Genetic changes that increase 5-hydroxymethyl furfural resistance in ethanol-producing Escherichia coli LY180.

The ability of a biocatalyst to tolerate furan inhibitors present in hemicellulose hydrolysates is important for the production of renewable chemicals. This study shows EMFR9, a furfural-tolerant mutant of ethanologenic E. coli LY180, has also acquired tolerance to 5-hydroxymethyl furfural (5-HMF). The mechanism of action of 5-HMF and furfural appear similar. Furan tolerance results primarily from lower expression of yqhD and dkgA, two furan reductases with a low K(m) for NADPH. Furan tolerance was also increased by adding plasmids encoding a NADPH/NADH transhydrogenase (pntAB). Together, these results support the hypothesis that the NADPH-dependent reduction of furans by YqhD and DkgA inhibits growth by competing with biosynthesis for this limiting cofactor.

In vivo uncoating and efficient expression of foreign mRNAs packaged in TMV-like particles.

The ribonucleocapsids of many plant viruses are extremely stable. The protein coat protects the RNA genome against degradation during the accumulation and spread of progeny virions. Chimeric single-stranded RNA molecules were transcribed in vitro from recombinant plasmids and later encapsidated, in vitro, into ribonucleoprotein particles (pseudoviruses) 60 nanometers long that resembled tobacco mosaic virus. Transcripts encoding an assayable enzyme, chloramphenicol acetyltransferase (CAT), were packaged into pseudovirus particles to assess the utility of this single-stranded RNA delivery system in a wide range of cell types. In all cases, packaged CAT messenger RNA was uncoated and transiently expressed. Significantly higher levels of CAT activity were detected with packaged than with naked CAT messenger RNA after inoculation of plant protoplasts in the presence of polyethylene glycol or abrasive inoculation of intact leaf surfaces. Structural events that lead to the uncoating and expression of CAT messenger RNA showed no cell specificity. This observation may support the view that the comparatively restricted host range of a true plant virus results from events that occur later during the infection cycle.

Lactobacillus rhamnosus strain GG restores alkaline phosphatase activity in differentiating Caco-2 cells dosed with the potent mycotoxin deoxynivalenol.

Deoxynivalenol (DON) contamination of cereal crops occurs frequently, and may cause acute exposure at high levels or chronic more moderate exposure. DON has proven toxicity including restriction of enterocyte differentiation, which may play a part in DON induced gastroenteritis. The probiotic bacteria Lactobacillus rhamnosus strain GG (GG) can bind DON, and therefore potentially restrict bioavailability of this toxin. Binding efficacy is not significantly altered by heat treatment, and therefore this in vitro study evaluated whether heat inactivated GG could restore the differentiation process in Caco-2 cells, using alkaline phosphatase (ALP) activity as a marker of differentiation. DON (200ng/mL) caused a significant (p<0.001) 36% reduction in ALP activity (1598+/-137U/mg protein) compared to untreated cells (2502+/-80U/mg). A dose dependant restoration of ALP activity was observed where DON treated cells were co-incubated with heat inactivated GG (1719+/-84; 2007+/-142; 2272+/-160U/mg for GG at 1x10(4) (p>0.9), 1x10(7) (p<0.001), and 1x10(10)CFU/mL (p<0.001), respectively). Co-incubation of the non-binding strain, LC-705 (1x10(10)CFU/mL), with DON did not significantly restore the ALP (1841+/-97U/mg, p<0.077) compared to DON only treated cells. When viable GG were co-incubated with DON a similar restoration of ALP activity was observed as seen for heat inactivated GG. These combined data suggest that the major effect of GG on restoring ALP activity, and therefore Caco-2 cell differentiation, was due to specific binding of DON, with possibly a more minor role of non-specific bacterial interference.

Barriers to implementing the NICE guidelines for early-onset neonatal infection: cross-sectional survey of neonatal blood culture reporting by laboratories in the UK.

