Irradiation influence on the phenoloxidase pathway and an anti-oxidant defense mechanism in Spodoptera litura (Lepidoptera: Noctuidae) and its implication in radio-genetic 'F 1 sterility' and biorational pest suppression tactics.

The present study was conducted to appraise the ontogenic radio-sensitivity of a serious tropical pest, Spodoptera litura (Fabr.). The molecular responses pertaining to the phenoloxidase (PO) pathway and an anti-oxidant defense mechanism were evaluated in order to understand its implication in pest control at pre-harvest and post-harvest intervals. Irradiation exhibited an inverse relationship with age with respect to impact on developmental and transcriptional responses. Transcript abundance of PO cascade enzymes, prophenoloxidase (slppo-2), its activating enzyme (slppae-1) and free-radical scavenging enzymes, superoxide dismutase (slsod) and catalase (slcat) was evaluated upon gamma irradiation alone and the dual-stress of radiation plus microbial challenge. The slppo-2, slppae-1, slsod and slcat transcripts were significantly up-regulated in F 1 L6 larvae (6th-instar) resulting from 100 Gy sub-sterilized male adults and unirradiated female moths. The extent of upregulation was relatively higher in comparison with L6 survivors (6th-instar larvae) developed from irradiated neonates (L1) treated with 100 Gy. Upon Photorhabdus challenge, the transcripts were down-regulated in irradiated L1 suggesting increased larval susceptibility to bacterial infections. Radioresistance increased with the age of the insect, and molecular responses (transcript abundance) of insect defense mechanism were less influenced when older age (F 1 progeny) were irradiated. These findings will help to optimize the gamma dose to be employed in inherited sterility technique for (pre-harvest) pest suppression and (post-harvest) phytosanitation and quarantine, and suggest compatible integration of biorational tactics including nuclear technology.

Uterine catecholamines and prostaglandins during the estrous cycle of the rat.

The uterine contents of norepinephrine, epinephrine, dopamine, and prostaglandins F and E were determined on each day of the rat estrous cycle. Catecholamines were determined by high performance liquid chromatography with electrochemical detection (HPLC-EC) as well as by a radioenzymatic technique; prostaglandins were quantitated by RIA. The norepinephrine and dopamine values obtained by the radioenzymatic assay were approximately 1.5 times as high as the values obtained by HPLC-EC (norepinephrine, 285 vs. 188 ng/g; dopamine, 11.0 vs. 7.5 ng/g). Despite this difference in levels, both analytic techniques showed a decline in uterine norepinephrine from diestrus to estrus, followed by a significant (P less than 0.01) increase in norepinephrine on the day of metestrus. A cyclic pattern was also revealed for uterine dopamine concentration. There was a decline in dopamine concentration from diestrus to proestrus (radioenzymatic, P less than 0.01), followed by a return to high levels at metestrus (HPLC-EC). Epinephrine levels were low (undetectable by radioenzymatic assay; 24 ng/g by HPLC-EC) and showed no variation during the estrous cycle. Prostaglandin F was uniformly higher than prostaglandin E (10 vs. 2.5 ng/uterus). Significant increases in the uterine contents of both prostaglandins were shown on the day of proestrus.

Recombinant enrichment by exploitation of restriction sites with interrupted palindromes: design, synthesis and incorporation of zero-background linkers in cloning and expression vectors.

A universal approach for improving the efficiency of cloning through a selective enrichment of recombinants has been developed. This was achieved by using novel polylinkers or multiple cloning sites (MCS) termed zero-background linkers (ZBL). These MCS have short-cutter site(s) incorporated in the wobble portion of the recognition sequence of the long-cutter(s) in such a fashion that cloning at the short-cutter site disrupts the continuity of the long-cutter-specific sequence. Consequently, digestion of the ligation mixture with the long-cutter, prior to transformation, essentially eliminates the insert negative clones from transformants. The usefulness of such background removal has been illustrated through the design and synthesis of a model ZBL molecule and then its incorporation into various popular cloning and expression vectors. We demonstrate that replacement of the residing MCS of a vector with ZBL leads to enhanced cloning efficiency, as evidenced by the marked increase in the ratio of recombinants to non-recombinants. Such vector improvement is nearly universal as zero backgrounding is an add-on feature and can be adapted to practically any vector system, without having to alter the essential features of the parent vectors.

Point mutation of a conserved arginine (104) to lysine introduces hypersensitivity to inhibition by glyphosate in the 5-enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis.

