Expression and characterization of a recombinant i-type lysozyme from the harlequin ladybird beetle Harmonia axyridis.

Lysozymes are enzymes that destroy bacterial cell walls by hydrolysing the polysaccharide component of peptidoglycan. In insects, there are two classes of lysozymes, the c-type with muramidase activity and the i-type whose prototypical members from annelids and molluscs possess both muramidase and isopeptidase activities. Many insect genes encoding c-type and i-type lysozymes have been identified during genome and transcriptome analyses, but only c-type lysozymes have been functionally characterized at the protein level. Here we produced one of five i-type lysozymes represented in the immunity-related transcriptome of the invasive harlequin ladybird beetle Harmonia axyridis as recombinant protein. This was the only one containing the serine and histidine residues that are thought to be required for isopeptidase activity. This i-type lysozyme was recombinantly expressed in the yeast Pichia pastoris, but the purified protein was inactive in both muramidase and isopeptidase assays. Transcription and immunofluorescence analysis revealed that this i-type lysozyme is produced in the fat body but is not inducible by immune challenge. These data suggest that i-type lysozymes in insects may have acquired novel and as yet undetermined functions in the course of evolution.

Influence of splenectomy on first set rejection reactions of C57BL/6 females to male skin isografts.

The tempo with which C57BL/6 females reject male skin isografts is determined in part by the immunogenicity of the H-Y antigen and in part by the capacity of the host to respond immunologically. Our studies indicate that the spleen plays an important role in determining the briskness of the rejection process in that splenectomy 7-30 days before grafting with male skin significantly shortens the survival time. The results of reconstitution experiments suggests that a population of cells is present in spleens of normal, but not specifically sensitized, females which can restore the conventional first set reaction in splenectomized females. It is inferred that this resident population normally operates in spleen-intact females to delay the development of specific effector responses. Lymphoid cells from H-Y antigen-sensitized, splenectomized females failed to evoke graft-vs.-host responses in males whereas similar cells from females with spleens intact did possess graft-vs.-host potential.

Differential stimulation of the Na+/H+ exchanger determines chloroquine uptake in Plasmodium falciparum.

Here we describe the identification and characterization of a physiological marker that is associated with the chloroquine-resistant (CQR) phenotype in the human malarial parasite Plasmodium falciparum. Single cell in vivo pH measurements revealed that CQR parasites consistently have an elevated cytoplasmic pH compared to that of chloroquine-sensitive (CQS) parasites because of a constitutively activated Na+/H+ exchanger (NHE). Together, biochemical and physiological data suggest that chloroquine activates the plasmodial NHE of CQS parasites, resulting in a transitory phase of rapid sodium/hydrogen ion exchange during which chloroquine is taken up by this protein. The constitutively stimulated NHE of CQR parasites are capable of little or no further activation by chloroquine. We propose that the inability of chloroquine to stimulate its own uptake through the constitutively activated NHE of resistant parasites constitutes a minimal and necessary event in the generation of the chloroquine-resistant phenotype.

Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.

A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.

Expression of var genes located within polymorphic subtelomeric domains of Plasmodium falciparum chromosomes.

Plasmodium falciparum var genes encode a diverse family of proteins, located on the surfaces of infected erythrocytes, which are implicated in the pathology of human malaria through antigenic variation and adhesion of infected erythrocytes to the microvasculature. We have constructed a complete representative telomere-to-telomere yeast artificial chromosome (YAC) contig map of the P. falciparum chromosome 8 for studies on the chromosomal organization, distribution, and expression of var genes. Three var gene loci were identified on chromosome 8, two of which map close to the telomeres at either end of the chromosome. Analysis of the previously described chromosome 2 contig map and random P. falciparum telomeric YAC clones revealed that most, if not all, 14 P. falciparum chromosomes contain var genes in a subtelomeric location. Mapping the chromosomal location of var genes expressed in a long-term culture of the P. falciparum isolate Dd2 revealed that four of the five different expressed var genes identified map within subtelomeric locations. Expression of var genes from a chromosomal domain known for frequent rearrangements has important implications for the mechanism of var gene switching and the generation of novel antigenic and adhesive phenotypes.

[Benzo[c][2,7]naphthyridine-5-yl-amines and benzo[h][1,6]naphthyridine-5-yl-amines--potential antimalarials]. / Benzo[c][2,7]naphthyridin-5-yl-amine und Benzo[h][1,6]naphthyridin-5-yl-amine--Potenzielle Antimalariamittel.

