In Bsg(-/-) mice, there is a severe reduction in accumulation of the MCT1 and -3 proteins in the RPE and a concomitant reduction in MCT1 and -4 in the neural retina supporting a role for 5A11/basigin in the targeting of these transporters to the plasma membrane. Decreased expression of MCT1 and -4 on the surfaces of Müller and photoreceptor cells may compromise energy metabolism in the outer retina, leading to abnormal photoreceptor cell function and degeneration.

The cellular localization of glutamine synthetase [

The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH Ϸ 3 units⅐mm ؊1 ) in which an extremely alkaline (pH Ϸ 11) anterior midgut lies between near-neutral posterior midgut and gastric cecum (pH 7-8). A plasma membrane H ؉ V-ATPase energizes this alkalinization but the ion carriers involved are unknown. Capillary zone electrophoresis of body samples with outlet conductivity detection showed a specific transepithelial distribution of chloride and bicarbonate͞carbonate ions, with high concentrations of both anions in the midgut tissue: 68.3 ؎ 5.64 and 50.8 ؎ 4.21 mM, respectively. Chloride was higher in the hemolymph, 57.6 ؎ 7.84, than in the lumen, 3.51 ؎ 2.58, whereas bicarbonate was higher in the lumen, 58.1 ؎ 7.34, than the hemolymph, 3.96 ؎ 2.89. Timelapse video assays of pH profiles in vivo revealed that ingestion of the carbonic anhydrase inhibitor acetazolamide and the ion exchange inhibitor DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid), at 10 ؊4 M eliminates lumen alkalinization. Basal application of these inhibitors in situ also reduced gradients recorded with self-referencing pH-sensitive microelectrodes near the basal membrane by Ϸ65% and 85% respectively. Self-referencing chlorideselective microelectrodes revealed a specific spatial profile of transepithelial chloride transport with an efflux maximum in anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes. These data suggest that an H ؉ V-ATPase-energized anion exchange occurs across the apical membrane of the epithelial cells and implicate an electrophoretic Cl ؊ ͞HCO 3 ؊ exchanger and carbonic anhydrase as crucial components of the steady-state alkalinization in anterior midgut of mosquito larvae.T he human stomach works at an acid pH near 1, whereas the larval mosquito midgut works at an alkaline pH near 11. The acid pH of mammalian stomachs has been thoroughly investigated and its mechanisms are understood in great detail. The alkaline pH of larval mosquito midguts has been little studied and remains largely a mystery. Recently developed techniques for direct analysis of the intact midgut in vivo and in semi-intact larvae, with a resolution that allows analytical assay of small patches of tissue, even of individual cells, have led to insights into the alkalinization mechanism.The principal function of the larval mosquito midgut is to digest and absorb nutrients. High pH values have been recorded in anterior midgut of several mosquito larvae, including Aedes aegypti (1), as well as in some lepidopteran larvae with high tannin diet (2). The alkaline environment of the anterior midgut lumen enhances dissociation of tannin-protein complexes and, therefore, would be advantageous for the nutrition of herbivorous and detritus-feeding larvae (3). The tannin-free protein could then be digested and absorbed at the near neutral pH of posterior midgut lumen (3, 4). This pattern of nutrient processing has led to the large longitudinal gradients of pH and ionic concentrat...

The specific and limited expression of 5A11/Basigin-2 explicitly within photoreceptor cells implies that this glycoprotein plays a fundamental role within the retina. However, its role remains to be determined.

In continuing efforts to identify cell-surface molecules involved in cell-cell interactions in the developing avian retina, we identified a monoclonal antibody, the 5 A l l antibody, which possessed the ability to interfere with contact-dependent glial cell maturation in vitro. We sought to determine the molecular and biochemical identity of the glycoprotein recognized by this antibody, and using additional criteria, establish whether the 5 A l l antigen is indeed a cell-recognition molecule in the developing retina. Immunohistochemical analyses demonstrate that in the hatchling chick retina and in live cultures of embryonic retina cells, the 5 A l l antigen is predominantly associated with Miiller glial cells whereas little is observed on neuronal elements. Microsequencing of the major component isolated by immunoaffinity chromatography identifies the HT7 antigen (Seulberger et al.: EMBO Journal 92151-2158,1990), a unique member of the immunoglobulin super gene family (IGSF), as a homologous if not identical protein to the 5 A l l antigen. The HT7 antibody, furthermore, recognizes affinity purified 5 A l l antigen, and both the HT7 antibody and additional probes generated against the 5 A l l antigen recognize a major polypeptide of 45.5 kDa and a minor band of 69 kDa on Western blots of membrane preparations from neural retina. To verify that the 5 A l l antigen mediates cell-cell recognition events in the developing neural retina, we examined the consequences of adding antibody to monolayer cultures of dissociated embryonic retina cells and to dissociated retina cells in rotation-mediated suspension culture. Addition of the 5 A l l antibody to monolayer cultures results in alteration in the development of the stereotypic arrangement of neurons and glia characterized by a reduction in the number and complexity of neural extensions upon the glial-derived flat cells, Similarly, addition of antibodies generated against the 5 A l l antigen to dissociated cells in rotation cultures significantly reduces retina cell reaggregation as monitored by computer-assisted image analysis of cell aggregate size. These data and the identification of the 5 A l l antigen as a member of the IGSF establish a role for the 5 A l l antigen as a novel recognition molecule in the developing neural retina.

SUMMARY We have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes(Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned,nor have the encoded proteins been localized or characterized. The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia. A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.

SUMMARYMosquito larvae live in dynamic aqueous environments, which can fluctuate drastically in salinity due to environmental events such as rainfall and evaporation. Larval survival depends upon the ability to regulate hemolymph osmolarity by absorbing and excreting ions. A major organ involved in ion regulation is the rectum, the last region for modification of the primary urine before excretion. The ultrastructure and function of culicine larval recta have been studied extensively; however, very little published data exist on the recta of anopheline larvae. To gain insight into the structure and functions of this organ in anopheline species, we used immunohistochemistry to compare the localization of three proteins [carbonic anhydrase (CA9), Na

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