The National Institute for Health and Care Excellence published guidelines for managing early-onset neonatal infections in 2012. It recommended provision for reporting blood cultures (BCs) with growth detected or not detected at 36 h. To determine if this was followed, a telephone survey was conducted amongst lead biomedical scientists based at microbiology laboratories (N = 209) in the UK. Overall, 202/209 responded and 139/202 had on-site facilities for BCs. BC results with growth detected or not detected at 36 h were available out-of-hours in 36/139 (26.6%) and 66/139 (47.5%) neonatal units, respectively. Early discontinuation of antibiotics should lead to improved antibiotic stewardship.

Respiratory-syncytial-virus- and rhinovirus-related bronchiolitis in children aged <2 years in an English district general hospital.

BACKGROUND: Bronchiolitis is the most common reason for hospitalization in young children. In addition to respiratory syncytial virus (RSV), other viruses have been increasingly implicated. Guidance on testing has also changed. AIMS: To compare clinicopathological outcomes in young children admitted with bronchiolitis due to RSV in comparison with rhinovirus (RV), and identify associated risk/epidemiological factors. METHODS: Children aged less than two years admitted to hospital with a clinical diagnosis of bronchiolitis with positive results for either RSV or RV were included in this study. Polymerase-chain-reaction-negative cases using an extended respiratory virus panel served as a control group. Retrospective data were collected on sex, risk factors, respiratory support, intravenous fluids and antibiotics. Outcomes such as length of stay (LOS) and need for transfer to the high-dependency unit/paediatric intensive care unit were included. FINDINGS: Two hundred and twenty-seven out of 437 nasopharyngeal aspirate samples were positive for either RSV (N = 162) or RV (N = 65). The median age of cases was three months and 75% had at least one risk factor. Risk factors were higher in the RV group (P = 0.004). RV accounted for the majority of cases outside the RSV season (P < 0.01). RV-associated bronchiolitis had a longer LOS (more than seven days) (P < 0.05) and increased need for chest X-rays and/or antibiotics (P < 0.05). Use of intravenous fluids and respiratory support were higher in the RV group, but the difference was not significant. CONCLUSIONS: RV is the second most common pathogen associated with bronchiolitis and is isolated all year round. This may be important in those with risk factors resulting in prolonged LOS. Further research is necessary to establish the exact role of RV in this common condition, particularly outside the traditional RSV season.

Reduction in exposure to carcinogenic aflatoxins by postharvest intervention measures in west Africa: a community-based intervention study.

BACKGROUND: Aflatoxins are fungal metabolites that frequently contaminate staple foods in much of sub-Saharan Africa, and are associated with increased risk of liver cancer and impaired growth in young children. We aimed to assess whether postharvest measures to restrict aflatoxin contamination of groundnut crops could reduce exposure in west African villages. METHODS: We undertook an intervention study at subsistence farms in the lower Kindia region of Guinea. Farms from 20 villages were included, ten of which implemented a package of postharvest measures to restrict aflatoxin contamination of the groundnut crop; ten controls followed usual postharvest practices. We measured the concentrations of blood aflatoxin-albumin adducts from 600 people immediately after harvest and at 3 months and 5 months postharvest to monitor the effectiveness of the intervention. FINDINGS: In control villages mean aflatoxin-albumin concentration increased postharvest (from 5.5 pg/mg [95% CI 4.7-6.1] immediately after harvest to 18.7 pg/mg [17.0-20.6] 5 months later). By contrast, mean aflatoxin-albumin concentration in intervention villages after 5 months of groundnut storage was much the same as that immediately postharvest (7.2 pg/mg [6.2-8.4] vs 8.0 pg/mg [7.0-9.2]). At 5 months, mean adduct concentration in intervention villages was less than 50% of that in control villages (8.0 pg/mg [7.2-9.2] vs 18.7 pg/mg [17.0-20.6], p<0.0001). About a third of the number of people had non-detectable aflatoxin-albumin concentrations at harvest. At 5 months, five (2%) people in the control villages had non-detectable adduct concentrations compared with 47 (20%) of those in the intervention group (p<0.0001). Mean concentrations of aflatoxin B1 in groundnuts in household stores in intervention and control villages were consistent with measurements of aflatoxin-albumin adducts. INTERPRETATION: Use of low-technology approaches at the subsistence-farm level in sub-Saharan Africa could substantially reduce the disease burden caused by aflatoxin exposure.

