1Section on Neurobiology, Leslie and Susan Gonda Department of Cell and Molecular Biology, House Ear Institute, Los Angeles, California; and 2Departments of Otolaryngology and Cell Biology, Emory University School of Medicine, Atlanta, Georgia
Submitted 14 July 2004 ; accepted in final form 8 November 2004
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ABSTRACT |
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cochlea; coassembly; deafness
The importance of Cxs in normal cochlear functions has been demonstrated by many genetic studies showing that about one-half of inherited childhood nonsyndromic deafness cases are caused by mutations in the Cx26 gene (21, 27, 28). Less frequently encountered are mutations in other Cx genes, e.g., Cx30 (10), Cx31 (22, 40), and Cx32 (2). It is clear that mutations in Cx genes are one of the most common forms of human genetic defects resulting in hearing loss for millions of patients with either autosomal recessive or dominant deafness (7, 17). The role of gap junction and the interactions of different subtypes of Cxs in the cochlea are currently unknown. Still less apparent is why mutations in Cx genes, which are widely expressed in a variety of tissues, can be mostly nonsyndromic. It has been speculated that gap junction networks in the cochlea provide intercellular conduits by which potassium ions are recycled from the base of hair cells to the endolymph (18, 32). However, experimental results directly testing this hypothesis are currently not available.
To understand how gap junctions contribute to the intercellular communication in the inner ear, it is essential to investigate the distribution and molecular assembly of different subtypes of Cxs in the cochlea. Earlier freeze-fracture studies have suggested an abundant presence of gap junctions in the inner ear (15, 16). Immunolabeling results demonstrated widespread expression of Cx26 and Cx30 in the supporting cells of the sensory epithelia, fibrocytes in the spiral ligament and spiral limbus, and also in vestibular systems (18, 19). Our recent findings showed that Cx26 and Cx30 are the two most abundantly expressed Cx subtypes, and that they coassemble to form gap junctions in the adult cochlea (1). However, it is not clear how extensive this coassembly occurs temporally and spatially in the cochlea, and whether wild-type gap junctions with different molecular configurations may exhibit functional differences. In this study, we first investigated coexpressions of Cx26 and Cx30 in the cochlea during development. The coassembly of Cx26 and Cx30 in most gap junctional plaques in the cochlea was shown by coimmunostaining and confirmed by coimmunoprecipitation. We further quantitatively compared differences in the kinetics of intercellular Ca2+ signaling across gap junctions reconstituted in vitro with either homo- or heteromeric configurations. Results demonstrated that heteromerically assembled gap junctions consisting of Cx26 and Cx30 had the advantage of allowing Ca2+ signaling to spread intercellularly twice as fast as their homomerically assembled counterparts.
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MATERIALS AND METHODS |
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In vitro reconstitution and verification of gap junctions with different molecular configurations.
HEK-293 cells (American Type Culture Collection, Manassas, VA) grown to 80% confluence on culture plates were transfected with plasmid DNA (purity >1.8) using Lipofectamine Plus 2000 (Invitrogen, Carlsbad, CA). Original Cx26 and Cx30 constructs were obtained from Dr. Howard Evans (Department of Medicine Biochemistry and Diagnostic Radiology, University of Wales College of Medicine) and Dr. Willecke (Institut für Genetik, Bonn, Germany), respectively. The Cx coding sequences were cut out of the multiple cloning sites (MCSs) and ligated into the MCSs of pEGFP vector (BD Biosciences, Palo Alto, CA) to create Cx-eGFP fusion protein (Cx-eGFP) after transfections. We also ligated Cx coding sequences into the pIRES2-DsRed2 vector (BD Biosciences) to create separate Cx and DsRed proteins (Cx-IRES-DsRed) in the same cell. Transfecting HEK-293 cells with the Cx-eGFP and Cx-IRES-DsRed constructs together allowed us to estimate the efficiency of cotransfection. We also checked colocalization and coassembly of Cx26 and Cx30 in the same reconstituted gap junction plaque (Figs. 4 and 5). Growth medium [DMEM+10% fetal bovine serum + penicillin (100 IU) + streptomycin (100 µg/ml)] was replaced with OptiMEM (Invitrogen, Carlsbad, CA) before transfection. The transfection protocol for cells grown in 60-mm culture dishes is described below. Reagent amount and volume were adjusted according to the surface area of the dish when other types of dishes were used. After all reagents were warmed to room temperature, DNA (8 µg) and Plus reagent (16 µl) were mixed in 500-µl final volume of OptiMEM. For cotransfection, two different plasmids (4 µg each) were mixed in the same tube. In a separate tube, 7.5 µl of Lipofectamine was diluted in the same volume of OptiMEM (500 µl). After 30 min, the contents of both tubes were mixed together to allow DNA and Lipofectamine complex formation by incubation at room temperature for another 20 min. This complex was applied dropwise to the culture dish in a final volume of 5 ml. The dishes were swirled to distribute the complex evenly and returned to a CO2 incubator (5%, 37°C). After 3 to 4 h of transfection, 500 µl of fetal bovine serum were added to the dish to reduce the toxicity of Lipofectamine and incubation was continued overnight. The transfection medium was replaced 15 h later with the regular growth medium.