The role of a conserved arginine (R104) in the putative phosphoenol pyruvate binding region of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis has been investigated. Employing site directed mutagenesis arginine was substituted by lysine or glutamine. Native and mutant proteins were expressed and purified to near homogeneity. Estimation of Michaelis and inhibitor constants of the native and mutant proteins exhibited altered substrate-inhibitor binding mode and constants. Mutation R104K hypersensitized the enzyme reaction to inhibition by glyphosate. The role of R104 in discriminating between glyphosate and phosphoenol pyruvate is discussed.

Phage display of Bacillus thuringiensis CryIA(a) insecticidal toxin.

The display of proteins or peptides on the surface of filamentous phages or phagemids has been shown to be a very powerful technology for the rescue of specific binders from large combinatorial libraries, as well as to select derivatives of known proteins with altered binding properties. The Bacillus thuringiensis (Bt) crystal proteins are a large family of insecticidal toxins which bind to receptors found on the brush border of larval midgut cells, different crystal toxins having different larval specificities. Here we describe the display of different CryIA(a) toxin regions on the surface of phagemids using the display vector pHEN1, the purpose being the identification of toxin sequences suitable for mutagenesis and selection using phage display. We show that CryIA(a) domain II, in which the receptor binding activity is located, is efficiently displayed as well as being secreted as soluble protein into the periplasm of bacterial cell. This forms the basis of a simple means for the modification of toxin specificity and the selection of toxin proteins with novel or expanded host ranges.

Age-related differences in the effect of chronic administration of naloxone on opiate binding in rat brain.

Infant and adult rats were injected chronically with either naloxone or saline for 21 consecutive days. At various intervals after cessation of the pretreatment with naloxone, animals were sacrificed and assessed for specific binding of [3H]naloxone in different regions of the CNS. Infants displayed an increase in opiate binding in the spinal cord, hypothalamus, striatum and cortex one day after cessation of the pretreatment with naloxone, but the increase in opiate binding was dissipated within one week after cessation of the pretreatment. The increase in opiate binding in infants was accompanied by an increase in the antinociceptive efficacy of morphine. In contrast to infants, adults failed to display any alteration in opiate binding following the chronic pretreatment with naloxone. Infants may be especially susceptible to naloxone-induced receptor supersensitivity because infants excrete naloxone more slowly than adults, and thus their opiate receptors may be blocked for a longer duration following an injection of naloxone.

Is domperidone a selective peripheral dopamine-receptor antagonist in vivo?

The ability of domperidone (DOM) to antagonize dopamine (DA) receptor agonist-induced hypothermia or hyperactivity was investigated in rats. Apomorphine (APO) or RDS-127 (2-di-n-propylamino-4,7-dimethoxyindane), two DA receptor agonists, produced dose-dependent hypothermia following subcutaneous (s.c.) administration. Also, RDS-127 produced hypothermia following lateral ventricular (i.c.v.) administration. The hypothermia produced by apomorphine or RDS-127 (given s.c. or i.c.v.) was antagonized by pretreatment with pimozide (0.25 mg/kg, i.p.) or domperidone (0.2, 1.0 and 5.0 mg/kg, i.p.). The hyperactivity produced by apomorphine was unaffected by pretreatment with domperidone. These data suggested that central DA receptors mediating temperature regulation, but not those mediating locomotor activity are accessible to peripherally administered domperidone. Therefore, domperidone may not be useful to differentiate hypothalamic (central) vs peripheral sites of action for DA receptor agonists in the rat.

Evidence that central dopamine receptors modulate sympathetic neuronal activity to the adrenal medulla to alter glucoregulatory mechanisms.

