Reduced expression of Basigin gene products in response to chronic inflammation may contribute to vision loss.

Chronic inflammation of the retina, like that of diabetic retinopathy, disrupts the blood-retina barrier (BRB). Disruption of the BRB increases vascular permeability and leads to vision loss. Basigin gene products, cell-adhesion molecules and members of the immunoglobulin superfamily, are expressed on endothelial cells, photoreceptor cells and Müller glial cells. Basigin variant-1 on photoreceptors interacts with Basigin variant-2 on Müller glial cells and to rod-derived cone viability factor (RdCVF) to form metabolic support mechanisms necessary for the survival of photoreceptor neurons. The goal of the current study was to determine the gene expression changes of Basigin gene products in ex vivo neonatal, adolescent, and adult retina when exposed to an inflammatory insult in acute and chronic phases. Retinas extracted from mice at postnatal day (P) 7, 30, and 180 were incubated with either phosphate-buffered saline (PBS), as a control, or lipopolysaccharide (LPS), an endotoxin, for 3, 6, 12, or 24 h. RNA was then extracted and Basigin gene products were quantified by qPCR. Analyses indicate both gene products are influenced by LPS exposure in a time and age dependent manner. Specifically, P180 retinas exposed to LPS showed significant decreases in both Basigin gene products, suggesting older retinas may be susceptible to chronic inflammation and subsequent vision loss.

The retina-specific basigin isoform does not induce IL-6 expression in mouse monocytes.

Purpose: Basigin gene products are positioned on adjacent cell types in the neural retina and are thought to compose a lactate metabolon important for photoreceptor cell function. The Ig0 domain of basigin isoform 1 (basigin-1) is highly conserved throughout evolution, which suggests a conserved function. It has been suggested that the Ig0 domain has proinflammatory properties, and it is hypothesized to interact with basigin isoform 2 (basigin-2) for cell adhesion and lactate metabolon formation. Therefore, the purpose of the present study was to determine whether the Ig0 domain of basigin-1 binds to basigin-2 and whether the region of the domain used for binding is also used to stimulate interleukin-6 (IL-6) expression. Methods: Binding was assessed using recombinant proteins corresponding to the Ig0 domain of basigin-1 and endogenously expressed basigin-2 from mouse neural retina and brain protein lysates. The proinflammatory properties of the Ig0 domain were analyzed with exposure of the recombinant proteins to the mouse monocyte RAW 264.7 cell line and subsequent measurement of the IL-6 concentration in the culture medium via enzyme-linked immunosorbent assay (ELISA). Results: The data indicate that the Ig0 domain interacts with basigin-2 through a region within the amino half of the domain and that the Ig0 domain does not stimulate the expression of IL-6 in mouse cells in vitro. Conclusions: The Ig0 domain of basigin-1 binds to basigin-2 in vitro. In addition, contrary to previous reports, there was no evidence that the Ig0 domain potentiates IL-6 expression in a mouse monocyte cell line in vitro. However, it is possible that the Ig0 domain stimulates the expression of proinflammatory cytokines other than IL-6, or that the potential involvement of the Ig0 domain of basigin-1 in the acute inflammatory response is dependent on species.

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.

Correction: The effects of fermented vegetable consumption on the composition of the intestinal microbiota and levels of inflammatory markers in women: A pilot and feasibility study.

[This corrects the article DOI: 10.1371/journal.pone.0275275.].

Deletion of the Basigin gene results in reduced mitochondria in the neural retina.

Basigin-null mice are characterized as blind from the time of eye opening, with degeneration of the retina beginning at 8weeks of age, and progressing until the entire photoreceptor cell layer is destroyed. It is likely that a metabolic deficiency underlies the blindness and degeneration phenotypes, as it has been determined that Basigin-null mice do not express the transporter protein monocarboxylate transporter one on the membrane of photoreceptor cells and inner segments, nor Müller cells of the neural retina, as is observed in normal mice. The purpose of the present study was to assess the health of mitochondria in normal and Basigin-null mice, specifically to determine if mitochondria within the Basigin-null mouse neural retina are metabolically active. This was achieved via a measurement of cytochrome C concentration and the expression of autophagy-specific proteins via ELISA analyses. Additionally, Mitotracker dyes were used to assess the number and relative activity of mitochondria. It was determined that cytochrome C concentrations and expression of autophagy-specific proteins were not increased in Basigin-null animals, as compared to control animals. Also, while Basigin-null mice do have metabolically active mitochondria, the amount of mitochondria was greatly reduced, when compared to control animals. The results suggest that a reduction in mitochondria is a result, rather than the cause, of the metabolic deficiency observed in Basigin-null mice, and likely occurs because of reduced metabolic activity in the absence of MCT1 expression.

