Nitrogen excretion in cockroaches: uric Acid is not a major product.

Thin-layer chromatographic and enzymatic spectrophotometric analyses have failed to detect uric acid in fecal extracts from American cockroaches on specified diets. Uric acid appears in the excreta when it is a dietary constituent, although most of it is apparently absorbed and stored internally. Surprisingly, ammonia is a major excretory product.

Comparison of the V1b antagonist, SSR149415, and the CRF1 antagonist, CP-154,526, in rodent models of anxiety and depression.

Vasopressin and corticotropin releasing factor (CRF) are both critical regulators of an animal's stress response and have been linked to anxiety and depression. As such, antagonists of the CRF1 and V1b receptor subtypes are being developed as potential treatments for affective disorders. The two most characterized V1b and CRF1 antagonists are SSR149415 and CP-154,526, respectively, and the present studies were designed to compare these two compounds in acute animal models of affective disorders. We employed five anxiety models: Separation-induced pup vocalizations (guinea pig and rat), elevated plus-maze (EPM), conditioned lick suppression (CLS), and marble burying (mouse); as well as three depression models: forced swim test (FST; mouse and rat) and tail suspension test (TST; mouse). SSR149415 (1-30 mg/kg) was active in the vocalization, EPM and CLS models, but inactive in marble burying. CP-154,526 (1-30 mg/kg) was active in vocalization models, but inactive in EPM, CLS, and marble burying. SSR149415 was inactive in all depression models; CP-154,526 was active in rat FST but inactive in mouse models. This work demonstrates the different profiles of V1b and CRF1 receptor antagonists and supports both approaches in the treatment of affective disorders.

Behavior and daily activity patterns of specialist and generalist predators of the hemlock woolly adelgid, Adelges tsugae.

The behavior and daily activity patterns of two specialist predators, Laricobius nigrinus Fender (Coleoptera: Derodontidae) and Sasajiscymnus tsugae, Sasaji and McClure (Coleoptera: Coccinellidae), and a generalist predator, Harmonia axyridis Pallas (Coleoptera: Coccinellidae), of hemlock woolly adelgid, Adelges tsugae (Hemiptera: Adelgidae), were examined using digital video recording in the laboratory. The two specialists are part of a biological control program for A. tsugae, and it is not known if competitive interactions with previously established generalist predators will negatively impact their effectiveness. The behavior and daily activity patterns of adult females of each species were documented in single- and paired-predator assays under simulated spring and summer conditions. Behavior varied qualitatively and quantitatively by species, and did not appear to be highly coordinated temporally or spatially. All species exhibited continuous activity patterns that were punctuated by longer periods of rest. Extensive and intensive searching behavior occurred in all species, with intensive searching being highly variable. Specialist predators appeared to be more selective of feeding and oviposition sites, and rested at more concealed locations than the generalist species. In spring conditions, L. nigrinus had greater activity and a more even behavior distribution than S. tsugae or H. axyridis, which were skewed towards resting. In summer, the latter two species showed increased activity at higher temperatures. Conspecifics significantly altered the time allocated to specific behaviors for L. nigrinus and H. axyridis, resulting in reduced predator effectiveness by reducing time and energy expenditure on activities that directly impact the adelgids. In contrast, S. tsugae conspecifics and all heterospecific combinations showed non-interference. The activity of each species varied with time of day; L. nigrinus was more active at night, while S. tsugae and H. axyridis were more active during the day. All predator groupings maintained a high degree of spatial separation relative to assay size. The use of multiple-predator species combinations that include the specialist predators, is recommended over single-species for biological control of A. tsugae, as temporal and spatial patterns were not highly coordinated. Low-density releases may reduce the potential negative effects of intraspecific competition.

Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate.

