Cloning and characterization of microbial activated Aedes aegypti†MEK4 (AaMEK4): influences of noncatalytic domains on enzymatic activity.

Protein kinases are known to be involved in a number of signal transduction cascades. Both the stress-activated Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) p38 pathways have been shown to correlate with the insect immune response to microbial infection. MAP kinase kinase 4 (MEK4) is an upstream kinase of JNK and p38 kinase. The cDNA of AaMEK4 was cloned and characterized. AaMEK4 was activated by microbial lysates of Gram-positive, Gram-negative bacteria and yeast. The conserved lysine (K112 ) and the putative phosphorylation sites (S238 and T242 ) were shown to be important for kinase activity by site-directed mutagenesis. A common MAPK docking site (MAPK_dsA) was found and in addition, a new nearby docking site, MAPK_dsB, was identified in the N-terminal noncatalytic domain of AaMEK4. MAPK_dsB was shown to be a unique element in the MEK4 family. In this study, both MAPK_dsA and _dsB were demonstrated to be important to AaMEK4 enzymatic activity for the downstream protein kinase, Aap38.

Blood-brain barrier impairment with enhanced SP, NK-1R, GFAP and claudin-5 expressions in experimental cerebral toxocariasis.

Infection by Toxocara canis in humans may cause cerebral toxocariasis (CT). Appreciable numbers of T. canis larvae cross the blood-brain barrier (BBB) to invade the brain thus causing CT. In the present studies, we evaluated the BBB permeability and BBB injury as assessed by the cerebral Evans blue (EB) concentration as well as by pathological changes and glial fibrillary acidic protein (GFAP) expression in T. canis -infected mice monitored from 3 days (dpi) to 8 weeks post-infection (wpi). The vasodilation neuropeptides, the expressions of substance P (SP) and its preferred binding neurokinin-1 receptor (NK-1R) as well as claudin-5 of tight-junction proteins associated with BBB impairment were also assessed by Western blotting and reverse-transcriptase polymerase chain reaction. Results revealed that BBB permeability increased as evidenced by a significantly elevated EB concentration in brains of infected mice. BBB injury appeared due to enhanced GFAP protein and mRNA expressions from 4 to 8 wpi. Leukocytes might have been unrelated to BBB impairment because there was no inflammatory cell infiltration despite T. canis larvae having invaded the brain; whereas markedly elevated SP protein and NK-1R mRNA expressions concomitant with enhanced claudin-5 expression seemed to be associated with persistent BBB impairment in this experimental CT model.

Comparison of one-tube multiplex PCR, automated ribotyping and intergenic spacer (ITS) sequencing for rapid identification of Acinetobacter baumannii.

Acinetobacter baumannii has emerged as a serious cause of nosocomial infections. Rapid identification of this pathogen is required so that appropriate therapy can be given and outbreaks controlled. This study evaluated a multiplex PCR and an automated ribotyping system for the rapid identification of Acinetobacter baumannii. In total, 22 different reference strains and 138 clinical isolates of Acinetobacter spp., identified by 16S-23S rRNA intergenic spacer (ITS) sequence analysis, were evaluated. All A. baumannii isolates (82 clinical isolates and one reference strain) were identified by the multiplex PCR method (specificity 100%). The sensitivity and specificity of the ribotyping system for identification of A. baumannii were 85.5% (71/83) and 93.5% (72/77), respectively. An additional 100 clinical isolates belonging to the Acinetobacter calcoaceticus-A. baumannii complex were used to compare these two methods for identification of A. baumannii, and this comparison revealed a level of disagreement of 14% (14 isolates). The accuracy of the multiplex PCR was 100%, which was confirmed by sequence analysis of the ITS and recA gene of these isolates. Thus, the multiplex PCR method dramatically increased the efficiency and speed of A. baumannii identification.

Analysis of mosquito vitellogenin cDNA. Similarity with vertebrate phosvitins and arthropod serum proteins.

