Punctate Appearance Research Articles

PH-Responsive Rhodamine Nanotube Capable of Self-Reporting the Assembly State.

Nanomaterials that respond to intracellular signals, such as pH, have the potential for many biomedical applications, such as drug delivery, because the assembly/disassembly process can be tailored to respond to a stimulus characteristic of a specific subcellular location. In this work, two rhodamine-peptides that form stable nanotubes at physiological pH but dissociate into highly fluorescent monomers within the acidified interior of endosomal/lysosomal cellular compartments have been developed. The rhodamine dipeptide conjugates, NH2-KK(RhB)-NH2 (RhB-KK) and NH2-EK(RhB)-NH2 (RhB-KE) with rhodamine B chromophores appended at the ε-amino position of a lysine residue, were shown to assemble into well-defined nanotubes at pH values above ∼4-5 and to dissociate into a fluorescent monomer state at lower pH values. The pH dependence of the assembly process was investigated using circular dichroism (CD) and fluorescence spectroscopy along with transmission electron microscopy (TEM), atomic force microscopy (AFM), and confocal imaging. Although the ring opening/closing transition of the rhodamine chromophore took place at pH 4.1 for both peptides, the onset of assembly began at pH 4.6 for RhB-KE and at a comparatively more basic pH (5.8) for RhB-KK. Accordingly, the rhodamine-peptides interconverted between three pH-dependent states: an open-ring, monomeric state (λmax 580 nm, λex 550 nm) at pH values at or below ∼4.6; a closed-ring, nanotube form that exhibits AIEE (λmax 460 nm, λex = 330 nm) at higher pH values; a closed-ring, nonemissive monomeric state that emerged below the critical micelle concentrations (CMC). The pH-responsive features of the peptides were evaluated by live-cell imaging in three cancer cell lines using confocal laser scanning microscopy (CLSM). Visualizing the cells after incubation with either RhB-KE or RhB-KK produced CLSM images with a punctate appearance in the Texas Red channel that colocalized with the lysosomes. These experiments indicate that the nanotubes were rapidly trafficked into the acidic lysosomal compartments within the cells, which induced dissociation into a monomeric, open state. Uptake inhibition studies suggested that cellular uptake was mediated by either caveolae- or clathrin-mediated endocytosis, depending on the cell line studied.

Strawberry Cervix- A Clinical Image of Trichomonas vaginalis: A Case Report

Trichomonas vaginalis is a common cause of symptomatic vaginitis in women. Trichomoniasis occurs more frequently in people with multiple sexual partners. Women often present with vaginal discharge, painful intercourse, urinary tract infection symptoms, vaginal itching, or pelvic pain. The strawberry cervix is a finding upon physical examination where the cervix has an erythematous, punctate, and papilliform appearance. The strawberry cervix is named because of its superficially similar appearance to a strawberry. As opposed to a more general inflammation of the cervix found in cervicitis, the strawberry cervix appearance is considered to be selectively associated with Trichomoniasis. The clinical image of Trichomonas vaginalis is rarely reported. We present a clinical image of the strawberry cervix in a woman diagnosed to have trichomonas vaginalis.

Osteopontin-induced vascular hyperpermeability through tight junction disruption in diabetic retina

Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.

Subcellular localization of D2 receptors in the murine substantia nigra.

