Biomedical subjects

B M Christensen

Publications and source records attributed to B M Christensen.

At least 19 recordsLinked to original sources

PubMed2003

A potential role for phenylalanine hydroxylase in mosquito immune responses.

In mosquitoes the melanotic encapsulation immune response is an important resistance mechanism against filarial worms and malaria parasites. The rate limiting substrate for melanin production is tyrosine that is hydroxylated by phenoloxidase (PO) to produce 3, 4-dihydroxyphenylalanine. The single pathway for endogenous production of tyrosine is by hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH). In this study we describe a potential role for PAH in melanotic immune responses in the yellow fever mosquito, Aedes aegypti. A 1.6 kb A. aegypti PAH cDNA, encoding a 51 kDa protein, was isolated and subsequently expressed in an Escherichia coli expression system. In developing mosquitoes, PAH transcript is present in all stages and it is differentially expressed in adult tissues. Following an immune-challenge with Dirofilaria immitis microfilariae (mf) or bacteria, PAH transcript is up-regulated in hemocytes. Likewise, western analysis of hemocytes collected from immune-activated mosquitoes show an increase in gene product over control samples. Like PO, ultrastructure observations provide verification that PAH is located in oenocytoid and granulocyte hemocytes. Our results offer the first data that suggest PAH is used in mosquito melanin synthesis and defense responses.

Amino Acid Sequence↗

PubMed2002

Glucosamine:fructose-6-phosphate aminotransferase: gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegypti.

Glucosamine:fructose-6-phosphate aminotransferase (GFAT) catalyses the formation of glucosamine 6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. The final product of the hexosamine pathway, UDP-N-acetyl glucosamine, is an active precursor of numerous macromolecules containing amino sugars, including chitin in fungi and arthropods. Chitin is one of the essential components of insect cuticle and peritrophic matrix. The peritrophic matrix is produced in the midgut of mosquitoes in response to bloodfeeding, and may affect vector competence by serving as a physical barrier to pathogens. It is hypothesized that GFAT plays a regulatory role in biosynthesis of chitin and peritrophic matrix formation in insects. We cloned and sequenced the GFAT gene (AeGfat-1) and its 5' regulatory region from Aedes aegypti. There is no intron in AeGfat-1 and there are two potential transcription start sites. AeGfat-1 cDNA is 3.4 kb in length and its putative translation product is 75.4 kDa. The amino acid sequence of GFAT is highly conserved in lower and higher eukaryotes, as well as in bacteria. AeGfat-1 message is constitutively expressed but is gradually up-regulated in the midgut after bloodfeeding. The putative regulatory region of the gene contains the ecdysone response element, E74, and Broad complex motifs, similar to what is found in the glutamine synthetase gene in Ae. aegypti. Results suggest that Ae. aegypti GFAT-1 may have a regulatory role in chitin biosynthesis and peritrophic matrix formation, and probably is under the regulation of ecdysteroids.

5' Untranslated Regions↗

PubMed2001

Aedes aegypti glutamine synthetase: expression and gene structure.

The peritrophic matrix (PM) is the first natural barrier a mosquito-borne parasite faces when ingested with a blood meal; consequently, understanding the biology of PM formation could provide novel transmission control strategies. Because the PM is composed of chitin (a molecule of repeating units of N-acetyl glucosamine), glycoproteins and glucose, characterizing the regulation of enzymes involved in chitin production should provide information concerning factors that influence PM formation. We previously have shown that glutamine synthetase (GS) provides the glutamine needed in the initial steps of chitin biosynthesis in the yellow fever mosquito, Aedes aegypti. In the present study we show that GS is encoded by a single 4.5 kb gene, designated mGS, containing three exons and two introns. Multiple transcripts are generated from mGS presumably by differential splicing of the introns. Sequences of two cDNAs encoding GS are identical at the protein level, but differ in their 5'-untranslated regions. GS message is constitutively expressed in all developmental stages and in most tissues, with an increase in GS transcription observed in midgut and fat body tissues of female mosquitoes following a blood meal. Transcripts are localized to the apical side of the mosquito midgut epithelium and data suggest that mGS transcription is regulated by an Oct-1 transcription factor.

Aedes↗

PubMed2001

Aedes aegypti peroxidase gene characterization and developmental expression.