Previous reports suggest that analogs of dopamine (DA) can produce hyperglycemia in rats by interacting with DA receptors. Experiments reported here indicate the site of action and describe the metabolic sequalae associated with the hyperglycemic effect of apomorphine (APO), produced in conscious unrestrained rats. Apomorphine was more potent when administered by intracerebroventricular (i.c.v.) injection than when given subcutaneously (s.c.). Very small doses of the DA receptor antagonist pimozide, given intraventricularly, blocked the hyperglycemic effect of apomorphine administered subcutaneously. Sectioning of the spinal cord at thoracic vertebra T1-2 or sectioning the greater splanchnic nerve blocked apomorphine-induced hyperglycemia; whereas section of the superior colliculus or section at T5-6 had no effect. A dose of apomorphine or epinephrine (EPI) producing a similar degree of hyperglycemia elevated the concentration of EPI in serum to a similar degree, and the increase in EPI in serum preceded the increase in glucose in serum. Fasting animals for 2 or 18 hr had no significant effect on EPI- or apomorphine-induced hyperglycemia despite a reduction (91-93%) of the glycogen content of liver and skeletal muscle during the 18 hr fast. 5-Methoxyindole-2-carboxylic acid (MICA), an inhibitor of gluconeogenesis, blocked EPI- and apomorphine-induced hyperglycemia in rats fasted for 18 hr. However, 5-methoxyindole-2-carboxylic acid was ineffective in blocking hyperglycemia in animals fasted for 2 hr. Changes in insulin or glucagon in serum alone cannot account for the hyperglycemic action of apomorphine. These data demonstrate that apomorphine interacts with central DA receptors located in the hindbrain to activate sympathetic neuronal activity to the adrenal gland which subsequently releases epinephrine to alter homeostasis of glucose. Epinephrine may then, depending on the nutritional status, facilitate glycogenolytic or gluconeogenic processes to produce hyperglycemia.

Conformationally restricted congeners of dopamine derived from 2-aminoindan.

Two series of N-substituted 2-aminoindan systems have been prepared: 4,5-dihydroxy-2-aminoindan (1) has a hydroxylation pattern analogous to the alpha conformer of dopamine, and 5,6-dihydroxy-2-aminoindan (2) has a hydroxylation pattern of the beta conformer of dopamine. All members of both series demonstrated only extremely weak binding to calf caudate homogenate. Certain N-alkylated 4,5-dihydroxyindans were violent emetics in the dog and were potent in blockade of the effect of stimulation of the cardioaccelerator nerve of the cat. In contrast, the 5,6-dihydroxy series displayed low or no activity/potency in these assays. Conformational analysis of the 2-aminoindan system is described and discussed.

Conformationally restricted congeners of dopamine derived from octahydrobenzo[g]quinoline and octahydrobenzo[f]quinoline.

Series of N-alkylated derivatives of trans-octahydrobenzo[g]quinoline and of cis- and trans-octahydrobenzo[f]quinoline were prepared for pharmacological testing as congeners of 2-amino-5,7-dihydroxytetralin, which elicits dopaminergic effects in a variety of assays. Trans-fused compounds bearing N-ethyl or N-n-propyl displayed high potency/activity in inhibition of effect of stimulation of the cat cardioaccelerator nerve. Certain N-alkyl homologues in the octahydrobenzo[f]quinoline series showed high potency in binding studies in rat caudate homogenate.

5-HT1A-receptor antagonism: N-alkyl derivatives of (R)-(-)-8,11-dimethoxynoraporphine.

Prompted by previous findings that a p-dimethoxy substitution pattern on an aromatic ring permits retention of dopaminergic agonist effects in certain ring systems, catechol derivatives of which are potent dopaminergic agonists, an 8,11-dimethoxy substitution pattern was introduced into the aporphine ring in place of the 10,11-dihydroxy moiety in apomorphine. Acid-catalyzed rearrangement of an appropriate morphine derivative provided the aporphine ring system with retention of the stereochemical integrity of the 6a asymmetric center. The hydroxyl group at position 10 was removed by catalytic hydrogenolysis of its phenyltetrazoyl ether. The methyl ether of the resulting 11-hydroxyaporphine was iodinated in high yield at position 8 with trifluoroacetyl hypiodite. This is the first account of synthesis of an iodinated aporphine derivative. The 8-iodo substituent was replaced with methoxyl by reaction with sodium methoxide and cuprous iodide. Neither the N-methyl target compound 7 nor the N-n-propyl derivative 8 demonstrated dopaminergic nor serotonergic agonism. However, 7 exhibited receptor-binding characteristics and other pharmacological properties suggesting that it may be a 5-HT1A receptor antagonist.

p-Dimethoxy-substituted trans-octahydrobenzo[f]- and -[g]quinolines: synthesis and assessment of dopaminergic agonist effects.

The N-n-propyl homologues of the title compounds were prepared for further assessment of the ability of the "p-dimethoxy" moiety to confer dopaminergic agonism upon a variety of ring systems. Both the angularly and the linearly annulated trans-benzoquinoline ring derivatives displayed prominent DA2 dopaminergic effects on the peripheral sympathetic nerve terminal and displayed postjunctional dopamine receptor agonist properties in the striatum. It is speculated that the angular octahydrobenzo[f]quinoline derivative (but not the linear octahydrobenzo[g]quinoline derivative) may owe its dopamine-like effects to metabolic activation phenomena. In contrast, the cis-fused isomer of the angularly annulated benzoquinoline was inactive, as was the simple benzene derivative N,N-di-n-propyl-beta-(2,6-dimethoxyphenyl)ethylamine.

