole susceptible and resistant isolates of five Candida species.

The in-vitro susceptibilities of 1380 isolates of five Candida species were determined in order to establish whether isolates resistant to fluconazole were cross-resistant to itraconazole. IC50 values were determined by a broth microdilution method. 690 Candida albicans isolates, seven Candida glabrata isolates, seven Candida krusei isolates, 120 Candida parapsilosis isolates and 37 Candida tropicalis isolates were susceptible to both fluconazole (IC50 < or = 32 mg/L) and itraconazole (IC50 < or = 4 mg/L). Twenty eight of 160 C. albicans isolates (17.5%), 180 of 293 C. glabrata isolates (61.4%), six of 48 C. krusei isolates (12.5%), and 10 of 18 C. tropicalis isolates (55.5%) resistant to fluconazole (IC50 > or = 64 mg/L) were also resistant to itraconazole (IC50 > or = 8 mg/L). In contrast, drug-specific resistance to itraconazole was not observed in any of the isolates tested. However, the itraconazole IC50s for fluconazole susceptible isolates were lower than those for fluconazole resistant isolates, which suggests that patients who fail fluconazole treatment might require itraconazole at higher dosages than usual.

Evaluation of the AUXACOLOR system, a new method of clinical yeast identification.

AIMS: To compare the AUXACOLOR yeast identification system with the API 20C system. METHODS: Yeast isolates (n = 215), comprising 16 species, were identified using the AUXACOLOR system and the API 20C system. Isolates that could not be identified with the API 20C system or which produced discrepant results in the two systems were identified by assimilation and fermentation procedures. RESULTS: AUXACOLOR correctly identified 150 (85.7%) of 175 germ tube negative isolates while API 20C identified 155 (88.6%). Incorrect identifications were more common with API 20C (7.4%) than with AUXACOLOR (3.7%). Of 110 isolates of four common pathogens (Candida glabrata, C parapsilosis, C tropicalis, and Cryptococcus neoformans), 82.7% (91/110) were identified by AUXACOLOR while API 20C identified 74.5% (82/110). Of 65 less common germ tube negative isolates, 55.4% (36/65) were identified by AUXACOLOR while API 20C identified 63.1% (41/65). CONCLUSION: Although it has a limited database of 26 species, the AUXACOLOR system is a useful method for identification of germ tube negative clinical yeast isolates. Compared with the API 20C, the AUXACOLOR system is simpler and quicker to set up, easier to interpret, and comparable in cost.