The effects of fermented vegetable consumption on the composition of the intestinal microbiota and levels of inflammatory markers in women: A pilot and feasibility study.

The primary objective of this pilot study was to investigate the feasibility of regular consumption of fermented vegetables for six weeks on markers of inflammation and the composition of the gut microflora in women (clinical trials ID: NTC03407794). Thirty-one women were randomized into one of three groups: 100 g/day of fermented vegetables (group A), 100 g/day pickled vegetables (group B), or no vegetables (group C) for six weeks. Dietary intake was assessed by a food frequency questionnaire and blood and stool samples were provided before and after the intervention for measurement of C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), and lipopolysaccharide binding protein (LBP). Next-generation sequencing of the V4 region of the 16S rRNA gene was performed on the Illumina MiSeq platform. Participants' ages ranged between 18 and 69 years. Both groups A and B had a mean daily consumption of 91g of vegetables for 32 and 36 days, respectively. Serum CRP ranged between 0.9 and 265 ng/mL (SD = 92.4) at baseline, while TNF-α and LBP concentrations ranged between 0 and 9 pg/mL (SD = 2.3), and 7 and 29 µg/mL (SD = 4.4), respectively. There were no significant changes in levels of inflammatory markers among groups. At timepoint 2, group A showed an increase in Faecalibacterium prausnitzii (P = 0.022), a decrease in Ruminococcus torques (P<0.05), and a trend towards greater alpha diversity measured by the Shannon index (P = 0.074). The findings indicate that consumption of ~100 g/day of fermented vegetables for six weeks is feasible and may result in beneficial changes in the composition of the gut microbiota. Future trials should determine whether consumption of fermented vegetables is an effective strategy against gut dysbiosis.

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.

Characterization of monocarboxylate transporter 1 (MCT1) binding affinity for Basigin gene products and L1cam.

The purpose of this study was to determine the binding affinities of Basigin gene products and neural cell adhesion molecule L1cam for monocarboxylate transporter-1 (MCT1). ELISA binding assays were performed in which recombinant proteins of the transmembrane domains of Basigin gene products and L1cam were incubated with MCT1 captured from mouse brain. It was determined that Basigin gene products bind MCT1 with moderate affinity, but L1cam does not bind MCT1. Despite a high degree of sequence conservation between Basigin gene products and L1cam, the sequences are different enough to prevent L1cam from interacting with MCT1.

Mesohaline conditions represent the threshold for oxidative stress, cell death and toxin release in the cyanobacterium Microcystis aeruginosa.

As aquatic ecosystems become increasingly affected by hydrologic alterations, drought and sea level rise a need exists to better understand the biological effects of elevated salinity on toxigenic cyanobacteria such as Microcystis aeruginosa. This study investigated the impacts of oligohaline/low mesohaline conditions and exposure time on selected physiological and biochemical responses in M. aeruginosa including cell viability, oxidative stress, antioxidant responses, in addition to microcystin synthesis and release into the surrounding environment. M. aeruginosa was able to grow in most test salinity treatments (1.4-10 ppt), as supported by cell abundance data and chlorophyll-a (chl-a) concentrations. Physiological data showed that after certain salinity thresholds (∼7ppt) were surpassed, salt stress had cascading effects, such as increased ROS production and lipid peroxidation, potentiating the decline in cellular viability. Furthermore, elevated salinity induced oxidative stress which was concomitant with a decrease in cell abundance, chl-a concentration and photochemical efficiency in the 7-10 ppt treatments. M. aeruginosa did not synthesize microcystins (MCs) in response to increased saline conditions, and mcy-D expression was not correlated with either salinity treatment or extracellular MC concentrations, indicating that salinity stress could inhibit toxin production and that released toxins were likely synthesized prior to exposure. Additionally, extracellular MC concentrations were not correlated with decreased cellular integrity, as evidenced by SYTOX analyses, suggesting that toxins may be released through mechanisms other than cellular lysis. Results from this study support that M. aeruginosa can survive with limited negative impacts to cellular structure and function up to a certain threshold between 7-10 ppt. However, after these thresholds are surpassed, there is radical decline in cell health and viability leading to toxin release. This work underscores the importance of understanding the balance between ROS production and antioxidant capacities when assessing the fate of M. aeruginosa under mesohaline conditions.

Loss of MCT1, MCT3, and MCT4 expression in the retinal pigment epithelium and neural retina of the 5A11/basigin-null mouse.