The cDNA coding for vitellogenin of the mosquito Aedes aegypti was cloned and sequenced. An immunological analysis of expressed deletions from the 5'-end of the vitellogenin cDNA clones using vitellogenin subunit-specific antibodies showed that the small vitellogenin subunit is located at the N terminus and the large one at the carboxy-portion of the pre-provitellogenin. The position of the cleavage between the vitellogenin subunits in the pre-provitellogenin was identified by locating the N terminus of the large subunit. The cleavage site has a consensus RXRR for the subtilisin-processing endoprotease. Mosquito vitellogenin is highly hydrophilic with 17 putative N-linked glycosylation sites and 13 potential tyrosine sulfation sites. In contrast to known invertebrate vitellogenins, mosquito vitellogenin contains three polyserine domains that are similar to those of phosvitins in vertebrate vitellogenins. These polyserine domains, originally presumed to be vertebrate-specific, have several phosphorylation consensus sites in their sequences. Unlike other known vitellogenins, mosquito vitellogenin is rich in aromatic amino acid residues, tyrosine and phenylalanine, and in this respect is similar to insect serum proteins, arylphorins. This similarity suggests that mosquito vitellogenin may supply aromatic amino acids to the cuticle of rapidly developing embryos.

A novel function of 20-hydroxyecdysone: translational repression of the lysosomal protease mRNA in the mosquito fat body.

In the female fat body of the mosquito Aedes aegypti, lysosomes play important roles during the cessation of vitellogenesis by degrading the biosynthetic machinery and aiding the remodeling of the fat body cells. A detailed study of a mosquito lysosomal aspartic protease (AaLAP) has shown a unique expression pattern in the vitellogenic fat body: the level of AaLAP mRNA dramatically rises and peaks at 24 h post blood meal (PBM) correlating with the high titer of ecdysteroids; however, there is a 12 h lag before peak levels of AaLAP protein and its enzymatic activity has been observed. These observations suggest that the high titer of 20-hydroxyecdysone (20E) may hinder translation of the AaLAP mRNA. Here, we used an in vitro organ culture to study the effect of 20E on the protein synthesis of AaLAP in the fat body. The increase in the AaLAP protein level in the fat body, dissected at 24 h PBM and incubated for 6 or 12 h, was inhibited by the presence of 10(-5) M 20E in the medium. Incubation in the hormone-free medium did not effect accumulation of the AaLAP protein which proceeded at the levels comparable to the intact insect. Furthermore, the effect of 10(-5) M 20E on the AaLAP accumulation was reversible. These experiments support the hypothesis of the 20E-mediated repression of lysosomal protease mRNAs at the translational level in the regulation of vitellogenic and postvitellogenic events in the mosquito fat body. Analysis of the 5' and 3' -end untranslated regions (UTR) of AaLAP mRNA form secondary structures suggest that they may also contribute to mRNA stability and 20E-mediated translational inhibition.

Mosquito ecdysteroid receptor: analysis of the cDNA and expression during vitellogenesis.

An insect steroid hormone, 20-hydroxyecdysone (20E), plays an important role in regulating egg maturation in mosquitoes. To better understand its role, we cloned the cDNA coding for the putative ecdysteroid receptor from the mosquito, Aedes aegypti (AaEcR). The 4158 bp AaEcR cDNA has an open reading frame of 675 amino acids with 10 potential glycosylation sites and a putative phosphorylation polyserine domain. The AaEcR has a DNA binding domain with two zinc fingers and a ligand binding domain characteristic of members of the steroid hormone receptor superfamily. These AaEcR domains share 97 and 87% identities with the respective domains of the Drosophila ecdysteroid receptor (DmEcR). However, the A/B region of the AaEcR shares 35% identity with that of DmEcR-B1 isoform. The F region, located at the carboxyl-terminal of the AaEcR, has only 9% identity with the corresponding region of DmEcR. Potential nuclear targeting and dimerization signals are also present in the AaEcR sequence. There are three AaEcR transcripts of 4.2 kb, 6 kb and 11 kb in adult mosquitoes. 4.2 kb mRNA is predominantly expressed in female mosquitoes during vitellogenesis. In both the fat body and ovaries of the female mosquito, the level of AaEcR mRNA is high at the previtellogenic period and after the onset of vitellogenesis (6 h post blood meal, PBM).

Cloning and characterization of cDNAs encoding the antibacterial peptide, defensin A, from the mosquito, Aedes aegypti.