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RESULTS |
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Coimmunolabeling and coimmunoprecipitation results demonstrated that Cx26 and Cx30 were coassembled in most cochlear gap junctions. Dense immunoreactivity to both Cx26 and Cx30 in conventionally prepared cochlear sections made it difficult to observe individual gap junction plaques even with confocal microscopy. To investigate the colocalization of the two Cxs at higher resolution, we used semithin cochlear cryosections cut at a thickness of 0.5 µm. These sections were coimmunolabeled with both Cx antibodies but visualized with secondary antibody conjugated to different fluorphors. One example of Cx26 and Cx30 colocalization in discrete gap junction plaques around a single cell in the lateral wall is given in Fig. 3, AC. The overlapping staining patterns around this cell were visualized by bright Cy2 (green, indicating Cx30 in Fig. 3A) and Cy3 (red, indicating Cx26 in Fig. 3B) fluorescent spots around the nucleus (shown in blue by DAPI staining). Most gap junction plaques around this cell showed colocalization of Cx26 and Cx30 (yellow spots in Fig. 3C), except for a few indicated by an arrow in Fig. 3C. The exact percentage of Cx26 and Cx30 colocalization varied in different regions of the cochlea, as indicated by examples of coimmunolabeling obtained from seven different regions of the cochlear cross sections (Fig. 3D). Percentages given in Fig. 3D indicate the overlapped Cx26 and Cx30 spots in that particular section. Average results from four cochlear sections are summarized in Table 1. Most regions in the cochlea showed overwhelming overlapping. One notable exception was the Deiter cells under the outer hair cells, where most gap junction plaques (86%) showed immunoreactivity for Cx30 only.
Each gap junction plaque contains hundreds of gap junction channels; therefore, colocalization of Cx26 and Cx30 in the similar gap junction plaque demonstrated by coimmunolabeling does not prove that the two proteins are assembled together into the same gap junction. Direct interactions of Cx26 and Cx30 by coassembling into the same gap junction were examined by coimmunoprecipitation. The experimental procedures were repeated eight times, and the same results were obtained. As pointed out earlier, the specificity of the antibodies we used against Cx26 and Cx30 was confirmed using Western blot analysis (Fig. 4, A and B). Membrane proteins isolated from cochlear tissues (P1 mouse) were immunoprecipitated with the antibody against Cx30, and the precipitated protein complex was analyzed in the Western blots using antibodies against both Cx26 and Cx30 (Fig. 4C). Results consistently yielded two bands corresponding to Cx26 and Cx30, suggesting that Cx26 and Cx30 physically interact with each other in the cochlea. To test whether Cx26 and Cx30 were coassembled in reconstituted gap junctions after transfections, we immunoprecipitated membrane protein extracted from co-transfected HEK-293 cells with the antibody against Cx30. Physical interactions of Cx26 with Cx30 were supported by subsequent Western blot analysis results showing that 1) Cx26 was present in the Cx30 immunoprecipitated protein complex (Fig. 4E); 2) two bands matching Cx30-eGFP and Cx26-eGFP fusion proteins were present if an antibody against eGFP was used in the Western blots of the Cx30 immunoprecipitated proteins (Fig. 4F). We compared the gap junction protein levels in the cell membrane by immunoblotting with an antibody against eGFP (Fig. 4G), which is common to both Cx26- and Cx30-eGFP fusion proteins produced with transfections. Loading differences in each lane were controlled by detecting the amount of a housekeeping protein (actin, Fig. 4H). Regarding transfections done with a single type of Cx [Cx30-eGFP (middle lane) and Cx26-eGFP (right lane) in Fig. 4G], a comparison of the intensities of the bands showed that co-transfection using half of the plasmid for each Cx (left lane, Fig. 4G) did not increase the total number of gap junctions in the cell membrane. Total amount of connexin in cotransfectant was comparable to that when only one type of connexin was used in transfections.
