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B M Christensen

Publications and source records attributed to B M Christensen.

At least 37 records · Page 2Linked to original sources

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↗

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↗

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↗

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.

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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.

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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↗

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↗

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↗

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↗

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.

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Human calumenin localizes to the secretory pathway and is secreted to the medium.

Calumenin belongs to a family of multiple EF-hand proteins that include reticulocalbin, ERC-55, and Cab45. Reticulocalbin and ERC-55 localize to the ER due to a C-terminal HDEL retrieval signal. Cab45 contains a HEEF C-terminal sequence and is localized to the Golgi apparatus. The murine homologue of calumenin is reported to be present in the ER due to a C-terminal HDEF retrieval signal. The human homologue differs from the murine at 7 amino acid positions but the HDEF signal is conserved. However, in the cultured human cell lines, HaCaT keratinocytes, normal and transformed MRC-5 fibroblasts, as well as in transfected COS-1 cells, human calumenin could be demonstrated in the ER as well as in the Golgi complex. Especially in MRC-5 cells, a certain heterogeneity was observed, with some of the cells having calumenin localized solely to the ER while in other cells calumenin could be demonstrated in the ER as well as in the Golgi complex. Immunoelectron microscopy of placental syncytiotrophoblast cells showed that a substantial fraction of calumenin is localized in close association with the ER membrane. In addition, the protein may be recovered from the medium of cultured cells in an endoglycosidase H-resistant form, suggesting that the glycosylated protein has been further modified in the Golgi apparatus and secreted to the medium.

Antibody Specificity↗

Mosquito-Plasmodium interactions in response to immune activation of the vector.

During the development of Plasmodium sp. within the mosquito midgut, the parasite undergoes a series of developmental changes. The elongated ookinete migrates through the layers of the midgut where it forms the oocyst under the basal lamina. We demonstrate here that if Aedes aegypti or Anopheles gambiae, normally susceptible to Plasmodium gallinaceum and P. berghei, respectively, are immune activated by the injection of bacteria into the hemocoel, and subsequently are fed on an infectious bloodmeal, there is a significant reduction in the prevalence and mean intensity of infection of oocysts on the midgut. Only those mosquitoes immune activated prior to, or immediately after, parasite ingestion exhibit this reduction in parasite development. Mosquitoes immune activated 2-5 days after bloodfeeding show no differences in parasite burdens compared with naive controls. Northern analyses reveal that transcriptional activity for mosquito defensins is not detected in the whole bodies of Ae. aegypti from 4 h to 10 days after ingesting P. gallinaceum, suggesting that parasite ingestion, passage from the food bolus through the midgut, oocyst formation, and subsequent release of sporozoites into the hemolymph do not induce the production of defensin. However, reverse transcriptase-PCR of RNA isolated solely from the midguts of Ae. aegypti indicates that transcription of mosquito defensins occurs in the midguts of naive mosquitoes and those ingesting an infectious or noninfectious bloodmeal. Bacteria-challenged Ae. aegypti showed high levels of mature defensin in the hemolymph that correlate with a lower prevalence and mean intensity of infection with oocysts. Because few oocysts were found on the midgut of immune-activated mosquitoes, the data suggest that some factor, induced by bacterial challenge, kills the parasite at a preoocyst stage.

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Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti.

Aedes aegypti were immune activated by injection with bacteria, and the expression of insect defensins was measured over time. Northern analyses indicated that defensin transcriptional activity continued for at least 21 days after bacterial injection, and up to 10 days after saline inoculation. Mature defensin levels in the haemolymph reached approximately 45 microM at 24 h post inoculation. cDNAs encoding the preprodefensins of three previously described mature Ae. aegypti defensins were amplified by PCR, cloned and sequenced. Genomic clones were amplified using primers designed against the cDNA sequence. Sequence comparison indicates that there is significant inter- and intra-isoform variability in the signal peptide and prodefensin sequences of defensin genes. Preprodefensin sequences of isoforms A and B are very similar, consisting of a signal peptide region of twenty amino acids, a prodefensin region of thirty-eight amino acids and a forty amino acid mature peptide domain. The sequence encoding isoform C is significantly different, comprising a signal peptide region of twenty-three amino acids, a prodefensin region of thirty-six amino acids, and the mature protein domain of forty amino acids. Analysis of the genomic clones of each isoform revealed one intron spatially conserved in the prodefensin region of all sequences. The intron in isoforms A and B is 64 nt long, and except for a 4 nt substitution in one clone, these intron sequences are identical. The intron in isoform C is 76 nt long and does not share significant identity with the intron sequences of isoforms A or B. The defensin gene mapped to chromosome 3, between two known loci, blt and LF168.

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Physiology and pathophysiology of renal aquaporins.

The discovery of aquaporin membrane water channels by Agre and coworkers answered a long-standing biophysical question of how water specifically crosses biologic membranes, and provided insight, at the molecular level, into the fundamental physiology of water balance and the pathophysiology of water balance disorders. Of nine aquaporin isoforms, at least six are known to be present in the kidney at distinct sites along the nephron and collecting duct. Aquaporin-1 (AQP1) is extremely abundant in the proximal tubule and descending thin limb, where it appears to provide the chief route for proximal nephron water reabsorption. AQP2 is abundant in the collecting duct principal cells and is the chief target for vasopressin to regulate collecting duct water reabsorption. Acute regulation involves vasopressin-regulated trafficking of AQP2 between an intracellular reservoir and the apical plasma membrane. In addition, AQP2 is involved in chronic/adaptational regulation of body water balance achieved through regulation of AQP2 expression. Importantly, multiple studies have now identified a critical role of AQP2 in several inherited and acquired water balance disorders. This concerns inherited forms of nephrogenic diabetes insipidus and several, much more common acquired types of nephrogenic diabetes insipidus where AQP2 expression and/or targeting are affected. Conversely, AQP2 expression and targeting appear to be increased in some conditions with water retention such as pregnancy and congestive heart failure. AQP3 and AQP4 are basolateral water channels located in the kidney collecting duct, and AQP6 and AQP7 appear to be expressed at lower abundance at several sites including the proximal tubule. This review focuses mainly on the role of AQP2 in water balance regulation and in the pathophysiology of water balance disorders.