G-protein-coupled D2 autoreceptors expressed on dopamine neurons (D2Rs) inhibit transmitter release and cell firing at axonal endings and somatodendritic compartments. Mechanistic details of somatodendritic dopamine release remain unresolved, partly due to insufficient information on the subcellular distribution of D2Rs. Previous studies localizing D2Rs have been hindered by a dearth of antibodies validated for specificity in D2R knockout animals and have been limited by the small sampling areas imaged by electron microscopy. This study utilized sub-diffraction fluorescence microscopy and electron microscopy to examine D2 receptors in a superecliptic pHlourin GFP (SEP) epitope-tagged D2 receptor knockin mouse. Incubating live slices with an anti-SEP antibody achieved the selective labeling of plasma membrane-associated receptors for immunofluorescent imaging over a large area of the substantia nigra pars compacta (SNc). SEP-D2Rs appeared as puncta-like structures along the surface of dendrites and soma of dopamine neurons visualized by antibodies to tyrosine hydroxylase (TH). TH-associated SEP-D2Rs displayed a cell surface density of 0.66puncta/µm2, which corresponds to an average frequency of 1 punctum every 1.50µm. Separate ultrastructural experiments using silver-enhanced immunogold revealed that membrane-bound particles represented 28% of total D2Rs in putative dopamine cells within the SNc. Structures immediately adjacent to dendritic membrane gold particles were unmyelinated axons or axon varicosities (40%), astrocytes (19%), other dendrites (7%), or profiles unidentified (34%) in single sections. Some apposed profiles also expressed D2Rs. Fluorescent and ultrastructural analyses also provided the first visualization of membrane D2Rs at the axon initial segment, a compartment critical for action potential generation. The punctate appearance of anti-SEP staining indicates there is a population of D2Rs organized in discrete signaling sites along the plasma membrane, and for the first time, a quantitative estimate of spatial frequency is provided.

Cellular Mechanism Underlying Highly-Active or Antiretroviral Therapy-Induced Lipodystrophy: Atazanavir, a Protease Inhibitor, Compromises Adipogenic Conversion of Adipose-Derived Stem/Progenitor Cells through Accelerating ER Stress-Mediated Cell Death in Differentiating Adipocytes.

Lipodystrophy is a common complication in human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy (HAART) or antiretroviral therapy (ART). Previous studies demonstrated that endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR) is involved in lipodystrophy; however, the detailed mechanism has not been fully described in human adipogenic cell lineage. We utilized adipose tissue-derived stem cells (ADSCs) obtained from human subcutaneous adipose tissue, and atazanavir (ATV), a protease inhibitor (PI), was administered to ADSCs and ADSCs undergoing adipogenic conversion. Marked repression of adipogenic differentiation was observed when ATV was administered during 10 days of ADSC culture in adipogenic differentiation medium. Although ATV had no effect on ADSCs, it significantly induced apoptosis in differentiating adipocytes. ATV treatment also caused the punctate appearance of CCAAT-enhancer-binding (C/EBP) protein homologous protein (CHOP), and altered expression of CHOP and GRP78/Bip, which are the representation of ER stress, only in differentiating adipocytes. Administration of UPR inhibitors restored adipogenic differentiation, indicating that ER stress-mediated UPR was induced in differentiating adipocytes in the presence of ATV. We also observed autophagy, which was potentiated in differentiating adipocytes by ATV treatment. Thus, adipogenic cell atrophy leads to ATV-induced lipodystrophy, which is mediated by ER stress-mediated UPR and accelerated autophagy, both of which would cause adipogenic apoptosis. As our study demonstrated for the first time that ADSCs are unsusceptible to ATV and its deleterious effects are limited to the differentiating adipocytes, responsible target(s) for ATV-induced lipodystrophy may be protease(s) processing adipogenesis-specific protein(s).

Subcellular Localization And Formation Of Huntingtin Aggregates Correlates With Symptom Onset And Progression In A Huntington'S Disease Model.

