In vivo structure-activity studies on the dark-color-inducing neurohormone of locusts.

In the 11-residue long dark-color-inducing neurohormone (DCIN = [His7]-corazonin), of locusts, from residue 2 to residue 11, one amino acid at each time was substituted by D-phenylalanine (D-Phe). The dark-color-inducing effect of these peptides was investigated in comparison with unaltered DCIN by a bioassay based on nymphs of a DCIN-deficient albino mutant of the migratory locust, Locusta migratoria. Substitution of any single amino acid by D-Phe always reduced the activity, but did not abolish it completely. Maximum inactivation was obtained after substitution of Gln4, Ser6, or Trp9. The latter two residues are within the partial sequence -Ser-Xxx-Gly-Trp- (Xxx = His in theDCIN) that seems to be important for the dark-color-inducing activity, as found also in another study (Insect Biochem. Mol. Biol.32, 2002, 909). GIn4, however, is outside of this partial sequence.Minimal, although still considerable, inactivation occurred after substitution of Gly8, Phe3, or Asn11, despite the fact that Gly8 is within the -Ser-Xxx-Gly-Trp- partial sequence. In conclusion, no single active core was found, indicating that the whole sequence of the DCIN is necessary to induce maximum darkening effect. No difference was found in the activity of the peptides in which Gly8was substituted by D-Phe or by L-Phe. Therefore the -Ser-Xxx-Gly-Trp- partial sequence does not seem to be stabilized by a type II beta-turn. Nevertheless, existence of another kind of turn that includes this partial sequence is feasible. A single unsuccessful attempt was made to discover an antagonist to the DCIN.     

Nuclear magnetic resonance (NMR) studies of the dark-color-inducing neurohormone of locusts and corazonin

The dark-color-inducing neurohormone (DCIN) of locusts and corazonin of a cockroach, both 11 residue-long peptides, induce dark coloration in albino nymphs of Locusta migratoria when injected after a nymphal molt. These peptides differ at position 7 (His in DCIN and Arg in corazonin) and elicit an almost identical darkening response. The three-dimensional structures of these peptides, dissolved in dimethylsulfoxide (DMSO), were determined by NMR. Structural elements determined at atomic resolution may provide insight into the biological activity of these two neurohormones. The calculated structures of DCIN and corazonin indicate clear, prevalent conformations with similar secondary features. The generated low-energy solution structures of each show structural elements within residues Phe3 to Trp9 with a turn situated at the core of the peptide from which the sidechains of residue 7 of each peptide protrude. A calculated negative electrostatic potential surface almost completely covers both neurohormones and only the 7th residue sidechains of each peptide emerge in their entirety. Within these residues there is a partial sequence seen in several neurohormones that control various physiological functions in Arthropods: -Ser-X-Gly-Trp- (X=His in DCIN and Arg in corazonin). This partial sequence may play a role in the physiological activity of some Arthropod neurohormones.     

In vivo studies on the release of hormones from the corpora cardiaca of locusts

Summary Sectioning of the afferent nerves (NCCl and NCCll) to the locust corpus cardiacum prevents thein vivo release of adipokinetic hormone from the glandular lobes. This failure to release the hormone during flight and the consequent lack of lipid mobilisation brings about an impairment of flight performance which can be corrected by injections of corpus cardiacum extracts. Sectioning of the NCCl and NCCll reduces markedly the activity of the corpora allata. However, the poor flight performance of allatectomised locusts is not related to an inability to mobilise lipid since injections of corpus cardiacum extract which will mobilise fat body lipid in these locusts have no effect on flight performance. The results of individual sectioning of the NCCl and NCCll suggest that a double innervation of the glandular lobes functionsin vivo to control adipokinetic hormone release but that the NCCl alone may control the release of the diuretic hormone.     