The results presented above showed that expressions of Cx26 and Cx30 were remarkably similar both temporally and spatially during cochlear development, and that the Cxs physically interacted with each other in cochlear gap junctions and reconstituted gap junctions in HEK-293 cells. Combining these results and our earlier demonstration that Cx26 and Cx30 were coimmunoprecipitated in the adult cochlea (1), these data suggested that the coassembly of the two Cxs occurred extensively during cochlear development. Because Cx26 and Cx30 are the two most abundantly expressed Cxs in the cochlea (1), our results also suggested that most gap junctions in the cochlea were formed by coassembly of Cx26 and Cx30.
Gap junctions with different molecular configurations show functional differences.
Gap junctions consisting of homomeric Cx26 (Cx26WT), homomeric Cx30 (Cx30WT), and heteromeric Cx26 and Cx30 (Cx26 and 30WT) were reconstituted in HEK-293 cells. On average, 3040% of cells showed bright GFP 24 h after transfecting cells with Cx-eGFP constructs. Among them, 5% of cell pairs formed gap junctions visible by bright GFP spots on the cell membrane (Fig. 5, C and D, Fig. 6, and Fig. 7A). Only the cell pairs that showed clear gap junction formation were used for functional studies of gap junctions. Cotransfection of the two Cxs in the same cell was confirmed by using Cx26-IRES-DsRed and Cx30-eGFP plasmids together for transfections (Fig. 5, A and B). Simultaneous presence of red (DsRed, Fig. 5A) and green (eGFP, Fig. 5B) fluorescence in the same cell was observed in almost all cells we counted from random views of three dishes (96%, n = 128). We also checked co-localization of Cx26 and Cx30 in the same gap junction plaques by immunolabeling (Fig. 5, C and D). Results demonstrated that Cx26-labeled gap junction plaques (Fig. 5C, arrow) overlapped with eGFP-tagged Cx30 (Fig. 5D, arrow) in most cell pairs we checked (89 of 91). The coimmunoprecipitation results presented above (Fig. 4, DF) further confirmed that Cx26 and Cx30 co-assembled in reconstituted gap junctions after cotransfections.