Huntington’s disease is caused by the expansion of a CAG repeat within exon 1 of the HTT gene, which is unstable, leading to further expansion, the extent of which is brain region and peripheral tissue specific. The identification of DNA repair genes as genetic modifiers of Huntington’s disease, that were known to abrogate somatic instability in Huntington’s disease mouse models, demonstrated that somatic CAG expansion is central to disease pathogenesis, and that the CAG repeat threshold for pathogenesis in specific brain cells might not be known. We have previously shown that the HTT gene is incompletely spliced generating a small transcript that encodes the highly pathogenic exon 1 HTT protein. The longer the CAG repeat, the more of this toxic fragment is generated, providing a pathogenic consequence for somatic expansion. Here, we have used the R6/2 mouse model to investigate the molecular and behavioural consequences of expressing exon 1 HTT with 90 CAGs, a mutation that causes juvenile Huntington’s disease, compared to R6/2 mice carrying ∼200 CAGs, a repeat expansion of a size rarely found in Huntington’s disease patient’s blood, but which has been detected in post-mortem brains as a consequence of somatic CAG repeat expansion. We show that nuclear aggregation occurred earlier in R6/2(CAG)90 mice and that this correlated with the onset of transcriptional dysregulation. Whereas in R6/2(CAG)200 mice, cytoplasmic aggregates accumulated rapidly and closely tracked with the progression of behavioural phenotypes and with end-stage disease. We find that aggregate species formed in the R6/2(CAG)90 brains have different properties to those in the R6/2(CAG)200 mice. Within the nucleus, they retain a diffuse punctate appearance throughout the course of the disease, can be partially solubilized by detergents and have a greater seeding potential in young mice. In contrast, aggregates from R6/2(CAG)200 brains polymerize into larger structures that appear as inclusion bodies. These data emphasize that a subcellular analysis, using multiple complementary approaches, must be undertaken in order to draw any conclusions about the relationship between HTT aggregation and the onset and progression of disease phenotypes.

Neuronal Repressor REST Controls Ewing Sarcoma Growth and Metastasis by Affecting Vascular Pericyte Coverage and Vessel Perfusion.

Survival rates for Ewing sarcoma (ES) patients with metastatic disease have not improved in over 20 years. Tumor growth and metastasis are dependent on tumor vasculature expansion; therefore, identifying the regulators that control this process in ES may provide new therapeutic opportunities. ES expresses high levels of repressor element 1 silencing transcription factor (REST), which is regulated by the EWS-FLI-1 fusion gene. However, the role of REST in ES growth and the regulation of the tumor vasculature have not been elucidated. To study this role, we established REST-knockout human TC71 ES cell lines through CRISPR/Cas9 recombination. While knockout of REST did not alter tumor cell proliferation in vitro, REST knockout reduced tumor growth and metastasis to the lung in vivo and altered tumor vascular morphology and function. Tumor vessels in the REST-knockout tumors had a punctate appearance with significantly decreased tumor vascular pericytes, decreased perfusion, and increased permeability. REST-knockout tumors also showed increased apoptosis and hypoxia. These results indicate that REST plays a critical role in ES vascular function, which in turn impacts the ability of ES tumors to grow and metastasize. These findings therefore provide a basis for the targeting of REST as a novel therapeutic approach in ES.

Modeling Parkinson's Disease With the Alpha-Synuclein Protein.

Alpha-synuclein (α-Syn) is a key protein involved in Parkinson's disease (PD) pathology. PD is characterized by the loss of dopaminergic neuronal cells in the substantia nigra pars compacta and the abnormal accumulation and aggregation of α-Syn in the form of Lewy bodies and Lewy neurites. More precisely, the aggregation of α-Syn is associated with the dysfunctionality and degeneration of neurons in PD. Moreover, mutations in the SNCA gene, which encodes α-Syn, cause familial forms of PD and are the basis of sporadic PD risk. Given the role of the α-Syn protein in the pathology of PD, animal models that reflect the dopaminergic neuronal loss and the widespread and progressive formation of α-Syn aggregates in different areas of the brain constitute a valuable tool. Indeed, animal models of PD are important for understanding the molecular mechanisms of the disease and might contribute to the development and validation of new therapies. In the absence of animal models that faithfully reproduce human PD, in recent years, numerous animal models of PD based on α-Syn have been generated. In this review, we summarize the main features of the α-Syn pre-formed fibrils (PFFs) model and recombinant adeno-associated virus vector (rAAV) mediated α-Syn overexpression models, providing a detailed comparative analysis of both models. Here, we discuss how each model has contributed to our understanding of PD pathology and the advantages and weakness of each of them.SignificanceHere, we show that injection of α-Syn PFFs and overexpression of α-Syn mediated by rAAV lead to a different pattern of PD pathology in rodents. First, α-Syn PFFs models trigger the Lewy body-like inclusions formation in brain regions directly interconnected with the injection site, suggesting that there is an inter-neuronal transmission of the α-Syn pathology. In contrast, rAAV-mediated α-Syn overexpression in the brain limits the α-Syn aggregates within the transduced neurons. Second, phosphorylated α-Syn inclusions obtained with rAAV are predominantly nuclear with a punctate appearance that becomes diffuse along the neuronal fibers, whereas α-Syn PFFs models lead to the formation of cytoplasmic aggregates of phosphorylated α-Syn reminiscent of Lewy bodies and Lewy neurites.