Structure-activity relations of the dark-colour-inducing neurohormone of locusts

The dark-colour-inducing effect of several peptides in comparison to that of the dark-colour-inducing neurohormone (DCIN, [His(7)]-corazonin) of locusts was investigated by a bioassay based on nymphs of a DCIN-deficient albino mutant of Locusta migratoria. The study was aimed at elucidating the active part of the DCIN and to explore the contribution of its amino acids to the activity. Graded doses of all peptides were injected in oil. [Arg(7)]-corazonin and DCIN were equally effective. Certain arthropod neuropeptides having the -SXGW- partial sequence (a part of the DCIN and of [Arg(7)]-corazonin; X=His and X=Arg, respectively) yielded the following findings: Scg-AKH-II (adipokinetic hormone II of Schistocerca gregaria X=Thr), Grb-AKH ( adipokinetic hormone of Gryllus bimaculatus X=Thr) and RPCH (red pigment concentrating hormone of crustaceans X=Pro) evoked a moderate darkening response, but Lom-AKH-II (adipokinetic hormone II of L. migratoria X=Ala) was ineffective. Step by step shortening of the sequence of the DCIN at the N-terminal, from pGlu-3-11DCIN to pGlu-9-11DCIN, resulted in a decreasing activity, but even pGlu-9-11DCIN induced a weak response with high doses. Shortening of the DCIN from the C-terminal revealed a moderate activity of 1-7DCIN-NH(2) and a weak activity of 1-5DCIN-NH(2). An octadecapeptide which induces dark colour in moth larvae, having the pentamer FTPRL-NH(2) at its C-terminal, evoked no darkening in the albino locusts. We conclude that although the -SXGW- partial sequence has some role in induction of darkening, for obtaining maximal effect the whole sequence of the DCIN (or of [Arg(7)]-corazonin) is necessary.     

The dark-colour-inducing neurohormone of locusts in relation to an albino mutant of Schistocerca gregaria

In the albino mutant of an Okinawa strain of Locusta migratoria (L.) (Orthoptera: Acrididae), albinism is caused by the absence of the dark‐colour‐inducing neurohormone (DCIN), which is present in the corpora cardiaca (CC) of normally coloured phenotypes. This study tests whether the absence of DCIN is responsible for albinism in an albino mutant of another locust, Schistocerca gregaria (Forsk.) (Orthoptera: Acrididae). This seemed feasible because a single Mendelian unit controls albinism in both species. However, implantation of CC, or injection of an extract of CC, from albino donors of S. gregaria, induce dark coloration in crowded nymph recipients of the Okinawa albino mutant of L. migratoria, as effectively as do implanted CC, or injections of extract of CC, from normal phenotype donors of S. gregaria. Therefore, DCIN is present in the albino mutant of S. gregaria, and consequently, the albinism in this mutant is not caused by its absence. Implantation of CC, or injection of extracts of CC, from albino donors of S. gregaria to conspecific albino nymphs does not induce darkening. Only extremely high doses of synthetic DCIN injected into albino nymphs of S. gregaria are effective, inducing some darkening. The dose to induce such darkening in albino nymphs of S. gregaria is 50 nmol, ≈ 5 × 10⁶ times higher than that (10 femtomol) needed to induce equivalent darkening in nymphs of the Okinawa albinos of L. migratoria. The results are discussed and some possible explanations of the observed effects outlined.     

The dark-colour-inducing neurohormone of locusts: strain-dependent and phase-independent effects on adults of the migratory locust, Locusta migratoria

Abstract.  An albino strain that had originated from Okinawa, Japan, and a normally coloured strain that had originated from West Africa, were used to study the darkening response to injection of graded doses of dark-colour-inducing neurohormone of locusts (DCIN) ([His⁷]-corazonin) of gregarious and solitarious adults of the migratory locust, Locusta migratoria (L.). By repeated crossings, congenic albinos and normal phenotypes were obtained, both with a 99.6% West African genome, and their darkening response was compared with the original Okinawa and West African strains. Within each of these four strains, no difference was found in DCIN-induced darkening between gregarious and solitarious adults despite previous publications in the literature claiming an absence of 'fire-darkening' in gregarious adults of this species. Okinawa albino adults showed a markedly higher darkening response than the other three strains, including albinos with a 99.6% West African genome. This finding demonstrates that the differential darkening response of the Okinawa albinos is caused not by albinism, but by the geographical origin (Okinawa) of the strain. This is the first report of geographical-strain-dependent differences in the response of an insect to a neurohormone. The darkening response of adults reached a maximum on day 10 after injection; subsequently, the dark colour faded slowly. Adults injected 1 day after their moult showed a greater darkening response than those injected after 14 or 28 days.     

Relaxin causes changes of the liver. In vivo studies in rats.

