Txn2 haplodeficiency does not affect cochlear antioxidant defenses or accelerate the progression of cochlear cell loss or hearing loss across the lifespan.

Thioredoxin 2 (TXN2) is a small redox protein found in nearly all organisms. As a mitochondrial member of the thioredoxin antioxidant defense system, TXN2 interacts with peroxiredoxin 3 (PRDX3) to remove hydrogen peroxide. Accordingly, TXN2 is thought to play an important role in maintaining the appropriate mitochondrial redox environment and protecting the mitochondrial components against oxidative stress. In the current study, we investigated the effects of Txn2 haplodeficiency on cochlear antioxidant defenses, auditory function, and cochlear cell loss across the lifespan in wild-type (WT) and Txn2 heterozygous knockout (Txn2+/-) mice backcrossed onto CBA/CaJ mice, a well-established model of age-related hearing loss. Txn2+/- mice displayed a 58% decrease in TXN2 protein levels in the mitochondria of the inner ears compared to WT mice. However, Txn2 haplodeficiency did not affect the thioredoxin or glutathione antioxidant defense in both the mitochondria and cytosol of the inner ears of young mice. There were no differences in the levels of mitochondrial biogenesis markers, mitochondrial DNA content, or oxidative DNA and protein damage markers in the inner ears between young WT and Txn2+/- mice. In a mouse inner ear cell line, knockdown of Txn2 did not affect cell viability under hydrogen peroxide treatment. Consistent with the tissue and cell line results, there were no differences in hair cell loss or spiral ganglion neuron density between WT and Txn2+/- mice at 3-5 or 23-25 months of age. Furthermore, Txn2 haplodeficiency did not affect auditory brainstem response threshold, wave I latency, or wave I amplitude at 3-5, 15-16, or 23-25 months of age. Therefore, Txn2 haplodeficiency does not affect cochlear antioxidant defenses, accelerate degeneration of cochlear cells, or affect auditory function in mice across the lifespan.

GSTA4 mediates reduction of cisplatin ototoxicity in female mice.

Cisplatin is one of the most widely used chemotherapeutic drugs for the treatment of cancer. Unfortunately, one of its major side effects is permanent hearing loss. Here, we show that glutathione transferase α4 (GSTA4), a member of the Phase II detoxifying enzyme superfamily, mediates reduction of cisplatin ototoxicity by removing 4-hydroxynonenal (4-HNE) in the inner ears of female mice. Under cisplatin treatment, loss of Gsta4 results in more profound hearing loss in female mice compared to male mice. Cisplatin stimulates GSTA4 activity in the inner ear of female wild-type, but not male wild-type mice. In female Gsta4-/- mice, cisplatin treatment results in increased levels of 4-HNE in cochlear neurons compared to male Gsta4-/- mice. In CBA/CaJ mice, ovariectomy decreases mRNA expression of Gsta4, and the levels of GSTA4 protein in the inner ears. Thus, our findings suggest that GSTA4-dependent detoxification may play a role in estrogen-mediated neuroprotection.

Ectopic expression of L1CAM ectodomain alters differentiation and motility, but not proliferation, of human neural progenitor cells.