Phospholipase D2 in prostate cancer: protein expression changes with Gleason score

BackgroundPhospholipases D1 and D2 (PLD1/2) are implicated in tumorigenesis through their generation of the signalling lipid phosphatidic acid and its downstream effects. Inhibition of PLD1 blocks prostate cell growth and colony formation. Here a role for PLD2 in prostate cancer (PCa), the major cancer of men in the western world, is examined.MethodsPLD2 expression was analysed by immunohistochemistry and western blotting. The effects of PLD2 inhibition on PCa cell viability and cell motility were measured using MTS, colony forming and wound-healing assays.ResultsPLD2 protein is expressed about equally in luminal and basal prostate epithelial cells. In cells from different Gleason-scored PCa tissue PLD2 protein expression is generally higher than in non-tumorigenic cells and increases in PCa tissue scored Gleason 6–8. PLD2 protein is detected in the cytosol and nucleus and had a punctate appearance. In BPH tissue stromal cells as well as basal and luminal cells express PLD2. PLD2 protein co-expresses with chromogranin A in castrate-resistant PCa tissue. PLD2 inhibition reduces PCa cell viability, colony forming ability and directional cell movement.ConclusionsPLD2 expression correlates with increasing Gleason score to GS8. PLD2 inhibition has the potential to reduce PCa progression.

Osler weber rendu syndrome: A rare case report

Osler weber rendu syndrome: A rare case report - IJCED- Print ISSN No: - 2581-4710 Online ISSN No:- 2581-4729 Article DOI No:- 10.18231/2581-4729.2019.0020, IP Indian Journal of Clinical and Experimental Dermatology-IP Indian J Clin Exp Dermatol

Presence of IL6 and IL8 receptors within mesenteric and skeletal muscle microvessels in mice

Metabolic diseases like inflammatory conditions result in an upregulation of proinflammatory cytokines (Diabetes Research and Clinical Practice: 105(2); 141–50, 2014). We have previously examined vasoactive response to IL6 and IL8 in mesenteric and skeletal muscle microvessels in normal wild‐type C57 BL/6J and leptin‐deficient db/db mice. Due to the apparent cross‐talk with endothelin related vasoactive responses (The FASEB Journal 27: 687.15, 2013), we confirmed the presence of these receptors within the tissues. Receptors were identified using immunofluorescence with specific antibodies directed towards the IL6 (ABCAM ab83053) and IL8 (CXCR2, ab14935) receptors. Mesenteric tissues and skeletal muscle tissues from C57BL/6J mice [obtained through a tissue sharing facility at SBU] were fixed in 4% paraformaldehyde overnight, then in 30% sucrose in saline overnight to 5 days, imbedded in freezing medium (optimal cutting temperature compound, OCT) and kept at −80C until use. Tissues were sliced 10 um thick using a cryotome. Tissues were first incubated at room temperature with blocking buffer (0.2% BSA). Primary antibodies were diluted to 1:50 to 1:200, and tissues incubated for 30–60 minutes, rinsed 3–6 times with PBS, and then incubated with FITC IgG secondary antibodies (1:50–1:200) for 30–60 minutes, with a final wash in PBS (3–6 times). Tissues were mounted with fluormount and kept cool in the dark until imaging. The results showed that in both mesenteric microvessels and skeletal muscle microvessels, there was a punctate appearance along the axial distribution of vessels indicative of positive staining to both IL6 and IL8 receptor antibodies. These results indicate that vasoactive responses to the IL6 and IL8 cytokines may be specific in nature, and not solely through the endothelin receptor system. We have not ruled out a potential interaction between separate receptor systems as being a requirement for vasoactive responses.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Balancing functions of annexin A6 maintain equilibrium between hypertrophy and apoptosis in cardiomyocytes.