The chloroimine 1a reacted with the novaldiamine-base to yield the 5-(2-methylpyrrolidinyl)-derivative 3. The 5-chloro-benzonaphthyridines 1 and 9 reacted with secondary aliphatic amines to give the amidines 5-8 and 10, while the aromatic amidines 11-14 were obtained with primary aromatic amines. Mixtures of the phenol Mannich bases 15 and 16 of the isoquine type were isolated from the aminomethylation of 13b. The amodiaquine analogues 19 and 20 were obtained from the reaction of 1b and 9a with 4-amino-2-piperidinomethyl-phenol dihydrochloride. The structure of the compounds 5a (potassium salt), 6b, 10a, 11e and 18 was proven by X-ray crystal analysis. Compounds 3, 6a-e, 7, 10a, 11a, 16, 19 and 20 were tested for in vitro antimalarial activity using a chloroquine-sensitive and -resistant Plasmodium falciparum strain. The highest activity against the sensitive strain was shown by the amodiaquine analogoue 20 with an IC50 value of 160 nM. The mixture of the isoquine derivatives 15a and 16a possessed the highest activity against the resistant strain with an IC50 value of 1100 nM.

[Benzo[c][2,7]naphthyridine-5-yl-arylamines-phenol Mannich bases of the amodiaquine-, cycloquine- and pyronaridine-type]. / Benzo[c][2,7]naphthyridin-5-yl-arylamine-Phenol-Mannich-Basen vom Amodiaquin-, Cycloquin- und Pyronaridin-Typ.

2,5-Dichloro-4-methyl-benzo[c][2,7]naphthyridine (1) reacted with aromatic amines selectively by substitution at the 5-position to yield the amidines 2. The 4-aminophenol 2c could also be synthesized by cleavage of the ether 2b. The structure of 2c was proved by X-ray crystal analysis. Aminomethylation of 2c yielded the amodiaquine analogue 3. The mono- and bisaminomethylated derivatives 4 and 5 were obtained by reaction of compound 1 with phenol Mannich base hydrochlorides. Compounds 3-5 were tested in vitro for antimalarial activity using chloroquine-sensitive and resistant Plasmodium-falciparum strains. The highest activities were shown by the pyronaridine-type compounds 5a and 5b with IC50 values of approximately 200 nM.

[Thieno[3,4-c]quinoline-4-yl-amines--synthesis and investigation of activity against malaria]. / Thieno[3,4-c]chinolin-4-yl-amine--Synthese und Prüfung auf Wirksamkeit gegen Malaria.

The 4-aryl derivative 3, obtained by Suzuki cross coupling of the methyl 4-bromothiophene-3-carboxylate (2) with 2-nitrophenylboronic acid cyclizes under reductive conditions pH-dependant to yield the tricyclic hydroxamic acid 4 or the lactam 5. The chlorothieno[3,4-c]quinoline 6 was formed by reaction of the lactam 5 with P,P-dichlorophenylphosphinoxide. The amines 7-14 were synthesized from the chloroimine 6. Compounds 7a,b, 8, 9, 10b, 11, 12 and 14a, b were tested for in vitro antimalarial activity using the chloroquine sensitive 3D7 and the chloroquine resistant Plasmodium falciparum strain Dd2. The highest activity were shown by 10b with IC50 values of 130 nM and 50 nM, respectively and by 11 with IC50 values of 190 nM and 44 nM, respectively.

[Thieno[3,2-c]quinoline-4-yl-amines--synthesis and investigation of activity against malaria]. / Thieno[3,2-c]chinolin-4-yl-amine--Synthese und Prufung auf Wirksamkeit gegen Malaria.