Adult human neural progenitor and stem cells have been implicated as a potential source of brain cancer causing cells, but specific events that might cause cells to progress towards a transformed phenotype remain unclear. The L1CAM (L1) cell adhesion/recognition molecule is expressed abnormally by human glioma cancer cells and is released as a large extracellular ectodomain fragment, which stimulates cell motility and proliferation. This study investigates the effects of ectopic overexpression of the L1 long ectodomain (L1LE; ˜180 kDa) on the motility, proliferation, and differentiation of human neural progenitor cells (HNPs). L1LE was ectopically expressed in HNPs using a lentiviral vector. Surprisingly, overexpression of L1LE resulted in reduced HNP motility in vitro, in stark contrast to the effects on glioma and other cancer cell types. L1LE overexpression resulted in a variable degree of maintenance of HNP proliferation in media without added growth factors but did not increase proliferation. In monolayer culture, HNPs expressed a variety of differentiation markers. L1LE overexpression resulted in loss of glutamine synthetase (GS) and ß3-tubulin expression in normal HNP media, and reduced vimentin and increased GS expression in the absence of added growth factors. When co-cultured with chick embryonic brain cell aggregates, HNPs show increased differentiation potential. Some HNPs expressed p-neurofilaments and oligodendrocytic O4, indicating differentiation beyond that in monolayer culture. Most HNP-L1LE cells lost their vimentin and GFAP (glial fibrillary acidic protein) staining, and many cells were positive for astrocytic GS. However, these cells rarely were positive for neuronal markers ß3-tubulin or p-neurofilaments, and few HNP oligodendrocyte progenitors were found. These results suggest that unlike for glioma cells, L1LE does not increase HNP cell motility, but rather decreases motility and influences the differentiation of normal brain progenitor cells. Therefore, the effect of L1LE on increasing motility and proliferation appears to be limited to already transformed cells.

Increased burden of mitochondrial DNA deletions and point mutations in early-onset age-related hearing loss in mitochondrial mutator mice.

Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in a variety of age-related neurodegenerative diseases, including age-related hearing loss (AHL). In the current study, we investigated the roles of mtDNA deletions and point mutations in AHL in mitochondrial mutator mice (Polgmut/mut) that were backcrossed onto CBA/CaJ mice, a well-established model of late-onset AHL. mtDNA deletions accumulated significantly with age in the inner ears of Polgmut/mut mice, while there were no differences in mtDNA deletion frequencies in the inner ears between 5 and 17 months old Polg+/+ mice or 5 months old Polg+/+ and Polgmut/mut mice. mtDNA deletions also accumulated significantly in the inner ears of CBA/CaJ mice during normal aging. In contrast, 5 months old Polgmut/mut mice displayed a 238-fold increase in mtDNA point mutation frequencies in the inner ears compared to age-matched Polg+/+ mice, but there were no differences in mtDNA point mutation frequencies in the inner ears between 5 and 17 months old Polgmut/mut mice. Seventeen-month-old Polgmut/mut mice also displayed early-onset severe hearing loss associated with a significant reduction in neural output of the cochlea, while age-matched Polg+/+ mice displayed little or no hearing impairment. Consistent with the physiological and mtDNA deletion test result, 17-month-old Polgmut/mut mice displayed a profound loss of spiral ganglion neurons in the cochlea. Thus, our data suggest that a higher burden of mtDNA point mutations from a young age and age-related accumulation of mtDNA deletions likely contribute to early-onset AHL in mitochondrial mutator mice.

Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases.

In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control strategies into the new millennium.

Characterization of the Expression of Basigin Gene Products Within the Pineal Gland of Mice.

The expression of Basigin gene products and monocarboxylate transporter-1 (MCT1) has been investigated within the mammalian neural retina and suggests a role for these proteins in cellular metabolism within that tissue. The purpose of the present study was to investigate the expression of these same proteins in the pineal gland of the mouse brain. Mouse pineal gland and neural retina RNA and protein were subjected to quantitative reverse transcription-polymerase chain reaction and immunoblotting analyses. In addition, paraffin-embedded sections of each tissue were analyzed for expression of Basigin gene products and MCT1 via immunohistochemistry. The results indicate that MCT1 and Basigin variant-2, but not Basigin variant-1, are expressed within the mouse pineal gland. The expression of Basigin variant-2 and MCT1 was localized to the capsule surrounding the gland. The position and relative amounts of the gene products suggest that they play a much less prominent role within the pineal gland than in the neural retina.

Organ-specific splice variants of aquaporin water channel AgAQP1 in the malaria vector Anopheles gambiae.