Animals↗

Potential for evolution of California serogroup bunyaviruses by genome reassortment in Aedes albopictus.

Aedes albopictus was introduced into the United States in used tires in 1985. Its successful colonization of the upper Midwest has potential to alter the current epidemiology of bunyaviruses that circulate in the region. It is permissive for the replication of several arboviruses, including La Crosse (LACV) and Jamestown Canyon (JCV) bunyaviruses. In this study, we demonstrate the ability of LACV and JCV to coinfect Ae. albopictus mosquitoes and to form all six possible reassortant genotypes. All reassortant viruses infect Ae. albopictus orally and can be transmitted to suckling mice. All reassortants are neurovirulent in mice. However, reassortant viruses carrying the LACV M segment in the foreign genetic background of JCV are more neuroinvasive than JCV, or any other reassortant genotype. In addition, these reassortants can replicate in gerbils and infect Ae. triseriatus, characteristics of LACV, but not JCV. Because Ae. albopictus is spreading into new geographic areas and feeds on a variety of mammals, including humans, it has the potential to transmit new, emerging bunyaviruses in nature.

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Automated measurement of oxygen consumption by the yellow fever mosquito, Aedes aegypti.

Oxygen consumption of single mosquitoes was measured using a differential pressure transducer (DPT) connected to two small chambers. A mosquito was placed in the experimental chamber (P1) and was separated from NaOH by 4 cm2 of marquisette mesh. The reference chamber (P2) contained the same amount of NaOH and the marquisette mesh but without a mosquito. When these two chambers were sealed, removed O2 from P1 resulted in a pressure decrease with respect to P2. This pressure differential was transduced into an output voltage that was directly proportional to the amount of O2 consumed by the mosquito. An array of eight DPTs was interfaced with an IBM 486 PC using an ADAC 5500MF analog to digital converter and software from ADAC (Direct View) to automate the recording procedure. We determined that our apparatus was sensitive enough to detect subtle differences in O2 consumption in mosquitoes subjected to different physiologic conditions.

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Biochemical analysis of a blood meal-induced Aedes aegypti glutamine synthetase gene.

Glutamine synthetase (GS) in the mosquito, Aedes aegypti, is induced in the midgut following a blood meal. Mosquito GS message is detected as soon as 1 h post-blood feeding and remains stable for 18 h. Using a PCR product encoding mosquito GS, a lambda gt10 adult female mosquito cDNA library was screened. A cDNA clone, pCl5A2, encoding the full translation product of mosquito GS was isolated and sequence analyses performed. Mosquito GS cDNA is 2.5 kb in length and its putative translation product shares all the conserved regions characteristic of the GS gene family, including the presumed ATP biding site. Glutamine synthetase activity in the mosquito midgut is highest at 18 h post-blood feeding. Activity can be detected over a broad pH range, from 6.0 to 7.5. Unlike other cellular GS enzymes, mosquito GS is not active in the presence of ATP. Very low dosages (0.05 mM) of L-methionine S-sulfoximine are sufficient to partially inhibit mosquito GS activity. Inhibition of GS disrupts the normal formation of the midgut peritrophic matrix, suggesting that GS enzyme might be involved in the initial pathway of chitin synthesis. The unique expression pattern and inducible nature of the mosquito GS gene make it an interesting candidate for studying promoter function. Additionally, the blood meal activation of the GS gene makes this a potentially valuable tool in mosquito transformation studies.

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Development of a comparative genetic linkage map for Armigeres subalbatus using Aedes aegypti RFLP markers.

One of the causative agents of lympahtic filariasis is the nematode parasite Brugia malayi that requires a competent mosquito vector for its development and transmission. Armigeres subalbatus mosquitoes rapidly destroy invading B. malayi microfilariae via a defense response known as melanotic encapsulation. We have constructed a genetic linkage map for this mosquito species using RFLP markers from Aedes aegypti. This heterologous approach was possible because of the conserved nature of the coding sequences used as markers and provided an experimental framework to evaluate the hypothesis that linkage and gene order are conserved between these mosquito species. Of the 56 Ae. aegypti markers tested, 77% hybridize to genomic DNA digests of Ar. subalbatus under stringent conditions, with 53% of these demonstrating strain-specific polymorphisms. Twenty-six Ae. aegypti markers have been mapped using an F2- segregating Ar. subalbatus population derived from a cross of strains originating in Japan and Malaysia. Linear order of these marker loci is highly conserved between the two species. Only 1 of these markers, LF92, was not linked in the manner predicted by the Ae. aegypti map. In addition, the autosomal sex-determination locus that occurs in linkage group 1 in Ae. aegypti resides in group 3 in Ar. subalbatus. The Ar. subalbatus map provides a basic genetic context that can be utilized in further genetic studies to clarify the genetic basis of parasite resistance in this mosquito and is a necessary precursor to the identification of genome regions that carry genes that determine the encapsulation phenotype. [The composite map and sequence database information for Ae. aegypti markers can be retrieved directly from the Ae. aegypti Genome Database through the World Wide Web: http://klab.agsci.colostate.edu.]

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