Insect defensins are cationic, inducible antibacterial peptides. Four full-length cDNAs encoding defensin A from the mosquito Aedes aegypti were cloned using polymerase chain reaction (PCR) and sequenced. All four cDNAs are 473 base pairs long, bearing an open reading frame of 98 amino acids with a few substitutions in the signal peptide domain. The deduced amino acid sequence of Aedes aegypti defensin (AaDef) contains a signal peptide sequence of 18 amino acids followed by a 40-amino acid putative propeptide domain and a 40-amino acid mature peptide domain. The mature peptide, with a predicted M(r) of 4148, shows 80% identity and 93% similarity to Phormia defensin A, and is identical to the peptide sequencing data for mosquito defensin A of Lowenberger et al. (1995) and B of Chalk et al. (1995). There are three potential phosphorylation sites but no glycosylation sites detected in AaDef. Three putative disulfide linkages between cysteines, characteristic of insect defensins, are conserved in AaDef. Aedes aegypti defensin mRNA is produced in response to a bacterial challenge; it is dramatically enhanced 6 h after bacterial injection, continues to increase through 24 h, and is maintained at high levels until at least 30 h post-bacterial injection.

Molecular characterization of a defensin gene from the mosquito, Aedes aegypti.

Insect immune proteins, defensins, are inducible anti-Gram-positive bacterial peptides. We report here the identification of two defensin genes from the mosquito, Aedes aegypti, which encode a large 541 bp transcript (AaDef Ala) and a small 473 bp transcript (AaDef Asm). The cDNA corresponding to AaDef Ala was cloned, sequenced, and compared with the previously reported AaDef Asm cDNA. The AaDef Ala gene was isolated through genomic library screening and characterized. It putative regulatory region contains a 64 bp intron, a TATA box and a putative arthropod initiator. Two 150 bp long direct and several palindromic repeats are present in this sequence. Similar to other insect immune peptide genes, the AaDef Ala gene contains numerous putative regulatory motifs with impressive similarity to elements of vertebrate acute phase response protein genes.

Aging effects on the secretion of corticosterone in male rats.

BACKGROUND: The effects of aging on zona fasciculatareticularis (ZFR) cell function in male rats were studied. METHODS: Male rats 3, 6, and 22 months of age were divided into three groups, and collagenase-dispersed ZFR cells were isolated and incubated with adrenocorticotropin (ACTH), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), ovine prolactin (oPRL), deoxycorticosterone (DOC), or 3-isobutyl-l-methylxanthine (IBMX) at 37 degrees C for 1 hour. Corticosterone concentrations in cell media and cAMP production in ZFR cells were measured by radioimmunoassay. Protein expression of PRL receptor in ZFR cells were analyzed by Western blot. RESULTS: The basal levels of plasma and medium corticosterone were higher in 22-month-old than in 3-month-old rats. In contrast, the release of corticosterone in response to ACTH, 8-Br-cAMP, and DOC was lower in 22-month-old than in 3- and 6-month-old rats. Aging decreased the oPRL-stimulated release of corticosterone but increased the protein expression of PRL receptor in ZFR cells. The basal levels of intracellular cAMP increased with age. However, the ACTH-stimulated production of intracellular cAMP decreased in 22-month-old compared with 3- or 6-month-old rats. The increment of cAMP accumulation in ZFR cells after administration of IBMX was greater in 22-month-old than in 3- or 6-month-old rats. CONCLUSIONS: These results suggest that the aging effects on the production of corticosterone in rat ZFR cells is associated with change of the generation of cAMP, the activity of 11 beta-hydroxylase and the protein expression of PRL receptor.

Age-related impairment of aldosterone secretion in zona glomerulosa cells of ovariectomized rats.

BACKGROUND: Clinical reports have revealed impaired sodium and water balance in elderly persons. The present studies were designed to investigate the effects and involved mechanisms of aging on aldosterone secretion in zona glomerulosa (ZG) cells of young and old ovariectomized (Ovx) rats. METHODS: Young (3 months) and old (24 months) female rats were Ovx for 4 days before decapitation. ZG cells of young and old rats were incubated with angiotensin II (Ang II), tetrandrine, nifedipine, adrenocorticotropic hormone (ACTH), forskolin, 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and precursors at 37 degrees C for 30 minutes. Aldosterone concentrations in plasma and cell media as well as 3':5'-cAMP production in ZG cells were determined by radioimmunoassay. The effects of aging on the activity of aldosterone synthase and the expression of cytochrome P450 side-chain cleavage enzyme (P450scc) in ZG cells were determined by thin-layer chromatography and Western blot analysis, respectively. RESULTS: Old rats had a lower plasma aldosterone level and a reduced basal aldosterone release from ZG cells than those in young rats. The conversions of steroidogenic precursors to aldosterone and the activity of aldosterone synthase as well as the expression of P450scc in ZG cells were lower in the old group than in the young group. Ang II-, ACTH-, forskolin- or 8-Br-cAMP-stimulated aldosterone secretion was attenuated in the old group as compared with the young group. Nifedipine decreased aldosterone secretion in the young group but not in the old group. The basal and forskolin-stimulated cAMP accumulations were lower in the old than in the young group. CONCLUSIONS: These results suggest that the age-related decline in aldosterone secretion is in part a consequence of the reduced activities of biosynthetic enzymes, adenylyl cyclase and L-type calcium channels, as well as the expression of P450scc protein in ZG cells.

