Nalin M. Kumar

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 β-catenin (cardamonin; 10 μmol/l) or GSK3-α/β (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.

Differences in expression patterns between mouse connexin-30.2 (Cx30.2) and its putative human orthologue, connexin-31.91

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.

Relation of Response to Treatment with Dorzolamide in X-Linked Retinoschisis to the Mechanism of Functional Loss in Retinoschisin

PURPOSEnTo 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.nnnDESIGNnRetrospective case series.nnnMETHODSnThirteen 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.nnnRESULTSnSignificant 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.nnnCONCLUSIONSnThis 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.

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 α3C×46 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 γ‐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.

High Glucose–Induced Downregulation of Connexin 30.2 Promotes Retinal Vascular Lesions: Implications for Diabetic Retinopathy

PURPOSEnTo 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).nnnMETHODSnWestern 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.nnnRESULTSnCx30.2 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.nnnCONCLUSIONSnThese 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.

The necessity of functional proteomics: protein species and molecular function elucidation exemplified by in vivo alpha A crystallin N-terminal truncation.

Summary.Ten years after the establishment of the term proteome, the science surrounding it has yet to fulfill its potential. While a host of technologies have generated lists of protein names, there are only a few reported studies that have examined the individual proteins at the covalent chemical level defined as protein species in 1997 and their function. In the current study, we demonstrate that this is possible with two-dimensional gel electrophoresis (2-DE) and mass spectrometry by presenting clear evidence of in vivo N-terminal alpha A crystallin truncation and relating this newly detected protein species to alpha crystallin activity regulation by protease cleavage in the healthy young murine lens. We assess the present state of technology and suggest a shift in resources and paradigm for the routine attainment of the protein species level in proteomics.

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.

Electroretinographic Findings in a Patient with Congenital Stationary Night Blindness Due to a Novel NYX Mutation

ABSTRACT 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 (819Gu2009>u2009A) 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 6.33–100u2009Hz. 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 12u2009Hz, 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 50u2009Hz 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.

Analysis of and expression of connexin 26 mutations

Problem: Mutations in the gap junction beta-2 (GJB2) gene, which encodes the connexin 26 protein, account for 50% of all the patients with autosomal recessive nonsyndromic hearing loss. Connexins are proteins that oligomerize to form a gap junction channel. Gap junctions enable intercellular communication between cell membranes of 2 neighboring cells. Mutations in these connexins lead to absent or malformed gap junctions and congenital deafness. The expression of different connexin mutations’ molecules has not been fully evaluated. n nMethods: Based on their prevalence and inheritance, 4 specific mutations were chosen (35delG, R184P, R184Q, and C202F). Primers were designed based on specific mutations and site-directed PCR mutagenesis was performed using Stratagene’s Quikchange site-directed mutagenesis kit. Mutations were confirmed via DNA sequencing. The mutated GJB2 genes and wild-type GJB2 were inserted into the EcoRI site of the pNUT vector and transfected into baby-kidney-cells (BHK) using Lipofectamine. Expression of mutated genes was analyzed using immunofluorescence. n nResults: Mutations (35delG, R184P, R184Q, C202F) were successfully created using site-directed PCR mutagenesis. DNA sequencing confirmed desired mutations. Mutations and wild-type GJB2 genes were successfully expressed in BHK cells and analyzed using immunofluorescence. Specific results and difference will be presented. n nConclusion: Specific GJB2 mutations (35delG, R184P, 184Q, C202F) were successfully created and expressed. Immunofluorescence results confirm specific difference between wild-type GJB2 and GJB2 mutations. This study was the first to successfully express and analyze mutations R184Q and C202F and to express mutations 35delG and R184P in a mouse in vitro system. n nSignificance: As noted above, mutations in the gap junction beta-2 (GJB2) gene, which encodes the connexin 26 protein, account for 50% of all the patients with autosomal recessive nonsyndromic hearing loss. Expression and analysis of these mutations are crucial to our understanding of this disease and to our ability to provide potential therapy via targeted gene therapy in the future. n nSupport: None reported.