Choice test to determine potential attractants and repellents for the sheep scab mite, Psoroptes ovis (Acari: Psoroptidae).

A choice test bioassay was devised to screen compounds as potential semiochemicals (e.g., kairomones or allomones that mediate aggregation, attraction or repellence) for the obligate parasitic mite, Psoroptes ovis. The choice test used filter paper discs in a 1:4 test:control ratio and was found to be a reliable, effective and efficient method. Four mammalian lipid components were assessed as potential attractants-linoleic acid, arachidonic acid, methyl myristate and squalene-, and the insect/tick repellent DEET for potential repellence. Linoleic acid was significantly attractive to P. ovis adult females and has the potential to act as an attractant. Identification of P. ovis semiochemicals, especially attractants, would be beneficial in the development of novel control methods and tools for this species. This is essential considering the increase in resistance to the limited prophylactic chemical treatments in the UK, and the high prevalence of scab infections.

Identification of T-cell epitopes from benzylpenicillin conjugated to human serum albumin and implication in penicillin allergy.

BACKGROUND: There is in vitro evidence that T cells from allergic patients react to benzylpenicillin-human serum albumin (BP-HSA) bioconjugates. Our group has recently shown the existence of naïve CD4+ T cells recognizing BP-HSA in healthy donors. However, BP-haptenated peptides from HSA participating in the immunization of allergic patients have never been identified. The purpose of the present study is to identify immunodominant BP-haptenated peptides from HSA involved in immunization of patients to BP and to refine the frequency calculation of naïve CD4+ T cells recognizing BP. METHODS: Co-cultures were established with CD4+ T cells from non-allergic donors and mature autologous dendritic cells (DCs) loaded with BP-HSA or BP-haptenated peptides from HSA. The CD4+ T-cell response specific for BP-HSA or for individual BP-haptenated peptides was measured using an interferon-γ (IFN-γ) ELISpot assay. The frequency of BP-specific CD4+ T cells was then calculated using the Poisson distribution. BP-HSA and BP-haptenated peptides recognition by allergic patients was evaluated on peripheral blood mononuclear cells (PBMCs) using a lymphocyte transformation test (LTT). RESULTS: Results showed that BP-HSA and BP-haptenated peptides were recognized by naïve T cells from 15/16 and 13/14 tested healthy donors, respectively. Most donors responded to 3 peptides with BP covalently bound on lysines 159, 212, and 525. Two of these benzylpenicilloylated peptides (lysines 159 and 525) were also found to induce PBMCs proliferation in patients with allergic reaction to penicillins. CONCLUSION: This study identifies and characterizes for the first time the BP-haptenated peptides from HSA involved in the immunization of patients to penicillins.

Effect of stress on heat shock protein levels, immune response and survival to fungal infection of Mamestra brassicae larvae.

Although the utilisation of fungal biological control agents to kill insect pests is desirable, it is known that the outcome of infection may be influenced by a number of criteria, including whether or not the target insect is stressed. In the current work, topical treatment of larvae of the lepidopteran pest, Mamestra brassicae, with conidia of Beauveria bassiana, followed by a heat stress (HS; 37°C for 1h) 48h later, resulted in a similar level of larval survival to that occurring for no heat stress (No-HS), fungus-treated larvae. By contrast, when the HS was applied 24h after fungal treatment, larval survival was significantly increased, indicating that the HS is protecting the larvae from B. bassiana. Similarly, exposure of larvae to a HS provided protection against Metarhizium brunneum (V275) at 48h (but not 24h) after fungal treatment. To elucidate the mechanism(s) that might contribute to HS-induced increases in larval survival against fungal infection, the effects of a HS on key cellular and humoral immune responses and on the level of selected heat shock proteins (HSP) were assessed. When larvae were kept under control (No HS) conditions, there was no significant difference in the haemocyte number per ml of haemolymph over a 24h period. However, exposure of larvae to a HS, significantly increased the haemocyte density immediately after (t=0h) and 4h after HS compared to the No HS controls, whilst it returned to control levels at t=24h. In addition, in vitro assays indicated that haemocytes harvested from larvae immediately after (0h) and 4h (but not 24h) after a HS exhibited higher rates of phagocytosis of FITC-labelled B. bassiana conidia compared to haemocytes harvested from non-HS larvae. Interestingly, the HS did not appear to increase anti-fungal activity in larval plasma. Western blot analysis using antibodies which cross react with Drosophila melanogaster HSP, resulted in a relatively strong signal for HSP 70 and HSP 90 from extracts of 50,000 and 100,000haemocytes, respectively, harvested from No-HS larvae. By contrast, for HSP 60, a lysate derived from 200,000haemocytes resulted in a relatively weak signal. When larvae were exposed to a HS, the level of all three HSP increased compared to the No HS control 4h and 16h after the HS. However, 24h after treatment, any heat stress-mediated increase in HSP levels was minimal and not consistently detected. Similar results were obtained when HSP 90, 70, and 60 levels were assessed in fat body harvested from heat stressed and non-heat stressed larvae. With regard to HSP 27, no signal was obtained even when a lysate from 200,000haemocytes or three times the amount of fat body were processed, suggesting that the anti-HSP 27 antibody utilised does not cross-react with the M. brassicae HSP. The results suggest that a HS-mediated increase in haemocyte density and phagocytic activity, together with an upregulation of HSP 90 and 70, may contribute to increasing the survival of M. brassicae larvae treated with B. bassiana and M. brunneum (V275).