BACKGROUND: Aquaporin (AQP) water channels are important for water homeostasis in all organisms. Malaria transmission is dependent on Anopheles mosquitoes. Water balance is a major factor influencing mosquito survival, which may indirectly affect pathogen transmission. METHODOLOGY/PRINCIPAL FINDINGS: We obtained full-length mRNA sequences for Anopheles gambiae aquaporin 1 (AgAQP1) and identified two splice variants for the gene. In vitro expression analysis showed that both variants transported water and were inhibited by Hg(2+). One splice variant (AgAQP1A) was exclusively expressed in adult female ovaries indicating a function in mosquito reproduction. The other splice variant (AgAQP1B) was expressed in the midgut, malpighian tubules and the head in adult mosquitoes. Immunolabeling showed that in malpighian tubules, AgAQP1 is expressed in principal cells in the proximal portion and in stellate cells in the distal portion. Moreover, AgAQP1 is expressed in Johnston's organ (the "ear"), which is important for courtship behavior. CONCLUSIONS AND SIGNIFICANCE: These results suggest that AgAQP1 may play roles associated with mating (courtship) and reproduction in addition to water homeostasis in this important African malaria vector.

Dose and developmental responses of Anopheles merus larvae to salinity.

Saltwater tolerance is a trait that carries both ecological and epidemiological significance for Anopheles mosquitoes that transmit human malaria, as it plays a key role in determining their habitat use and ecological distribution, and thus their local contribution to malaria transmission. Here, we lay the groundwork for genetic dissection of this trait by quantifying saltwater tolerance in three closely related cryptic species and malaria vectors from the Afrotropical Anopheles gambiae complex that are known to differ starkly in their tolerance to salinity: the obligate freshwater species A. gambiae and A. coluzzii, and the saltwater-tolerant species A. merus. We performed detailed comparisons of survivorship under varying salinities, using multiple strains of A. gambiae, A. coluzzii and A. merus, as well as F1 progeny from reciprocal crosses of A. merus and A. coluzzii. Additionally, using immunohistochemistry, we compared the location of three ion regulatory proteins (Na(+)/K(+)-ATPase, carbonic anhydrase and Na(+)/H(+)-antiporter) in the recta of A. coluzzii and A. merus reared in freshwater or saline water. As expected, we found that A. merus survives exposure to high salinities better than A. gambiae and A. coluzzii. Further, we found that exposure to a salinity level of 15.85 g NaCl l(-1) is a discriminating dose that kills all A. gambiae, A. coluzzii and A. coluzzii-A. merus F1 larvae, but does not negatively impact the survival of A. merus. Importantly, phenotypic expression of saltwater tolerance by A. merus is highly dependent upon the developmental time of exposure, and based on immunohistochemistry, salt tolerance appears to involve a major shift in Na(+)/K+-ATPase localization in the rectum, as observed previously for the distantly related saline-tolerant species A. albimanus.

New model of action for mood stabilizers: phosphoproteome from rat pre-frontal cortex synaptoneurosomal preparations.

BACKGROUND: Mitochondrial short and long-range movements are necessary to generate the energy needed for synaptic signaling and plasticity. Therefore, an effective mechanism to transport and anchor mitochondria to pre- and post-synaptic terminals is as important as functional mitochondria in neuronal firing. Mitochondrial movement range is regulated by phosphorylation of cytoskeletal and motor proteins in addition to changes in mitochondrial membrane potential. Movement direction is regulated by serotonin and dopamine levels. However, data on mitochondrial movement defects and their involvement in defective signaling and neuroplasticity in relationship with mood disorders is scarce. We have previously reported the effects of lithium, valproate and a new antipsychotic, paliperidone on protein expression levels at the synaptic level. HYPOTHESIS: Mitochondrial function defects have recently been implicated in schizophrenia and bipolar disorder. We postulate that mood stabilizer treatment has a profound effect on mitochondrial function, synaptic plasticity, mitochondrial migration and direction of movement. METHODS: Synaptoneurosomal preparations from rat pre-frontal cortex were obtained after 28 daily intraperitoneal injections of lithium, valproate and paliperidone. Phosphorylated proteins were identified using 2D-DIGE and nano LC-ESI tandem mass spectrometry. RESULTS: Lithium, valproate and paliperidone had a substantial and common effect on the phosphorylation state of specific actin, tubulin and myosin isoforms as well as other proteins associated with neurofilaments. Furthermore, different subunits from complex III and V of the electron transfer chain were heavily phosphorylated by treatment with these drugs indicating selective phosphorylation. CONCLUSIONS: Mood stabilizers have an effect on mitochondrial function, mitochondrial movement and the direction of this movement. The implications of these findings will contribute to novel insights regarding clinical treatment and the mode of action of these drugs.