PURPOSE: The neural retina expresses multiple monocarboxylate transporters (MCTs) that are likely to play a key role in the metabolism of the outer retina. Recently, it was reported that targeting of MCT1 and -4 to the plasma membrane requires association with 5A11/basigin (CD147). In the present study, the hypothesis that reduced amplitudes in the electroretinograms in the 5A11/basigin null mouse (Bsg(-/-)) may be linked to altered expression of MCTs was studied. METHODS: The expression and subcellular distribution of MCTs in Bsg(-/-) mice was analyzed by immunofluorescence microscopy with isoform-specific antibodies. Protein expression was analyzed by Western blot analysis, and mRNA expression was examined with RT-PCR. RESULTS: Immunofluorescence labeling of tissue sections from the Bsg(-/-) mice revealed a dramatic reduction in labeling with MCT antibodies. There was a loss of MCT1 labeling in the apical membrane of the RPE and in the neural retina. MCT3, which is expressed in the basolateral membrane of the RPE wild-type mouse, was expressed at very low levels in both the apical and basolateral membranes of the Bsg(-/-) mouse. There was no change in expression or distribution of the glucose transporter (GLUT)-1 in the RPE and retina of the Bsg(-/-) mouse. Western blot analysis of detergent-soluble lysates prepared from wild-type and Bsg(-/-) eyes confirmed that the levels of MCT1, MCT3, and MCT4 protein were severely reduced in Bsg(-/-) mice. RT-PCR analyses of mRNA levels from wild-type and Bsg(-/-) mice demonstrated that the MCT1 transcript was expressed at normal levels in Bsg(-/-) mice. CONCLUSIONS: 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.

Retina-specific expression of 5A11/Basigin-2, a member of the immunoglobulin gene superfamily.

PURPOSE: 5A11/Basigin has recently been identified as a critical glycoprotein for full maturity and function of the mouse retina. However, the biological function of 5A11/Basigin has yet to be determined. Previous reports indicate the presence of multiple 5A11/Basigin polypeptides within the retina. Therefore, in an effort to determine the function of 5A11/Basigin, the molecular diversity of its expression was evaluated. METHODS: Northern blot and immunoblot techniques were used to evaluate the number of forms of 5A11/Basigin in the mouse retina. cDNA cloning, using a mouse retina library or RT-PCR from rat, chicken, zebrafish, and human retina, was performed to determine the sequence of 5A11/Basigin transcripts. A peptide was generated, based on the deduced amino acid sequence, for subsequent antibody production. Localization of 5A11/Basigin expression was evaluated by immunoblot, immunohistochemistry, and real-time RT-PCR. RESULTS: Two 5A11/Basigin transcripts of approximately 1.5 kb and approximately 1.8 kb, which correspond to glycosylated proteins of approximately 45 and approximately 55 kDa, respectively, were identified in mouse retina. The shorter form was previously cloned. However, the longer form, a splice variant of mouse 5A11/Basigin, is a member of the immunoglobulin gene superfamily and has been named 5A11/Basigin-2. Homologous transcripts were also cloned from rat, chicken, zebrafish, and human retina. 5A11/Basigin-2 expression was limited to the retina, specifically to photoreceptor cells, where it appeared to be most concentrated in the inner segments. CONCLUSIONS: 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.

Inactivation of the Basigin gene impairs normal retinal development and maturation.

5A11/Basigin is an immunoglobulin-like glycoprotein expressed on the surface of Müller cells, the apical and basal surfaces of the retinal pigmented epithelium, and photoreceptor cell bodies and their inner segments. Disruption of the 5A11/Basigin gene in the mouse results in photoreceptor degeneration and a corresponding decrease in electroretinogram amplitudes in mature mice. The purpose of this study was to examine the electrophysiology of the 5A11/Basigin null mouse retina at earlier ages than previously examined. Although the architecture of the 5A11/Basigin null mouse retina appears normal, the ERG amplitudes are severely depressed at eye opening, indicating failure in retinal maturation.

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.

Carbonic anhydrase XIV identified as the membrane CA in mouse retina: strong expression in Müller cells and the RPE.

The presence of carbonic anhydrase (CA) activity in the neural retina has been known for several decades. CA-II, a soluble cytoplasmic isoform expressed by Müller cells and a subset of amacrine cells, was thought to be the sole source of CA activity in the neural retina. However, CA-II deficient mice retain CA activity in the neural retina, which implies that another isoform must be present in that tissue. Recently CA-XIV, an integral membrane protein, was cloned and characterized. We, therefore, sought to determine whether CA-XIV is expressed in the neural retina, and hence is responsible for the CA activity observed in CA-II null animals. Immunohistochemical analyses of histological sections from CA-II null, CA-XIV null, and control mice were performed to localize the CA-XIV isoform, as well as other known retinal markers. Immunoblotting and real-time RT-PCR analyses were also performed to test for CA-XIV expression in retina and other mouse tissues. We determined herein that CA-XIV, a approximately 45kDa membrane protein, is expressed in retina, as it is in kidney. In the retina, CA-XIV is expressed on the plasma membrane of Müller cells. CA-XIV is also found on both the apical and basal membranes of the retinal pigmented epithelium. The data presented here indicate that like CA-II, CA-XIV is highly expressed in the neural retina and, like CA-II, more specifically by the Müller cells. The cellular compartmentalization of the two isoforms in the Müller cell-one cytoplasmic and the other on the plasma membrane-suggest that the two enzymes have specific and unique functions. Future studies will be necessary to assign functions to CA-II and CA-XIV in the mouse neural retina.