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.

The activation of multiple interleukin-1beta converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

Cloning and characterisation of a U6 small nuclear RNA gene from potato.

Using a mixed U6 snRNA gene probe and a low stringency hybridization procedure we have isolated a U6 snRNA containing clone from a potato genomic library in lambda EMBL 3. This clone contains a single U6 snRNA gene which has been subcloned and sequenced. Southern blotting experiments using this gene and the heterologous U6 genes as probes indicate that the potato U6 gene family consists of more than 20 members. The potato U6 gene sequence shows high identity to previously characterised plant U6 snRNA gene sequences and possesses correctly positioned and spaced transcription control elements suggesting that it is an active gene.

Functional redundancy of promoter elements ensures efficient transcription of the human 7SK gene in vivo.

Deletion and mutation studies of the human 7SK gene transfected into HeLa cells have identified three functional regions of the promoter corresponding to the TATA box at -25, the proximal sequence element (PSE) between -49 and -65 and the distal sequence element (DSE) between -243 and -210. These elements show sequence homology to equivalent regions in other snRNA genes and are functionally analogous. Unlike the DSEs of many snRNA genes however, the 7SK DSE does not contain a consensus binding site for the transcription factor Oct-1 but rather, contains two non-consensus Oct-1 binding sites that can function independently of one another to enhance transcription. Unusually, the 7SK PSE can retain function even after extensive mutation and removal of the conserved TGACC of the PSE has little effect in the context of the whole promoter. However, the same mutation abolishes transcription in the absence of the DSE suggesting that protein/protein interactions between DSE and PSE binding factors can compensate for a mutant PSE. Mutation of the 7SK TATA box allows snRNA type transcription by RNA polymerase II to occur and this is enhanced by the DSE, indicating that both the DSE and the PSE can also function with pol II. In addition, mutation of the TATA box does not abolish pol III dependent transcription, suggesting that other sequence elements may also play a role in the determination of polymerase specificity. Although the human 7SK gene is transcribed efficiently in Xenopus oocytes, analysis of the 7SK wild-type gene and mutants in Xenopus oocytes gives significantly different results from the analysis in HeLa cells indicating that the recognition of functional elements is not the same in the two systems.

Dietary exposure to aflatoxin from maize and groundnut in young children from Benin and Togo, West Africa.

Aflatoxins are a family of fungal toxins that are carcinogenic to man and cause immunosuppression, cancer and growth reduction in animals. We conducted a cross-sectional study among 480 children (age 9 months to 5 years) across 4 agro-ecological zones (SS, NGS, SGS and CS) in Benin and Togo to identify the effect of aflatoxin exposure on child growth and assess the pattern of exposure. Prior reports on this study [Gong, Y.Y.,Cardwell, K., Hounsa, A., Egal, S., Turner, Hall, A.J., Wild, C.P., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. British Medical Journal 325, 20-21, Gong, Y.Y., Egal, S., Hounsa, A., Turner, P.C., Hall, A.J., Cardwell, K., Wild, C.P., 2003. Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning and weaning foods. International Journal of Epidemiology, 32, 556-562] showed that aflatoxin exposure among these children is widespread (99%) and that growth faltering is associated with high blood aflatoxin-albumin adducts (AF-alb adducts), a measure of recent past exposure. The present report demonstrates that consumption of maize is an important source of aflatoxin exposure for the survey population. Higher AF-alb adducts were correlated with higher A. flavus (CFU) infestation of maize (p=0.006), higher aflatoxin contamination (ppb) of maize (p<0.0001) and higher consumption frequencies of maize (p=0.053). The likelihood of aflatoxin exposure from maize was particularly high in agro-ecological zones where the frequency of maize consumption (SGS and CS), the presence of aflatoxin in maize (SGS) or the presence of A. flavus on maize (NGS and SGS) was relatively high. Socio-economic background did not affect the presence of A. flavus and aflatoxin in maize, but better maternal education was associated with lower frequencies of maize consumption among children from the northernmost agro-ecological zone (SS) (p=0.001). The impact of groundnut consumption on aflatoxin exposure was limited in this population. High AF-alb adduct levels were correlated with high prevalence of A. flavus and aflatoxin in groundnut, but significance was weak after adjustment for weaning status, agro-ecological zone and maternal socio-economic status (resp. p=0.091 and p=0.083). Ingestion of A. flavus and aflatoxin was high in certain agro-ecological zones (SS and SGS) and among the higher socio-economic strata due to higher frequencies of groundnut consumption. Contamination of groundnuts was similar across socio-economic and agro-ecological boundaries. In conclusion, dietary exposure to aflatoxin from groundnut was less than from maize in young children from Benin and Togo. Intervention strategies that aim to reduce dietary exposure in this population need to focus on maize consumption in particular, but they should not ignore consumption of groundnuts.