AgCad2 cadherin in Anopheles gambiae larvae is a putative receptor of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan.

In an effort to study the mode of action of Cry11Ba, we identified toxin binding proteins in Anopheles gambiae larval midgut and investigated their receptor roles. Previously, an aminopeptidase (AgAPN2) and an alkaline phosphatase (AgALP1) were identified as receptors for Cry11Ba toxin in A. gambiae. However, an A. gambiae cadherin (AgCad1) that bound Cry11Ba with low affinity (K(d) = 766 nM) did not support a receptor role of AgCad1 for Cry11Ba. Here, we studied a second A. gambiae cadherin (AgCad2) that shares 14% identity to AgCad1. Immunohistochemical study showed that the protein is localized on A. gambiae larval midgut apical membranes. Its cDNA was cloned and the protein was analyzed as a transmembrane protein containing 14 cadherin repeats. An Escherichia coli expressed CR14MPED fragment of AgCad2 bound Cry11Ba with high affinity (K(d) = 11.8 nM), blocked Cry11Ba binding to A. gambiae brush border vesicles and reduced Cry11Ba toxicity in bioassays. Its binding to Cry11Ba could be completely competed off by AgCad1, but only partially competed by AgALP1. The results are evidence that AgCad2 may function as a receptor for Cry11Ba in A. gambiae larvae.

High affinity (3)H-phenylalanine uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBBMVw).

Brush border membrane vesicles from whole Aedes aegypti larvae (AaBBMVw) are confirmed to be valid preparations for membrane transport studies. The Abdul-Rauf and Ellar method was used to isolate AaBBMVw that were frozen, stored for several months, transported to a distant site, thawed and used to study Na(+)-coupled, (3)H-labeled, phenylalanine (Phe) uptake. The affinity for all components of the uptake was very high with half maximal values in the sub-micromolar range. By contrast a K(0.5)(Phe) of 0.2mM and a K(0.5)(Na) of 26 mM were calculated from Phe-induced electrical currents in Xenopus oocytes that were heterologously expressing the Anopheles gambiae symporter (co-transporter), AgNAT8, in a buffer with 98 mM Na(+). What accounts for the >1000-fold discrepancy in affinity for substrates between the BBMV and oocyte experiments? Is it because Ae. aegypti were used to isolate BBMVw whereas An. gambiae were used to transfect oocytes? More likely, it is because BBMVw were exposed to [Na(+)] in the micromolar range with the transporter(s) being surrounded by native lipids. By contrast, the oocyte measurements were made at [Na(+)] 100,000 times higher with AgNAT8 surrounded by foreign frog lipids. The results show that AaBBMVw are osmotically sealed; the time-course has a Na(+)-induced overshoot, the pH optimum is ∼7 and the K(0.5) values for Phe and Na(+) are very low. The transport is virtually unchanged when Na(+) is replaced by K(+) or Li(+) but decreased by Rb(+). This approach to resolving discrepancies between electrical data on solute transporters such as AgNAT8 that are over-expressed in oocytes and flux data on corresponding transporters that are highly expressed in native membrane vesicles, may serve as a model for similar studies that add membrane biochemistry to molecular biology in efforts to identify targets for the development of new methods to control disease-vector mosquitoes.