Pathological cardiac hypertrophy is a major risk factor associated with heart failure, a state concomitant with increased cell death. However, the mechanism governing progression of hypertrophy to apoptosis at the single-cell level remains elusive. Here, we demonstrate annexin A6 (Anxa6), a calcium (Ca2+)-dependent phospholipid-binding protein critically regulates the transition of chronic hypertrophied cardiomyocytes to apoptosis. Treatment of the H9c2(2-1) cardiomyocytes with hypertrophic agonists upregulates and relocalizes Anxa6 with increased cytosolic punctate appearance. Live cell imaging revealed that chronic exposure to hypertrophic agonists such as phenylephrine (PE) compromises the mitochondrial membrane potential (ΔΨm) and morphological dynamics. Such chronic hypertrophic induction also activated the caspases 9 and 3 and induced cleavage of the poly-(ADP-ribose) polymerase 1 (Parp1), which are the typical downstream events in the mitochondrial pathways of apoptosis. An increased rate of apoptosis was evident in the hypertrophied cardiomyocytes after 48–72 h of treatment with the hypertrophic agonists. Anxa6 was progressively associated with the mitochondrial fraction under chronic hypertrophic stimulation, and Anxa6 knockdown severely abrogated mitochondrial network and dynamics. Ectopically expressed Anxa6 protected the mitochondrial morphology and dynamics under PE treatment, and also increased the cellular susceptibility to apoptosis. Biochemical analysis showed that Anxa6 interacts with Parp1 and its 89 kDa cleaved product in a Ca2+-dependent manner through the N-terminal residues (1–28). Furthermore, expression of Anxa6S13E, a mutant dominant negative with respect to Parp1 binding, served as an enhancer of mitochondrial dynamics, even under chronic PE treatment. Chemical inhibition of Parp1 activity released the cellular vulnerability to apoptosis in Anxa6-expressing stable cell lines, thereby shifting the equilibrium away from cell death. Taken together, the present study depicts a dual regulatory function of Anxa6 that is crucial for balancing hypertrophy with apoptosis in cardiomyocytes.

Mitochondrial properties in Rhabdomyosarcoma: An insight into the molecular mechanisms of muscle differentiation

Rhabdomyosarcoma (RMS) is a type of cancer that arises in muscle tissue and primarily affects children. RMS cells remain immature and thus, their lack of differentiation into mature muscle cells is one of the hallmarks of this disease. The cause of this lack of differentiation remains unknown. In this study, we hope to gain an insight into the molecular mechanisms of RMS cell differentiation and how mitochondrial properties may be involved in this process, particularly related to mitochondrial movement, morphology and protein markers of mitochondrial biogenesis. For the purpose of this study, human skeletal muscle cells (HSKM) and rhabdomyosarcoma (RD) were induced to differentiate for a period of 7 days. While no difference was observed in mitochondrial speed, acceleration and distance travelled from the origin in HSKM cells after 7 days of differentiation, RD cells exhibited an approximate 2‐ fold increase in speed and acceleration after 7 days of differentiation. Moreover, mitochondria in RD cells had a more punctate appearance after 7 days of differentiation. p53, a regulator of cell cycle arrest, was markedly elevated in RD cells. However, BAX, a regulator of apoptosis, remained highly expressed in HSMK cells throughout the 7 day differentiation period. While these changes occurred in HSMK and RD cells, no difference in PGC1‐α, a marker of mitochondrial biogenesis, was observed. This study suggests that mitochondrial properties and motility are altered in RD cells when induced to differentiate, and this could be related to the degree of tumorigenesis. Supported by NSERC and Mitacs.