A transcriptional analysis of the gene encoding mouse U7 small nuclear RNA.

Expression of the U7 gene, encoding mouse U7 snRNA, following microinjection into Xenopus oocytes is both accurate and efficient, giving rise to mature U7 snRNA and a precursor with an 8-nucleotide (nt) 3' extension. The mouse U7 gene promoter, which is similar to that of the vertebrate major U genes comprising a DSE, a PSE and a 3' box, with the same spatial arrangement, is as efficient as the Xenopus U2 gene promoter in this assay. A deletion analysis of the mouse U7 gene identified sequences downstream from the 3' box, within the region (nt +74 to +196), which seem to have a negative regulatory effect upon the frequency of transcription initiation and are also required for accurate 3' end formation. Sequences in the nt -1699 to -431 region also seemed to have a negative effect on the level of transcription. In addition, sequences upstream from the PSE, within the nt -65 to -421 region, are necessary for accurate and efficient synthesis of mature U7 snRNA. Finally, the mouse U7 snRNA may not form a functional snRNP in Xenopus oocytes due to defective snRNP assembly and/or nuclear import.

The Xenopus U7 snRNA-encoding gene has an unusually compact structure.

A subclone containing a single Xenopus borealis U7 snRNA-encoding gene has been microinjected into X. laevis oocyte nuclei to examine its expression using [32P]GTP as an in vivo label. Only two U7 snRNA bands were detected after incubation, and subsequent fractionation of the oocyte showed that only the larger transcript is present in the nucleus. The sequence of this functional U7 gene shows that, in addition to the coding region, it contains, in the appropriate locations, the 3'-box and proximal sequence element (PSE) which are typical of Pol II-transcribed snRNA genes. Surprisingly, the Xenopus U7 gene contains two adjacent octamer-binding motifs located only 12 and 24 bp upstream from the PSE, instead of the usual location around 150-200 bp upstream. No other cis-acting elements appear to be present. A 5' deletion analysis shows that the transcription level of this U7 gene remains constant if sequences upstream of the two octamer motifs are removed, yet is undetectable when an additional 34 bp containing both octamers and the PSE are removed. This confirms that the Xenopus U7 gene is the most compact snRNA-encoding gene isolated to date. A comparison of U7 sequences shows there is a much greater conservation in the 5' half of the molecule, which contains sequences that base-pair with target pre-mRNA, than in the 3' half which can form a single stem-loop structure that varies in size.

Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression.