During pregnancy, the liver undergoes metabolic adjustments directed to fulfil the needs of the mother and the growing fetus. This study was designed to verify whether relaxin, a hormone related to pregnancy, may induce histochemical and ultrastructural modifications of hepatocytes which can be related to metabolic changes. Estrogen-primed female rats were treated with relaxin (10 microg in repository vehicle) for 18 h. Additional male rats were treated with relaxin (10 microg/day in PBS) for 4 days. Appropriate vehicle-treated rats were used as controls. After fasting, the rats were killed and liver fragments were processed for light and electron microscopy and for computer-assisted morphometry of PAS-positive glycogen deposits and acid phosphatase-reactive organelles. In both sexes, the relaxin-treated rats underwent a significant decrease in the amount of glycogen in the hepatocytes as compared with the controls. These changes were accompanied by an increase in smooth endoplasmic reticulum, endocytosis vesicles and lysosomes. These findings show that relaxin promotes glycogen depletion and induces morphological changes of hepatocytes which are consistent with functional activation. It is suggested that relaxin might play an important role in hepatic metabolic adjustments occurring during pregnancy.     

In vivo studies on the interaction of RecBCD enzyme and lambda Gam protein.

The interaction between the RecBCD enzyme of Escherichia coli and the lambda Gam protein was investigated. Two types of experiments were done. In one type, Gam protein was produced by transient induction of the cells lysogenic for lambda cI857gam+. The presence of Gam protein, which inhibits RecBCD nuclease, enabled these cells to support the growth of a gene 2 mutant of bacteriophage T4 (T4 2). The lysogens overproducing the RecB subunit of RecBCD enzyme could titrate Gam protein and thus prevent the growth of T4 2. In contrast, the lysogens overproducing either RecC or RecD retained their capacity for growth of T4 2. It is therefore concluded that the RecB subunit is capable of binding Gam protein. In the second type of experiments, Gam protein was provided by derepressing the gamS gene on the plasmid pSF117 (S. A. Friedman and J. B. Hays, Gene 43:255-263, 1986). The presence of this protein did not interfere with the growth of wild-type cells (which were F-). Gam protein had a certain effect on recF mutants, whose doubling time became significantly longer. This suggests that the recF gene product plays an important role in maintenance of viability of the Gam-expressing cells. Gam protein exerted the most striking effect on growth of Hfr bacteria. In its presence, Hfr bacteria grew extremely slowly, but their ability to transfer DNA to recipient cells was not affected. We showed that the effect on growth of Hfr resulted from the interaction between the RecBCD-Gam complex and the integrated F plasmid.     

The response of a homochrome grasshopper, Oedipoda miniata, to the dark-colour-inducing neurohormone (DCIN) of locusts

The effect of the dark-colour-inducing neurohormone (DCIN=[His(7)]-corazonin) of locusts was investigated in nymphs of the grasshopper, Oedipoda miniata, which exhibit strong homochromy, but neither green-brown, nor phase colour polymorphism. Graded doses of synthetic DCIN were injected in 1 μl of olive oil into 0-24-h-old penultimate nymphs. DCIN induced dose-dependent darkening in the recipients, seen 4 days after injection (still in the penultimate nymphal instar), as well as in the subsequent last-instar nymphs. The dose-range obtained between discernible and maximum (almost black) darkening extended over three orders of magnitude, from 1 pmol to 1 nmol of DCIN. In spite of the darkening observed already in the penultimate nymphal instar, the exuviae in the moult from this to the last nymphal instar did not show marked dark patches. However, the exuviae of the next moult (from last-instar nymph to adult) showed such dark patches, which increased with the increase of the dose, revealing the presence of exocuticular melanin. We conclude that DCIN, or a very close peptide, is the endogenous hormone which plays the major or sole role in the control of homochromy of O. miniata and possibly also of other acridids which exhibit homochromy.     

Structure-function studies on neurohormone D: activity of naturally-occurring hormone analogues

Summary The relative potencies of 11 naturally-occurring peptides of the adipokinetic hormone/red pigmentconcentrating hormone family (AKH/RPCH-family) have been assessed with respect to increase in heart rate in adult, female American cockroaches,Periplaneta americana, in in vitro and in vivo bioassays. In addition, analogues that lacked the N-terminal pyroglutamate residue or had a free threonine acid at the C-terminus were also investigated. In both bioassays the N- or C-terminal-modified analogues give no or little response suggesting that blocked termini are essential for receptor-binding. In both bioassays the naturally-occurring peptide from the cockroach corpus cardiacum Pea-CAH-I (neurohormone D) is more potent than the second endogenous peptide, Pea-CAH-II. On the basis of this result and previous data it is proposed that neurohormone D is the only physiologically important true cardioactive peptide. The dose-response curves of the other peptides indicate that in octapeptides, amino acid residues at positions 2, 6, and 7 are important for receptor-recognition, and that decapeptides are not as effective as octapeptides (exception: the peptide Rom-CC-I isolated from the grasshopperRomalea microptera).     

