Cx30.2 deletion causes imbalances in testicular Cx43, Cx46, and Cx50 and insulin receptors. Reciprocally, diabetes/obesity alters Cx30.2 in mouse testis.

Cx30.2 protein content and localization were assessed during development. An account of Cx30.2, Cx43, Cx46, and Cx50, and insulin receptor (IR) responses to Cx30.2, Cx46, or Cx50 deficiency in mouse interstitial tissue (ITf)- and seminiferous tubule-enriched fractions (STf) is given. The impact of high glucose/insulin on Cx30.2 was investigated in spontaneously diabetic and obese db/db and ob/ob mouse testis and anterior pituitary (AP). Cx30.2 labeled contacts in vascular endothelial and Leydig cells and Sertoli cell junctions in stage V-VII. Cx30.2 expression is regulated differently in the interstitium and tubules. Cx30.2 at 30-kDa levels peaked by 28 days in ITf and by 14 days in STf. In STf, deleting Cx30.2 decreased Cx43 and Cx50, whereas deleting Cx50 downregulated Cx30.2. The opposite occurred in ITf. In STf, deleting Cx30.2 upregulated Cx46 except the full-length reciprocally, deleting Cx46 upregulated Cx30.2. In ITf, Cx30.2 deficiency upregulated full-length and phosphorylated Cx46, whereas deleting Cx46 downregulated 48- to 50-kDa Cx30.2. The db/db and ob/ob mouse ITf, STf, and AP showed imbalanced Cx30.2 levels. IRα levels at 135 kDa declined in Cx30.2-/- and Cx50-/- mouse ITf and Cx46-/- and Cx50-/- STf. IRß at 98 to 110 kDa dropped in Cx30.2-/- and Cx46-/- mice STf suggesting that Cx30.2 deficiency decreases active IR sites. The results show the connexins interdependence and interaction and that altering a single connexin changes the remaining connexins expression, which can modify gap junction-mediated glucose exchanges in contacting cells. Data suggest that glucose/insulin influences Cx30.2 turnover in testis and AP and, reciprocally, that connexins modulate testis glucose uptake and response to insulin.

Complementary expression and phosphorylation of Cx46 and Cx50 during development and following gene deletion in mouse and in normal and orchitic mink testes.

Gap junction-mediated communication helps synchronize interconnected Sertoli cell activities. Besides, coordination of germ cell and Sertoli cell activities depends on gap junction-mediated Sertoli cell-germ cell communication. This report assesses mechanisms underlying the regulation of connexin 46 (Cx46) and Cx50 in mouse testis and those accompanying a "natural" seasonal and a pathological arrest of spermatogenesis, resulting from autoimmune orchitis (AIO) in mink. Furthermore, the impact of deleting Cx46 or Cx50 on the expression, phosphorylation of junction proteins, and spermatogenesis is evaluated. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. During the mink active spermatogenic phase, Cx50 became phosphorylated and localized to the site of the blood-testis barrier. By contrast, Cx46 was dephosphorylated and associated with annular junctions, suggesting phosphorylation/dephosphorylation of Cx46 and Cx50 involvement in the barrier dynamics. Cx46-positive annular junctions in contact with lipid droplets were found. Cx46 and Cx50 expression and localization were altered in mink with AIO. The deletion of Cx46 or Cx50 impacted on other connexin expression and phosphorylation and differently affected tight and adhering junction protein expression. The level of apoptosis, determined by ELISA, and a number of Apostain-labeled spermatocytes and spermatids/tubules were higher in mice lacking Cx46 (Cx46-/-) than wild-type and Cx50-/- mice, arguing for life-sustaining Cx46 gap junction-mediated exchanges in late-stage germ cells secluded from the blood by the barrier. The data show that expression and phosphorylation of Cx46 and Cx50 are complementary in seminiferous tubules.

Mesenchymal stem cells improve cardiac conduction by upregulation of connexin 43 through paracrine signaling.