To investigate whether gap junctions with different molecular configurations show any difference for intercellular signaling, we first compared their permeability to two charged fluorescent dyes. The two fluorescent dyes we used have similar molecular weights [668 for propidium iodide (PI) and 643 for AlexaFluor 488] but opposite valences. We found that the Cx26WT (Fig. 6D), Cx30WT (Fig. 6E), and Cx26&30WT (Fig. 6F) gap junctions were all readily permeable to the positive charge PI (n = 18). In contrast, homomeric Cx30WT gap junctions were not permeable to the negatively charged AlexaFluor 488 (Fig. 6B). The other two types of reconstituted gap junctions were readily permeable to AlexaFluor 488 (n > 10 for each test) (Fig. 6, A and C). More functional differences between the three types of wild-type gap junctions were shown by quantitatively comparing the rate of intercellular Ca2+ signaling through Cx26WT, Cx30WT, and Cx26&30WT gap junctions. Slight deformation of the cell membrane with a microelectrode (tip size 1 µm) always elicited rapid increases in [Ca2+]i in the touched cell (Fig. 7, cell 1 or cell touched). Increases of [Ca2+]i spread to neighboring cells apparently by two mechanisms: 1) by propagating transient [Ca2+]i increases mediated by purinergic receptors; 2) by gap junction-mediated intercellular signaling. The purinergic receptor-mediated intercellular signaling was easily distinguishable from that mediated through gap junctions by their longer time delay of onsets and more transient responses. Purinergic receptor-mediated [Ca2+]i responses in surrounding cells usually returned to baseline completely in
50 s (Fig. 7B, data curves encircled by curved arrow), compared with >300 s needed for gap junction-mediated Ca2+ responses to return (Fig. 7, BD). In addition, adding suramin (200 µM, Fig. 7C) to block or adding ATP (20 µM, Fig. 7D) to saturate purinergic receptors eliminated the purinergic receptor-mediated intercellular signaling. The suramin and ATP treatments, however, left the gap junction-mediated intercellular Ca2+ signaling intact (Fig. 7, C and D). Only the cell formed gap junctions with the mechanically stimulated cell, followed with an increase in [Ca2+]i (cell 2 in Fig. 7, C and D). All other surrounding cells that did not form gap junctions with the source cell showed no increases in [Ca2+]i (data curves encircled by curved arrows in Fig. 7, C and D). These results indicated that the Ca2+ signaling spread intercellularly through the reconstituted gap junction channels could be studied separately from that mediated by the purinergic receptors.
Rising phases of [Ca2+]i increases in both source (Fig. 7A, cell 1) and follower (Fig. 7A, cell 2) cells could be well fitted by single exponential increases (red smooth curves in Fig. 7E). The Ca2+ signaling between the two cells was characterized by three parameters: 1) a time delay in the onset of Ca2+ rises in the follower cells (dT); 2) a faster time constant (1) and a much slower time constant (
2) in the curve fits describing the [Ca2+]i increases in the cell touched and cell followed, respectively; 3) ratio of the amplitude of peak [Ca2+]i rises in the cell touched (P1) and cell followed (P2). Figure 8 presents results comparing the three parameters among the Cx26WT, Cx30WT, and Cx26&30WT gap junctions. Three panels in Fig. 8A give examples of exponential curve fits to the three types of reconstituted gap junctions with different molecular configurations, and another panel gives raw data of time constants (
1 and
2) gathered from all cell pairs we tested. While no significant differences were found for
1,
2s fitted for Cx26WT (solid squares) and Cx30WT (open triangles) gap junctions were significantly slower than those of Cx26&30WT gap junctions (Fig. 7A, open circles). The averaged results given in Fig. 8B showed that
1 (in ms) of three types of gap junctions were 284.5 ± 14.9 (n = 15), 275.9 ± 11.3 (n = 13), and 305.6 ± 19.8 (n = 12) for Cx26WT, Cx30WT, and Cx26&30WT gap junctions, respectively. The differences were not statistically significant for
1, indicating that there were no differences in mechanically elicited [Ca2+]i responses in the source cells. The ratio of the peak [Ca2+]i in the two cells (P2/P1) was also similar (Fig. 8B), indicating that an equilibrium of [Ca2+]i between the two cells was reached by all three types of gap junctions. In contrast, statistically significant differences were observed in time delays of the onset of Ca2+ rises (dT) in the follower cells. The dT were (in ms) 1,268 ± 205 (n = 15), 1,045 ± 186 (n = 13), and 574 ± 102 (n = 12) for Cx26WT, Cx30WT, and Cx26&30WT gap junctions, respectively. In addition, the time constant describing the [Ca2+]i increases in the follower cells (
2) formed heteromeric gap junctions with the source cell was 1,197 ± 67 (n = 12). In comparison, the
2 values for gap junctions consisting of homomeric Cx26 or Cx30 were 3,011 ± 196 (n = 15) and 2,758 ± 153 (n = 13), respectively. Both dT and
2 for the intercellular Ca2+ signaling across heteromeric gap junctions were about half of those obtained from the homomeric gap junction channels. As demonstrated by results shown in Fig. 4G, these rate differences (dT and
2) were unlikely to be caused by increased number of gap junction channels after cotransfections of Cx26 and Cx30.