The functions of insect peroxidases include detoxification, stabilization of extracellular matrices, and possible involvement in insect immunity. The current study describes the isolation of a peroxidase gene, AePox, and its cDNA from the mosquito, Aedes aegypti. AePox codes for a protein that is homologous to various heme-peroxidases from vertebrates and invertebrates, with highest identity to Drosophila melanogaster peroxidase (62%). Sequence comparison identified several functionally and structurally conserved domains in the mosquito peroxidase, including a heme environment, a calcium binding site, and five possible disulfide bridges. These results imply that AePOX may likely have a similar structure and catalytic mechanism as those described for the mammalian myeloperoxidase superfamily. Expression studies demonstrate that AePox is transcribed in mosquito larvae and pupae, but not in adults, in ovaries, or in early embryos. However, AePOX protein is present in all mosquito stages and possibly has a maturation process that is similar to that of human myeloperoxidase. Unlike most human peroxidases, the AePox gene contains a TATA box and an ecdysone response element (EcRE).

Aedes↗

PubMed2001

Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedes aegypti.

In this study we describe the purification and molecular cloning of a dopachrome conversion enzyme (DCE) from the yellow fever mosquito, Aedes aegypti. DCE catalyzes the conversion of L-dopachrome to 5,6-dihydroxyindole in the melanization pathway. Melanin biosynthesis is involved with crucial protective phenomena in mosquitoes, including egg chorion and cuticular tanning, wound healing, and the melanotic encapsulation immune response. The enzyme was purified to homogeneity by various chromatographic techniques from A. aegypti larvae and has a relative molecular mass of 51 kDa as-revealed by SDS-PAGE analysis. Physiochemical analysis of DCE revealed a pH optimum of 7.5-8.0 and substrate activity for L-dopachrome and aminochromes generated from dopa methyl ester, alpha-methyl dopa and dopamine. Trypsin digestion of the isolated DCE and subsequent reverse-phase separation resulted in the isolation of several polypeptide fragments, from which two partial internal amino acid sequences were obtained by Edman degradation. PCR amplification, using a degenerate primer based on one internal amino acid sequence and an oligo-dT primer, produced a 650 bp DNA fragment. Subsequent screening of an A. aegypti pupal cDNA library resulted in the isolation of a 1.6 kb clone containing coding sequence for both internal DCE amino acid sequences, thereby confirming the identity of the isolated gene product (pAaDce1) as DCE. Northern analysis revealed the constitutive expression of DCE message in developmental stages and adults, with the majority of transcript localized in the fat body and ovaries of adult females. AaDce1 mRNA increased in abundance above constitutive levels in adult females when a melanotic encapsulation immune response was initiated by the intrathoracic inoculation of Dirofilaria immitis microfilariae.

Aedes↗

PubMed2001

Effect of prophenoloxidase expression knockout on the melanization of microfilariae in the mosquito Armigeres subalbatus.

Melanization is an effective defence reaction used by mosquito hosts to kill malarial and filarial worm parasites. Although phenoloxidase (PO) has long been considered to be the key enzyme in the biosynthesis of melanotic material in insects, there is no direct evidence verifying its role in parasite melanization. To elucidate the role of PO in the melanization of microfilariae (mf) by mosquitoes, a double subgenomic Sindbis (dsSIN) recombinant virus was used to transduce Armigeres subalbatus mosquitoes with a 600 base antisense RNA targeted to the highly conserved copper-binding region of an Ar. subalbatus PO gene. Compared with controls, haemolymph PO activity in mosquitoes transduced with antisense RNA was significantly reduced. When these mosquitoes were challenged with Dirofilaria immitis mf, the melanization of mf was almost completely inhibited. These data verify that PO is an essential component of the biochemical pathway required for the melanization of parasites, and that the dsSIN expression system represents a useful tool in the functional analysis of endogenous gene expression in mosquitoes.

Animals↗

PubMed2001

Molecular cloning of a second prophenoloxidase cDNA from the mosquito Armigeres subalbatus: prophenoloxidase expression in blood-fed and microfilariae-inoculated mosquitoes.