Mdm20 stimulates polyQ aggregation via inhibiting autophagy through Akt-Ser473 phosphorylation.

Mdm20 is an auxiliary subunit of the NatB complex, which includes Nat5, the catalytic subunit for protein N-terminal acetylation. The NatB complex catalyzes N-acetylation during de novo protein synthesis initiation; however, recent evidence from yeast suggests that NatB also affects post-translational modification of tropomyosin, which is involved in intracellular sorting of aggregated proteins. We hypothesized that an acetylation complex such as NatB may contribute to protein clearance and/or proteostasis in mammalian cells. Using a poly glutamine (polyQ) aggregation system, we examined whether the NatB complex or its components affect protein aggregation in rat primary cultured hippocampal neurons and HEK293 cells. The number of polyQ aggregates increased in Mdm20 over-expressing (OE) cells, but not in Nat5-OE cells. Conversely, in Mdm20 knockdown (KD) cells, but not in Nat5-KD cells, polyQ aggregation was significantly reduced. Although Mdm20 directly associates with Nat5, the overall cellular localization of the two proteins was slightly distinct, and Mdm20 apparently co-localized with the polyQ aggregates. Furthermore, in Mdm20-KD cells, a punctate appearance of LC3 was evident, suggesting the induction of autophagy. Consistent with this notion, phosphorylation of Akt, most notably at Ser473, was greatly reduced in Mdm20-KD cells. These results demonstrate that Mdm20, the so-called auxiliary subunit of the translation-coupled protein N-acetylation complex, contributes to protein clearance and/or aggregate formation by affecting the phosphorylation level of Akt indepenently from the function of Nat5.

Characterization of two pathogenic mutations in cystathionine beta-synthase: Different intracellular locations for wild-type and mutant proteins

Cystathionine β-synthase (CBS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine with serine to generate cystathionine. Homocystinuria is an autosomal recessive disorder commonly caused by a deficiency of CBS activity. Here, we characterized a novel CBS mutation (c.260C>A (p.T87N)) and a previously reported variant (c.700G>A (p.D234N)) found in Venezuelan homocystinuric patients, one nonresponsive and one responsive to vitamin B6. Both mutant proteins were expressed in vitro in prokaryotic and eukaryotic cells, finding lower soluble expression in HEK-293 cells (19% T87N and 23% D234N) compared to wild-type CBS. Residual activities obtained for the mutant proteins were 3.5% T87N and 43% D234N. Gel exclusion chromatography demonstrated a tendency of the T87N mutant to aggregate while the distribution of the D234N mutant was similar to wild-type enzyme. Using immunofluorescence microscopy, an unexpected difference in intracellular localization was observed between the wild-type and mutant proteins. While the T87N mutant exhibited a punctate appearance, the wild-type protein was homogeneously distributed inside the cell. Interestingly, the D234N protein showed both distributions. This study demonstrates that the pathogenic CBS mutations generate unstable proteins that are unable (T87N) or partially unable (D234N) to assemble into a functional enzyme, implying that these mutations might be responsible for the homocystinuria phenotype.

Distinct synaptic localization patterns of brefeldin A‐resistant guanine nucleotide exchange factors BRAG2 and BRAG3 in the mouse retina