Slc4-like anion transporters of the larval mosquito alimentary canal.

Mosquito larvae exhibit luminal pH extremes along the axial length of their alimentary canal that range from very alkaline (pH>10) in the anterior midgut to slightly acid in the hindgut. The principal buffer in the system is thought to be bicarbonate and/or carbonate, because the lumen is known to contain high levels of bicarbonate/carbonate and is surrounded by various epithelial cell types which express a variety of carbonic anhydrases. However, the precise mechanisms responsible for the transport of bicarbonate/carbonate into and out of the lumen are unclear. In the present study, we test the hypothesis that SLC4-like anion transporters play a role in bicarbonate/carbonate accumulation in the larval mosquito alimentary canal. Molecular, physiological and immnuohistochemical characterizations of Slc4-like transporters in the gut of larval mosquitoes (Aedes aegypti and Anopheles gambiae) demonstrate the presence of both a Na(+)-independent chloride/bicarbonate anion exchanger (AE) as well as a Na(+)-dependent anion exchanger (NDAE). Notably, immunolocalization experiments in Malpighian tubules show that the two proteins can be located in the same tissue, but to different cell types. Immunolabeling experiments in the gastric caecae show that the two proteins can be found in the same cells, but on opposite sides (basal vs. apical). In summary, our results indicate that the alimentary canal of larval mosquitoes exhibits robust expression of two SLC4-like transporters in locations that are consistent with a role in the regulation of luminal pH. The precise physiological contributions of each transporter remain to be determined.

Localization of two Na+- or K+-H+ antiporters, AgNHA1 and AgNHA2, in Anopheles gambiae larval Malpighian tubules and the functional expression of AgNHA2 in yeast.

The newly identified metazoan Na(+)/H(+) antiporter (NHA) family is represented by two paralogues, AgNHA1 and AgNHA2, in the genome of the African malaria mosquito, Anopheles gambiae. Both antiporters are postulated to be electrophoretic i.e. voltage-driven. AgNHA1 was first cloned from An. gambiae larvae and immunolocalized with respect to the H(+) V-ATPase by the Harvey laboratory. Little is known about the properties of NHA1s; attempts to characterize AgNHA1 in Na(+)/H(+) exchanger (NHE)-lacking Chinese hamster ovary cells and in yeast cells or frog oocytes were unsuccessful. Even less is known about AgNHA2. It is predicted to have a relative molecular mass of ∼60 kDa and shares 30.5% amino acid identity with AgNHA1. Immunolocalization images show AgNHA2 on the apical plasma membrane of stellate cells in Malpighian tubules of An. gambiae larvae and adults. When heterologously expressed in a mutant strain of the yeast, Saccharomyces cerevisiae, which lacks endogenous cation/proton antiporters and pumps, AgNHA2 enhanced repression of growth by the alkali metal cations, Li(+), Na(+), or K(+) and enhanced Li(+) accumulation. The yeast growth studies invite the speculation that AgNHA2 is an electrophoretic antiporter with a stoichiometry of nNa(+) to 1H(+) with n > 1. Immunolocalization images provide direct evidence that H(+) V-ATPase is co-localized with AgNHA1 on the apical membrane of principal cells but it is not present in the stellate cells where AgNHA2 is localized apically. These results are consistent with the notion that the outside positive voltage that the H(+) V-ATPase generates across the apical membrane of principal cells appears with but little attenuation across the apical membrane of stellate cells. This immunolocalization pattern is consistent with the hypothesis that the voltage acts via AgNHA1 to drive nH(+) into the principal cells and Na(+) out to the lumen and acts via AgNHA2 to drive nNa(+) into the stellate cells and H(+) out to the lumen. Precious Na(+) is then retained by ejection into the blood via a basal Na(+)/K(+)-ATPase. Localizations of anion transporters and their functions in stellate and principal cells are described by Linser, Romero and associates in this volume. The role that the electrogenic H(+) V-ATPase and the electrophoretic cationic and anionic transporters play in ion homeostasis is incorporated into a model for Malpighian tubule cells of larval mosquitoes.