Primary screen for potential sheep scab control agents.

The efficacy of potential acaricidal agents were assessed against the sheep scab mite Psoroptes ovis using a series of in vitro assays in modified test arenas designed initially to maintain P. ovis off-host. The mortality effects of 45 control agents, including essential oils, detergents, desiccants, growth regulators, lipid synthesis inhibitors, nerve action/energy metabolism disruptors and ecdysteroids were assessed against adults and nymphs. The most effective candidates were the desiccants (diatomaceous earth, nanoclay and sorex), the growth regulators (buprofezin, hexythiazox and teflubenzuron), the lipid synthesis inhibitors (spirodiclofen, spirotetramat and spiromesifen) and the nerve action and energy metabolism inhibitors (fenpyroximate, spinosad, tolfenpyrad, and chlorantraniliprole).

Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR(-)) rats.

Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km=3.2±0.8µM (SD), 9.0±3.1µM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km=192.1±291.5µM (SD), 69.2±36.2µM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h)=2.0±0.5) and vesicles (h=1.6±0.2) expressing Mrp2 and from SD (h=1.6±0.4) and Wistar (h=4.0±0.6) hepatocytes suggests transport involves more than one binding site. In TR(-) hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km=100.7±36.0µM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.

Identification and frequency of circulating CD4(+) T lymphocytes specific to Benzylpenicillin in healthy donors.

BACKGROUND: Drug hypersensitivity is known to rely on a drug-specific T-cell response. Amplitude of antigen-specific T-cell response is partly controlled by the size of the antigen-specific naïve CD4(+) T-cell repertoire, but estimate of this repertoire has never been investigated for allergenic drugs. The purpose of this study was to evaluate the frequency of benzylpenicillin-specific CD4(+) T lymphocytes in healthy donors. METHODS: Co-cultures were established with CD4(+) T lymphocytes from healthy donors and mature autologous dendritic cells loaded with benzylpenicillin coupled to human serum albumin. CD4(+) T lymphocytes were stimulated once a week for 4 weeks with benzylpenicillin coupled to human serum albumin. The CD4(+) T-cell response was measured using an interferon-γ ELISPOT assay. Frequency of benzylpenicillin-specific naive CD4(+) T lymphocytes was then calculated using the Poisson distribution law. RESULTS: Results showed the presence of benzylpenicillin-specific CD4(+) T lymphocytes in 9 of 10 tested healthy donors irrespective of their HLA typing, with a mean frequency of 0.29 cells per million of CD4(+) T cells. Experiments performed on naive (CD45RA(+) ) and on memory (CD45RO(+) ) CD4(+) T lymphocytes showed that these benzylpenicillin-specific CD4(+) T lymphocytes belonged to the naive T-cell subpopulation. CONCLUSION: This study showed for the first time the existence of naive CD4(+) T lymphocytes specific to benzylpenicillin in healthy donors.