Hemicholinium-3 congeners as potential antagonists to organophosphate-induced toxicity.

A series of congeners of hemicholinium-3, in which the 1,4-oxazinium rings of hemicholinium are replaced by pyrrolidine, piperidine, 1,3-dioxane, or 1,4-oxazine rings, is described. Several of the target compounds produced blockade of neuromuscular transmission in the rabbit, and three heterocyclic derivatives, 10, 11, and 13, significantly antagonized paraoxon-induced lethality in mice. 1,3-Dioxane derivative 11 was an extremely potent antagonist of paraoxon-induced toxicity in mice, compared with prototypical protective agents physostigmine and pyridostigmine. Compound 11 exhibited a much more favorable therapeutic ratio than the reference drugs. The mechanism of action of 11 has not been elucidated, although it is concluded that it differs from that of hemicholinium-3 (inhibition of high-affinity, sodium-dependent uptake of choline into nerve terminals).

Structure-activity studies on a potent antagonist to organophosphate-induced toxicity.

Molecular modifications have been made on a highly potent, active antagonist to organophosphate-induced toxicity, 4,4'-bis[1,3-dioxan-2-ylmethyl)methylamino]acetyl]biphenyl dimethobromide (1). Stepwise removal of the oxygen atoms from the dioxane rings, as well as changing the position of attachment of substituents on the 1,3-dioxane rings and decreasing the ring size from six-membered to five-membered caused drastic or complete loss of pharmacological effect. Partial structures of 1 were all inactive. Thus, the structure of 1 seems to be remarkably specific. Additional pharmacological data are reported for 1.

Introduction of a putative dopaminergic prodrug moiety into a 6,7-substitution pattern characteristic of certain 2-aminotetralin dopaminergic agonists.

On the basis of the premise that the dopaminergic agonist profile of 2-(di-n-propylamino)-5-hydroxy-6-methyltetralin (1a) is due to in vivo oxidation of the 6-methyl moiety and that 1a may represent a novel prodrug strategy, the vicinal methyl-hydroxyl substitution pattern was incorporated into the 6- and 7-positions of 2-(di-n-propylamino)tetralin to give the 6-methyl-7-hydroxy and 6-hydroxy-7-methyl isomers 8 and 9, respectively. A multistep synthetic approach was devised which permitted preparation of target molecules 8 and 9. Pharmacological data revealed that both target compounds exhibit modest dopamine-like effects in the cardioaccelerator nerve assay in the cat, but neither appeared to be metabolically activated as was the case with 1a. The effects of 9 (but not of 8) were antagonized by pretreatment with haloperidol. Thus, the 5-hydroxy-6-methyl substitution pattern in the 2-aminotetralins remains unique as a dopaminergic agonist prodrug structure.

(R)-(-)-10-methyl-11-hydroxyaporphine: a highly selective serotonergic agonist.

Prior work in these laboratories identified (+/-)-5-hydroxy-6-methyl-2- (di-n-propylamino)tetralin as a dopaminergic agonist prodrug. The ortho methyl hydroxy aromatic substitution pattern in this molecule has now been incorporated into the aporphine ring system to give a congener of the dopaminergic agonist apomorphine in which the position 10 OH group has been replaced by methyl. Preparation of the target compound involved acid-catalyzed rearrangement of the 3-(1-phenyltetrazolyl) ether of morphine and subsequent molecular modification of the product, the 10-(1-phenyltetrazolyl) ether of (R)-(-)-apomorphine. Surprisingly, the target compound elicited no responses in any assays for effects at dopamine receptors, but rather it displayed pharmacological properties consistent with its being a serotonergic agonist with a high degree of selectivity for 5-HT1A receptors similar to the serotonergic agonist 8-hydroxy-2-(di-n-propylamino)tetralin.

Resorcinol congeners of dopamine derived from benzocycloheptene and indan.