Countering a bioterrorist introduction of pathogen-infected mosquitoes through mosquito control.

The release of infected mosquitoes or other arthropods by bioterrorists, i.e., arboterrorism, to cause disease and terror is a threat to the USA. A workshop to assess mosquito control response capabilities to mount rapid and effective responses to eliminate an arboterrorism attack provided recommendations to improve capabilities in the USA. It is essential that mosquito control professionals receive training in possible responses, and it is recommended that a Council for Emergency Mosquito Control be established in each state to coordinate training, state resources, and actions for use throughout the state.

The 2M6 antigen is a Müller cell-specific intracellular membrane-associated protein of the sarcolemmal-membrane-associated protein family and is also TopAP.

PURPOSE: The differentiation marker 2M6 has been used to identify Müller cells within the developing chick retina for several years, although the molecular identity of 2M6 was not known. This study was aimed at determining the identity of the protein antigen recognized by the 2M6 monoclonal antibody. METHODS: Affinity chromatography and subsequent mass spectrometry were used to determine the molecular identity of the 2M6 antigen. Immunohistochemistry of monolayer preparations and paraffin-embedded sections of chick retina were performed to localize expression of the 2M6 antigen within cells of the chick retina. RESULTS: Mass spectrometry analyses revealed that the 2M6 antigen is identical (with 95% probability) to the protein known as Top(AP), which is a member of the sarcolemmal membrane-associated protein family of proteins. The 2M6 polypeptide is expressed by Müller glial cells as well as boundary cells within the chick retina. Expression localizes to intracellular membrane structures within those cells. CONCLUSIONS: Members of the sarcolemmal membrane-associated protein family of proteins have been implicated in structural and functional roles related to the cytoskeleton and Ca(+2) release from internal stores. It is thought that 2M6 plays a similar role in Müller cells of the vertebrate retina.

Physiological and pharmacological characterizations of the larval Anopheles albimanus rectum support a change in protein distribution and/or function in varying salinities.

Ion regulation is a biological process crucial to the survival of mosquito larvae and a major organ responsible for this regulation is the rectum. The recta of anopheline larvae are distinct from other subfamilies of mosquitoes in several ways, yet have not yet been characterized extensively. Here we characterize the two major cell types of the anopheline rectum, DAR and non-DAR cells, using histological, physiological, and pharmacological analyses. Proton flux was measured at the basal membrane of 2%- and 50%-artificial sea water-reared An. albimanus larvae using self-referencing ion-selective microelectrodes, and the two cell types were found to differ in basal membrane proton flux. Additionally, differences in the response of that flux to pharmacological inhibitors in larvae reared in 2% versus 50% ASW indicate changes in protein function between the two rearing conditions. Finally, histological analyses suggest that the non-DAR cells are structurally suited for mediating ion transport. These data support a model of rectal ion regulation in which the non-DAR cells have a resorptive function in freshwater-reared larvae and a secretive function in saline water-reared larvae. In this way, anopheline larvae may adapt to varying salinities.

H(+) V-ATPase-energized transporters in brush border membrane vesicles from whole larvae of Aedes aegypti.