Mesenchymal stem cells (MSCs) were shown to improve cell survival and alleviate cardiac arrhythmias when transplanted into cardiac tissue; however, little is known about the mechanism by which MSCs modify the electrophysiological properties of cardiac tissue. We aimed to distinguish the influence of cell-cell coupling between myocytes and MSCs from that of MSC-derived paracrine factors on the spontaneous activity and conduction velocity (θ) of multicellular cardiomyocyte preparations. HL-1 cells were plated on microelectrode arrays and their spontaneous activity and θ was determined from field potential recordings. In heterocellular cultures of MSCs and HL-1 cells the beating frequency was attenuated (t(0h): 2.26 ± 0.18 Hz; t(4h): 1.98 ± 0.26 Hz; P < 0.01) concomitant to the intercellular coupling between MSCs and cardiomyocytes. In HL-1 monolayers supplemented with MSC conditioned media (ConM) or tyrode (ConT) θ significantly increased in a time-dependent manner (ConT: t(0h): 2.4 cm/s ± 0.2; t(4h): 3.1 ± 0.4 cm/s), whereas the beating frequency remained constant. Connexin (Cx)43 mRNA and protein expression levels also increased after ConM or ConT treatment over the same time period. Enhanced low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation after ConT treatment implicates the Wnt signaling pathway. Suppression of Wnt secretion from MSCs (IWP-2; 5 µmol/l) reduced the efficacy of ConT to induce phospho-LRP6 and to increase θ. Inhibition of ß-catenin (cardamonin; 10 µmol/l) or GSK3-α/ß (LiCl; 5 mmol/l) also suppressed changes in θ, further supporting the hypothesis that MSC-mediated Cx43 upregulation occurs in part through secreted Wnt ligands and activation of the canonical Wnt signaling pathway.

Omicron BA.2 lineage predominance in severe acute respiratory syndrome coronavirus 2 positive cases during the third wave in North India.

Background: Recent studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reveal that Omicron variant BA.1 and sub-lineages have revived the concern over resistance to antiviral drugs and vaccine-induced immunity. The present study aims to analyze the clinical profile and genome characterization of the SARS-CoV-2 variant in eastern Uttar Pradesh (UP), North India. Methods: Whole-genome sequencing (WGS) was conducted for 146 SARS-CoV-2 samples obtained from individuals who tested coronavirus disease 2019 (COVID-19) positive between the period of 1 January 2022 and 24 February 2022, from three districts of eastern UP. The details regarding clinical and hospitalized status were captured through telephonic interviews after obtaining verbal informed consent. A maximum-likelihood phylogenetic tree was created for evolutionary analysis using MEGA7. Results: The mean age of study participants was 33.9 ± 13.1 years, with 73.5% accounting for male patients. Of the 98 cases contacted by telephone, 30 (30.6%) had a travel history (domestic/international), 16 (16.3%) reported having been infected with COVID-19 in past, 79 (80.6%) had symptoms, and seven had at least one comorbidity. Most of the sequences belonged to the Omicron variant, with BA.1 (6.2%), BA.1.1 (2.7%), BA.1.1.1 (0.7%), BA.1.1.7 (5.5%), BA.1.17.2 (0.7%), BA.1.18 (0.7%), BA.2 (30.8%), BA.2.10 (50.7%), BA.2.12 (0.7%), and B.1.617.2 (1.3%) lineages. BA.1 and BA.1.1 strains possess signature spike mutations S:A67V, S:T95I, S:R346K, S:S371L, S:G446S, S:G496S, S:T547K, S:N856K, and S:L981F, and BA.2 contains S:V213G, S:T376A, and S:D405N. Notably, ins214EPE (S1- N-Terminal domain) mutation was found in a significant number of Omicron BA.1 and sub-lineages. The overall Omicron BA.2 lineage was observed in 79.5% of women and 83.2% of men. Conclusion: The current study showed a predominance of the Omicron BA.2 variant outcompeting the BA.1 over a period in eastern UP. Most of the cases had a breakthrough infection following the recommended two doses of vaccine with four in five cases being symptomatic. There is a need to further explore the immune evasion properties of the Omicron variant.

Global gene expression analysis of lenses from different mouse strains and in the alpha3Cx46 knockout mouse.

PURPOSE: Disruption of the mouse gene encoding the gap junction subunit alpha3 connexin 46 (alpha3Cx46) results in the formation of lens cataracts that have a severity affected by the genetic background of the mouse strain. To identify the genes that influence the severity of the nuclear opacity, global gene expression was analyzed in lenses from the 129SvJae strain and compared to the C57BL/6J strain. METHODS: Lens transcripts were subjected to cDNA microarray analysis. Results on selected genes were confirmed by real-time PCR. RESULTS: GENES THAT WERE DETERMINED TO BE ALTERED IN EXPRESSION LEVELS AS A RESULT OF STRAIN DIFFERENCES COULD BE CLUSTERED INTO THREE GROUPS: energy metabolism, stress response, and cell growth. CONCLUSIONS: There were no observed changes in gene expression as a result of the lack of alpha3Cx46 in the different mouse strains, suggesting that the pathways mediated by this connexin do not influence gene transcription in the lens. Analysis of the transcript changes due to strain differences provides new insights into potential genetic modifiers of cataractogenesis. More detailed experimentation will be needed to determine if these observed changes do indeed affect cataractogenesis.