Series of N-alkylated derivatives of 2-amino-4,6-dihydroxyindan 3 and 6-amino-1,3-dihydroxybenzocycloheptene 2 were prepared for pharmacological testing as congeners of 2-amino-5,7-dihydroxytetralin, which elicits dopaminergic effects in a variety of assays. All of the subject compounds demonstrated a lower order of dopamine-like activity than the tetralin derivatives. Some of the subject compounds showed weak interactions with alpha 1- and beta 1-adrenoceptors, but the major determinant of activity seemed to be the nature of the N-alkyl substituent rather than ring size.

2-Amino-4,7-dimethoxyindan derivatives: synthesis and assessment of dopaminergic and cardiovascular actions.

N-Alkylated derivatives of 2-amino-4,7-dimethoxyindan were prepared for evaluation of central and peripheral dopaminergic activity using biochemical and behavioral tests in the rat and cardiovascular responses in the cat. 2-(Di-n-propylamino)-4,7-dimethoxyindan (4e) demonstrated equal activity with apomorphine to activate peripheral presynaptic dopamine receptors. Central pre- and postsynaptic dopamine receptors were also activated with 4e. In contrast to the intense long-acting sympathomimetic actions previously reported for the 2-amino-5,8-dimethoxytetralins, these compounds produced weak, transient effects in heart rate and blood pressure. The majority of 2-amino-4,7-dimethoxyindan derivatives tested are weak or inactive pre- and postsynaptic dopamine receptor agonists.

Assessment of a potential dopaminergic prodrug moiety in several ring systems.

The ortho hydroxy/methyl, hydroxy/hydroxymethyl, hydroxy/formyl, and hydroxy/carboxy substitution patterns, some of which confer dopaminergic agonist effects upon 2-aminotetralin ring systems, have been incorporated into beta-phenethylamine, 2-aminoindan, and trans-octahydrobenzo[f]quinoline rings. Certain of the 2-aminoindan derivatives displayed pharmacologic properties consistent with their being dopaminergic agonists. The beta-phenethylamine derivative did not show any significant dopamine-like activity. The 7-hydroxy-8-methyloctahydrobenzo[f]quinoline derivative 4a was a moderately potent, short-acting DA2 receptor antagonist. All of the carboxylic acid derivatives were inert. Of the ortho hydroxy/methyl derivatives, only the 5-hydroxy-6-methyl-2-aminotetralin derivative displayed pharmacological properties consistent with its being a dopaminergic prodrug. It is concluded that 5-hydroxy-6-methyl-2-(di-n-propylamino)tetralin (1a) is structurally unique for a dopaminergic drug.

Regional development of norepinephrine, dopamine-beta-hydroxylase and tyrosine hydroxylase in the rat brain subsequent to neonatal treatment with subcutaneous 6-hydroxydopamine.

Neonatal rats were injected subcutaneously with 100 mg/kg 6-hydroxydopamine (6-OHDA), or vehicle, on postnatal days 1, 2 and 3. At several times thereafter, determinations of tyrosine hydroxylase (TOH) and dopamine-beta-hydroxylase (DBH) activities, and norepinephrine (NE) concentration were made in the parietal cortex, cerebellum and pons-medulla in order to assess the extent of initial noradrenergic degeneration induced, and the rate of any ensuing regeneration. By the day following completion of the treatment (postnatal day 4), degeneration of noradrenergic terminals in the parietal cortex and cerebellum was very extensive, with NE levels and DBH activities reduced by more than 80%, and TOH activities reduced by 50%. In the parietal cortex noradrenergic degeneration remained virtually complete; and 9 and 70 days postnatal NE concentration and DBH and TOH activities were all decreased by more than 90--95%. In the cerebellum a progressive regeneration and apparent sprouting of NE fibers was observed. By postnatal day 9, NE, DBH and TOH in this tissue had all recovered to near control levels, and by day 70 these measures exceeded control levels by 95%, 115% and 50% respectively. In the pons-medulla, the initial effect of 6-OHDA on any of the measured parameters was negligible. By postnatal day 9 an increase in NE concentration was apparent, which increased further by day 70 to surpass the control level by 70%. At this same time DBH activity was increased by only 15% and TOH activity was unchanged. Separate analysis of the rostral half of the pons, which contains the locus coeruleus, revealed that on day 70 NE and DBH levels were increased much more substantially than in the whole pons-medulla, and TOH activity was also significantly elevated. This data indicates that the initial amount of degeneration induced by the 6-OHDA treatment is similar in both the parietal cortex and cerebellum, but regeneration proceeds only in the cerebellum. This suggests that noradrenergic fiber growth and regeneration in each target tissue is under independent regulation, possibly by the individual target neurons themselves.