Tumoral presence of human cytomegalovirus is associated with shorter disease-free survival in elderly patients with colorectal cancer and higher levels of intratumoral interleukin-17.

Infectious diseases are closely related to cancer. Human cytomegalovirus (HCMV) has been implicated in the promotion of tumour growth, and is present in the tumour specimens of colorectal cancer (CRC). This study aimed to investigate whether tumoral presence of HCMV is associated with a different clinical outcome in elderly patients with CRC. We analysed archived tumour specimens from 95 CRC patients aged ≥65 years. HCMV was detected by PCR. Clinical, pathological, disease-free and overall survival data were compared between patients with HCMV-positive and HCMV-negative tumours. A quantitative RT-PCR array was used to evaluate the expression levels of cytokines genes of T-helper subpopulations in tumours. In the Kaplan-Meier analysis of the 81 patients who underwent curative surgery, 39 patients with HCMV-positive tumours had a lower disease-free survival rate (p 0.024). For patients with stage II or stage III tumours, tumoral HCMV status correlated with disease-free survival more closely than the traditional histopathological staging methods. In a multivariate Cox proportional hazard model, tumoral presence of HCMV independently predicted tumour recurrence in 5 years (hazard ratio 4.42; 95% CI 1.54-12.69, p 0.006). The qRT-PCR analysis of ten stage II tumours showed that the gene expression levels of interleukin-17-the signature cytokine of T-helper 17 cells-and its receptor, interleukin-17 receptor C, were higher in the five HCMV-positive tumours. Our results suggest that the presence of HCMV in CRC is associated with poorer outcome in elderly patients. How the virus interacts with the tumour microenvironment should be further investigated.

Dissemination of multidrug-resistant, class 1 integron-carrying Acinetobacter baumannii isolates in Taiwan.

In this study, 283 multidrug-resistant Acinetobacter baumannii (MDR-AB) bloodstream isolates were collected between 1996 and 2004, from three teaching hospitals located in different regions of Taiwan. Susceptibility data showed that strains carrying class 1 integrons were significantly more resistant (p <0.01) to all tested antibiotics (except aztreonam and chloramphenicol) than strains lacking integrons, Seven types of gene cassette were identified among these strains, including two that have not been previously reported. The vast majority of the cassettes encoded aminoglycoside resistance genes, including aacA4, aacC1, aac(6')-II, aadA1, aadA2, aadA4 and aadDA1. Sixteen distinct ribotypes were identified in MDR-AB isolates carrying class 1 integrons. Only one strain was found to produce an extended-spectrum beta-lactamase, i.e. VEB-3. In the 18 imipenem-resistant strains, two carbapenenmase genes, bla(VIM-11) and bla(OXA-58), were found concomitantly in one isolate. An island-wide epidemic clone and an endemic clone from a hospital located in the northern region were identified by ribotyping. On the basis of the susceptibility data among the different ribogroups, the epidemic clone was associated more significantly with resistance to cefepime and ampicillin-sulbactam than was the endemic clone. In conclusion, the presence of class 1 integrons was significantly associated with resistance in MDR-AB, and the epidemic, class 1 integron-carrying MDR-AB clone was found to be widespread in Taiwan.

A p38 MAP kinase regulates the expression of the Aedes aegypti defensin gene in mosquito cells.