Development and reproduction of crustaceans is regulated by a combination of neuropeptide hormones, ecdysteroids (moulting hormones) and the isoprenoid, methyl farnesoate (MF), the unepoxidised analogue of insect juvenile hormone-III (JH-III). MF and the ecdysteroids are respectively synthesised under the negative control of the sinus gland-derived mandibular organ-inhibiting hormones (MO-IHs) and moult-inhibiting hormone (MIH) that are produced in eyestalk neural ganglia. Previous work has demonstrated the existence of two isoforms of MO-IH, called MO-IH-1 and -2, that differ by a single amino acid in the mature peptide and one in the putative signal peptide. To study the structural organisation of the crab MIH and MO-IH genes, a genomic DNA library was constructed from DNA of an individual female crab and screened with both MO-IH and MIH probes. The results from genomic Southern blot analysis and library screening indicated that the Cancer pagurus genome contains at least two copies of the MIH gene and three copies of the MO-IH genes. Upon screening, two types of overlapping genomic clone were isolated. Each member of one type of genomic clone contains a single copy of each of the convergently transcribed MO-IH-1 and MIH genes clustered within 6.5kb. The other type contains only the MO-IH-2 gene, which is not closely linked to an MIH gene. There are three exons and two introns in all MIH and MO-IH genes analysed. The exon-intron boundary of the crab MIH and MO-IH genes follows Chambon's rule (GT-AG) for the splice donor and acceptor sites. The first intron occurs within the signal peptide region and the second intron occurs in the coding region of the mature peptide. Sequence analysis of upstream regions of MO-IH and MIH genes showed that they contained promoter elements with characteristics similar to other eukaryotic genes. These included sequences with high degrees of similarity to the arthropod initiator, TATA box and cAMP response element binding protein. Additionally, putative CF1/USP and Broad Complex Z2 transcription factor elements were found in the upstream regions of MIH and MO-IH genes respectively. The implications of the presence of the latter two putative transcription factor binding-elements for control of expression of MIH and MO-IH genes is discussed. Phylogenetic analysis and gene organisation show that MO-IH and MIH genes are closely related. Their relationship suggests that they represent an example of evolutionary divergence of crustacean hormones.

A Xenopus borealis homeobox gene expressed preferentially in posterior ectoderm.

We have isolated a new homeobox-encoding gene (XhoxB.1) from Xenopus borealis which has a homeobox exon identical to that of the murine Hox1.7 gene, except for one amino acid. XhoxB.1 transcripts of 2.1 and 7.0 kb are first detected at the late gastrula stage, accumulate until the tailbud stage and are most abundant in the posterior third of the dorsal part of the embryo.

Characterization of cDNA encoding molt-inhibiting hormone of the crab, Cancer pagurus; expression of MIH in non-X-organ tissues.

Synthesis of ecdysteroids (molting hormones) by crustacean Y-organs is regulated by a neuropeptide, molt-inhibiting hormone (MIH), produced in eyestalk neural ganglia. We report here the molecular cloning of a cDNA encoding MIH of the edible crab, Cancer pagurus. Full-length MIH cDNA was obtained by using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides based upon the amino acid sequence of MIH, in conjunction with 5'- and 3'-RACE. Full-length clones of MIH cDNA were obtained that encoded a 35 amino acid putative signal peptide and the mature 78 amino acid peptide. Of various tissues examined by Northern blot analysis, the X-organ was the sole major site of expression of the MIH gene. However, a nested-PCR approach using non-degenerate MIH-specific primers indicated the presence of MIH transcripts in other tissues. Southern blot analysis indicated a simple gene arrangement with at least two copies of the MIH gene in the genome of C. pagurus. Additional Southern blotting experiments detected MIH-hybridizing bands in another Cancer species, Cancer antennarius and another crab species, Carcinus maenas.

Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro.

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega' sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega'. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence. These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.

Detectable levels of serum aflatoxin B1-albumin adducts in the United Kingdom population: implications for aflatoxin-B1 exposure in the United Kingdom.