The BRAG/IQSEC is a family of guanine nucleotide exchange factors for ADP ribosylation factors, small GTPases that regulate membrane trafficking and actin cytoskeleton, and comprises three structurally related members (BRAG1-3) generated from different genes. In the mouse retina, BRAG1 (also known as IQSEC2) was previously shown to localize at synaptic ribbons of photoreceptor terminals and to form a protein complex with RIBEYE. In this study, we examined the immunohistochemical localization of BRAG2 (IQSEC1) and BRAG3 (IQSEC3) in the adult mouse retina at the light and electron microscopic levels. In the outer plexiform layer, BRAG2 showed a punctate distribution in intimate association with dystrophin and β-dystroglycan. Immunoelectron microscopic analysis revealed that BRAG2 localized at specific subcompartments of photoreceptor terminals in both rod spherules and cone pedicles. In the inner plexiform layer, immunolabeling for both BRAG2 and BRAG3 had a punctate appearance, suggestive of synaptic labeling. Double immunostaining demonstrated that BRAG2 colocalized preferentially with PSD-95 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptors (AMPARs). By contrast, BRAG3 colocalized with gephyrin and a subpopulation of inhibitory synapses expressing glycine receptors or γ-aminobutyric acid type A receptors (GABA(A) Rs). Immunoelectron microscopic analysis revealed that BRAG2 localized to postsynaptic processes at bipolar dyads, while BRAG3 localized to postsynaptic components at conventional synapses. These findings suggest that BRAG/IQSEC family members have key roles in the function and organization of distinct excitatory and inhibitory synapses in the retina.

Generation of striatal projection neurons extends into the neonatal period in the rat brain

Substantial advances have been made in the last decade on our understanding of the basic physiology underlying neurogenesis in the postnatal mammalian brain. The bulk of the work in this area has been based on analysis of the adult brain. Relatively less is known about the capacity for neurogenesis in specific structures within the neonatal brain. Here we report that the production of medium spiny striatal projection neurons extends into the early neonatal period under normal physiological conditions in the rat brain. Birth-dating of newborn cells with bromodeoxyuridine at postnatal days 0, 2 and 5 showed a peak production close to birth, which sharply declined at the later time-points. Additionally, there was a low-level but stable contribution of neurons with interneuron identity over the same time-period. Importantly, retroviral labelling of new striatal projection neurons with green fluorescent protein showed long-term survival and terminal differentiation with characteristic morphology, including highly elaborated spiny dendrites, and appropriate axonal targeting of the globus pallidus and midbrain. This latent period of striatal neurogenesis in the early neonatal brain represents an interesting target for regenerative approaches aimed at restoring striatal circuitry in perinatal pathologies, such as hypoxic and ischaemic damage associated with cerebral palsy.

Biophysical and Functional Analyses Suggest That Adenovirus E4-ORF3 Protein Requires Higher-order Multimerization to Function against Promyelocytic Leukemia Protein Nuclear Bodies

The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT·L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity.

Abstract 1114: Loss of OPA1 expression results in apoptosis for cells overexpressing c-Myc

Abstract c-Myc (Myc) is a transcription factor that controls the expression of a vast array of genes that function in many essential cellular pathways, and high levels of Myc have been implicated in the development of many human cancers. One pathway that Myc controls is mitochondrial biogenesis. We have previously shown that Myc does this is in part via the regulation of the expression of proteins involved in mitochondrial fusion, including Opa1, whose levels are increased upon the over-expression of Myc. This results in a highly interconnected mitochondrial network in cells with high levels of Myc. This fused mitochondrial network is present despite the fact that Myc also increases the expression of proteins involved in mitochondrial fission as well. We sought to understand how Myc controls the expression of Opa1 and to determine if the high levels of mitochondrial fusion in Myc over-expressing cells are important in cellular transformation. We hypothesized that the increased mitochondrial fusion driven by the Myc-mediated increase in Opa1 may reduce apoptosis. Rat fibroblasts with inducible Myc expression were transfected with an OPA1-specific shRNA. These cells were characterized by a series of assays that measured different aspects of mitochondrial structure and function. After only 48h of Myc induction, mitochondrial mass increased by approximately two-fold compared to control cells; however, when Opa1 levels were reduced in this background (Myc+/Opa-), mitochondrial mass was decreased four-fold. The cells with decreased Opa1 expression but minimal Myc activity (Myc-/Opa-) had a punctate appearance to the mitochondria, which became even more dramatic in Myc+/Opa- cells. This was likely related to the ability of Myc to increase the proteins involved in fission. There was an approximate 50% decrease in the ability to perform oxidative phosphorylation (OXPHOS) when Myc-/Opa- cells were compared to Myc+/Opa- cells. This was consistent with the four-fold decrease in net ATP levels when Myc-/Opa- cells were compared to Myc+/Opa- cells. Finally, prolonged activation of Myc in cells with decreased expression of OPA1 leads to apoptosis. After 48 hours of Myc induction the Myc+/Opa- cells the cells gradually began to die and cultures were nearly devoid of viable cells within 10 days, whereas Myc-/Opa- cells remained almost completely viable. We believe that the unopposed mitochondrial fission in Myc+/Opa- cells results in an increase susceptibility to an early apoptotic death. The mechanisms involved in this phenomenon are being explored, as is the possibility of exploiting this consequence of Opa1 reduction as a means of selectively eliminating tumor cells that display Myc over-expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1114. doi:1538-7445.AM2012-1114