An Aedes aegypti p38 (Aap38) mitogen-activated protein kinase was isolated and characterized in this study. The 1761 bp long full-length Aap38 cDNA encodes an open reading frame of 358 amino acids, exhibiting characteristics of Thr/Tyr dual kinase specificities. We showed that bacteria activate both the kinase activity of Aap38 and the expression of the Aedes aegypti defensin A (AaDefA) gene, which is inhibited by a p38 kinase inhibitor SB203580 and dsRNA interference of Aap38. A similar result was obtained by a reporter construct containing the AaDefA regulatory region linked to Ds-Red. The lipopolysaccharide-activated reporter gene was inhibited by SB203580. In addition, Aap38 translocated to the nucleus after lipopolysaccharide induction. Our findings suggest that the p38 protein kinase pathway is involved in the antibacterial peptide synthesis in mosquitoes.

Cloning of cDNA for mosquito lysosomal aspartic protease. Sequence analysis of an insect lysosomal enzyme similar to cathepsins D and E.

A cDNA coding for the lysosomal aspartic protease from the mosquito (mLAP) was cloned and sequenced. The mLAP cDNA is 1420 base pairs long with an open reading frame of 387 amino acids. The deduced amino acid sequence contains a signal pre-propeptide sequence of 18 amino acids followed by 369 amino acids with a 35-amino acid putative pro-enzyme domain in the NH2-terminal. The amino acid sequence of mLAP is 92 and 81% similar to human cathepsin D and cathepsin E, respectively. Typical cleavage sites for cathepsin D processing into light and heavy chains are lacking in mLAP. A single glycosylation site occurs in the mLAP sequence at a position corresponding to the first glycosylation site of cathepsins D. The mLAP sequence shares putative phosphorylation determinants, which in cathepsins D are linked to the formation of mannose 6-phosphate. In the mosquito fat body, lysosomal enzymes specifically degrade organelles involved in the biosynthesis and secretion of vitellogenin. The mLAP mRNA accumulates to its highest level 24 h after initiation of vitellogenin synthesis and 12 h before the peak of mLAP protein accumulation and its enzymatic activity. Translational regulation of mLAP mRNA may occur. The 5'-untranslated region of mLAP mRNA is similar to elements conferring negative translational control by steroids.

An extraovarian protein accumulated in mosquito oocytes is a carboxypeptidase activated in embryos.

We report a phenomenon previously unknown for oviparous animals; in Aedes aegypti mosquitoes a serine carboxypeptidase is synthesized extraovarially and then internalized by oocytes. The cDNA encoding mosquito vitellogenic carboxypeptidase (VCP) was cloned and sequenced. The VCP cDNA hybridizes to a 1.5-kilobase mRNA present only in the fat body of vitellogenic females. The deduced amino acid sequence of VCP shares significant homology with members of the serine carboxypeptidase family. Binding assays using a serine protease inhibitor, [3H]diisopropyl fluorophosphate, showed that VCP is activated in eggs at the onset of embryonic development. Activation of VCP is associated with the reduction in its size from 53 kDa (inactive proenzyme) to 48 kDa (active enzyme). The active, 48-kDa, form of VCP is maximally present at the middle of embryonic development and disappears by the end.

Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti.

Progress in molecular genetics makes possible the development of alternative disease control strategies that target the competence of mosquitoes to transmit pathogens. We tested the regulatory region of the vitellogenin (Vg) gene of Aedes aegypti for its ability to express potential antipathogen factors in transgenic mosquitoes. Hermes-mediated transformation was used to integrate a 2.1-kb Vg-promoter fragment driving the expression of the Defensin A (DefA) coding region, one of the major insect immune factors. PCR amplification of genomic DNA and Southern blot analyses, carried out through the ninth generation, showed that the Vg-DefA transgene insertion was stable. The Vg-DefA transgene was strongly activated in the fat body by a blood meal. The mRNA levels reached a maximum at 24-h postblood meal, corresponding to the peak expression time of the endogenous Vg gene. High levels of transgenic defensin were accumulated in the hemolymph of bloodfed female mosquitoes, persisting for 20-22 days after a single blood feeding. Purified transgenic defensin showed antibacterial activity comparable to that of defensin isolated from bacterially challenged control mosquitoes. Thus, we have been able to engineer the genetically stable transgenic mosquito with an element of systemic immunity, which is activated through the blood meal-triggered cascade rather than by infection. This work represents a significant step toward the development of molecular genetic approaches to the control of vector competence in pathogen transmission.

Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor.