Neuropeptide regulators of juvenile hormone synthesis: structures, functions, distribution, and unanswered questions.

Juvenile hormones (JH), produced by the corpora allata, have an essential role in growth and development, morphogenesis, and reproductive processes of insects. The output of JH and circulating titer are required to be precisely regulated throughout the insect's life in response to developmental requirements and environmental factors. The synthesis of JH must be periodically turned off and on, or finely tuned, in a highly coordinated way. Except for a few key or intensely studied insect species, the control of synthesis of JH by regulatory peptides remains largely undefined and many of the details remain obscure. Several different classes of neuropeptide are believed to be involved in the regulation of corpus allatum function and hence JH output. In different insect species and at different stages of development, these regulatory peptides may include at least three types of inhibitory allatostatins, at least one type of stimulatory allatotropin, and perhaps several other, as yet largely undefined, additional neuropeptides. The details of how each of these peptides acts to affect JH production and their relationship to each other in the coordination of JH synthesis remain to be established. There are several insect orders for which almost nothing is known concerning the regulation of JH synthesis and the peptides that might be involved. Current proteomic and genomic studies are helping to redress this balance but at the same time posing new questions. Other neuropeptides are implicated in the regulation of JH production, and there is new evidence concerning the mode of action of allatotropins.

Degradation of leucomyosuppressin by enzymes in the hemolymph and midgut of Lacanobia oleracea and Spodoptera littoralis (Lepidoptera: Noctuidae) larvae.

The degradation of 2 nmol synthetic leucomyosuppressin (LMS) by enzymes of the hemolymph, midgut lumen and midgut tissues of both Lacanobia oleracea and Spodoptera littoralis was investigated using reversed-phase high-performance liquid chromatography together with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Degradation of LMS in diluted hemolymph of L. oleracea and S. littoralis was not rapid, t(1/2)=65.4 and 13.1 min, respectively, due to carboxypeptidase(s) and endopeptidase(s) present in the hemolymph. There was also some aminopeptidase activity in the hemolymph of both species. However, LMS was rapidly degraded by the diluted contents of the midgut lumen of L. oleracea and S. littoralis, t(1/2)=0.5 and 2.2 min, respectively. The enzymes most likely responsible were trypsin-like serine protease(s) and carboxypeptidase(s). Degradation of LMS by midgut tissues containing 5 microg protein was not rapid in L. oleracea or S. littoralis, t(1/2)=40.7 and 69.8 min, respectively. The most abundant degradation products probably resulted from carboxypeptidase activity, but some aminopeptidase activity was also detected.

Neuropeptides associated with the regulation of feeding in insects.

The stomatogastric nervous system plays a pivotal role in feeding behaviour. Central to this system is the frontal ganglion, which is responsible for foregut motor activity, and hence the passage of food through the gut. Many insect peptides, which exhibit myoactivity on the visceral muscles of the gut in vitro, have been detected in the stomatogastric nervous system by immunochemical or mass spectrometric techniques. This localisation of myoactive peptides, particularly in the frontal ganglion, implies roles for these peptides in the neural control and modulation of feeding in insects. Insect sulfakinins, tachykinins, allatotropin and proctolin have all been shown to stimulate the foregut muscles, whereas myosuppressins, myoinhibitory peptides and allatostatins all inhibited spontaneous contractions of the foregut in a variety of insects. Some of these peptides, when injected, inhibited feeding in vivo. Both the A-type and B-type allatostatins suppressed feeding activity when injected into the cockroach, Blattella germanica and the Manduca sexta C-type allatostatin and allatotropin inhibited feeding when injected into the larvae of two noctuid moths, Lacanobia oleracea and Spodoptera frugiperda, respectively. Injection of sulfakinins into the fly Phormia regina, the locust Schistocera gregaria and the cockroach B. germanica also suppressed feeding, whereas silencing the sulfakinin gene through the injection of double stranded RNA resulted in an increase in food consumption in the cricket Gryllus bimaculatus. The regulation of feeding in insects is clearly very complex, and involves the interaction of a number of mechanisms, one of which is the release, either centrally or locally, of neuropeptides. However, the role of neuropeptides, their mechanisms of action, interactions with each other, and their release are still poorly understood. It is also unclear why insects possess such a number of different peptides, some with multiples copies or homologues, which stimulate or inhibit gut motility, and how their release, sometimes from the same neurone, is regulated. These neuropeptides may also act at sites other than visceral muscles, such as centrally through the brain or on gut stretch receptors.