Melanization constitutes an important component in various aspects of insect life, including cuticular sclerotization, egg-shell tanning, melanization of parasites and wound healing. Recently, a cDNA encoding prophenoloxidase (pro-PO), a key enzyme in the biosynthesis of melanotic material in insects, was cloned from microfilariae (mf)-inoculated mosquitoes, Armigeres subalbatus. However, results of Northern blot analyses indicated that two pro-POs might be present in Ar. subalbatus and these pro-POs might be responsible for two distinct physiological functions, egg-shell tanning and melanization of parasites. Subsequently, the second pro-PO cDNA (As-pro-PO II) was cloned from blood-fed Ar. subalbatus by rapid amplification of cDNA ends polymerase chain reaction. The 2210 bp As-pro-PO II cDNA contains a 41 bp 5'-non-coding region, a 2064 bp open reading frame and a 105 bp 3'-non-coding region. A hydrophobic signal peptide for endoplasmic reticulum targeting is not found in the N-terminal region. The deduced amino acid sequence of As-pro-PO II shares a high degree of identity (81.5%) with that of the As-pro-PO I obtained from mf-inoculated Ar. subalbatus. Both Northern blot and reverse transcription polymerase chain reaction analysis demonstrated that these two mosquito pro-POs are persistently expressed in mosquito haemocytes and not in fat body, midgut, or ovaries. The expression of As-pro-PO I and As-pro-PO II in mosquitoes is associated with melanization of mf and blood feeding, respectively.

Amino Acid Sequence↗

PubMed2001

Molecular cloning of two prophenoloxidase genes from the mosquito Aedes aegypti.

The biosynthesis of melanotic materials is an important process in the life of a mosquito. Melanin production is critical for many diverse processes such as egg chorion tanning, cuticular sclerotization, and melanotic encapsulation of metazoan parasites. Prophenoloxidase plays a critical role in this biochemical cascade. Two cDNAs, one full length and one partial clone, and two genomic clones encoding prophenoloxidase (pro-PO) were isolated from the yellow fever mosquito, Aedes aegypti. The full-length cDNA, pAaProPO1, is 2286 bp long with a 2055 bp open reading frame encoding a 685 amino acid protein that shares 89% identity with Armigeres subalbatus pro-PO. It contains two putative copper binding domains (amino acids 197-243 and 346-423) that are homologous to other insect pro-POs. AaProPO1 messenger RNA (mRNA) was detected by reverse transcription polymerase chain reaction (RT-PCR) only from third-stage larvae and not in adult mosquitoes after blood feeding, during the melanotic encapsulation of Dirofilaria immitis microfilariae or following exposure to bacteria. A 750 bp fragment of the second cDNA (pAaProPO2) was cloned using RT-PCR from mRNA obtained from 14-day postovipostional eggs. AaProPO2 mRNA was not found in any other life stages, and may be in low abundance or transiently expressed. AaProPO2 and AaProPO1 each contain three introns that are 60, 68 and 58 bp and 61, 69 and 59 bp long, respectively, and the intron sequences of these two genes are not similar.

Aedes↗

PubMed2001

Short report: A practical technique for the cryopreservation of Dirofilaria immitis, Brugia malayi, and Wuchereria bancrofti microfilariae.

A technique to cryopreserve microfilariae has been developed. This method was used to cryopreserve microfilariae of Dirofilaria immitis, Brugia malayi, and Wuchereria bancrofti at a controlled rate of 1 degree C/min by use of a freezing tank. Microfilariae of each of these species retained their ability to infect susceptible mosquito species and develop to the infective stage after cryopreservation. The method presented here is quickly and easily carried out with inexpensive equipment.

Animals↗

PubMed2000

Effects of medium hypertonicity on water permeability in the mammalian rectum: ultrastructural and molecular correlates.

Minute-by-minute net water fluxes (Jw) were measured across the isolated rectal epithelium in rats and rabbits. Five minutes after a serosal (but not mucosal) hypertonic challenge (plus 200 mosmol/l) a significant increase in the basal Jw was recorded in both species [deltaJw, microl min(-1) cm(-2): 0.40+/-0.06 (rats); 0.45+/-0.10 (rabbits)]. At the same time, most epithelial cells shrank markedly while the intercellular spaces were wide open (electron microscopy studies). In freeze-fracture studies multi-strand tight-junction structures (only slightly modified by serosal hypertonicity in rabbits) were observed in control conditions. No structural changes were observed after mucosal hypertonicity (both in rats and rabbits). Immunohistochemical studies showed the expression of aquaporin 3 (AQP3) at the basolateral membrane of epithelial cells in the rat. A first conclusion is that the epithelium of the mammalian rectum is a highly polarized, aquaporin-3-containing, water permeability structure. The Jw increase induced by serosal hypertonicity was sensitive to mercurial agents in both species and no changes in unidirectional [14C]mannitol fluxes (Ps) or transepithelial resistance (RT) were observed during this Jw increase. These observations suggest a transcellular route for the osmotically induced increase in water fluxes. In the rabbit rectum the initial Jw response, associated with serosal hypertonicity, was a transient one. It was followed by a second, slow and HgCl2-sensitive Jw increase (a transient peak in paracellular mannitol permeability was also observed). A second conclusion is that serosal hypertonicity induces an increase in transcellular water permeability in both rat and rabbit rectum.