The activity of the serine protease plasminogen activator (PA), which correlates with tumorigenicity and metastatic capacity, was examined using the 125I-labeled fibrin plate assay in cell extracts from four human glioma lines as a function of growth in vitro. Cell-associated inhibitory activity to plasmin and urokinase-type PA was also measured concurrently. The relative PA activities differed markedly among the lines, whereas inhibitory activities did not. Two lines, SNB-19 and SNB-75, exhibited maximal PA activities (1-6 m Plough units/micrograms protein) as cultures approached confluence, whereas two other lines, SNB-56 and SNB-78, expressed low PA activity at all times (less than 0.2 m Plough units/micrograms protein). The PA of SNB-19 cell extracts was predominantly urokinase-type PA. In addition to having the highest PA levels, SNB-19 and SNB-75 were the most clonogenic in soft agar and tumorigenic in nude mice. In contrast, SNB-56 and SNB-78 were poorly clonogenic in soft agar and were not tumorigenic in nude mice. Measured directly, inhibitory activities to plasmin, urokinase-type PA, and tissue-type PA were detected in SNB-19 (high PA) and SNB-56 (low PA) cell extracts. However, there were no qualitative or quantitative differences in inhibitor effects between SNB-19 and SNB-56 suggesting that the differences in PA activity between these lines resulted from changes in PA activity and were not due to differential plasminogen activator inhibitor effects. The ability of the differentiating agent sodium butyrate (NaB) to modulate total PA activity was also examined. Peak SNB-19 cell PA activity was decreased in a concentration (Ki, 0.75 mM) and time-dependent manner by the addition of nontoxic amounts of NaB. The dose-dependent decrease in PA activity induced by NaB was most likely due to an effect on PA itself, since the action of inhibitor on urokinase was unchanged in response to NaB. These results suggest that net cellular PA activity in glioma cells is a balance between relative PA activity and inhibitor(s) effects and that this balance can be modulated by sodium butyrate.

Human leucocyte urokinase inhibitor--purification, characterization and comparative studies against different plasminogen activators.

A plasminogen activator inhibitor (PA-I) which inhibits primarily plasminogen activator of the urokinase type (u-PA) was isolated from the cytosol of human peripheral leukocytes. The inhibitor was isolated using ion exchange chromatography, gel filtration and FPLC. This inhibitor has an apparent molecular weight of 45 kDa, determined by SDS-PAGE, and a pI of 5.5-5.7. The inhibitor is a fast reacting inhibitor, is thermally unstable and is inactivated outside the pH range 7-9. Treatment of cytosol to pH 9 for 30 min at 37 degrees C resulted in a large increase in inhibitory activity. Antibodies against human placental UK-I completely quenched the inhibitory activity of human leucocyte UK-I.

[D-Trp(34)] neuropeptide Y is a potent and selective neuropeptide Y Y(5) receptor agonist with dramatic effects on food intake.

The neuropeptide Y (NPY) Y(5) receptor has been proposed to mediate several physiological effects of NPY, including the potent orexigenic activity of the peptide. However, the lack of selective NPY Y(5) receptor ligands limits the characterization of the physiological roles of this receptor. Screening of several analogs of NPY revealed that [D-Trp(34)]NPY is a potent and selective NPY Y(5) receptor agonist. Unlike the prototype selective NPY Y(5) receptor agonist [D-Trp(32)]NPY, [D-Trp(34)]NPY markedly increases food intake in rats, an effect that is blocked by the selective NPY Y(5) receptor antagonist CGP 71683A. These data demonstrate that [D-Trp(34)]NPY is a useful tool for studies aimed at determining the physiological roles of the NPY Y(5) receptor.

Pharmacological characterization of the cloned neuropeptide Y y(6) receptor.

Neuropeptide Y has potent appetite stimulating effects which are mediated by hypothalamic receptors believed to be of the neuropeptide Y Y(1) and/or neuropeptide Y Y(5) subtype. In mice, the neuropeptide Y y(6) receptor is also expressed in the hypothalamus, suggesting that it too may function as a feeding receptor in this species. Several laboratories have studied the pharmacology of the neuropeptide Y y(6) receptor, but their results are not in agreement. Using neuropeptide Y and a variety of peptide analogs and small molecule antagonists, we have determined that the pharmacology of the cloned mouse neuropeptide Y y(6) receptor is distinct from that of the other known neuropeptide Y receptors. The rank order of binding affinity for the mouse neuropeptide Y y(6) receptor is [(Ile, Glu,Pro,Dpr,Tyr,Arg,Leu,Arg,Tyr-NH(2))(2)human peptide YY=human, rat neuropeptide Y=human, rat neuropeptide Y-(2-36)=human, rat [Leu(31), Pro(34)porcine (Cys(2))-neuropeptide Y-(1-4)-8-aminooctanoyl-(D-Cys(27)porcine [D-Trp(32)rat pancreatic polypeptide=human pancreatic polypeptide. A similar rank order of potency is seen for inhibition of forskolin-stimulated cyclic AMP. The neuropeptide Y Y(5) receptor antagonist trans-naphthalene-1-sulfonic acid ¿4-[4-amino-quinazolin-2-ylamino)-methyl]-cyclohexylmethy l¿-amide hydrochloride (CGP 71683A) and the neuropeptide Y Y(1) receptor antagonist ((R)-N(2)-diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininam ide) (BIBP3226) bind weakly to the neuropeptide Y y(6) receptor (K(i)10, 000 nM, respectively). Although the function of the neuropeptide Y y(6) receptor remains to be elucidated, its pharmacology is not consistent with a role in appetite regulation.