In vitro and in vivo effects of myo-active peptides on larvae of the tomato moth Lacanobia oleracea and the cotton leaf worm Spodoptera littoralis (Lepidoptera; Noctuidae).

Neuropeptides from five different neuropeptide families [Manduca sexta allatostatin (Manse-AS), and Manse-AS deletion analogue(5-15), M. sexta allatotropin (Manse-AT), leucomyosuppressin, perisulfakinin, and myoinhibitory peptide I (MIP I)] were assayed for their ability to affect the development and food consumption of penultimate and last larval instars of two lepidopteran species, L. oleracea and S. littoralis. Injections of Manse-AS deletion analogue(5-15), Manse-AT, perisulfakinin, and MIP I had no observable effects on development, food consumption, or mortality compared to controls. Single injections of Manse-AS significantly reduced the weight gain and increased mortality of L. oleracea and S. littoralis larvae compared to controls. By contrast, feeding Manse-AS to L. oleracea had no such effects. These differences were probably due to the degradation of the peptide by digestive enzymes in the foregut of L. oleracea. In studies in vitro, perisulfakinin, and MIP I had no effect on the spontaneous foregut contractions of L. oleracea larvae. Leucomyosuppressin, however, had myoinhibitory effects on the foregut. Single injections of leucomyosuppressin significantly reduced the weight gain and food consumption of L. oleracea and S. littoralis larvae and increased mortality. These data suggest that the deleterious effects observed in vivo were due to the myoinhibition by Manse-AS and leucomyosuppressin of the normal peristaltic movements of the gut either by the intact peptide or by its cleavage products resulting from degradation in the haemolymph.

Allatoregulatory peptides in Lepidoptera, structures, distribution and functions.

Allatoregulatory peptides either inhibit (allatostatins) or stimulate (allatotropins) juvenile hormone (JH) synthesis by the corpora allata (CA) of insects. However, these peptides are pleitropic, the regulation of JH biosynthesis is not their only function. There are currently three allatostatin families (A-, B-, and C-type allatostatins) that inhibit JH biosynthesis, and two structurally unrelated allatotropins. The C-type allatostatin, characterised by its blocked N-terminus and a disulphide bridge between its two cysteine residues, was originally isolated from Manduca sexta. This peptide exists only in a single from in Lepidoptera and is the only peptide that has been shown to inhibit JH synthesis by the CA in vitro in this group of insects. The C-type allatostatin also inhibits spontaneous contractions of the foregut. The A-type allatostatins, which exist in multiple forms in a single insect, have also been characterised from Lepidoptera. This family of peptides does not appear to have any regulatory effect on JH biosynthesis, but does inhibit foregut muscle contractions. Two structurally unrelated allatotropins stimulate JH biosynthesis in Lepidoptera. The first was identified in M. sexta (Manse-AT) and occurs in other moths. The second (Spofr AT2) has only been identified in Spodoptera frugiperda. Manduca sexta allatotropin also stimulates heart muscle contractions and gut peristalsis, and inhibits ion transport across the midgut of larval M. sexta. The C-terminal (amide) pentapeptide of Manse-AT is important for JH biosynthesis activity. The most active conformation of Manse-AS requires the disulphide bridge, although the aromatic residues also have a significant effect on biological activity. Both A- and C-type allatostatins and Manse-AT are localised in neurosecretory cells of the brain and are present in the corpora cardiaca, CA and ventral nerve cord, although variations in localisation exist in different moths and at different stages of development. The presence of Manse-AS and Manse-AT in the CA correlates with the biological activity of these peptides on JH biosynthesis. There is currently no explanation for the presence of A-type allatostatins in the CA. The three peptide types are also co-localised in neurosecretory cells of the frontal ganglion, and are present in the recurrent nerve that supplies the muscles of the gut, particularly the crop and stomodeal valve, in agreement with their role in the regulation of gut peristalsis. There is also evidence that they are expressed in the midgut and reproductive tissues.