In vivo studies of dopamine receptor ontogeny

Studies of the ontogeny of dopamine and neuroleptic receptors in the central nervous system of the rat were carried out $\text{in vivo}$ using ³H-spiperone as ligand. It was demonstrated that intraperitoneal injections can be successfully used to label these receptors in rat pups up to at least 30 days of age. The time course and characteristics of ³H-spiperone binding in the brain of 5, 15 and 30 day old rat pups were determined and found to include appropriate regional distribution, saturability and appropriate pharmacology. The developmental pattern of ³H-spiperone binding paralleled what has been seen using $\text{in vitro}$ techniques. In addition preliminary autoradiographic studies describe the neuroanatomical pattern of dopamine receptor ontogeny in the striatum.     

Spiroimidazolidinone NPC1L1 inhibitors. Part 2: structure-activity studies and in vivo efficacy

Ezetimibe (Zetia®), a cholesterol-absorption inhibitor (CAI) approved by the FDA for the treatment of hypercholesterolemia, is believed to target the intestine protein Niemann-Pick C1-Like 1 (NPC1L1) or its pathway. A spiroimidazolidinone NPC1L1 inhibitor identified by virtual screening showed moderate binding activity but was not efficacious in an in vivo rodent model of cholesterol absorption. Synthesis of analogs established the structure–activity relationships for binding activity, and resulted in compounds with in vivo efficacy, including 24, which inhibited plasma cholesterol absorption by 67% in the mouse, thereby providing proof-of-concept that non-β-lactams can be effective CAIs.     

In vivo studies on mouse erythrocytes linked to transferrin

Transferrin (Tf), a plasma protein with numerous, highly specific receptors in proliferating and differentiating cells was already discussed as a targeting ligand for drugs and liposomes in previous studies. In this paper, we deal with erythrocytes linked to Tf as possible physiological targeting carrier systems for delivering anticancer drugs. For that purpose we have used glutaraldehyde (0.1%) as a coupling agent between Tf and erythrocytes. The highest amount of Tf linked to erythrocytes turned out to be 0.76 +/- 0.13 microg Tf/10(6) cells, while reaching 65% of cell recovery. After 13 days, the Tf-erythrocytes hemolysis reached 50%, with transferrin still coupled to erythrocytes. The in vivo kinetic behaviour of intravenously injected 51Cr-Tf-erythrocytes showed a reduced half-life to hours as compared to days of controls. However, a considerable percentage of Tf-erythrocytes (close to 20%) remained circulating for a relatively long period (around 2 days), which made possible the specific targeting by these carrier systems. In vivo biodistribution studies indicated that 51Cr-Tf-erythrocytes rapidly accumulated in the different studied organs (liver, spleen, lungs, kidneys, femur-tibia, and heart), suggesting a selective removal of Tf-erythrocytes by the cells of the mononuclear phagocytic system present mainly in liver and spleen. On the other hand, Tf-erythrocytes showed a poor targeting of heart tissue, therefore a reduced cardiac toxicity should be expected after administration of erythrocyte-encapsulated drugs. The presence of Tf-erythrocytes in femur-tibia and spleen could be related to the Tf-specific binding to the hematopoietic cells containing Tf receptors. The final results of this study encourage additional research on Tf-erythrocyte to investigate the relationship between transferrin-mediated targeting by carrier erythrocytes and uptake of different erythrocyte-encapsulated drugs. Consequently, the current study showed possible use of these carriers as a potential therapeutic tool for drug targeting in animal models with alterations affecting mononuclear phagocytic system or carcinomas of various origins whose cells show elevated number of Tf receptors.     

In vivo imaging studies shed light on germinal-centre development

Affinity maturation of antibodies during the course of an adaptive immune response requires germinal centre (GC) formation within B-cell follicles. Much of the current understanding of GC function has been derived from histology, but these static views have left unresolved many questions about cell movement in GCs. In this Progress article, we describe how several recent studies using time-resolved multiphoton microscopy to track GC B-cell movement within lymph nodes have shed light on the processes that influence GC B-cell dynamics.