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DISCUSSION |
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Cxs are classified into at least three subgroups based on their sequence similarities. Cxs26, 31, and 30 belong to the group, Cxs43 and 50 belong to the
group, and Cx29 belongs to the putative
group. Generally Cxs from different subgroups are not able to form functional heterotypic gap junctions (3). Thus gap junction-mediated intercellular communications are restricted among clusters of cells expressing compatible Cxs. Intercellular communication along a selective pathway can thus result in a compartmentalization for intercellular signal exchanges within the cochlea. Cx31 was found in a subpopulation of fibrocytes near spiral prominence where both Cx26 and 30 are not present (a region indicated by asterisks in Fig. 1) (9). Although the exact locations of Cx43 and Cx50 expressions are currently controversial (5, 9, 20), it is clear that they do not generally overlap with Cx26 and Cx30. Cx45 was expressed in the inner ear vascular system (5). Expression pattern of Cx29 in the cochlea has not been checked yet. However, it is generally found in myelin-forming glial cells (31). We have shown new data in this work demonstrating that both Cx26 and 30 were expressed early in development and that their expressions in the cochlea were developmentally regulated. The expression of the two Cxs was limited to regions outside the developing organ of Corti before birth and gradually increased in both supporting cells and fibrocytes in the lateral wall. Our result demonstrates for the first time that a band of cells bordering stria vascularis expressed high levels of both Cx26 and Cx30 before the onset of hearing in mice. This pattern of Cx expression changed to a more homogeneous distribution in the lateral wall after P12 (Fig. 1). This dramatic change in Cx expression patterns may reflect an underlying transformation in the demand for both biochemical and ionic couplings in the maturing cochlea. In the adult stage both Cx26 and Cx30 were found in many parts of the cochlea along the proposed K+ recycling pathway (18, 32). Therefore, alteration of their functions is expected to have severe consequences for ionic or biochemical homeostasis. On the other hand, the maintenance of cochlear homeostasis requires appropriate driving force in terms of chemical or electrical potential gradients in the right amount and direction, and directional rectifications of gap junction to help both ionic and biochemical fluxes to go in the appropriate direction. Presence of multiple types of Cxs may help to fine tune the gap junction channels both temporally and spatially appropriate for the physiological demands of the cochlea.
Our finding that most gap junction plaques along the proposed K+ recycling pathway in the cochlea were formed by co-assembly of Cx26 and Cx30 raises some interesting issues. Co-assembly of Cx26 and Cx30 indicates redundancy in building gap junction intercellular channels, and suggests that deleting one Cx gene does not necessarily eliminate gap junction channels in the cochlea. Instead, homomeric gap junctions might be able to replace heteromeric gap junctions where they are normally found in the cochlea. Indeed, the expression pattern of Cx26 in the cochleae of Cx30 knockout mice is indistinguishable from that in the wild-type mice (33). Homomeric gap junctions constituted by either Cx26 or Cx30 have good permeability to K+ (24, 34); therefore, it is puzzling why such a seemingly subtle change (at least for ionic permeation) resulted in deafness and reduction in endolymphatic potential in mice (33). One hint is that gap junctions consisting of homomeric Cx26 or Cx30 have very different permeation profiles for certain larger molecules, despite the fact that the two Cxs share the most similar sequences in the Cx family. It is known that gap junctions consisting of homomeric Cx30 are not permeable to Lucifer yellow (LY; molecular weight = 457 atomic mass unit, charge = 2), whereas homomeric Cx26 gap junctions allow LY to pass readily (24). We expanded this line of work by showing that all three types of gap junctions we studied were permeable to propidium iodide, which is a fluorescent tracer molecule with a molecular weight of 668 and a charge of +2. In contrast, permeability of heteromeric gap junctions measured by diffusion of AlexaFluor 488 was similar only to that of homomeric Cx26 gap junctions. Considering that AlexaFluor 488 and propidium iodide have similar molecular weight (643 vs. 668 atomic mass unit) but different charges (3 vs. +2), the results suggested that the pore of heteromeric gap junction channels in the cochlea might filter out negatively charged molecules more selectively than positively charged molecules. It was recently suggested that affinity binding sites may exist in the pores of gap junctions to facilitate passage of specific molecules (35). This model of gap junction permeation suggests that gap junctions may not act like inert and passive channels as traditionally believed.