GR231118 (1229U91) and other analogues of the C-terminus of neuropeptide Y are potent neuropeptide Y Y1 receptor antagonists and neuropeptide Y Y4 receptor agonists.

GR231118, BW1911U90, Bis(31/31')[[Cys31, Trp32, Nva34] neuropeptide Y(31-36)] (T-190) and [Trp-Arg-Nva-Arg-Tyr]2-NH2 (T-241) are peptide analogs of the C-terminus of neuropeptide Y that have recently been shown to be antagonists of the neuropeptide Y Y1 receptor. In this study, the activity of these peptides at each of the cloned neuropeptide Y receptor subtypes is determined in radioligand binding assays and in functional assays (inhibition of forskolin-stimulated cAMP formation). GR231118 is a potent antagonist at the human and rat neuropeptide Y Y1 receptors (pA2 = 10.5 and 10.0, respectively; pKi = 10.2 and 10.4, respectively), a potent agonist at the human neuropeptide Y Y4 receptor (pEC50 = 8.6; pKi = 9.6) and a weak agonist at the human and rat neuropeptide Y Y2 and Y5 receptors. GR231118 also has high affinity for the mouse neuropeptide Y Y6 receptor (pKi = 8.8). Therefore, GR231118 is a relatively selective neuropeptide Y Y1 receptor antagonist, but has appreciable activity at the neuropeptide Y Y4 and Y6 receptors as well. BW1911U90, T-190 and T-241 are moderately potent neuropeptide Y Y1 receptor antagonists (pA2 = 7.1, 5.8 and 6.5, respectively; pKi = 8.3, 6.5 and 6.8, respectively) and neuropeptide Y Y4 receptor agonists (pEC50 = 6.8, 6.3 and 6.6, respectively; pKi; 8.3, 7.7 and 8.3, respectively). These data suggest that the C-terminus of neuropeptide Y and related peptides is sufficient for activation of the neuropeptide Y Y4 receptor, but is not sufficient for activation of the neuropeptide Y Y1 receptor. Because BW1911U90, T-190 and T-241 are significantly less potent at the cloned human neuropeptide Y Y1 receptor than at the neuropeptide Y receptor in human erythroleukemia cells, these cells may express a novel neuropeptide Y receptor with high affinity for these peptides.

Purification and biological activities of an angiogenesis factor from human placenta.

Several crude angiogenesis preparations, as well as a purified angiogenesis factor from human placenta, were tested for their ability to stimulate the production of plasminogen activator (PA) and collagenase activities, motility, chemotaxis, DNA synthesis and clonal growth in cultured endothelial cells. Treatment of capillary endothelial cells resulted in a stimulation of all the above activities, whereas endothelial cells derived from large vessels did not respond. These cellular activities are postulated to be responsible for capillary growth in vivo.

Human placental tissue stimulates bovine capillary endothelial cell growth, migration and protease production.

A crude extract of human placenta has been demonstrated to stimulate growth, motility and the production of the proteases plasminogen activator and collagenase in cultured bovine capillary endothelial cells. These data are in keeping with the presence of an angiogenic factor(s) in human placenta.

Apparatus for evaluating vapor-induced dispersal of Blattella germanica (Dictyoptera: Blattellidae).

An apparatus for evaluating vapor-induced dispersal of Blattella germanica (L) is described. It is made of two 750-ml crystallizing dishes connected by a glass and wire mesh tube. Its use is illustrated by experiments on exposure of late instars to vapors of a propoxur in oil formulation and those of its solvent system.

Characterization of the V1a antagonist, JNJ-17308616, in rodent models of anxiety-like behavior.