Identification of proteins that modify cataract of mouse eye lens.

The occurrence of a nuclear cataract in the eye lens due to disruption of the alpha3Cx46 connexin gene, Gja3, is dependent on strain background in a mouse model, implicating factors that modify the pathology. The differences upon cataractogenesis in the urea soluble proteins of the lens of two mouse strains, C57BL/6J and 129/SvJ, were analyzed by a comparative proteomics approach. Determination of the complete proteome of an organ offers the opportunity to characterize at a molecular level, differences in gene expression and PTMs occurring during pathology and between individuals. The abundance of 63 protein species was altered between the strains. A unique aspect of this study is the identification of chaperonin subunit 6A, mortalin, ERp29, and syntaxin-binding protein 6 in the eye lens. DNA polymorphisms resulting in nonconservative amino acid changes that led to altered physicochemical properties of the proteins were detected for mortalin, chaperonin subunit 6A, annexin A1, and possibly gamma-N crystallin. The results show HSP27/25 and/or ERp29 are the likely major modifying factors for cataractogenesis. Extension of the results suggests that small heat-shock proteins have a major role for influencing cataract formation in humans.

Age-related cataracts in alpha3Cx46-knockout mice are dependent on a calpain 3 isoform.

PURPOSE: Previous studies have demonstrated that in 129alpha3Cx46-/- mice, age-related nuclear cataract is formed. In the present study, a more in vivo-relevant model was generated to test the hypothesis that the calpain 3 gene is involved in age-related nuclear cataractogenesis in alpha3Cx46 knockout mice. METHODS: To test the hypothesis that the calpain 3 gene is involved in age-related nuclear cataractogenesis in alpha3Cx46 knockout mice, 129alpha3Cx46-/- and CAPN3-/- mice were mated to generate homozygous double-knockout (dKO) mice. Lenses from the mice were examined by visual observation, laser scan analysis, and histologic and biochemical methods. RESULTS: In the absence of the CAPN3 gene, the formation of a cataract was delayed, and its appearance was changed to a more diffuse, pulverulent type. Unlike in the 129alpha3Cx46-/- mouse, cleavage of gamma-crystallin was not detected in the dKO mouse. In both 129alpha3Cx46-/- and dKO mice, total Ca2+ increased. CONCLUSIONS: The present study shows for the first time that calpain 3 is necessary for the formation of age-dependent nuclear cataracts in alpha3Cx46-/- mice. Evidence that the calpain 3 gene is directly involved in, or part of the pathway that leads to, gamma-crystallin cleavage is presented. These results are consistent with the hypothesis that the loss of alpha3Cx46 leads to increased levels of Ca2+ ions, and this increase activates the CAPN3 isoform, Lp82/85, which results in the formation of a nuclear cataract.

Lens connexins alpha3Cx46 and alpha8Cx50 interact with zonula occludens protein-1 (ZO-1).

Connexin alpha1Cx43 has previously been shown to bind to the PDZ domain-containing protein ZO-1. The similarity of the carboxyl termini of this connexin and the lens fiber connexins alpha3Cx46 and alpha8Cx50 suggested that these connexins may also interact with ZO-1. ZO-1 was shown to be highly expressed in mouse lenses. Colocalization of ZO-1 with alpha3Cx46 and alpha8Cx50 connexins in fiber cells was demonstrated by immunofluorescence and by fracture-labeling electron microscopy but showed regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate with alpha3Cx46 and alpha8Cx50, and pull-down experiments showed that the second PDZ domain of ZO-1 was involved in this interaction. Transiently expressed alpha3Cx46 and alpha8Cx50 connexins lacking the COOH-terminal residues did not bind to the second PDZ domain but still formed structures resembling gap junctions by immunofluorescence. These results indicate that ZO-1 interacts with lens fiber connexins alpha3Cx46 and alpha8Cx50 in a manner similar to that previously described for alpha1Cx43. The spatial variation in the interaction of ZO-1 with lens gap junctions is intriguing and is suggestive of multiple dynamic roles for this association.