Brush border membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs) contain an H(+) V-ATPase (V), a Na(+)/H(+) antiporter, NHA1 (A) and a Na(+)-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles. AgNAT8 was cloned from Anopheles gambiae, localized in BBMs and characterized in Xenopus laevis oocytes. AgNHA1 was cloned and localized in BBMs but characterization in oocytes was compromised by an endogenous cation conductance. AeBBMVWs complement Xenopus oocytes for characterizing membrane proteins, can be energized by voltage from the V-ATPase and are in their natural lipid environment. BBMVs from caterpillars were used in radio-labeled solute uptake experiments but approximately 10,000 mosquito larvae are needed to equal 10 caterpillars. By contrast, functional AeBBMVWs can be prepared from 10,000 whole larvae in 4h. Na(+)-coupled (3H)phenylalanine uptake mediated by AeNAT8 in AeBBMVs can be compared to the Phe-induced inward Na(+) currents mediated by AgNAT8 in oocytes. Western blots and light micrographs of samples taken during AeBBMVW isolation are labeled with antibodies against all of the VAN components. The use of AeBBMVWs to study coupling between electrogenic V-ATPases and the electrophoretic transporters is discussed.

Organ-associated muscles in Aedes albopictus (Diptera: Culicidae) respond differentially to Sindbis virus.

Differential host cell responses to the alphavirus Sindbis were observed in visceral muscles of the adult female mosquito Aedes albopictus. Following intrathoracic inoculation with SIN, muscles associated with the midgut, hindgut, and ovary resulted in clearance, persistence, and refractoriness to virus, respectively. Prominent sarcomeres characteristic of myofilaments were identified in muscles associated with these three organs by phalloidin labeling of actin, confirming these cells as muscle. The location of virus antigen mimicked the distribution of actin in both mid- and hindgut-associated muscles. Furthermore, these myofilaments remained intact following virus clearance from midgut muscles and during virus persistence in hindgut muscles. Changes in the temporal onset of virus antigen following high titer inoculum compared with standard titer inoculum was observed in anterior midgut muscles, but not in muscles associated with the posterior midgut or hindgut. Muscle bundles closely approximated the gut surface, while a wispy association was displayed at the ovary surface. Prominent ultrastructural differences were observed in the basal lamina attached to the gut compared with the ovary. Additionally, ultrastructural evidence for virus-associated pathology was observed in gut-associated muscles and gut epithelium. Visceral muscles, all composed of the same tissue type, but associated to three different organs in the insect abdomen, responded differentially to Sindbis. We speculate that variations in structure, function or physiology and ultrastructure inherent to insect host cells or organs interactions reflect the complicated milieu of the organism and contribute to differential virus phenotypic expression in muscle cells.

Hydrophobic interactions stabilize the basigin-MCT1 complex.

Previous reports demonstrated that monocarboxylate transporter-1 (MCT1) interacts with Basigin. It was hypothesized that the two proteins interact via the transmembrane domain of Basigin, specifically through the glutamate residue within the domain. We therefore sought to test this hypothesis and determine which amino acids of the Basigin protein are necessary for the interaction with MCT1. Probes consisting of the full-length putative transmembrane domain, as well as small regions of the domain, were generated for use in ELISA binding assays using endogenous mouse MCT1. Site directed mutagenesis of candidate residues was performed and probes were generated for ELISA analyses to determine the specific residues involved. The data suggest that hydrophobic residues at the N- and C-termini of the putative transmembrane domain of Basigin interact with MCT1, but the glutamate plays no role. The previously proposed hypothesis is partially correct, in that the putative transmembrane domain of Basigin does interact with MCT1.

Silencing of carbonic anhydrase in an Anopheles gambiae larval cell line, Ag55.

RNAi has been used extensively to down-regulate proteins in adult mosquitoes; however, it is not well adapted for use in larvae. Larval mosquitoes can generate a pH as high as 10.5 in the anterior region of their midgut. The mechanisms responsible for the generation and maintenance of this pH are not entirely understood, but members of the carbonic anhydrase (CA) family of enzymes have been implicated. Here we use an An. gambiae larval cell line, Ag55 cells, to demonstrate that application of full-length double-stranded RNA specific to one CA, AgCA9, is sufficient to silence AgCA9 mRNA and down-regulate the corresponding protein. This is a first step towards determining the role(s) of these enzymes in pH regulation.