Besides possible differences in biochemical permeability, our data demonstrated a clear distinction between heteromeric and homomeric gap junctions with regard to their efficiency in spreading intercellular Ca2+ signals. Results showed that [Ca2+]i increases in the source cell passed only to neighboring cells formed wild-type gap junctions with the source cell to reach an eventual equilibrium (P2/P1 in Fig. 8B). The Ca2+ signaling did not spread to those cells that formed no gap junctions (Fig. 7, C and D) or nonfunctional mutant gap junctions (e.g., Cx26R75W; data not shown) with the source cell. These results assured us that we had measured gap junction-mediated intercellular Ca2+ signaling. Shorter time delay (dT) of [Ca2+]i rises in the follower cells through heteromeric gap junctions also suggested that changes in gap junction molecular assembly, but not the total number of gap junction channels, were responsible for kinetic alterations. Transfers of a variety of signaling molecules through gap junction channels have been investigated using cellular imaging methods similar to those used in this study. Previous work shows that mechanical stimuli quickly increase [Ca2+]i and Ca2+ rises propagate to neighboring cells by either simple diffusion of Ca2+ or secondarily as a result of IP3 diffusion through gap junctions (4, 30). It is unclear which signaling molecule (or both of them) crossed gap junctions in the current study. However, based on our observations that 1) we can fit [Ca2+]i rises in the follower cells (cell 2 in Fig. 5) with a single exponential rising curve and 2) the average amplitude of mechanically elicited Ca2+ responses obtained in Ca2+-free HBSS were less than one-half of those obtained in normal HBSS (data not shown), we believe that direct transfer of Ca2+ across the reconstituted gap junction channels constituted a significant source for the increase in [Ca2+]i in follower cells. Regardless of which molecules are the major ones to cross the gap junctions, faster intercellular Ca2+ signaling shown by heteromerically assembled gap junctions suggested that transient behaviors of intercellular exchanges through gap junctions of different molecular assembly may be an important factor to consider for the function of gap junctions in the cochlea. In addition, our finding suggest that, in addition to recycling K+, gap junctions in the cochlea may have other vital roles in transiently regulating the needs for intercellular communications.
Both Cx26 and Cx30 are found in many different types of tissues; therefore, it is unclear why Cx mutations mostly cause nonsyndromic deafness. If heteromeric assembly of Cx30 and Cx26 is unique to the cochlea and the gap junction properties of this particular combination are vitally suited to the demands of homeostasis in the cochlea, this could provide one simple explanation for the nonsyndromic feature of human Cx26 mutations found in most patients. Our demonstration that heteromeric gap junctions consisting of Cx26 and Cx30 mediated intercellular Ca2+ signaling faster than their homomeric counterparts is consistent with this theory. Alternatively, different tissues may have different tolerance thresholds for quantitative changes in gap junction-mediated intercellular ionic or biochemical couplings. The cochlea, with its unique ionic environment in fluid compartments tightly coupled to mechanotransduction, may happen to be the most sensitive to gap junction mutations. Heteromeric assembly of Cx26 and Cx30 also has some functional implications for Cx mutation studies. It suggests that Cx26 mutations, recessive or dominant, must have transdominant effects on gap junction channels composed of Cx26 and Cx30. This was recently confirmed by others (26). In this work we studied gap junction-mediated Ca2+ signaling through gap junctions of different molecular configurations as an example of functional specificity of native gap junctions in the cochlea. Considering the diversity in the clinical syndrome of many Cx26 mutations, much remains to be discovered to answer the special biophysical properties and functional roles of gap junctions in the cochlea.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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