Thieno[3,2-c]quinoline-4-yl-amines - synthesis and investigation of activity against malaria pH-Dependant reduction of the methyl 2-(2-nitrophenyl)thiophene-3-carboxylate 3, formed by Suzuki coupling of methyl 2-iodothiophene-3-carboxylate (2) with 2-nitrophenylboronic acid, yielded the cyclic hydroxamic acid 4 and the lactam 5, respectively. The 4-chlorothieno[3,2-c]quinoline 6 was formed from the lactam 5 by heating with POCI3/PCI5s. Melting of 6 with the novaldiamine base in phenol gave the chloroquine analogue 7, whereas the amodiaquine and the pyronaridine analogues 8 and 9 were obtained using phenol Mannich bases. The reaction of 6 with putrescine and N,N'-bis(3-aminopropyl)piperazine as spacer formed the bisquinoline derivatives 10 and 11 as well as the monosubstituted quinoline 12. In the same manner the isomeric 4-chlorothieno[2,3-c]quinoline 13 reacted to yield the quinoline-4-yl-amines 14-16. The compounds 7-12 and 14-16 were tested for in vitro growth inhibition of the malaria parasite Plasmodium falciparum. As most active compound the pyronaridine derivative 9 displayed an IC50 value of 210 nM with the chloroquine sensitive P. falciparum strain 3D7 and 750 nM with the chloroquine resistant P. falciparum strain Dd2. The N,N'-bis(3-aminopropyl)piperazine derivative 11 displayed in vivo activity in Plasmodium vinckei infected mice with an ED50 value of 30 mg/kg after i.p. administration.

[Benzo[c][2,7]naphthyridine-2-yl-, 5-yl- and 2,5-diyl novaldiamines--synthesis and investigation of anti-malarial activity]. / Benzo[c][2,7]naphthyridin-2-yl-, 5-yl- und 2,5-diyl-novaldiamine--synthese und prüfung auf wirksamkeit gegen Malaria.

The 2,5-dichlorobenzo[c][2,7]naphthyridine 6 was synthesized starting from the 2-pyridone 1 in four or five steps, respectively. The 5-yl amine 7 and the 2,5-diyl amines 8 and 9 were isolated by the reaction of compound 6 with the novaldiamine base. Starting with the reaction of the 6-chloropyridine 3 with the novaldiamine base to yield the 6-aminopyridine 11, the 2-yl amine 13, isomeric to 7, was obtained. Compounds 7-13 were tested for in vitro antimalarial activity using a chloroquine sensitive and resistant Plasmodium falciparum strain. The highest activity was shown by 8 with IC50 values of 90 nM and 190 nM, respectively.

Novel lead structures for antimalarial farnesyltransferase inhibitors.

Through the combination of nitrophenylfurylacryloyl moiety which has been designed to occupy an aryl binding site of farnesyltransferase with several AA(X)-peptidomimetic substructures some novel farnesyltransferase inhibitors were obtained. Evaluation of their antimalarial activity and some initial modifications yielded a 4-benzophenone- and a sulfonamid-based novel lead for antimalarial farnesyltransferase inhibitors.

2-(aminoacylamino)benzophenones: farnesyltransferase inhibition and antimalarial activity.

The use of amino acids as acyl substitutents at the 2-amino group of our benzophenone core structure yielded compounds with mainly good to moderate farnesyltransferase inhibitory and moderate antimalarial activity. However, these farnesyltransferase inhibitors display some degree of selectivity towards malarial parasites since there was no cytotoxic activity observed at 70-80 microM.

Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

Possible delta receptor mediation of the effect of beta-endorphin on luteinizing hormone (LH) release, but not on prolactin (PRL) release, in the ovariectomized rat.

Administration of opioid receptor antagonists was utilized to determine the opioid receptor type involved in the suppression of LH release by beta-endorphin (beta-END). Long-term (three to four weeks) ovariectomized rats with chronic third ventricular cannulae were fitted with jugular catheters and received treatment with vehicle or one of three opioid antagonists. The delta antagonist ICI 154, 129, but not the mu1 or mu antagonists naloxazone or beta-funaltrexamine, respectively, blocked the suppressive effect of beta-END on plasma LH levels and transiently but significantly increased LH levels above preinfusion value. None of the antagonists significantly reduced the beta-END-induced release of PRL. These results provide evidence that the inhibitory effect of beta-END on LH release may be mediated by delta receptors.

LytB, a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli.

The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli.

The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.

Adenosine kinase inhibitors. 1. Synthesis, enzyme inhibition, and antiseizure activity of 5-iodotubercidin analogues.