Structural and immunocytochemical alterations in eye lens fiber cells from Cx46 and Cx50 knockout mice.

In the current study we describe the changes of overall organization of lens fiber cells in connexin 46 (Cx46) and connexin 50 (Cx50) knockout mice. Morphometric analyses and the application of immunocytochemical techniques revealed that in Cx46 knockout lens (Cx46 -/-), where Cx50 is expressed alone, the postnatal differentiation of secondary fiber cells proceeds faster and is characterized by an increased number of smaller fiber cells. Conversely, in Cx50 knockout mice (Cx50 -/-), the lenticular mass is considerably reduced and characterized by a small number of fiber cells added during the postnatal period. The process of terminal differentiation was impaired and generated larger fiber cells still possessing cytoplasmic organelles. Freeze-fracture and fracture labeling revealed that the junctional assembly, packing organization and topographic interactions between connexons and MP26 differed when Cx46 and Cx50 were co-assembled in the wild-type or expressed separately in the two distinct knockout phenotypes. Filipin cytochemistry provided indirect evidence that Cx46 and Cx50 expressed alone are recruited into different lipid environments. Our results represent the structural proof that interaction of connexins and MP26 contributes to the overall organization of the fiber cells.

Differences in expression patterns between mouse connexin-30.2 (Cx30.2) and its putative human orthologue, connexin-31.9.

A novel gap junction forming protein, mouse connexin-30.2 (Cx30.2) contains 278 amino acid residues, and is 79% identical to human Cx31.9 (GJA11). Northern analysis showed that Cx30.2 is ubiquitously expressed, most prominently in testis. Polyclonal antibodies against Cx30.2 detected a 30 kDa protein in cells overexpressing Cx30.2, and in mouse testis. Immunofluorescence showed that Cx30.2 was expressed in vascular smooth muscle, but also in cell types where Cx31.9 was not detected. These data demonstrate that Cx30.2 is a bona fide gene, and suggest that it is the orthologue of Cx31.9, but that it may have additional roles compared with Cx31.9.

Projection structure of full length connexin 43 by electron cryo-crystallography.

We previously used electron cryo-crystallography to determine the three-dimensional structure of recombinant gap junction channels formed by a C-terminal truncation mutant of Cx43 (11). The dodecameric channel is formed by the end-to-end docking of two hexameric connexons, each comprised of 24 transmembrane alpha-helices. We have now generated two-dimensional crystals of the recombinant, full-length channel, as well as crystals in which the C-tail has been completely removed by trypsin digestion. Projection density maps at 7.5 A resolution closely resemble our previous analysis of the C-terminal truncation mutant (9). A difference map between the full length and trypsin-treated channels suggests that there are small but significant shifts in protein density upon removal of the C-tail.

RNA interference targeting transforming growth factor-beta type II receptor suppresses ocular inflammation and fibrosis.

PURPOSE: Transforming growth factor-beta(TGF-beta) is an important mediator of wound healing responses. In the eye, TGF-beta activity has been implicated in causing corneal haze after laser surgery and subconjunctival scarring following glaucoma surgery. The purpose of the study was to determine whether small interference RNAs (siRNAs) targeting the type II receptor of TGF-beta (TbetaRII) could be used to suppress the TGF-beta action. METHODS: TbetaRII specific siRNAs designed from the human gene sequence were transfected into cultured human corneal fibroblasts. The protein and transcript levels of the receptor were determined by immunofluorescence, western blotting, and real time PCR. Immunofluorescence and immunoblotting were carried out to examine fibronectin assembly. A wound closure assay was used to assess cell migration in in vitro fibroblast cultures. In addition, the in vivo effects of TbetaRII siRNA were evaluated in a mouse model of ocular inflammation and fibrosis generated by subconjunctival injection of phosphate buffered saline and latex beads. Mouse TbetaRII siRNA was introduced into experimental eyes. Cellularity on tissue sections was evaluated after staining with hematoxylin and eosin. Collagen deposition was visualized by picrosirius red staining. RESULTS: TbetaRII siRNAs abrogated the receptor transcript and protein expression in cultured corneal fibroblasts. The gene knockdown inhibited fibronectin assembly and retarded cell migration. In the mouse model, introduction of TbetaRII specific siRNA significantly reduced the inflammatory response and matrix deposition. CONCLUSIONS: TbetaRII specific siRNAs were efficacious both in vitro and in vivo in knocking down the TGF-beta action. A direct application of siRNA into eyes to downregulate TbetaRII expression may provide a novel therapy for preventing ocular inflammation and scarring.