First Report of Neocosmospora striata Causing Peanut Pod Rot in China.

In 2008, an outbreak of pod rot of peanut (Arachis hypogaea L.) occurred on most of the peanut cultivars in the Old Yellow River drainage area, the largest peanut-growing region in China. Disease incidence reached as high as 90% in some fields, causing severe yield losses. The black rot of pods and blackened, nonrotting taproots is similar to symptoms of peanut black rot caused by Cylindrocladium parasiticum, but the reddish orange perithecia of C. parasiticum were not found on the taproots close to the surface of the soil. The foliage of affected plants was generally asymptomatic, but some plants turned greener. This pod rot disease was further investigated in 2008 and 2010. Twenty-three Fusarium-like isolates were obtained from symptomatic, surface-disinfested pods with a frequency of 82%. These isolates were fast growing, with flat, thin, and grayish white colonies when cultured on potato dextrose agar (PDA) at 28°C for 3 to 4 days. The hyaline, elongated to cylindrical conidia, aggregated in slimy heads on conidiogenous cells developed from undifferentiated hyphae when observed with the light microscope. The size of conidia (single celled or one septum) varied from 3 to 9 μm long and 1.5 to 3.5 μm wide on the basis of the measurement of 50 spores. Some conidia appeared slightly curved. Ascomata formed within 10 to 14 days, with a punctate appearance on the colony. The cerebriform ascomata were dark brown, pyriform, ostiolate, glabrous, 120 to 170 × 90 to 130 μm, and with necks 30 to 50 μm long. Asci measured 60 to 90 × 6 to 10 μm, were cylindrical to cylindric-clavate, thin walled, and had an apical ring. Ascospore arrangement was obliquely uniseriate or partially biseriate, very pale yellow to hyaline, ellipsoidal, and measured 8 to 12 × 4.5 to 6 μm. Some spores had a median transverse straight or curved septum and were slightly constricted at the septum, with 6 to 10 thin, transverse, hyaline flanges. Morphological characteristics of the isolates with ascomata dark brown and ascospores with 6 to 10 transverse hyaline flanges matched the description for Neocosmospora striata (1). The internal transcribed spacer (ITS) region of rDNA was amplified from extracted template DNA with primer pairs ITS4/ITS5 and sequenced. A 591-bp amplicon (GenBank Accession No. HM461900) had 99% sequence identity with Fusarium solani (HQ607968 and HQ608009) and N. vasinfecta (GU213063), which indicated that these fungi belong to the genus Neocosmospora or Fusarium, although there is no direct sequence evidence that they are N. striata. N. striata has only been previously reported in Japan (2). This species is unique because of the dark brown ascomata and there is no comparable species (1). Koch's postulates were completed by surface-disinfesting 80 peanut pods of cv. Jihua 9813 and soaking them in conidial suspensions (105 conidia/ml) for 2 min. Another 80 other pods soaked in sterile water served as controls. All peanuts were incubated in moist petri dishes under darkness at 28°C. Symptoms similar to those originally observed in the field formed within 10 days on all inoculated peanut pods and not the controls. N. striata was reisolated from all affected peanut pods. To our knowledge, this is first report of N. striata causing peanut pod rot in China and the first description of the anamorph of the fungus.