Adenosine receptor agonists produce a wide variety of therapeutically useful pharmacologies. However, to date they have failed to undergo successful clinical development due to dose-limiting side effects. Adenosine kinase inhibitors (AKIs) represent an alternative strategy, since AKIs may raise local adenosine levels in a more site- and event-specific manner and thereby elicit the desired pharmacology with a greater therapeutic window. Starting with 5-iodotubercidin (IC50 = 0.026 microM) and 5'-amino-5'-deoxyadenosine (IC50 = 0.17 microM) as lead inhibitors of the isolated human AK, a variety of pyrrolo[2,3-d]pyrimidine nucleoside analogues were designed and prepared by coupling 5-substituted-4-chloropyrrolo[2,3-d]pyrimidine bases with ribose analogues using the sodium salt-mediated glycosylation procedure. 5'-Amino-5'-deoxy analogues of 5-bromo- and 5-iodotubercidins were found to be the most potent AKIs reported to date (IC50S < 0.001 microM). Several potent AKIs were shown to exhibit anticonvulsant activity in the rat maximal electric shock (MES) induced seizure assay.

Adenosine kinase inhibitors. 2. Synthesis, enzyme inhibition, and antiseizure activity of diaryltubercidin analogues.

In the preceding article (Ugarkar et al. J. Med. Chem. 2000, 43) we reported that analogues of tubercidin are potent adenosine kinase (AK) inhibitors with antiseizure activity in the rat maximum electroshock (MES) model. Despite the discovery of several highly potent AK inhibitors (AKIs), e.g., 5'-amino-5'-deoxy- 5-iodotubercidin (1c) (IC50 = 0.0006 microM), no compounds were identified that exhibited a safety, efficacy, and side effect profile suitable for further development. In this article, we demonstrate that substitution of the tubercidin molecule with aromatic rings at the N4- and the C5-positions not only retains AKI potency but also improves in vivo activity. Synthesis of such compounds entailed transformation of 4-arylamino-5-iodotubercidin analogues to their corresponding 5-aryl derivatives via the Suzuki reaction. Alternatively, 4-N-arylamino-5-arylpyrrolo[2,3-d]pyrimidine bases were constructed and then glycosylated with appropriately protected alpha-ribofuranosyl chlorides using a phase-transfer catalyst. Several compounds exhibited potent activity in the rat MES seizure assay with ED50s < or = 2.0 mg/kg, ip, and showed relatively mild side effects.

Seroquel (ICI 204 636), a putative "atypical" antipsychotic, in schizophrenia with positive symptomatology: results of an open clinical trial and changes of neuroendocrinological and EEG parameters.

Preclinical data indicated that seroquel (ICI 204 636), a dibenzothiazepine with 5-HT2 and D2-like receptor antagonistic properties, might be an effective antipsychotic agent, causing fewer extrapyramidal side effects than typical neuroleptics. In the present study, 12 patients suffering from schizophrenia or schizophreniform disorder with predominantly positive symptomatology were treated in an open clinical trial for 4 weeks with seroquel at a maximum dosage of 750 mg/day. The drug was generally well tolerated, and virtually no adverse extrapyramidal side effects such as acute dystonia, parkinsonism or akathisia were observed. Total scores for BPRS (item score 0-6; baseline: 42.0 +/- 2.3; mean +/- SEM), SAPS (64.5 +/- 4.8) and SANS (55.0 +/- 4.3) showed a moderate decrease at the end of treatment (BPRS: 30.0 +/- 3.5; SAPS: 36.1 +/- 6.7; SANS: 42.5 +/- 5.9), when intention-to-treat analysis was applied. There were considerable interindividual differences in treatment response, with some subjects showing almost full remission of positive symptoms, in contrast to about half of the patients who showed no satisfactory clinical improvement. Interestingly, patients showing good antipsychotic response reported slight initial side effects like mild sedation. Prolactin and TSH levels were not altered during seroquel administration. As to pharmaco-EEG investigations, seroquel caused a moderate increase of the absolute power in the alpha, theta, and beta frequency bands, paralleled by a decrease of delta activity. There were no signs of paroxysmal EEG activity under seroquel.(ABSTRACT TRUNCATED AT 250 WORDS)

Tools for discovery of inhibitors of the 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase: an approach with enzymes from the pathogenic bacterium Pseudomonas aeruginosa.

Two Pseudomonas aeruginosa genes encoding the enzymes 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase, both involved in the mevalonate-independent biosynthesis of isoprenoids, have been expressed as recombinant enzymes in Escherichia coli. The purified P. aeruginosa DXP reductoisomerase was inhibited by submicromolar concentrations of the antibiotics fosmidomycin and FR-900098 in a well established method. A novel and convenient spectrophotometric assay was developed to determine activity and inhibition of P. aeruginosa DXP synthase. Fluoropyruvate is described as a first inhibitor of DXP synthase.