Complete bilateral gemination of maxillary incisors with separate root canals.

Developmental anomalies in the hard tissue are seen frequently in dental practice. Gemination and fusion are the most commonly encountered anomalies, and distinction between the two is always challenging. Gemination, also called double tooth, is an anomaly exhibiting two joined crowns and usually a single root. It represents an incomplete attempt of a single tooth germ to split. It is considered multifactorial in etiology, with genetic and environmental causes. This paper discusses a rare example of bilateral gemination (prevalence 0.04%) of maxillary central incisors with completely separated roots. Multidisciplinary care ensured a successful esthetic and functional outcome.

High glucose-induced downregulation of connexin 30.2 promotes retinal vascular lesions: implications for diabetic retinopathy.

PURPOSE: To investigate whether high glucose (HG) alters expression of connexin 30.2 (Cx30.2) and influences gap junction intercellular communication (GJIC) in retinal endothelial cells and promotes vascular lesions characteristic of diabetic retinopathy (DR). METHODS: Western blot analysis and immunostaining were performed to determine Cx30.2 protein expression and localization in rat retinal endothelial cells (RRECs) grown in normal (N; 5 mM) or HG (30 mM) medium for 7 days. Concurrently, GJIC was assessed in cells grown in N or HG medium and in cells transfected with Cx30.2 siRNA. Similarly, retinal Cx30.2 expression was assessed in nondiabetic and diabetic rats. Additionally, the effect of reduced Cx30.2 on development of acellular capillaries (ACs) and pericyte loss (PL) was studied in retinas of Cx30.2 knockout mice. RESULTS: Cx30.2 was identified in RRECs in vitro and in vascular cells of retinal capillaries. RRECs grown in HG exhibited significantly reduced Cx30.2 protein levels consistent with decreased Cx30.2 immunostaining compared with those grown in N medium. Cells grown in HG and cells transfected with Cx30.2 siRNA exhibited significantly diminished dye transfer compared with N or nontransfected cells. Importantly, Cx30.2 protein level and immunostaining were decreased in diabetic retinas compared with nondiabetic retinas. Retinal capillaries of Cx30.2 knockout mice exhibited increased numbers of ACs and PL compared with those of wild-type mice. CONCLUSIONS: These results indicate that HG- or diabetes-induced downregulation of Cx30.2 expression and decrease in GJIC activity play a critical role in the development of retinal vascular lesions in early DR.

Complex composite odontoma with characteristic histology.

Odontomas are the most commonly occurring odontogenic tumors, which are considered by many to be hamartomas rather than neoplasms. These clinically asymptomatic tumors are classified into complex and compound odontomas. They are usually discovered in radiographs and rarely cause bony expansion or infection. This paper discusses a case report of a complex odontoma exhibiting all the structural features and defects of enamel, dentine, and cementum in succession, with an overview on its etiology.

Electroretinographic findings in a patient with congenital stationary night blindness due to a novel NYX mutation.

PURPOSE: To document a novel NYX gene mutation in a patient with X-linked complete congenital stationary night blindness and to describe this patient's electroretinogram (ERG) characteristics. METHODS: ERGs were recorded from a 17-year-old male with a previously unreported NYX mutation (819G > A) that results in a missense codon change (Trp237Ter). ERGs were recorded in response to brief-flash stimuli, 6.33-Hz sawtooth flicker, and sinusoidal flicker ranging from 6.33-100 Hz. The omitted stimulus response (OSR) of the flicker ERG, which is thought to be generated within the ON-pathway, was also assessed. RESULTS: The patient's single-flash responses were consistent with previously documented NYX ERG characteristics, including a high-luminance flash response that was electronegative under dark-adapted conditions and a square-like a-wave followed by an abnormally shaped positive potential under light-adapted conditions, both of which are consistent with an ON-pathway deficit. Further evidence for an ON-pathway deficit included: (1) ERGs to rapid-on sawtooth flicker in which b-wave amplitude was reduced more than a-wave amplitude, and (2) responses to sinusoidal flicker that lacked the normal amplitude minimum and phase inflection near 12 Hz, ERG characteristics that are like those of patients with other NYX mutations. Novel findings included a pronounced amplitude attenuation for sinusoidal flicker at frequencies above approximately 50 Hz and an absent OSR, suggesting ON-pathway dysfunction at high frequencies. CONCLUSION: The substantial loss of ERG amplitude and apparent ON-pathway dysfunction at high temporal frequencies distinguish this patient with a Trp237Ter NYX mutation from those with other previously reported NYX mutations.