This study aimed to estimate aflatoxin B1 (AFB1) exposure in the United Kingdom population by measuring levels of serum AFB1-albumin (alb), using immunoassay and high-performance liquid chromatography (HPLC) with fluorescence detection. A self-questionnaire on dietary habits from 104 volunteers (47 men and 57 women) in York was completed, and blood samples were collected. Serum alb was extracted, and AFB1-lysine (lys), the digest product of AFB1-alb, was isolated and measured. A sensitive ELISA (detection limit, approximately 1.4 pg of AFB1-lys) was developed. A good correlation was found between calibration of ELISA results and scintillation counting, for rats dosed with [3H]AFB1 (r = 0.972; P < 0.001). This ELISA was subsequently used to analyze human serum alb. For United Kingdom human sera, the mean adduct levels were 29.3 +/- 14.8 pg AFB1-lys equivalents (eq) mg albumin (males) and 26.9 +/- 14.4 pg AFB1-lys eq/mg alb (females). Confirmation of the ELISA data was sought using reversed-phase HPLC with fluorescence detection. HPLC chromatograms of digested York serum alb were compared to digested serum alb for humans from Qidong County, People's Republic of China, and from AFB1-dosed rats. These all gave similar HPLC profiles. Each sample contained fluorescent material that coeluted with and just before the AFB1-lys standard. Fluorescent fractions were found to be inhibitory in a separate anti-AFB1-lys ELISA, indicating that these earlier fluorescent peaks contained AFB1 residues. Our results suggest that measurable internal AFB1 exposure may be occurring in some United Kingdom individuals, albeit at lower levels than those seen for areas with high AFB1 exposure. The source of this exposure may reflect the known difficulties in accurately monitoring regulated imported foodstuffs and/or the lack of regulations on other potentially contaminated imports. However, no positive correlations were found between our AFB1-lys measurements and any dietary questionnaire information. Animal studies, as well as human studies, have been important in developing exposure and internal adduct relationships in humans. Based on this literature, our AFB1-alb data indicate a mean daily exposure of 3 microg of AFB1 and a mean internal dose in liver DNA of 5.9 adducts/10(7) nucleotides. We believe this may be an overestimate of the AFB1 exposure level in the United Kingdom, and further studies are needed to accurately relate external dose and internal AFB1 biomarkers in humans.

A PCR-based method for manipulation of the vaccinia virus genome that eliminates the need for cloning.

A general method is described for altering specific genes of vaccinia virus (VV). We demonstrate and evaluate the procedure by gene inactivation, using a dominant selectable marker in conjunction with recombinant polymerase chain reaction (PCR). Primers based on the sequence of the target gene enable amplification of flanking arms and their subsequent attachment to the gpt cassette that confers resistance to mycophenolic acid. Linear PCR constructs are transfected into cells infected with wild-type vaccinia virus. Mutant viruses with gpt inserted into the target gene by homologous recombination are then selected by growth in the presence of MPA. This technique was applied to the vaccinia virus thymidine kinase gene and compared to the traditional method of constructing gpt-containing plasmids by cloning. The PCR scheme was found to be highly efficient and could theoretically be used to insert any foreign DNA element into any nonessential target gene for which partial or complete sequence information is available. The procedure can potentially be used for a wide variety of genetic modifications, including the insertion of foreign genes, with poxviruses and other DNA viruses. Genomes of microorganisms, such as bacteria and yeast that can be transformed with linear DNA, are also candidates for manipulation by this methodology.

A novel neuropeptide-endocrine interaction controlling ecdysteroid production in ixodid ticks.

Ixodid (hard) ticks are blood-feeding arthropods that require a blood meal to complete each stage of development. However, the hormonal events coordinating aspects of feeding and development are only poorly understood. We have delineated a new neuropeptide-endocrine interaction in the adult tick, Amblyomma hebraeum, that stimulates the synthesis of the moulting hormones, the ecdysteroids. In adult female ticks, ecdysteroid synthesis could be demonstrated in integumental tissue incubated in vitro with a synganglial (central nervous system) extract, but not in its absence. Stimulation by the synganglial extract is both time- and dose-dependent, but is completely abolished by trypsin treatment, suggesting that the activity is due to a peptide/protein. Integumental tissue ecdysteroidogenesis is also stimulated by elevation of the cAMP concentration using forskolin and 3-isobutyl-l-methyl-xanthine, or by 8-bromo-cAMP. This suggests the involvement of at least a cAMP second messenger system in the neuropeptide-ecdysteroidogenesis axis, without precluding a role for other second messengers as well. Despite involving a quite different steroidogenic tissue, the foregoing system has some parallels with the known prothoracicotropic hormone (neuropeptide)-prothoracic gland endocrine axis of insects.