Excess mortality in two-year rodent carcinogenicity studies.

This paper considers the impacts of various patterns of differential or excess mortality on the biological and statistical interpretation of 2-year rodent carcinogenicity studies. It provides suggestions on experimental design that are intended to maximize the value of such studies for carcinogenic risk assessment. Specifically, it recommends dose reduction, possibly to the level of dose cessation, when biologically feasible and considers the merits of termination of the entire study as alternatives to the commonly employed strategy of terminating particular dose groups. It then recommends statistical analysis modifications that are appropriate when these suggestions on experimental design are adopted. One of the recommended modifications is a new statistical test to determine whether a dose group exceeds the maximum tolerated dose (MTD) on the basis of mortality. While the authors provide recommendations for the most commonly occurring exigencies, they acknowledge the need for and strongly support the practice of active engagement of the appropriate regulatory agency, e.g., the FDA, prior to any action.

Interactions between allatostatins and allatotropin on spontaneous contractions of the foregut of larval Lacanobia oleracea.

The interactions between the activity of three neuropeptides, Manduca sexta allatostatin (Manse-AS), M. sexta allatotropin (Manse-AT) and cydiastatin 4, on the spontaneous foregut contractions of the tomato moth, Lacanobia oleracea, were investigated. Bioassays revealed that application of Manse-AS to the foregut at high concentrations (10(-7)M) stopped contractions completely, and this inhibition could not be reversed by Manse-AT. Conversely, Manse-AS could inhibit a Manse-AT stimulated tissue. In contrast, Manse-AT reversed the inhibition of foregut peristalsis by cydiastatin 4 (10(-7)M), and cydiastatin 4 counteracted the stimulation by Manse-AT. These results imply that the Manse-AS inhibitory effect is dominant over the stimulatory action of Manse-AT. However, when two peptides with opposing actions were added together, the overall effect on foregut peristalsis was determined by the relative concentrations of each peptide, suggesting that in these experiments, no peptide was dominant over the other. When Manse-AS and cydiastatin 4 were applied to foregut tissues simultaneously the overall effect was not significantly different to the individual peptides, i.e. there was no additive effect. This suggests that the individual activities of Manse-AS and cydiastatin 4 are suppressed by an undetermined mechanism in the presence of the other peptide. These results question the need for two structurally different allatostatins that have the same physiological effect on foregut peristalsis in L. oleracea larvae.

Alanine substitution and deletion analogues of Manduca sexta allatostatin: structure-activity relationship on the spontaneous contractions of the foregut of larval Lacanobia oleracea.

Manduca sexta allatostatin (Manse-AS) is a 15-residue non-amidated peptide with a blocked N-terminus and a disulphide bridge between the cysteine residues at positions 7 and 14. Analogues of Manse-AS were used to examine the structural requirements of Manse-AS for inhibitory activity on spontaneous foregut contractions of larval tomato moth (Lacanobia oleracea). Breaking the disulphide bond between C(7) and C(14) by reduction reduced the potency of the peptide, suggesting that the conformation of Manse-AS is important for its biological activity. When either of the cysteine residues were replaced with alanine the Manse-AS analogue had no measurable bioactivity. Alanine substitution at Q(6) was as potent as Manse-AS, all other alanine substitution analogues (R(5), Y(8), F(9), N(10), P(11), I(12) and S(13)), were myoinhibitory but less potent than native Manse-AS to varying degrees. Analogues with alanine substitution at amino acids with aromatic side chains (Y(8) and F(9)) were the least active. Amino-terminal deletion analogues Manse-AS(6-15) and Manse-AS(7-15) were inactive whereas Manse-AS(5-15) was fully active but not as potent as Manse-AS. The results show that amino acid residues both inside and outside the disulphide ring are important for biological activity.

Angiotensin I-converting enzyme (ACE) activity of the tomato moth, Lacanobia oleracea: changes in levels of activity during development and after copulation suggest roles during metamorphosis and reproduction.

Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that removes C-terminal dipeptides from relatively short oligopeptides, usually smaller than 15 amino acids. In mammals, the enzyme has several important roles in the metabolism of vasoactive peptides, but its physiological role in insects is not fully understood. We now report the properties of an ACE in a lepidopteran species (the tomato moth, Lacanobia oleracea) and suggest new physiological roles for the enzyme in this insect. ACE activity increases four-fold during the last stadium and in early pupae, a rise which, in its timing, is similar to what has been observed previously in the transition of larva to pupa in Drosophila melanogaster. This suggests that the increase in ACE activity might be of general importance for peptide metabolism during metamorphosis in holometabolous insects. High levels of ACE activity were found in the haemolymph of sixth stadium larvae and adult insects, and in the reproductive tissues of both male and female adults. Almost all of the ACE activity in the reproductive tissues was found in the accessory glands of the male and the spermatheca and bursa copulatrix of the female. The decline in accessory gland ACE in mated males and the concomitant rise in ACE activity in the spermatheca and bursa copulatrix of the female suggested the transfer of ACE from the male to the female during copulation. Using several convenient peptides as substrates, we have shown that the spermatophore/bursa copulatrix taken from mated female insects possess an aminopeptidase, a carboxypeptidase and a dipeptidase, in addition to high levels of ACE. These peptidases might be involved in the breakdown of proteins to peptides and eventually to amino acids in the spermatophore. Evidence for such a proteolytic pathway and its role in providing substrates for the TCA cycle has been obtained previously in a study of reproduction in Bombyx mori.

Human apolipoprotein E4 accelerates beta-amyloid deposition in APPsw transgenic mouse brain.

The human apolipoprotein E4 (ApoE4) isoform is associated with genetic risk for Alzheimer's disease. To assess the effects of different ApoE isoforms on amyloid plaque formation, human ApoE3 and ApoE4 were expressed in the brains of transgenic mice under the control of the human transferrin promoter. Mice were crossed with transgenic mice expressing human amyloid precursor protein containing the Swedish mutation (APPsw), which facilitates amyloid beta peptide (A beta) production. The following progeny were selected for characterization: APPsw+/- x ApoE3+/- and APPsw+/-, APPsw+/- x ApoE4+/- and APPsw+/- littermates. All mice analyzed were wild type for the endogenous mouse APP and ApoE genes. Mice expressing ApoE4 in combination with APPsw have accelerated A beta deposition in the brain as assessed by enzyme immunoassay for A beta40 and A beta42 extractable in 70% formic acid, by assessment of amyloid plaque formation using thioflavin-S staining, and by immunohistochemical staining with antibodies specific for A beta40 or A beta42 and the 4G8 monoclonal or 162 polyclonal antibody. No difference in the rate of A beta deposition in the brain was seen in mice expressing ApoE3 in combination with APPsw. Thus, our data are consistent with the observation in Alzheimer's disease that ApoE4 is associated with increased accumulation of A beta in the brain relative to ApoE3.

Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites.

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C18 analytical column (10 cm x 0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran-acetonitrile-water (10:10:80, v/v) changing to tetrahydrofuran-acetonitrile-water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic separation of eight metabolites of testosterone is 15 min. Detection by UV is linear between 300 ng/ml and 10 microg/ml with a limit of detection on column of 300 ng/ml. A method for the direct HPLC analysis of liver microsomal incubates of [14C]testosterone is also briefly described and when combined with the HPLC method, offers a distinct advantage over previously reported methods for the rapid screening of testosterone hydroxylase activity in rat and human liver microsomes.

Assessment of drug-drug interactions: concepts and approaches.