Animals↗

PubMed2000

Cloning and characterization of a chitin synthase cDNA from the mosquito Aedes aegypti.

Characterization of the enzymes involved in the chitin biosynthetic pathway in mosquitoes is critical due to the importance of chitin in the formation of the peritrophic matrix [PM] and its potential impact on vector competence. Chitin is the homopolymer of the amino sugar N-acetyl-D glucosamine [GlcNAc]. The final step of incorporation of GlcNAc into the chitin polymer is catalyzed by the enzyme chitin synthase [CS]. CS is a membrane bound enzyme, but the mechanism of its action in the biosynthesis of the PM is not understood. We have isolated and sequenced a CS-encoding cDNA clone from the mosquito Aedes aegypti, compared its sequence with CS from other organisms and studied its RNA expression. The cDNA is 3.5 kb in length with an open reading frame of 2.6 kb that encodes a protein of 865 amino acids with a predicted molecular mass of 99.5 kDa. The putative translation product shares 90% similarity to two CS proteins from Caenorhabditis elegans and 50% similarity to Saccharomyces cerevisiae in the catalytic domain of CS enzymes. Data suggest that CS is a single copy gene. RT-PCR analysis shows CS message in whole non-blood-fed females, whole blood-fed females, non-blood-fed midguts and in midguts dissected at different time points post-blood-feeding. In situ hybridization studies of midgut samples revealed that CS mRNA increases following a bloodmeal and is localized to the periphery of the epithelial cells facing the midgut lumen.

Aedes↗

PubMed2000

Aedes aegypti dopa decarboxylase: gene structure and regulation.

Dopa decarboxylase converts L-dopa to dopamine, a precursor molecule for diverse biological activities in insects including neurotransmission and a variety of tanning reactions required for development, reproduction and defence against parasites. Herein, we report the cloning and sequencing of the Aedes aegypti Ddc gene, including 2.1 kb of the upstream promoter region. The transcribed region of the gene spans more than 16 kb and contains five exons. In situ hybridization localizes the blood-meal-induced ovarian transcription of this gene to the follicular epithelial cells surrounding individual oocytes. Ovary tissue transcription of Ddc is increased in response to injection of 20-hydroxyecdysone to levels equal to those observed for blood-fed controls, however coinjection with the translational inhibitor cycloheximide negates the effect, indicating an indirect regulatory role for this hormone. Clusters of putative ecdysone-responsive elements and zinc-finger binding domains for the products of Broad-Complex gene family are identified in the 5'-promoter region. These elements are discussed in the context of common insect Ddc regulatory mechanisms.

Aedes↗

PubMed2000

A novel member of the RING-finger gene family associated with reproductive tissues of the mosquito, Aaedes aegypti.

The RING finger is a zinc-binding domain that is found in proteins from viruses, plants and animals. Here we report the characterization and tissue-specific expression of a mosquito gonadal protein gene, mgp, from the mosquito, Aedes aegypti. The putative gene product, MGP, contains two RING fingers, a B-box, and a hydrophobic core. These mosquito MGP structural motifs are highly conserved in proteins found in mouse and nematode. Northern blot analysis and in situ hybridization demonstrated the presence of multiple mgp RNA transcripts in male and female reproductive tissues. Expression of mgp in the ovary is constitutive, but an increase in message was observed in the ovaries of female mosquitoes previously exposed to a blood meal. These results suggest that MGP is a protein that might play a role(s) in mosquito gametogenesis.

Aedes↗

PubMed2000

Genetics of mosquito vector competence.

Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Efforts to control mosquito-borne diseases have been impeded, in part, by the development of drug-resistant parasites, insecticide-resistant mosquitoes, and environmental concerns over the application of insecticides. Therefore, there is a need to develop novel disease control strategies that can complement or replace existing control methods. One such strategy is to generate pathogen-resistant mosquitoes from those that are susceptible. To this end, efforts have focused on isolating and characterizing genes that influence mosquito vector competence. It has been known for over 70 years that there is a genetic basis for the susceptibility of mosquitoes to parasites, but until the advent of powerful molecular biological tools and protocols, it was difficult to assess the interactions of pathogens with their host tissues within the mosquito at a molecular level. Moreover, it has been only recently that the molecular mechanisms responsible for pathogen destruction, such as melanotic encapsulation and immune peptide production, have been investigated. The molecular characterization of genes that influence vector competence is becoming routine, and with the development of the Sindbis virus transducing system, potential antipathogen genes now can be introduced into the mosquito and their effect on parasite development can be assessed in vivo. With the recent successes in the field of mosquito germ line transformation, it seems likely that the generation of a pathogen-resistant mosquito population from a susceptible population soon will become a reality.

Animals↗

PubMed2000

Localization and regulation of PKA-phosphorylated AQP2 in response to V(2)-receptor agonist/antagonist treatment.

Phosphorylation of Ser(256), in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser(256) were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys(1),D-Arg(8)]vasopressin (DDAVP) treatment or V(2)-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V(2)-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser(256) is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V(2) receptors by altering phosphorylation and/or dephosphorylation of Ser(256) in AQP2.

Amino Acid Sequence↗

PubMed2000

Prostaglandin E(2) interaction with AVP: effects on AQP2 phosphorylation and distribution.

Prostaglandin E(2) (PGE(2)) antagonizes the action of arginine vasopressin (AVP) on collecting duct water permeability. To investigate the mechanism of this antagonism, rat renal inner medulla (IM) was incubated with the two hormones, and the phosphorylation and subcellular distribution of the water channel, aquaporin-2 (AQP2) were studied. Using a phosphorylation state-specific AQP2 antibody, we demonstrated that AVP stimulates AQP2 phosphorylation at the Ser(256) protein kinase A consensus site in a time- and dose-dependent manner. In parallel studies using a differential centrifugation technique, we demonstrated that AVP induced translocation of AQP2 from an intracellular vesicle-enriched fraction to a plasma membrane-enriched fraction. PGE(2) (10(-7) M) added after AVP (10(-8) M) did not decrease AQP2 phosphorylation but reversed AVP-induced translocation of AQP2 to the plasma membrane. Preincubation of IM with PGE(2) did not prevent the effects of AVP on AQP2 phosphorylation and trafficking. PGE(2) alone did not influence AQP2 phosphorylation and subcellular distribution. Our data indicate that 1) recruitment of AQP2 to the plasma membrane and its retrieval to a pool of intracellular vesicles may be regulated independently, 2) PGE(2) may counteract AVP action by activation of AQP2 retrieval, 3) dephosphorylation of AQP2 is not a prerequisite for its internalization.

Animals↗

PubMed1999

Heterotetrameric composition of aquaporin-4 water channels.

Aquaporin (AQP) water channel proteins are tetrameric assemblies of individually active approximately 30 kDa subunits. AQP4 is the predominant water channel protein in brain, but immunoblotting of native tissues has previously yielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four polypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa. The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the M1 isoform, and 32 and 36 kDa correspond to the shorter M23 isoform. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astrocyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergent-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus laevis oocytes demonstrated that heterotetramer formation reflects the relative expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers are formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influence water permeability but may contribute to membrane trafficking or assembly of arrays.

Amino Acid Sequence↗

PubMed1999

Antimicrobial activity spectrum, cDNA cloning, and mRNA expression of a newly isolated member of the cecropin family from the mosquito vector Aedes aegypti.

An antimicrobial peptide belonging to the cecropin family was isolated from the hemolymph of bacteria-challenged adult Aedes aegypti. This new peptide, named cecropin A, was purified to homogeneity and fully characterized after cDNA cloning. The 34-residue A. aegypti cecropin A is different from the majority of reported insect cecropins in that it is devoid of a tryptophan residue and C-terminal amidation. The importance of these two structural features on the activity spectrum was investigated using a chemically synthesized peptide. A comparison of the antimicrobial activity spectrum of A. aegypti and Drosophila cecropin A showed a lower activity for the mosquito molecule. A. aegypti cecropin mRNA expression was not detected by Northern blot or reverse transcription-polymerase chain reaction analysis in any immature stage of the mosquito, nor in naïve adults, but it was observed in challenged adults 6 h after bacteria inoculation, and it continued over 7-10 days.

Aedes↗