The mosquito (Aedes aegypti) vitellogenin receptor (AaVgR) is a large membrane-bound protein (214 kDa when linearized) that mediates internalization of vitellogenin, the major yolk-protein precursor, by oocytes during egg development. We have cloned and sequenced two cDNA fragments encompassing the entire coding region of AaVgR mRNA, to our knowledge the first insect VgR sequence to be reported. The 7.3-kb AaVgR mRNA is present only in female germ-line cells and is abundant in previtellogenic oocytes, suggesting that the AaVgR gene is expressed early in oocyte differentiation. The deduced amino acid sequence predicts a 202.7-kDa protein before posttranslational processing. The AaVgR is a member of the low density lipoprotein receptor superfamily, sharing significant homology with the chicken (Gallus gallus) VgR and particularly the Drosophila melanogaster yolk protein receptor, in spite of a very different ligand for the latter. Distance-based phylogenetic analyses suggest that the insect VgR/yolk protein receptor lineage and the vertebrate VgR/low density lipoprotein receptor lineage diverged before the bifurcation of nematode and deuterostome lines.

Identification of immune-related protein kinases from mosquitoes (Aedes aegypti).

Protein kinases are known to be involved in signal transduction for numerous physiological events. However, little is known about the roles of protein kinases in insect immunity. A fragment around 150 bp was amplified by polymerase chain reaction using cDNA templates from bacterial inoculated mosquitoes and primers corresponding to the conserved domain of protein kinases. Based on sequence analysis, 11 groups of protein kinases were characterized including 3 nonreceptor tyrosine kinases, 3 receptor tyrosine kinases, 3 serine/threonine kinases, and 2 novel protein kinases. The most abundant kinase obtained in this study reveals a high degree of similarity to human cholinesterase-related cell division controller (CHED) protein kinase. The expression of this mosquito CHED-like kinase is not detectable in normal female mosquitoes, but induced only after bacterial inoculation and trauma. A mosquito protein kinase was demonstrated to share homology with a plant Tousled gene, but has not yet been characterized in the animal system. In addition, analysis of the sequences of several protein kinases cloned from mosquitoes suggests that they might be involved in the regulation of cellular or humoral immunity.

From mosquito to man: identification of a novel protein kinase, HsHPK, which is highly expressed in human hepatoma tissues.

Protein kinases play an important role in the signaling pathway of growth factors in most of the higher organisms. During the study of protein kinase profiles of mosquitoes using RT-PCR and degenerate primers for consensus catalytic domain motifs to amplify protein kinase genes, we have noticed that a novel mosquito kinase, AaPK-38, shares a stretch of amino acids identical to the corresponding domain in Tousled gene of Arabidopsis thaliana that is required for leaf and flower development. A 2.1-kb cDNA encoding human HsHPK gene, which is a homolog of AaPK-38, was isolated from human testis cDNA library. This cDNA contains an open reading frame of 563 amino acids, with a complete kinase domain in its carboxyl terminus. The expressed Flag-tagged HsHPK was shown to have kinase activity based on in vitro autophosphorylation. Northern blot analysis revealed that human HsHPK mRNA is most abundant in testes, much less in heart and skeletal muscle and almost undetectable in liver and lung. Finally, we found that the expression of HsHPK in 4 out of 6 human hepatoma tissues is much higher than that in the adjacent normal counterpart. This result suggests HsHPK may play a role in the development of human hepatoma.

Molecular cloning, characterization and tissue expression of prophenoloxidase cDNA from the mosquito Armigeres subalbatus inoculated with Dirofilaria immitis microfilariae.

A cDNA encoding mosquito Armigeres subalbatus prophenol oxidase (As-pro-PO) was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) after Dirofilaria immitis inoculation. The 2205 bp As-pro-PO cDNA contains a 32 bp 5'-noncoding region, a 2055 bp open reading frame (685 amino acids), and a 118 bp 3'-noncoding region. Hydrophobic signal peptide for the endoplasmic reticulum targeting is not found in the NH2-terminal region. Two potential copper-binding domains, amino acids 197-245 and 345-412, are highly homologous to those of the other insect pro-POs. A 2.2 kb As-pro-PO transcript was identified by Northern blot analysis using D. immitis microfilariae-inoculated A. subalbatus. Both in situ hybridization and Northern blot analysis demonstrated that As-pro-PO mRNA was synthesized in mosquito haemocytes but not in other tissues, i.e. fat bodies, midguts and ovaries, etc.