1. A priori knowledge of the enzyme inhibitory potential of new drug entities and the drug-metabolizing enzymes involved can be used in support of important decisions on the future progress of a drug in clinical development. 2. Important advances in the knowledge of human drug-metabolizing enzymes have largely fuelled the integration of in vitro drug metabolism and clinical drug interaction studies for use in drug development programmes. 3. The likelihood of correctly predicting in vivo drug-drug interactions appears highly dependent on selecting the correct enzyme inhibition model for use in deriving the inhibitor constant (Ki) and correctly determining the available concentration of inhibitor at the active site of the enzyme(s) of interest. 4. The uncertainty and inaccuracy of predicting the extent and duration of in vivo drug interactions currently stems from a lack of definitive models by which to assess likely substrate and inhibitor concentrations at the active site of metabolism. Additional issues contributing to the uncertainty of predicting drug interactions include assumptions of the contribution of presystemic drug extraction and the effect of inhibitors on the processes involved. 5. This review considers the practical aspects of in vitro enzyme inhibition studies and the use of in vitro-in vivo correlation approaches described in the literature to predict in vivo drug-drug interactions.

The role of allatostatic and allatotropic neuropeptides in the regulation of juvenile hormone biosynthesis in Lacanobia oleracea (Lepidoptera: Noctuidae).

In the sphinghid moth Manduca sexta, two allatoactive neuropeptides appear to be responsible for regulating juvenile hormone (JH) production by the corpora allata (CA). These peptides (M. sexta allatostatin, Mas-AS, and M. sexta allatotropin, Mas-AT) respectively inhibit and stimulate in vitro JH biosynthesis by CA in this insect. However, although Mas-AS inhibits CA in both larval and adult insects, Mas-AT is active only in adult M. sexta. The situation in other lepidopteran species is less clear-cut and, although both peptides have been detected (usually by immunologic and/or molecular techniques) in several other moths (including noctuids), their function as regulators of JH production remains uncertain. In the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae), we have previously demonstrated the occurrence of Mas-AS and/or Mas-AT in extracts of CA, brain and other organs, and have shown that both peptides are present in larval and adult forms. However, in L. oleracea, although Mas-AS inhibits larval and adult CA in vitro, it does so only at relatively high concentrations, and to a maximum of only approximately 70%. By contrast, Mas-AT (which is also present in larval and adult L. oleracea) stimulates larval and adult CA, but is substantially more potent ( approximately 100 fold) than the allatostatin. In this paper we present the results of paired, concurrent measurements (using ELISA) of levels of Mas-AS and Mas-AT in brains, CA and hemolymph (plasma and hemocytes) of L. oleracea at times when there are marked changes in JH titers. We also present data on the in vitro rates of JH biosynthesis by isolated CA, and on hemolymph JH esterase activity measured at the same critical developmental times, and discuss all of these data in relation to the putative allatoregulatory roles of the M. sexta allatotropic and allatostatic neuropeptides in L. oleracea.

Morphological and physiological comparisons of two types of allatostatin in the brain and retrocerebral complex of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae).

The cellular localisation of two types of allatostatin in the brain and retrocerebral complex has been studied in larvae of Lacanobia oleracea (Noctuidae) using antisera against Manduca sexta allatostatin (Mas-AS) and two members of the Y/FXFGL-NH(2) allatostatin family. The axons of two groups of Mas-AS-immunoreactive neurosecretory cells in the pars lateralis form part of the nervi corporis cardiaci (NCC 1). They exit the brain as the combined NCC 1 and NCC 2 and pass through the corpora cardiaca (CC), where they divide to innervate the corpora allata (CA) and the mandibular (salivary) gland. The presence of Mas-AS immunoreactivity in the CA is consistent with the inhibitory action of this peptide on juvenile hormone (JH) biosynthesis in L. oleracea. Immunoreactivity in the mandibular gland nerve suggests an additional, as yet unidentified role for this peptide. Cells of the pars intermedialis, the main contributors to NCC 2, do not show Mas-AS immunoreactivity. The distribution of Y/FXFGL-NH(2) immunoreactivity is different from that of Mas-AS. Although there are fewer cells in the pars lateralis, immunoreactivity is observed in certain neurones of the pars intermedialis and the tritocerebrum. Axons of these latter neurones contribute to NCC 2 and NCC 3, respectively, and, combined with those from NCC 1, result in the prominent occurrence of Y/FXFGL-NH(2) immunoreactivity in the CC, particularly in the storage lobe. The CA has far less Y/FXFGL-NH(2) immunoreactivity compared with Mas-AS. In bioassays, the Y/FXFGL-NH(2) allatostatins did not inhibit JH synthesis by CA of L. oleracea.