5A11/Basigin gene products are necessary for proper maturation and function of the retina.

5A11/Basigin gene products are important membrane glycoproteins for development and maturation of the retina. The gene encodes two immunoglobulin-like, membrane-bound glycoproteins as a result of splice variation. The smaller protein product, named 5A11/Basigin, is expressed by many tissues within the mouse, whereas the larger protein product, named 5A11/Basigin-2, is expressed only by the photoreceptor cells (PCs) of the retina. Mice in which the gene for 5A11/Basigin has been deleted have several abnormalities, including blindness from the time of eye opening with subsequent degeneration of the PCs. Studies by this laboratory suggest that a developmental deficiency is the underlying cause of the blindness. However, definitive biological functions for 5A11/Basigin and 5A11/Basigin-2 have yet to be defined. It is known that 5A11/Basigin is multifunctional and can interact with several different proteins. Preliminary studies indicate that 5A11/Basigin-2 may be multifunctional as well. Studies by this laboratory and others have demonstrated that 5A11/Basigin (and probably 5A11/Basigin-2) acts as a chaperone for monocarboxylate transporter-1 (MCT-1) translocation to the cell membrane. We have proposed that a lactate metabolon exists within the retina and functions to shuttle lactate, an energy source, from glial cells to the PCs. This metabolon is not present within 5A11/Basigin null mouse retinas, which may be the underlying cause of the retinal dysfunction and subsequent degeneration. Further studies will be necessary to determine which of the functions are critical for proper development of the retina.

Developmental analyses of 5A11/Basigin, 5A11/Basigin-2 and their putative binding partner MCT1 in the mouse eye.

Recent reports by this laboratory and others have demonstrated an association between 5A11/Basigin, a member of the immunoglobulin gene superfamily, and monocarboxylate transporter-1 (MCT1), a lactose transporter. Indeed, it was determined in the 5A11/Basigin null mouse retina that MCT1 does not properly integrate into the cell membranes of Müller cells (MCs) or the retinal-pigmented epithelium, where the two are colocalized. The purpose of this study was to elucidate the association of 5A11/Basigin and MCT1 in the developing mouse retina. Immunocytochemical localization and real-time RT-PCR were used to evaluate the expression and localization of 5A11/Basigin and MCT1 at embryonic days 12, 15, and 18, as well as post-natal days 1, 7, 14, and 21. Expression of both proteins progressed from a more generalized distribution throughout the undifferentiated neural retina to specific staining of retina-pigmented epithilia, the MCs, photoreceptor cells and the ciliary apparatus. Although these two membrane glycoproteins were often colocalized, distinct differences in the location and magnitude of their expression over time was observed. These findings suggest that although 5A11/Basigin and MCT1 can associate within the cell membrane, their expression is not always associated and colocalized.

Carbonic anhydrase in the midgut of larval Aedes aegypti: cloning, localization and inhibition.

The larval mosquito midgut exhibits one of the highest pH values known in a biological system. While the pH inside the posterior midgut and gastric caeca ranges between 7.0 and 8.0, the pH inside the anterior midgut is close to 11.0. Alkalization is likely to involve bicarbonate/carbonate ions. These ions are produced in vivo by the enzymatic action of carbonic anhydrase. The purpose of this study was to investigate the role of this enzyme in the alkalization mechanism, to establish its presence and localization in the midgut of larval Aedes aegypti and to clone and characterize its cDNA. Here, we report the physiological demonstration of the involvement of carbonic anhydrase in midgut alkalization. Histochemistry and in situ hybridization showed that the enzyme appears to be localized throughout the midgut, although preferentially in the gastric caeca and posterior regions with specific cellular heterogeneity. Furthermore, we report the cloning and localization of the first carbonic anhydrase from mosquito larval midgut. A cDNA clone from Aedes aegypti larval midgut revealed sequence homology to alpha-carbonic anhydrases from vertebrates. Bioinformatics indicates the presence of at least six carbonic anhydrases or closely related genes in the genome of another dipteran, the fruit fly Drosophila melanogaster. Molecular analyses suggest that the larval mosquito may also possess multiple forms.