RATIONALE: Vasopressin (AVP) plays a role in regulating anxiety, which is thought to be partially mediated through the V1a receptor. Recently, JNJ-17308616 was identified as a V1a antagonist. OBJECTIVES: The purpose of this work was to assess V1a receptor affinity and selectivity of JNJ-17308616 and in vivo efficacy in animal models of anxiety-like behavior. MATERIALS AND METHODS: The affinity of JNJ-17308616 for the human and rat V1a, V1b, V2, and oxytocin receptors was determined. Central administration of AVP induces a scratching response mediated through the V1a receptor. Inhibition of scratching was used as a behavioral measure of in vivo potency. JNJ-17308616 was tested in five models of anxiety: rat elevated plus-maze (EPM), rat-elevated zero-maze (EZM), rat-conditioned lick suppression (CLS), rat pup separation-induced ultrasonic vocalizations (USV), and mouse marble burying (MMB). RESULTS: High affinity for the human V1a receptor (K (i) 5.0 nM) was confirmed. However, the rat V1a receptor affinity was more modest (K (i) 216 nM), and the compound was not selective over the rat V2 receptor (K (i) 276 nM). At 100 mg/kg, JNJ-17308616 significantly reduced anxiety-like behavior in EPM, USV, and MMB; at 30 mg/kg, it was effective in EZM and CLS. JNJ-17308616 neither impaired social recognition nor reduced locomotor activity. CONCLUSIONS: These results demonstrate the potential for V1a receptor antagonists as novel anxiolytics. Tool compounds that have greater V1a receptor selectivity than JNJ-17308616 are necessary to make precise conclusions about the role of the V1a receptor in affective disorders.

Quantitative high-performance thin-layer chromatography of dansyl derivatives of biogenic amines.

A procedure for the separation, detection, and quantification of picomole levels of dansyl derivatives of the biogenic amines, dopamine, norepinephrine, octopamine, and serotonin, has been developed using high-performance thin-layer chromatography. The detection limit is 1 to 2 pmol. Each of the amine derivatives has been detected in insect brain tissue and a solvent system has been developed for the separation and quantification of octopamine in insect tissue samples.

Stimulation of motility in cultured bovine capillary endothelial cells by angiogenic preparations.

Several angiogenic preparations that have been shown to stimulate plasminogen activator (PA) and collagenase production by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate BCE cell motility in the phagokinetic track assay. Bovine retinal extract, medium conditioned by 3T3-F442A differentiated mouse adipocytes, SK HEP-1 human hepatoma cell lysate, mouse sarcoma 180 cell lysate, and medium conditioned by mouse sarcoma 180 cells stimulated motility 68.7%, 48.5%, 140.9%, 56.5%, and 102.1%, respectively, relative to untreated cells. The motility-stimulating activity of these preparations was dose dependent and linear over the 16-h assay period. Several hormones and growth factors were tested for BCE cell motility-stimulating activity, including insulin, vasopressin, fibroblast growth factor, and a partially purified preparation of sarcoma growth factor, and were found to be ineffective. 12-0-tetradecanoyl-phorbol-acetate (TPA), a potent stimulator of both PA and collagenase activities in BCE cells, also did not stimulate motility, indicating that protease production is not sufficient to stimulate BCE cell motility in this assay. Neither SK HEP-1 hepatoma cell lysate nor TPA was effective in stimulating motility in bovine aortic endothelial (BAE) cells. The inability of SK HEP-1 hepatoma cell lysate to stimulate movement in BAE cells is consistent with the observation that angiogenesis occurs by sprouting of capillaries, not large vessels.

Maternal and paternal nitrogen investment in Blattella germanica (L.) (Dictyoptera; Blattellidae).

The investment of nitrogenous materials by female and male German cockroaches Blattella germanica (L.) into their progeny was examined. Adult females maintained on dog food invested 34% of their dry mass and 26% of their nitrogen into an oothecae during their first gonadotrophic cycle. Females maintained on a low- (5%) protein diet and injected simultaneously with [3H]leucine and [14C]hypoxanthine incorporated less [3H]leucine-derived radiolabel in their oothecae than those on a dog food diet (25% crude protein). Females on the low-protein diet incorporated more [14C]hypoxanthine-derived material (primarily as [14C]urates) into their oothecae than they retained in their bodies. Stored [14C]urates were metabolized more readily by females on the low-protein diet. Oothecae obtained from females provided with an [15N]urate-amended diet contained at least four 15N-enriched amino acids, which supports the hypothesis that urates are utilized as a nitrogen resource in these insects. Dietary effects on paternal investment were also found to be significant. Females fed a low-protein diet and their oothecae contained 63% of the radiolabel made available to them at mating when paired with males injected simultaneously with [3H]leucine and [14C]hypoxanthine, whereas dog-food-fed females and their oothecae contained only 17% of the total radiolabel made available to them at mating.

Evaluating environmental hazards of land applying composted diazinon using earthworm bioassays.