Calpain expression and activity during lens fiber cell differentiation.

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was alphaII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.

Relation of response to treatment with dorzolamide in X-linked retinoschisis to the mechanism of functional loss in retinoschisin.

PURPOSE: To determine if a positive response of macular cysts to treatment with dorzolamide eye drops in patients with juvenile X-linked retinoschisis (XLRS) can occur with mutations that result in different types of retinoschisin protein dysfunction. DESIGN: Retrospective case series. METHODS: Thirteen eyes of seven patients seen at the University of Illinois at Chicago with a known diagnosis of XLRS were included. Each patient had received or currently was receiving treatment with topical dorzolamide. One patient from each family was screened for a genetic mutation. Using the method of cell transfection and protein preparation, the mutation in each patient was analyzed further and was categorized into one of three groups: 1) total absence of retinoschisin protein secretion, 2) decreased expression of the secreted protein, or 3) secretion of a nonfunctional protein. The response to dorzolamide was observed using optical coherence tomography. RESULTS: Significant improvement in the foveal zone thickness was observed with the use of dorzolamide in three of four patients with absence of protein secretion, in two patients with a lack of protein expression, and in one patient with a nonfunctional protein secretion. CONCLUSIONS: This study showed that the response of macular cysts to dorzolamide in patients with XLRS may be observed independent of the mechanism responsible for retinoschisin protein dysfunction. Hence, treatment with dorzolamide may be effective in patients with different mechanisms of dysfunction in retinoschisin.

Eye lens proteomics: from global approach to detailed information about phakinin and gamma E and F crystallin genes.

Exploration of the lenticular proteome poses a challenging and worthwhile undertaking as cataracts, the products of a disease phenotype elicited by this proteome, remains the leading cause of vision impairment worldwide. The complete ten day old lens proteome of Mus musculus C57BL/6J was resolved into 900 distinct spots by large gel carrier ampholyte based 2-DE. The predicted amino acid sequences of all 16 crystallins ubiquitous in mammals were corroborated by mass spectrometry (MS). In detailed individual spot analyses, the primary structure of the full murine C57BL/6J beaded filament component phakinin CP49 was sequenced by liquid chromatography/electrospray ionization-tandem MS and amended at two positions. This definitive polypeptide sequence was aligned to the mouse genome, thus identifying the entire C57BL/6J genomic coding region. Also, two murine C57/6J polypeptides, both previously classified as gamma F crystallin, were clearly distinguished by MS and electrophoretic mobility. Both were assigned to their respective genes, one of the polypeptides was reclassified as C57BL/6J gamma E crystallin. Building on these data and previous investigations an updated crystallin reference map was put forth and several non crystallin lenticular components were examined. These results represent the first part of a comprehensive investigation of the mouse lens proteome (http://www.mpiib-berlin.mpg.de/2D-PAGE) with emphasis on understanding genetic effects on proteins and disease development.

Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1.

A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the Cx31.9 gene localized to chromosome 17q21.2. Northern analysis of Cx31.9 showed a major 4.4-kilobase transcript, which was expressed at varying levels in all tissues analyzed. Two monoclonal antibodies generated against different domains of Cx31.9 recognized a 30-33-kDa protein from cells overexpressing Cx31.9. Immunofluorescence of overexpressing cells indicated the presence of Cx31.9 between adjacent cells, consistent with its localization to gap junctions. Double voltage clamp analyses of Cx31.9-overexpressing cells, and of paired Xenopus oocytes injected with Cx31.9 cRNA, demonstrated junctional currents indicative of gap junction channel formation. In contrast to previously characterized connexins, Cx31.9 showed no voltage-dependent gating within a physiologically relevant range. Cx31.9 was detected in human tissues by immunoblot analysis, and immunofluorescence localized Cx31.9 expression to vascular smooth muscle cells. Furthermore, it was demonstrated that Cx31.9 interacted with ZO-1. Thus, Cx31.9 represents a novel connexin gene that in vivo generates a protein with unique voltage gating properties.