Environmental hazards resulting from land application of composted pesticide residue have not been rigorously evaluated. This study was conducted to examine the toxicity of a composted pesticide residue using earthworms (Eisenia foetida Savigny) as a microinvertebrate model in a soil bioassay system. Diazinon, which was used in these experiments as a test pesticide, was removed from simulated rinsate (wastewater) by sorption onto peat moss. Following the rinsate clean-up phase, diazinon-laden peat moss was placed into bioreactors and composted for either 30 or 60 days. Earthworms were then exposed to soil amended with the composted material. Mortality and symptomatic effects characteristic of acetylcholinesterase inhibition, including weight loss, reduced burying ability and curling, occurred in earthworms exposed to soil amended with either uncomposted or 30-day composted diazinon, but not in those exposed to soil amended with 60-day composted diazinon. The amount of solvent-extractable diazinon from compost was not directly related to acute earthworm toxicity based on the selected criteria. These results indicated a reduction in diazinon bioavailability during latter 30 d of composting that did not correspond to a reduction in solvent-extractable diazinon concentrations. Measuring symptomatic effects of xenobiotics as described in this study may increase the sensitivity and diagnostic ability of earthworm bioassays.

Fate of atrazine and chlorpyrifos during solid state fermentation--examination of processes.

Solid state fermentation (SSF) was investigated as a means to dispose of two commonly used pesticides, chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine). SSF experiments were carried out in bench-scale bioreactors (equipped with CO2 and volatile organic traps) containing a mixture of lignocellulosic materials and a radiolabeled pesticide. Ethyl acetate-extractable, alkali soluble, and alkali insoluble fractions were evaluated for radioactivity following a 60-d incubation period at 40 degrees C. The majority of the [2,6-pyridyl-14C]chlorpyrifos was associated with the ethyl acetate extract (about 74%), 17% was trapped as organic volatiles by polyurethane foam traps and < 0.5% of the chlorpyrifos was mineralized to CO2. Only small amounts of the radioactivity were associated with alkali soluble (0.0003%) and alkali insoluble (0.3%) fractions. In the [14C-U-ring]atrazine bioreactors, very little of the radioactivity volatilized (<0.5%) and less than 0.5% was mineralized to CO2. Approximately 57% of the applied radioactivity was associated with the ethyl acetate extract while 9% and 24% of the radioactivity was associated with the alkali soluble (humic and fulvic acids) and alkali insoluble fractions, respectively. Possible reaction mechanisms by which covalent bonds could be formed between atrazine (or metabolites) and humic substances were investigated. The issue of bound atrazine residue (alkali soluble fraction) was at least partially resolved. Oxidative coupling experiments revealed that formation of covalent bond linkages between amino substituent groups of atrazine residue and humic substances is highly unlikely.

Inhibition of PDGF receptor binding and PDGF-stimulated biological activity in vitro and of intimal lesion formation in vivo by 2-bromomethyl-5-chlorobenzene sulfonylphthalimide.

The proliferation of vascular smooth muscle cells (SMCs) is a key event in the development of atherosclerotic lesions and in the restenosis of arteries after angioplasty. Polypeptide growth factors are potent SMC mitogens in vitro and are believed to be involved in SMC proliferation in vivo. Strong data exist linking platelet-derived growth factor (PDGF) activity to human atherosclerosis. However, no low-molecular-weight antagonists of this growth factor have been discovered. We identified a compound, SCH 13929 (2-bromomethyl-5-chlorobenzene sulfonylphthalimide), which inhibits binding of 125I-PDGF BB to cell surface receptors with an IC50 of 0.13 mumol/L. This compound has a lesser effect on the binding of 125I-epidermal growth factor (EGF), 125I-basic fibroblast growth factor (bFGF), or 125I-endothelin to specific cell surface receptors. Exposure of cultured SMCs to SCH 13929 inhibits PDGF BB- and PDGF AA-stimulated DNA synthesis but not EGF- or bFGF-stimulated DNA synthesis. PDGF BB-stimulated SMC division is also inhibited by exposure to SCH 13929. Chemotaxis assays indicate that SCH 13929 inhibits PDGF-stimulated directional migration and suggest that the compound interacts with PDGF rather than with the receptor. Oral administration of SCH 13929 (100 mg/kg per day) to Sprague-Dawley rats or spontaneously hypertensive rats results in significant inhibition of lesion formation in the balloon catheter-deendothelialized carotid artery. These results suggest that SCH 13929 may be a useful tool for understanding the role of PDGF in intimal lesion formation.